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Sample records for cells control mouse

  1. Control of cell division and radiation injury in mouse skin

    International Nuclear Information System (INIS)

    Yamaguchi, Takeo

    1974-01-01

    The method for determining the inhibitors of cell division (chalone-adrenalin system) in the irradiated epidermis and blood was developed using the epidermis of mouse ear conch during the cure of wounds (in vivo), and the epidermis cultured for a long period (in vitro). The whole body was irradiated with 200KV, 20 mA x-rays of 96 R/min filtered by 0.5 mmCu + 0.5 mmAl. Chalone, which is a physiologically intrinsic substance to control the proliferation, inhibits the DNA synthesis. From changes in cell division with time, chalone in the epidermis is considered to inhibit each process from G 2 to M, from G 2 to S, from G 1 to S. Adrenalin is indispensable when epidermal chalone acts the inhibition of cell division. Chalone activities in the epidermis irradiated with almost lethal doses were decreased. Factors to inhibit the proliferation of the epidermis by the potentiation of chalone and adrenalin are present in sera of animals irradiated to x-rays. (Serizawa, K.)

  2. EBI3 regulates the NK cell response to mouse cytomegalovirus infection

    DEFF Research Database (Denmark)

    Jensen, Helle; Chen, Shih-Yu; Folkersen, Lasse Westergaard

    2017-01-01

    Natural killer (NK) cells are key mediators in the control of cytomegalovirus infection. Here, we show that Epstein-Barr virus-induced 3 (EBI3) is expressed by human NK cells after NKG2D or IL-12 plus IL-18 stimulation and by mouse NK cells during mouse cytomegalovirus (MCMV) infection. The induc......Natural killer (NK) cells are key mediators in the control of cytomegalovirus infection. Here, we show that Epstein-Barr virus-induced 3 (EBI3) is expressed by human NK cells after NKG2D or IL-12 plus IL-18 stimulation and by mouse NK cells during mouse cytomegalovirus (MCMV) infection....... The induction of EBI3 protein expression in mouse NK cells is a late activation event. Thus, early activation events of NK cells, such as IFNγ production and CD69 expression, were not affected in EBI3-deficient (Ebi3-/-) C57BL/6 (B6) mice during MCMV infection. Furthermore, comparable levels of early viral...... replication in spleen and liver were observed in MCMV-infected Ebi3-/- and wild-type (WT) B6 mice. Interestingly, the viral load in salivary glands and oral lavage was strongly decreased in the MCMV-infected Ebi3-/- B6 mice, suggesting that EBI3 plays a role in the establishment of MCMV latency. We detected...

  3. Establishment of mouse neuron and microglial cell co-cultured models and its action mechanism.

    Science.gov (United States)

    Zhang, Bo; Yang, Yunfeng; Tang, Jun; Tao, Yihao; Jiang, Bing; Chen, Zhi; Feng, Hua; Yang, Liming; Zhu, Gang

    2017-06-27

    The objective of this study is to establish a co-culture model of mouse neurons and microglial cells, and to analyze the mechanism of action of oxygen glucose deprivation (OGD) and transient oxygen glucose deprivation (tOGD) preconditioning cell models. Mouse primary neurons and BV2 microglial cells were successfully cultured, and the OGD and tOGD models were also established. In the co-culture of mouse primary neurons and microglial cells, the cell number of tOGD mouse neurons and microglial cells was larger than the OGD cell number, observed by a microscope. CCK-8 assay result showed that at 1h after treatment, the OD value in the control group is lower compared to all the other three groups (P control group compared to other three groups (P neurons cells were cultured. In the meantime mouse BV2 microglia cells were cultured. Two types of cells were co-cultured, and OGD and tOGD cell models were established. There were four groups in the experiment: control group (OGD), treatment group (tOGD+OGD), placebo group (tOGD+OGD+saline) and minocycline intervention group (tOGD+OGD+minocycline). CCK-8 kit was used to detect cell viability and flow cytometry was used to detect apoptosis. In this study, mouse primary neurons and microglial cells were co-cultured. The OGD and tOGD models were established successfully. tOGD was able to effectively protect neurons and microglial cells from damage, and inhibit the apoptosis caused by oxygen glucose deprivation.

  4. Growth and production kinetics of human x mouse and mouse hybridoma cells at reduced temperature and serum content.

    Science.gov (United States)

    Borth, N; Heider, R; Assadian, A; Katinger, H

    1992-09-01

    The growth and production kinetics of a mouse hybridoma cell line and a human-mouse heterohybridoma were analyzed under conditions of reduced temperature and serum content. The mouse hybridoma P24 had a constant cell specific production rate and RNA content, while the heterohybridoma 3D6-LC4 showed growth associated production kinetics and an increased RNA content at higher growth rates. This behaviour of 3D6-LC4 cells can be explained by the unusual cell cycle kinetics of this line, which can be arrested in any phase under growth limiting conditions, so that a low growth rate does not result in a greater portion of high producing G1-phase cells. Substrate limitation changes the cell cycle distribution of this cell line to a greater extent than low temperature or serum content, which indicates that this stress factor exerts a greater physiological control than assumed.

  5. Inference of Transcriptional Network for Pluripotency in Mouse Embryonic Stem Cells

    International Nuclear Information System (INIS)

    Aburatani, S

    2015-01-01

    In embryonic stem cells, various transcription factors (TFs) maintain pluripotency. To gain insights into the regulatory system controlling pluripotency, I inferred the regulatory relationships between the TFs expressed in ES cells. In this study, I applied a method based on structural equation modeling (SEM), combined with factor analysis, to 649 expression profiles of 19 TF genes measured in mouse Embryonic Stem Cells (ESCs). The factor analysis identified 19 TF genes that were regulated by several unmeasured factors. Since the known cell reprogramming TF genes (Pou5f1, Sox2 and Nanog) are regulated by different factors, each estimated factor is considered to be an input for signal transduction to control pluripotency in mouse ESCs. In the inferred network model, TF proteins were also arranged as unmeasured factors that control other TFs. The interpretation of the inferred network model revealed the regulatory mechanism for controlling pluripotency in ES cells

  6. Retinoic acid combined with spermatogonial stem cell conditions facilitate the generation of mouse germ-like cells

    DEFF Research Database (Denmark)

    Dong, Guoyi; Shang, Zhouchun; Liu, Longqi

    2017-01-01

    Spermatogenic lineage has been directly generated in spermatogonial stem cell (SSC) conditions from human pluripotent stem cells (PSCs). However, it remains unknown whether mouse embryonic stem cells (ESCs) can directly differentiate into advanced male germ cell lineage in the same conditions. Here......, we showed rather low efficiency of germ-like cell generation from mouse ESCs in SSC conditions. Interestingly, addition of retinoic acid (RA) into SSC conditions enabled efficient differentiation of mouse ESCs into germ-like cells, as shown by the activation of spermatogenesis-associated genes...... such as Mvh, Dazl, Prdm14, Stella, Scp1, Scp3, Stra8 and Rec8. In contrast, for cells cultured in control medium, the activation of the above genes barely occurred. In addition, RA with SSC conditions yielded colonies of Acrosin-expressing cells and the positive ratio reached a peak at day 6. Our work thus...

  7. Exogenous lactate interferes with cell-cycle control in BALB/3T3 mouse fibroblasts

    International Nuclear Information System (INIS)

    Rutz, H. Peter; Little, John B.

    1995-01-01

    Purpose: Previous studies have shown that exogenous lactate may influence proliferation rates, radiation sensitivity, and postirradiation repair capacity of mammalian cells. In the present study, we addressed the question of potential underlying mechanisms and, therefore, examined effects of exogenous lactate on proliferation rates and cell-cycle distribution in immortal but nontumorigenic mammalian cells. Methods and Materials: Cells were grown at 37 deg. C in an incubator with 5% CO 2 and 95% air, in a culture medium supplemented or not with lactate at a 10 mM concentration. Daily, we changed the culture medium and counted cells per dish. On selected days, cell-cycle distribution was determined by flow cytometry. Balb/3T3 mouse fibroblasts were used. Results: During the exponential phase of cell proliferation, mean population doubling time was significantly increased from 17.7 to 19.9 h, due to selective prolongation of G 2 /M. However, in density-inhibited cultures, exogenous lactate stimulated entry into S and proliferation to a significantly higher saturation density. Conclusions: These findings indicate that exogenous lactate interferes with mechanisms of cell-cycle control at two different points in the cell-cycle, depending on cell density and the resulting absence or presence of inhibition of cell proliferation. Interference with cell-cycle control may underlay the modification by exogenous lactate of radiosensitivity and postirradiation repair capacity in mammalian cells

  8. Epithelial cell kinetics in mouse and rat skin irradiated with electrons

    International Nuclear Information System (INIS)

    McMaster-Schuyler, L.

    1984-02-01

    Experiments were performed to examine the kinetic responses of mouse and rat epidermal cells in vivo after single doses of ionizing radiation including responses of hair follicles at times after irradiation. The labeling indices in both species were reduced to 30 to 50% of control values immediately following irradiation at all the doses. In the rat, the labeling indices recovered and overshot control values within the first three days after 300 to 1200 rads. The mouse labeling indices continued to be suppressed for up to 10 days after 300 to 2400 rads. This indicated that rat G 1 phase epidermal cells recovered three times faster than those of the mouse with respect to the ability to maintain or increase control level cell proliferation after irradiation. After 1800 and 2400 rads, doses which produce skin ulceration, both species showed a reduction in their labeling indices for up to 7 days, indicating that a dose-dependent mechanism of recovery may be operable in the rat. 99 refs., 15 figs., 6 tabs

  9. Mel-18 controls the enrichment of tumor-initiating cells in SP fraction in mouse breast cancer.

    Science.gov (United States)

    Janakiraman, Harinarayanan; Nobukiyo, Asako; Inoue, Hiroko; Kanno, Masamoto

    2011-06-01

    Side population (SP) cell analysis has been used to identify and isolate a minor population of cells with stem cell properties in normal tissues and in many cancers including breast cancer cells. However, the molecular mechanisms that operate in tumor-initiating cells (TICs) in SP fraction remain unclear. The Polycomb group genes, including Bmi1 and Mel-18, have been implicated in the maintenance of hematopoietic stem cells (HSCs) and suggested to be oncogenic and tumor suppressive, respectively, in breast cancer. In this study, we determined the critical role of Mel-18 in the enrichment mechanisms of TICs with the SP phenotype in a mouse breast cancer cell line, MMK3, that was established from a breast cancer developed spontaneously in Mel-18+/- mice. The Mel-18 protein expression level significantly correlates to the percentage of SP fraction in the mouse breast cancer cell line MMK3 series. The comparison between MMK3V3 (V3) cells containing one copy of the Mel-18 gene and MMK3S2 (S2) cells having twice the amount of Mel-18 expression clearly demonstrates the above relationship. Similar results obtained with the percentage of ALDH+ cells in V3 and S2 further confirmed the correlation between protein expression level of Mel-18 and the TICs. More importantly, transplantation of SP and non-SP cells of V3 and S2 cells into the NOD/SCID mice clearly showed that the heterozygous level of Mel-18 leads to the disappearance of enrichment of TICs into SP fraction in vivo. Stem cell pathway focused gene expression profiling of V3 and S2 cells revealed that the genes Abcg2, Aldh1a1 and Dhh were highly down-regulated in V3 compared to S2. These results indicate that the precise Mel-18 expression level controls TIC enrichment mechanisms through the regulation of channel molecule of Abcg2 and functional TIC marker of Aldhlal. In conclusion, our findings revealed the significance of fine-tuning mechanisms for Mel-18 protein expression level in the maintenance of TIC into SP

  10. Mouse endometrial stromal cells produce basement-membrane components

    DEFF Research Database (Denmark)

    Wewer, U M; Damjanov, A; Weiss, J

    1986-01-01

    During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations. The dec......During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations....... Mouse decidual cells isolated from 6- to 7-day pregnant uteri explanted in vitro continue to synthesize basement-membrane-like extracellular matrix. Using immunohistochemistry and metabolic labeling followed by immunoprecipitation, SDS-PAGE, and fluorography, it was shown that the decidual cells...... to undergo pseudodecidualization. We thus showed that stromal cells from pregnant and nonpregnant mouse uteri synthesize significant amounts of basement-membrane components in vitro, and hence could serve as a good model for the study of normal basement-membrane components....

  11. The Role of Controlled Surface Topography and Chemistry on Mouse Embryonic Stem Cell Attachment, Growth and Self-Renewal.

    Science.gov (United States)

    Macgregor, Melanie; Williams, Rachel; Downes, Joni; Bachhuka, Akash; Vasilev, Krasimir

    2017-09-14

    The success of stem cell therapies relies heavily on our ability to control their fate in vitro during expansion to ensure an appropriate supply. The biophysical properties of the cell culture environment have been recognised as a potent stimuli influencing cellular behaviour. In this work we used advanced plasma-based techniques to generate model culture substrates with controlled nanotopographical features of 16 nm, 38 nm and 68 nm in magnitude, and three differently tailored surface chemical functionalities. The effect of these two surface properties on the adhesion, spreading, and self-renewal of mouse embryonic stem cells (mESCs) were assessed. The results demonstrated that physical and chemical cues influenced the behaviour of these stem cells in in vitro culture in different ways. The size of the nanotopographical features impacted on the cell adhesion, spreading and proliferation, while the chemistry influenced the cell self-renewal and differentiation.

  12. Assessment of plasminogen synthesis in vitro by mouse tumor cells using a competition radioimmunoassay for mouse plasminogen

    International Nuclear Information System (INIS)

    Roblin, R.O.; Bell, T.E.; Young, P.L.

    1978-01-01

    A sensitive, specific competition radioimmunoassay for mouse plasmin(ogen) has been developed in order to determine whether mouse tumor cells can synthesize plasminogen in vitro. The rabbit anti-BALB/c mouse plasminogen antibodies used in the assay react with the plasminogen present in serum from BALB/c, C3H, AKR and C57BL/6 mice, and also recognized mouse plasmin. The competition radiommunoassay can detect as little as 50 ng of mouse plasminogen. No competition was observed with preparations of fetal calf, human and rabbit plasminogens. A variety of virus-transformed and mouse tumor cell lines were all found to contain less than 100 ng mouse plasminogen/mg of cell extract protein. Thus, if the plasminogen activator/plasmin system is important in the growth or movement of this group of tumor cells, the cells will be dependent upon the circulatory system of the host for their plasminogen supply. (Auth.)

  13. CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

    Science.gov (United States)

    Cai, Na; Kurachi, Masashi; Shibasaki, Koji; Okano-Uchida, Takayuki; Ishizaki, Yasuki

    2012-03-01

    Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in

  14. In Vitro Chondrogenesis Transformation Study of Mouse Dental Pulp Stem Cells

    Directory of Open Access Journals (Sweden)

    Shahrul Hisham Zainal Ariffin

    2012-01-01

    Full Text Available A major challenge in the application of mesenchymal stem cells in cartilage reconstruction is that whether the cells are able to differentiate into fully mature chondrocytes before grafting. The aim of this study was to isolate mouse dental pulp stem cells (DPSC and differentiate them into chondrocytes. For this investigation, morphological, molecular, and biochemical analyses for differentiated cells were used. To induce the chondrocyte differentiation, DPSC were cultured in chondrogenic medium (Zen-Bio, Inc.. Based on morphological analyses using toluidine blue staining, proteoglycan products appear in DPSC after 21 days of chondrocyte induction. Biochemical analyses in differentiated group showed that alkaline phosphatase activity was significantly increased at day 14 as compared to control (P<0.05. Cell viability analyses during the differentiation to chondrocytes also showed that these cells were viable during differentiation. However, after the 14th day of differentiation, there was a significant decrease (P<0.05 in the viability proportion among differentiated cells as compared to the control cells. In RT-PCR molecular analyses, mouse DPSC expressed Cd146 and Cd166 which indicated that these cells belong to mesenchymal stem cells. Coll I and Coll II markers showed high expression after 14 and 21 days, respectively. In conclusion, this study showed that DPSC successfully differentiated into chondrocytes.

  15. JMJD1C Ensures Mouse Embryonic Stem Cell Self-Renewal and Somatic Cell Reprogramming through Controlling MicroRNA Expression.

    Science.gov (United States)

    Xiao, Feng; Liao, Bing; Hu, Jing; Li, Shuang; Zhao, Haixin; Sun, Ming; Gu, Junjie; Jin, Ying

    2017-09-12

    The roles of histone demethylases (HDMs) for the establishment and maintenance of pluripotency are incompletely characterized. Here, we show that JmjC-domain-containing protein 1c (JMJD1C), an H3K9 demethylase, is required for mouse embryonic stem cell (ESC) self-renewal. Depletion of Jmjd1c leads to the activation of ERK/MAPK signaling and epithelial-to-mesenchymal transition (EMT) to induce differentiation of ESCs. Inhibition of ERK/MAPK signaling rescues the differentiation phenotype caused by Jmjd1c depletion. Mechanistically, JMJD1C, with the help of pluripotency factor KLF4, maintains ESC identity at least in part by regulating the expression of the miR-200 family and miR-290/295 cluster to suppress the ERK/MAPK signaling and EMT. Additionally, we uncover that JMJD1C ensures efficient generation and maintenance of induced pluripotent stem cells, at least partially through controlling the expression of microRNAs. Collectively, we propose an integrated model of epigenetic and transcriptional control mediated by the H3K9 demethylase for ESC self-renewal and somatic cell reprogramming. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Resistance of human and mouse myeloid leukemia cells to UV radiation

    International Nuclear Information System (INIS)

    Poljak-Blazi, M.; Osmak, M.; Hadzija, M.

    1989-01-01

    Sensitivity of mouse bone marrow and myeloid leukemia cells and sensitivity of human myeloid leukemia cells to UV light was tested. Criteria were the in vivo colony-forming ability of UV exposed cells and the inhibition of DNA synthesis during post-irradiation incubation for 24 h in vitro. Mouse bone marrow cells irradiated with a small dose of UV light (5 J/m 2 ) and injected into x-irradiated animals did not form hemopoietic colonies on recipient's spleens, and recipients died. However, mouse leukemia cells, after irradiation with higher doses of UV light, retained the ability to form colonies on the spleens, and all recipient mice died with typical symptoms of leukemia. In vitro, mouse bone marrow cells exhibited high sensitivity to UV light compared to mouse myeloid leukemia cells. Human leukemia cells were also resistant to UV light, but more sensitive than mouse leukemia cells. (author)

  17. Guiding the osteogenic fate of mouse and human mesenchymal stem cells through feedback system control.

    Science.gov (United States)

    Honda, Yoshitomo; Ding, Xianting; Mussano, Federico; Wiberg, Akira; Ho, Chih-Ming; Nishimura, Ichiro

    2013-12-05

    Stem cell-based disease modeling presents unique opportunities for mechanistic elucidation and therapeutic targeting. The stable induction of fate-specific differentiation is an essential prerequisite for stem cell-based strategy. Bone morphogenetic protein 2 (BMP-2) initiates receptor-regulated Smad phosphorylation, leading to the osteogenic differentiation of mesenchymal stromal/stem cells (MSC) in vitro; however, it requires supra-physiological concentrations, presenting a bottleneck problem for large-scale drug screening. Here, we report the use of a double-objective feedback system control (FSC) with a differential evolution (DE) algorithm to identify osteogenic cocktails of extrinsic factors. Cocktails containing significantly reduced doses of BMP-2 in combination with physiologically relevant doses of dexamethasone, ascorbic acid, beta-glycerophosphate, heparin, retinoic acid and vitamin D achieved accelerated in vitro mineralization of mouse and human MSC. These results provide insight into constructive approaches of FSC to determine the applicable functional and physiological environment for MSC in disease modeling, drug screening and tissue engineering.

  18. Automatic detection and quantitative analysis of cells in the mouse primary motor cortex

    Science.gov (United States)

    Meng, Yunlong; He, Yong; Wu, Jingpeng; Chen, Shangbin; Li, Anan; Gong, Hui

    2014-09-01

    Neuronal cells play very important role on metabolism regulation and mechanism control, so cell number is a fundamental determinant of brain function. Combined suitable cell-labeling approaches with recently proposed three-dimensional optical imaging techniques, whole mouse brain coronal sections can be acquired with 1-μm voxel resolution. We have developed a completely automatic pipeline to perform cell centroids detection, and provided three-dimensional quantitative information of cells in the primary motor cortex of C57BL/6 mouse. It involves four principal steps: i) preprocessing; ii) image binarization; iii) cell centroids extraction and contour segmentation; iv) laminar density estimation. Investigations on the presented method reveal promising detection accuracy in terms of recall and precision, with average recall rate 92.1% and average precision rate 86.2%. We also analyze laminar density distribution of cells from pial surface to corpus callosum from the output vectorizations of detected cell centroids in mouse primary motor cortex, and find significant cellular density distribution variations in different layers. This automatic cell centroids detection approach will be beneficial for fast cell-counting and accurate density estimation, as time-consuming and error-prone manual identification is avoided.

  19. Isolation and characterization of neural stem cells from dystrophic mdx mouse

    International Nuclear Information System (INIS)

    Annese, Tiziana; Corsi, Patrizia; Ruggieri, Simona; Tamma, Roberto; Marinaccio, Christian; Picocci, Sabrina; Errede, Mariella; Specchia, Giorgina; De Luca, Annamaria; Frassanito, Maria Antonia; Desantis, Vanessa; Vacca, Angelo; Ribatti, Domenico; Nico, Beatrice

    2016-01-01

    The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and β-dystroglycan (αDG, βDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier

  20. Isolation and characterization of neural stem cells from dystrophic mdx mouse

    Energy Technology Data Exchange (ETDEWEB)

    Annese, Tiziana [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Corsi, Patrizia [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Physiology, University of Bari Medical School, Bari (Italy); Ruggieri, Simona; Tamma, Roberto; Marinaccio, Christian [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Picocci, Sabrina [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Physiology, University of Bari Medical School, Bari (Italy); Errede, Mariella [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); Specchia, Giorgina [Department of Emergency and Transplantation, Section of Hematology, University of Bari Medical School, Bari (Italy); De Luca, Annamaria [Department of Bioscience, Biotechnology and Pharmacological Sciences, Section of Pharmacology, University of Bari (Italy); Frassanito, Maria Antonia; Desantis, Vanessa; Vacca, Angelo [Department of Internal Medicine and Oncology, University of Bari Medical School, Bari (Italy); Ribatti, Domenico, E-mail: domenico.ribatti@uniba.it [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy); National Cancer Institute “Giovanni Paolo II”, Bari (Italy); Nico, Beatrice, E-mail: beatrice.nico@uniba.it [Department of Basic Medical Sciences, Neurosciences and Sensory Organs, Section of Human Anatomy and Histology, University of Bari Medical School, Bari (Italy)

    2016-05-01

    The blood-brain barrier (BBB) is altered in mdx mouse, an animal model to study Duchenne muscular dystrophy (DMD). Our previous work demonstrated that perivascular glial endfeet control the selective exchanges between blood and neuropil as well as the BBB development and integrity; the alterations of dystrophin and dystrophin-associated protein complex (DAPs) in the glial cells of mdx mouse, parallel damages of the BBB and increase in vascular permeability. The aim of this study was to improve our knowledge about brain cellular components in the mdx mouse through the isolation, for the first time, of the adult neural stem cells (ANSCs). We characterized them by FACS, electron microscopy, confocal immunofluorescence microscopy, Real Time-PCR and western blotting, and we studied the expression of the DAPs aquaporin-4 (AQP4), potassium channel Kir4.1, α- and β-dystroglycan (αDG, βDG), α-syntrophin (αSyn), and short dystrophin isoform Dp71 proteins. The results showed that the mdx ANSCs expressed CD133 and Nestin receptor as the control ones, but showed a reduction in Notch receptor and altered cell proliferation with an increment in the apoptotic nuclei. Ultrastructurally, they appeared 50% size reduced compared to control ones, with a few cytoplasmic organelles. Moreover, the mdx ANSCs are devoid in full length dystrophin 427, and they expressed post-transcriptional reduction in the Dp71 in parallel with the ubiquitin proteasome activation, and decrement of DAPs proteins which appeared diffused in the cytoplasm and not polarized on the stem cells plasmamembrane, as prevalently observed in the controls. Overall, these results indicate that structural and molecular alterations affect the neural stem cells in the dystrophic brain, whose increased apoptosis and reduced Dp71 and DAPs proteins expression, together with loss in Dp427 dystrophin, could be responsible of the altered mdx glial maintenance and differentiation and consequent failure in the vessels barrier

  1. Intraocular Injection of ES Cell-Derived Neural Progenitors Improve Visual Function in Retinal Ganglion Cell-Depleted Mouse Models

    Directory of Open Access Journals (Sweden)

    Mundackal S. Divya

    2017-09-01

    Full Text Available Retinal ganglion cell (RGC transplantation is a promising strategy to restore visual function resulting from irreversible RGC degeneration occurring in glaucoma or inherited optic neuropathies. We previously demonstrated FGF2 induced differentiation of mouse embryonic stem cells (ESC to RGC lineage, capable of retinal ganglion cell layer (GCL integration upon transplantation. Here, we evaluated possible improvement of visual function by transplantation of ES cell derived neural progenitors in RGC depleted glaucoma mice models. ESC derived neural progenitors (ES-NP were transplanted into N-Methyl-D-Aspartate (NMDA injected, RGC-ablated mouse models and a pre-clinical glaucoma mouse model (DBA/2J having sustained higher intra ocular pressure (IOP. Visual acuity and functional integration was evaluated by behavioral experiments and immunohistochemistry, respectively. GFP-expressing ES-NPs transplanted in NMDA-injected RGC-depleted mice differentiated into RGC lineage and possibly integrating into GCL. An improvement in visual acuity was observed after 2 months of transplantation, when compared to the pre-transplantation values. Expression of c-Fos in the transplanted cells, upon light induction, further suggests functional integration into the host retinal circuitry. However, the transplanted cells did not send axonal projections into optic nerve. Transplantation experiments in DBA/2J mouse showed no significant improvement in visual functions, possibly due to both host and transplanted retinal cell death which could be due to an inherent high IOP. We showed that, ES NPs transplanted into the retina of RGC-ablated mouse models could survive, differentiate to RGC lineage, and possibly integrate into GCL to improve visual function. However, for the survival of transplanted cells in glaucoma, strategies to control the IOP are warranted.

  2. Introduction of the human proα1(I) collagen gene into proα1(I)-deficient Mov-13 mouse cells leads to formation of functional mouse-human hybrid type I collagen

    International Nuclear Information System (INIS)

    Schnieke, A.; Dziadek, M.; Bateman, J.; Mascara, T.; Harbers, K.; Gelinas, R.; Jaenisch, R.

    1987-01-01

    The Mov-13 mouse strain carries a retroviral insertion in the proα1(I) collagen gene that prevents transcription of the gene. Cell lines derived from homozygous embryos do not express type I collagen although normal amounts of proα2 mRNA are synthesized. The authors have introduced genomic clones of either the human or mouse proα1(I) collagen gene into homozygous cell lines to assess whether the human or mouse proα1(I) chains can associate with the endogenous mouse proα2(I) chain to form stable type I collagen. The human gene under control of the simian virus 40 promoter was efficiently transcribed in the transfected cells. Protein analyses revealed that stable heterotrimers consisting of two human α1 chains and one mouse α2 chain were formed and that type I collagen was secreted by the transfected cells at normal rates. However, the electrophoretic migration of both α1(I) and α2(I) chains in the human-mouse hybrid molecules were retarded, compared to the α(I) chains in control mouse cells. Inhibition of the posttranslational hydroxylation of lysine and proline resulted in comigration of human and mouse α1 and α2 chains, suggesting that increased posttranslational modification caused the altered electrophoretic migration in the human-mouse hybrid molecules. Amino acid sequence differences between the mouse and human α chains may interfere with the normal rate of helix formation and increase the degree of posttranslational modifications similar to those observed in patients with lethal perinatal osteogenesis imperfecta. The Mov-13 mouse system should allow the authors to study the effect specific mutations introduced in transfected proα1(I) genes have on the synthesis, assembly, and function of collagen I

  3. Derivation of mouse embryonic stem cell lines from tyrosine hydroxylase reporter mice crossed with a human SNCA transgenic mouse model of Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Margarita Chumarina

    2017-03-01

    Full Text Available Mouse embryonic stem cell (mESC lines were derived by crossing heterozygous transgenic (tg mice expressing green fluorescent protein (GFP under the control of the rat tyrosine hydroxylase (TH promoter, with homozygous alpha-synuclein (aSYN mice expressing human mutant SNCAA53T under the control of the mouse Prion promoter (MoPrP, or wildtype (WT mice. The expression of GFP and human aSYN was validated by immunocytochemistry in midbrain neuron cultures upon differentiation of mESC lines using stromal cell-derived inducing activity. These mESC lines can help to study the impact of human aSYN expression in neurons and oligodendrocytes, and also trace GFP-expressing midbrain neurons.

  4. Cell-specific cre recombinase expression allows selective ablation of glutamate receptors from mouse horizontal cells.

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    Sebastian Ströh

    Full Text Available In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57, a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99% and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl. In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼ 50% in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼ 75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less

  5. Cell-Specific Cre Recombinase Expression Allows Selective Ablation of Glutamate Receptors from Mouse Horizontal Cells

    Science.gov (United States)

    Janssen-Bienhold, Ulrike; Schultz, Konrad; Cimiotti, Kerstin; Weiler, Reto; Willecke, Klaus; Dedek, Karin

    2013-01-01

    In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input

  6. Nuclear DNA but not mtDNA controls tumor phenotypes in mouse cells

    International Nuclear Information System (INIS)

    Akimoto, Miho; Niikura, Mamoru; Ichikawa, Masami; Yonekawa, Hiromichi; Nakada, Kazuto; Honma, Yoshio; Hayashi, Jun-Ichi

    2005-01-01

    Recent studies showed high frequencies of homoplasmic mtDNA mutations in various human tumor types, suggesting that the mutated mtDNA haplotypes somehow contribute to expression of tumor phenotypes. We directly addressed this issue by isolating mouse mtDNA-less (ρ 0 ) cells for complete mtDNA replacement between normal cells and their carcinogen-induced transformants, and examined the effect of the mtDNA replacement on expression of tumorigenicity, a phenotype forming tumors in nude mice. The results showed that genome chimera cells carrying nuclear DNA from tumor cells and mtDNA from normal cells expressed tumorigenicity, whereas those carrying nuclear DNA from normal cells and mtDNA from tumor cells did not. These observations provided direct evidence that nuclear DNA, but not mtDNA, is responsible for carcinogen-induced malignant transformation, although it remains possible that mtDNA mutations and resultant respiration defects may influence the degree of malignancy, such as invasive or metastatic properties

  7. Zinc stimulates glucose oxidation and glycemic control by modulating the insulin signaling pathway in human and mouse skeletal muscle cell lines.

    Science.gov (United States)

    Norouzi, Shaghayegh; Adulcikas, John; Sohal, Sukhwinder Singh; Myers, Stephen

    2018-01-01

    Zinc is a metal ion that is an essential cell signaling molecule. Highlighting this, zinc is an insulin mimetic, activating cellular pathways that regulate cellular homeostasis and physiological responses. Previous studies have linked dysfunctional zinc signaling with several disease states including cancer, obesity, cardiovascular disease and type 2 diabetes. The present study evaluated the insulin-like effects of zinc on cell signaling molecules including tyrosine, PRSA40, Akt, ERK1/2, SHP-2, GSK-3β and p38, and glucose oxidation in human and mouse skeletal muscle cells. Insulin and zinc independently led to the phosphorylation of these proteins over a 60-minute time course in both mouse and human skeletal muscle cells. Similarly, utilizing a protein array we identified that zinc could active the phosphorylation of p38, ERK1/2 and GSK-3B in human and ERK1/2 and GSK-3B in mouse skeletal muscle cells. Glucose oxidation assays were performed on skeletal muscle cells treated with insulin, zinc, or a combination of both and resulted in a significant induction of glucose consumption in mouse (pzinc alone. Insulin, as expected, increased glucose oxidation in mouse (pzinc and insulin did not augment glucose consumption in these cells. Zinc acts as an insulin mimetic, activating key molecules implicated in cell signaling to maintain glucose homeostasis in mouse and human skeletal muscle cells. Zinc is an important metal ion implicated in several biological processes. The role of zinc as an insulin memetic in activating key signaling molecules involved in glucose homeostasis could provide opportunities to utilize this ion therapeutically in treating disorders associated with dysfunctional zinc signaling.

  8. The PD-1/B7-H1 pathway modulates the natural killer cells versus mouse glioma stem cells.

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    Bo Yuan Huang

    Full Text Available Glioblastoma multiforme (GBM is the most malignant primary type of brain tumor in adults. There has been increased focus on the immunotherapies to treat GBM patients, the therapeutic value of natural killer (NK cells is still unknown. Programmed death-1 (PD-1 is a major immunological checkpoint that can negatively regulate the T-cell-mediated immune response. We tested the combination of the inhibiting the PD-1/B7H1 pathway with a NK-cell mediated immune response in an orthotopic mouse model of GBM.Mouse glioma stem cells (GL261GSCs and mouse NK cells were isolated and identified. A lactate dehydrogenase (LDH assay was perfomed to detect the cytotoxicity of NK cells against GL261GSCs. GL261GSCs were intracranially implanted into mice, and the mice were stratified into 3 treatment groups: 1 control, 2 NK cells treatment, and 3 PD-1 inhibited NK cells treatment group. Overall survival was quantified, and animal magnetic resonance imaging (MRI was performed to determine tumor growth. The brains were harvested after the mice were euthanized, and immunohistochemistry against CD45 and PCNA was performed.The mouse NK cells were identified as 90% CD3- NK1.1+CD335+ by flow cytometric analysis. In the LDH assay, the ratios of the damaged GL261GSCs, with the E:T ratios of 2.5:1, 5:1, and 10:1, were as follows: 1 non-inhibited group: 7.42%, 11.31%, and 15.1%, 2 B7H1 inhibited group: 14.75%, 18.25% and 29.1%, 3 PD-1 inhibited group: 15.53%, 19.21% and 29.93%, 4 double inhibited group: 33.24%, 42.86% and 54.91%. In the in vivo experiments, the mice in the PD-1 inhibited NK cells treatment group and IL-2-stimulated-NK cells treatment group displayed a slowest tumor growth (F = 308.5, P<0.01 and a slower tumor growth compared with control group (F = 118.9, P<0.01, respectively. The median survival of the mice in the three groups were as follows: 1 conrol group: 29 days, 2 NK cells treatment group: 35 days (P = 0.0012, 3 PD-1 inhibited NK cells treatment group

  9. [Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species].

    Science.gov (United States)

    Li, Cheng-xue; Zhao, Xin; Qian, Jing; Yan, Jie

    2012-07-01

    To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species. The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay. β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets. There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

  10. Differences between Mycobacterium-Host Cell Relationships in Latent Tuberculous Infection of Mice Ex Vivo and Mycobacterial Infection of Mouse Cells In Vitro

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    Elena Ufimtseva

    2016-01-01

    Full Text Available The search for factors that account for the reproduction and survival of mycobacteria, including vaccine strains, in host cells is the priority for studies on tuberculosis. A comparison of BCG-mycobacterial loads in granuloma cells obtained from bone marrow and spleens of mice with latent tuberculous infection and cells from mouse bone marrow and peritoneal macrophage cultures infected with the BCG vaccine in vitro has demonstrated that granuloma macrophages each normally contained a single BCG-Mycobacterium, while those acutely infected in vitro had increased mycobacterial loads and death rates. Mouse granuloma cells were observed to produce the IFNγ, IL-1α, GM-CSF, CD1d, CD25, CD31, СD35, and S100 proteins. None of these activation markers were found in mouse cell cultures infected in vitro or in intact macrophages. Lack of colocalization of lipoarabinomannan-labeled BCG-mycobacteria with the lysosomotropic LysoTracker dye in activated granuloma macrophages suggests that these macrophages were unable to destroy BCG-mycobacteria. However, activated mouse granuloma macrophages could control mycobacterial reproduction in cells both in vivo and in ex vivo culture. By contrast, a considerable increase in the number of BCG-mycobacteria was observed in mouse bone marrow and peritoneal macrophages after BCG infection in vitro, when no expression of the activation-related molecules was detected in these cells.

  11. Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA).

    Science.gov (United States)

    Narayan, Malathi; Seeley, Kent W; Jinwal, Umesh K

    2016-06-01

    Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032.

  12. The effect of the melatonin on cryopreserved mouse testicular cells

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    Ghasem Saki

    2016-01-01

    Full Text Available Background: After improvements in various cancer treatments, life expectancy has been raised, but success in treatment causes loss of fertility in many of the survived young men. Cryopreservation of immature testicular tissues or cells introduced as the only way to preserve fertility. However, freezing has some harmful effects. Melatonin, a pineal gland hormone, has receptors in reproductive systems of different species. It is assumed that melatonin has free radical scavenger properties. Objective: The aim of this study was to evaluate the effects of melatonin on the cryopreserved testicular cells in mouse. Materials and Methods: Cells from 7- 10 days old NMRI mice testes were isolated using two step enzymatic digestion. The testicular cells were divided into two groups randomly and cryopreserved in two different freezing media with and without the addition of 100 μm melatonin. Finally, apoptosis of the cells was assayed by flow cytometry. Also, lactate dehydrogenase activity test was performed to assess the cytotoxicity. Results: The results of lactate dehydrogenase showed the nearly cytotoxic effect of melatonin. The results of flow cytometry showed increase in apoptosis in the cryopreserved cells in the media containing melatonin compared to the control group. Conclusion: The present study shows that melatonin has an apoptotic effect on cryopreserved mouse testicular cells.

  13. Eliminating Glutamatergic Input onto Horizontal Cells Changes the Dynamic Range and Receptive Field Organization of Mouse Retinal Ganglion Cells.

    Science.gov (United States)

    Ströh, Sebastian; Puller, Christian; Swirski, Sebastian; Hölzel, Maj-Britt; van der Linde, Lea I S; Segelken, Jasmin; Schultz, Konrad; Block, Christoph; Monyer, Hannah; Willecke, Klaus; Weiler, Reto; Greschner, Martin; Janssen-Bienhold, Ulrike; Dedek, Karin

    2018-02-21

    In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however, how horizontal cell signals also affect the temporal, spatial, and contrast tuning in retinal output neurons, the ganglion cells. To study this, we generated a genetically modified mouse line in which we eliminated the light dependency of feedback by deleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actual mode of feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-α retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse. SIGNIFICANCE STATEMENT Horizontal cells represent a major neuronal class in the mammalian retina and provide lateral feedback and feedforward signals to photoreceptors and bipolar cells, respectively. The mode of signal transmission remains controversial and, moreover, the contribution of horizontal cells to visual processing is still elusive. To address the question of how horizontal cells affect retinal output signals, we recorded the light

  14. Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA

    Directory of Open Access Journals (Sweden)

    Malathi Narayan

    2016-06-01

    Full Text Available Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA on a mouse microglial (N9 cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD003032.

  15. Oxygen-controlled automated neural differentiation of mouse embryonic stem cells.

    Science.gov (United States)

    Mondragon-Teran, Paul; Tostoes, Rui; Mason, Chris; Lye, Gary J; Veraitch, Farlan S

    2013-03-01

    Automation and oxygen tension control are two tools that provide significant improvements to the reproducibility and efficiency of stem cell production processes. the aim of this study was to establish a novel automation platform capable of controlling oxygen tension during both the cell-culture and liquid-handling steps of neural differentiation processes. We built a bespoke automation platform, which enclosed a liquid-handling platform in a sterile, oxygen-controlled environment. An airtight connection was used to transfer cell culture plates to and from an automated oxygen-controlled incubator. Our results demonstrate that our system yielded comparable cell numbers, viabilities, metabolism profiles and differentiation efficiencies when compared with traditional manual processes. Interestingly, eliminating exposure to ambient conditions during the liquid-handling stage resulted in significant improvements in the yield of MAP2-positive neural cells, indicating that this level of control can improve differentiation processes. This article describes, for the first time, an automation platform capable of maintaining oxygen tension control during both the cell-culture and liquid-handling stages of a 2D embryonic stem cell differentiation process.

  16. Isolation, culture and adenoviral transduction of parietal cells from mouse gastric mucosa

    International Nuclear Information System (INIS)

    Gliddon, Briony L; Nguyen, Nhung V; Gunn, Priscilla A; Gleeson, Paul A; Driel, Ian R van

    2008-01-01

    Here we describe a method for the isolation of intact gastric glands from mice and primary culture and transfection of mouse gastric epithelial cells. Collagenase digestion of PBS-perfused mouse stomachs released large intact gastric glands that were plated on a basement membrane matrix. The heterogeneous gland cell cultures typically contain ∼60% parietal cells. Isolated mouse parietal cells remain viable in culture for up to 5 days and react strongly with an antibody specific to the gastric H + /K + ATPase. Isolated intact mouse gastric glands and primary cultures of mouse parietal cells respond to the secretagogue, histamine. Typical morphological changes from a resting to an acid-secreting active parietal cell were observed. In resting cultures of mouse parietal cells, the H + /K + ATPase displayed a cytoplasmic punctate staining pattern consistent with tubulovesicle element structures. Following histamine stimulation, an expansion of internal apical vacuole structures was observed together with a pronounced redistribution of the H + /K + ATPase from the cytoplasm to the apical vacuoles. A reproducible procedure to express genes of interest exogenously in these cultures of mouse parietal cells was also established. This method combines recombinant adenoviral transduction with magnetic field-assisted transfection resulting in ∼30% transduced parietal cells. Adenoviral-transduced parietal cells maintain their ability to undergo agonist-induced activation. This protocol will be useful for the isolation, culture and expression of genes in parietal cells from genetically modified mice and as such will be an invaluable tool for studying the complex exocytic and endocytic trafficking events of the H + /K + ATPase which underpin the regulation of acid secretion

  17. The Nanos3-3'UTR is required for germ cell specific NANOS3 expression in mouse embryos.

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    Hitomi Suzuki

    Full Text Available BACKGROUND: The regulation of gene expression via a 3' untranslated region (UTR plays essential roles in the discrimination of the germ cell lineage from somatic cells during embryogenesis. This is fundamental to the continuation of a species. Mouse NANOS3 is an essential protein required for the germ cell maintenance and is specifically expressed in these cells. However, the regulatory mechanisms that restrict the expression of this gene in the germ cells is largely unknown at present. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we show that differences in the stability of Nanos3 mRNA between germ cells and somatic cells is brought about in a 3'UTR-dependent manner in mouse embryos. Although Nanos3 is transcribed in both cell lineages, it is efficiently translated only in the germ lineage. We also find that the translational suppression of NANOS3 in somatic cells is caused by a 3'UTR-mediated mRNA destabilizing mechanism. Surprisingly, even when under the control of the CAG promoter which induces strong ubiquitous transcription in both germ cells and somatic cells, the addition of the Nanos3-3'UTR sequence to the coding region of exogenous gene was effective in restricting protein expression in germ cells. CONCLUSIONS/SIGNIFICANCE: Our current study thus suggests that Nanos3-3'UTR has an essential role in translational control in the mouse embryo.

  18. In vitro three-dimensional coculturing poly3-hydroxybutyrate-co-3-hydroxyhexanoate with mouse-induced pluripotent stem cells for myocardial patch application.

    Science.gov (United States)

    Shijun, Xu; Junsheng, Mu; Jianqun, Zhang; Ping, Bo

    2016-03-01

    Identifying a suitable polymeric biomaterial for myocardial patch repair following myocardial infarction, cerebral infarction, and cartilage injury is essential. This study aimed to investigate the effect of the novel polymer material, poly3-hydroxybutyrate-co-3-hydroxyhexanoate, on the adhesion, proliferation, and differentiation of mouse-induced pluripotent stem cells in vitro. Mouse-induced pluripotent stem cells were isolated, expanded, and cultured on either two-dimensional or three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films (membranes were perforated to imitate three-dimensional space). Following attachment onto the films, mouse-induced pluripotent stem cell morphology was visualized using scanning electron microscopy. Cell vitality was detected using the Cell Counting Kit-8 assay and cell proliferation was observed using fluorescent 4',6-diamidino-2-phenylindole (DAPI) staining. Mouse-induced pluripotent stem cells were induced into cardiomyocytes by differentiation medium containing vitamin C. A control group in the absence of an inducer was included. Mouse-induced pluripotent stem cell survival and differentiation were observed using immunofluorescence and flow cytometry, respectively. Mouse-induced pluripotent stem cells growth, proliferation, and differentiation were observed on both two-dimensional and three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Vitamin C markedly improved the efficiency of mouse-induced pluripotent stem cells differentiation into cardiomyocytes on poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Three-dimensional culture was better at promoting mouse-induced pluripotent stem cell proliferation and differentiation compared with two-dimensional culture. © The Author(s) 2016.

  19. Mouse cell culture - Methods and protocols

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    CarloAlberto Redi

    2010-12-01

    Full Text Available The mouse is, out of any doubt, the experimental animal par excellence for many many colleagues within the scientific community, notably for those working in mammalian biology (in a broad sense, from basic genetic to modeling human diseases, starting at least from 1664 Robert Hooke experiments on air’s propertyn. Not surprising then that mouse cell cultures is a well established field of research itself and that there are several handbooks devoted to this discipline. Here, Andrew Ward and David Tosh provide a necessary update of the protocols currently needed. In fact, nearly half of the book is devoted to stem cells culture protocols, mainly embryonic, from a list of several organs (kidney, lung, oesophagus and intestine, pancreas and liver to mention some........

  20. The PD-1/B7-H1 pathway modulates the natural killer cells versus mouse glioma stem cells.

    Science.gov (United States)

    Huang, Bo Yuan; Zhan, Yi Ping; Zong, Wen Jing; Yu, Chun Jiang; Li, Jun Fa; Qu, Yan Ming; Han, Song

    2015-01-01

    Glioblastoma multiforme (GBM) is the most malignant primary type of brain tumor in adults. There has been increased focus on the immunotherapies to treat GBM patients, the therapeutic value of natural killer (NK) cells is still unknown. Programmed death-1 (PD-1) is a major immunological checkpoint that can negatively regulate the T-cell-mediated immune response. We tested the combination of the inhibiting the PD-1/B7H1 pathway with a NK-cell mediated immune response in an orthotopic mouse model of GBM. Mouse glioma stem cells (GL261GSCs) and mouse NK cells were isolated and identified. A lactate dehydrogenase (LDH) assay was perfomed to detect the cytotoxicity of NK cells against GL261GSCs. GL261GSCs were intracranially implanted into mice, and the mice were stratified into 3 treatment groups: 1) control, 2) NK cells treatment, and 3) PD-1 inhibited NK cells treatment group. Overall survival was quantified, and animal magnetic resonance imaging (MRI) was performed to determine tumor growth. The brains were harvested after the mice were euthanized, and immunohistochemistry against CD45 and PCNA was performed. The mouse NK cells were identified as 90% CD3- NK1.1+CD335+ by flow cytometric analysis. In the LDH assay, the ratios of the damaged GL261GSCs, with the E:T ratios of 2.5:1, 5:1, and 10:1, were as follows: 1) non-inhibited group: 7.42%, 11.31%, and 15.1%, 2) B7H1 inhibited group: 14.75%, 18.25% and 29.1%, 3) PD-1 inhibited group: 15.53%, 19.21% and 29.93%, 4) double inhibited group: 33.24%, 42.86% and 54.91%. In the in vivo experiments, the mice in the PD-1 inhibited NK cells treatment group and IL-2-stimulated-NK cells treatment group displayed a slowest tumor growth (F = 308.5, Pmouse NK cells to kill the GL261GSCs, and the PD-1-inhibited NK cells could be a feasible immune therapeutic approach against GBM.

  1. Cryo-imaging of fluorescently labeled single cells in a mouse

    Science.gov (United States)

    Steyer, Grant J.; Roy, Debashish; Salvado, Olivier; Stone, Meredith E.; Wilson, David L.

    2009-02-01

    We developed a cryo-imaging system to provide single-cell detection of fluorescently labeled cells in mouse, with particular applicability to stem cells and metastatic cancer. The Case cryoimaging system consists of a fluorescence microscope, robotic imaging positioner, customized cryostat, PC-based control system, and visualization/analysis software. The system alternates between sectioning (10-40 μm) and imaging, collecting color brightfield and fluorescent blockface image volumes >60GB. In mouse experiments, we imaged quantum-dot labeled stem cells, GFP-labeled cancer and stem cells, and cell-size fluorescent microspheres. To remove subsurface fluorescence, we used a simplified model of light-tissue interaction whereby the next image was scaled, blurred, and subtracted from the current image. We estimated scaling and blurring parameters by minimizing entropy of subtracted images. Tissue specific attenuation parameters were found [uT : heart (267 +/- 47.6 μm), liver (218 +/- 27.1 μm), brain (161 +/- 27.4 μm)] to be within the range of estimates in the literature. "Next image" processing removed subsurface fluorescence equally well across multiple tissues (brain, kidney, liver, adipose tissue, etc.), and analysis of 200 microsphere images in the brain gave 97+/-2% reduction of subsurface fluorescence. Fluorescent signals were determined to arise from single cells based upon geometric and integrated intensity measurements. Next image processing greatly improved axial resolution, enabled high quality 3D volume renderings, and improved enumeration of single cells with connected component analysis by up to 24%. Analysis of image volumes identified metastatic cancer sites, found homing of stem cells to injury sites, and showed microsphere distribution correlated with blood flow patterns. We developed and evaluated cryo-imaging to provide single-cell detection of fluorescently labeled cells in mouse. Our cryo-imaging system provides extreme (>60GB), micron

  2. The PPARδ Ligand GW501516 Reduces Growth but Not Apoptosis in Mouse Inner Medullary Collecting Duct Cells

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    Jordan Clark

    2009-01-01

    Full Text Available The collecting duct (CD expresses considerable amounts of PPARδ. While its role is unknown in the CD, in other renal cells it has been shown to regulate both growth and apoptosis. We thus hypothesized that PPARδ reduces apoptotic responses and stimulates cell growth in the mouse CD, and examined the effect of GW501516, a synthetic PPARδ ligand, on these responses in mouse IMCD-K2 cells. High doses of GW501516 decreased both DNA and protein synthesis in these cells by 80%, but had no overall effect on cell viability. Although anisomycin treatment resulted in an increase of caspase-3 levels of about 2.59-fold of control, GW501516 did not affect anisomycin-induced changes in active caspase-3 levels. These results show that a PPARδ ligand inhibits growth but does not affect anisomycin-apoptosis in a mouse IMCD cell line. This could have therapeutic implications for renal diseases associated with increased CD growth responses.

  3. Genotoxicity of 3-nitrobenzanthrone and 3-aminobenzanthrone in MutaMouse and lung epithelial cells derived from MutaMouse.

    Science.gov (United States)

    Arlt, Volker M; Gingerich, John; Schmeiser, Heinz H; Phillips, David H; Douglas, George R; White, Paul A

    2008-11-01

    FE1 lung epithelial cells derived from MutaMouse are a new model system to provide in vitro mutagenicity data with the potential to predict the outcome of an in vivo MutaMouse test. 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and urban air pollution. We investigated the mutagenicity and DNA binding of 3-NBA and its main metabolite 3-aminobenzanthrone (3-ABA) in vitro and in vivo in the MutaMouse assay. Mice were treated with 3-NBA or 3-ABA (0, 2 or 5 mg/kg body weight/day) by gavage for 28 days and 28 days later lacZ mutant frequency (MF) was determined in liver, lung and bone marrow. For both compounds, dose-related increases in MF were seen in liver and bone marrow, but not in lung; mutagenic activity was approximately 2-fold lower for 3-ABA than for 3-NBA. With 3-NBA, highest DNA adduct levels (measured by (32)P-post-labelling) were found in liver (approximately 230 adducts per 10(8) nucleotides) with levels 20- to 40-fold lower in bone marrow and lung. With 3-ABA, DNA adduct levels were again highest in the liver, but approximately 4-fold lower than for 3-NBA. FE1 cells were exposed to up to 10 microg/ml 3-NBA or 3-ABA for 6 h with or without exogenous activation (S9) and harvested after 3 days. For 3-NBA, there was a dose-related increase in MF both with and without S9 mix, which was >10 times higher than observed in vivo. At the highest concentration of 3-ABA (10 microg/ml), we found only around a 2-fold increase in MF relative to controls. DNA adduct formation in FE1 cells was dose-dependent for both compounds, but 10- to 20-fold higher for 3-NBA compared to 3-ABA. Collectively, our data indicate that MutaMouse FE1 cells are well suited for cost-effective testing of suspected mutagens with different metabolic activation pathways as a guide for subsequent in vivo MutaMouse testing.

  4. Lentiviral Vector-Mediated GFP/fluc gene introduction into primary mouse NK cells

    International Nuclear Information System (INIS)

    L, Thi Thanh Hoa; Tae, Seong Ho; Min, Jung Joon

    2007-01-01

    NK cell is a type of lymphocyte that has ability in defense against virus infection and some kinds of cancer diseases. Recently, using genetic engineering, studies about the roles and functions of NK cells have been developing. In this study, we used lentivirus-based vector encoding GFP/Fluc gene to transfer into primary mouse NK cells. This model is a tool in studying characteristics of NK cells. The lentivirus used in this study was a commercial one, named LentiM1.3-Fluc, encoding GFP and Flue reporter genes under the control of the murine cytomegalovirus (MCMV) promoter. LentiM1.3-Fluc was infected into freshly isolated mouse NK cells at 2 20 MOl by incubating or using spin infection. In the spin infection, we gently suspended NK cells in viral fluid, then centrifuged at 2000 rpm, 20 minutes at room temperature and incubated for 1 day. After 1 day, virus was discarded and NK cells were cultured in IL-2 with or without IL-12 supplemented media. Infected NK cells were monitored by using fluorescent microscope for GFP and IVIS machine for Fire-fly luciferase expression. The results showed that using spin infection had much effect on introducing lentiviral vector-mediated reporter gene into NK cells than the way without spin. Also, NK cells which were cultured in IL-2 and IL-12 added media expressed higher fluorescent and luminescent signals than those cultured in only IL-2 supplemented media. When these NK cells were injected subcutaneously in Balb/C mice, the imaging signal was observed transiently. Our study demonstrates that by using a simple method, mouse NK cells can be transfected by lentivirus. And this will be useful in studying biology and therapeutic potential of NK cells. However, we require developing alternative lentiviral vectors with different promoter for in vivo application

  5. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  6. In vitro differentiation and maturation of mouse embryonic stem cells into hepatocytes

    International Nuclear Information System (INIS)

    Ishii, Takamichi; Yasuchika, Kentaro; Fujii, Hideaki; Hoppo, Toshitaka; Baba, Shinji; Naito, Masato; Machimoto, Takafumi; Kamo, Naoko; Suemori, Hirofumi; Nakatsuji, Norio; Ikai, Iwao

    2005-01-01

    It is difficult to induce the maturation of embryonic stem (ES) cells into hepatocytes in vitro. We previously reported that Thy1-positive mesenchymal cells derived from the mouse fetal liver promote the maturation of hepatic progenitor cells. Here, we isolated alpha-fetoprotein (AFP)-producing cells from mouse ES cells for subsequent differentiation into hepatocytes in vitro by coculture with Thy1-positive cells. ES cells expressing green fluorescent protein (GFP) under the control of an AFP promoter were cultured under serum- and feeder layer-free culture conditions. The proportion of GFP-positive cells plateaued at 41.6 ± 12.2% (means ± SD) by day 7. GFP-positive cells, isolated by flow cytometry, were cultured in the presence or absence of Thy1-positive cells as a feeder layer. Isolated GFP-positive cells were stained for AFP, Foxa2, and albumin. The expression of mRNAs encoding tyrosine amino transferase, tryptophan 2,3-dioxygenase, and glucose-6-phosphatase were only detected following coculture with Thy1-positive cells. Following coculture with Thy1-positive cells, the isolated cells produced and stored glycogen. Ammonia clearance activity was also enhanced following coculture. Electron microscopic analysis indicated that the cocultured cells exhibited the morphologic features of mature hepatocytes. In conclusion, coculture with Thy1-positive cells in vitro induced the maturation of AFP-producing cells isolated from ES cell cultures into hepatocytes

  7. the production of mouse embryonic stem cells

    Indian Academy of Sciences (India)

    MADU

    What history tells us VII. Twenty-five years ago: the production of mouse embryonic stem cells ... cells into the cavity of the blastocyst, it will be possible to test the effect of .... to the use of efficient immunosuppressive drugs like cyclosporin – was ...

  8. Induction of neural stem cell-like cells (NSCLCs) from mouse astrocytes by Bmi1

    International Nuclear Information System (INIS)

    Moon, Jai-Hee; Yoon, Byung Sun; Kim, Bona; Park, Gyuman; Jung, Hye-Youn; Maeng, Isaac; Jun, Eun Kyoung; Yoo, Seung Jun; Kim, Aeree; Oh, Sejong; Whang, Kwang Youn; Kim, Hyunggee; Kim, Dong-Wook; Kim, Ki Dong; You, Seungkwon

    2008-01-01

    Recently, Bmi1 was shown to control the proliferation and self-renewal of neural stem cells (NSCs). In this study, we demonstrated the induction of NSC-like cells (NSCLCs) from mouse astrocytes by Bmi1 under NSC culture conditions. These NSCLCs exhibited the morphology and growth properties of NSCs, and expressed NSC marker genes, including nestin, CD133, and Sox2. In vitro differentiation of NSCLCs resulted in differentiated cell populations containing astrocytes, neurons, and oligodendrocytes. Following treatment with histone deacetylase inhibitors (trichostatin A and valproic acid), the potential of NSCLCs for proliferation, dedifferentiation, and self-renewal was significantly inhibited. Our data indicate that multipotent NSCLCs can be generated directly from astrocytes by the addition of Bmi1

  9. Strain preservation of experimental animals: vitrification of two-cell stage embryos for multiple mouse strains.

    Science.gov (United States)

    Eto, Tomoo; Takahashi, Riichi; Kamisako, Tsutomu

    2015-04-01

    Strain preservation of experimental animals is crucial for experimental reproducibility. Maintaining complete animal strains, however, is costly and there is a risk for genetic mutations as well as complete loss due to disasters or illness. Therefore, the development of effective vitrification techniques for cryopreservation of multiple experimental animal strains is important. We examined whether a vitrification method using cryoprotectant solutions, P10 and PEPeS, is suitable for preservation of multiple inbred and outbred mouse strains. First, we investigated whether our vitrification method using cryoprotectant solutions was suitable for two-cell stage mouse embryos. In vitro development of embryos exposed to the cryoprotectant solutions was similar to that of fresh controls. Further, the survival rate of the vitrified embryos was extremely high (98.1%). Next, we collected and vitrified two-cell stage embryos of 14 mouse strains. The average number of embryos obtained from one female was 7.3-33.3. The survival rate of vitrified embryos ranged from 92.8% to 99.1%, with no significant differences among mouse strains. In vivo development did not differ significantly between fresh controls and vitrified embryos of each strain. For strain preservation using cryopreserved embryos, two offspring for inbred lines and one offspring for outbred lines must be produced from two-cell stage embryos collected from one female. The expected number of surviving fetuses obtained from embryos collected from one female of either the inbred or outbred strains ranged from 2.9 to 19.5. The findings of the present study indicated that this vitrification method is suitable for strain preservation of multiple mouse strains. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Cerebellar stem cells do not produce neurons and astrocytes in adult mouse

    International Nuclear Information System (INIS)

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui

    2014-01-01

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1 + or nestin + stem cells exist, and most of them are quiescent. • Cell proliferation rate is diversified among cerebellar regions and decreases over time. - Abstract: Although previous studies implied that cerebellar stem cells exist in some adult mammals, little is known about whether these stem cells can produce new neurons and astrocytes. In this study by bromodeoxyuridine (BrdU) intraperitoneal (i.p.) injection, we found that there are abundant BrdU + cells in adult mouse cerebellum, and their quantity and density decreases significantly over time. We also found cell proliferation rate is diversified in different cerebellar regions. Among these BrdU + cells, very few are mash1 + or nestin + stem cells, and the vast majority of cerebellar stem cells are quiescent. Data obtained by in vivo retrovirus injection indicate that stem cells do not produce neurons and astrocytes in adult mouse cerebellum. Instead, some cells labeled by retrovirus are Iba1 + microglia. These results indicate that very few stem cells exist in adult mouse cerebellum, and none of these stem cells contribute to neurogenesis and astrogenesis under physiological condition

  11. Characterization of mitomycin-C-sensitive mouse lymphoma L5178Y cell mutants

    International Nuclear Information System (INIS)

    Inaba, Hiroko; Shiomi, Naoko; Shiomi, Tadahiro; Sato, Koki; Yoshida, Michihiro.

    1985-01-01

    Twenty-six mutants showing high sensitivity to mytomicin-C (MMC) were isolated from mouse lymphoma L5178Y cells by a replica-plating technique. Twenty-five of the mutants were 5 - 10 times more sensitive to MMC than were parental cells, and showed normal sensitivity to U.V. light and x-rays. From a complementation analysis, 5 mutants (MC s ) isolated from independently mutagenized cell populations were classified into two groups. These mutants possessed recessive character for MMC-sensitivity and there were at least two genes involved in the MMC-sensitivity. As for DNA-damaging factors, such as photoadducts of 8-methoxypsoralen (8-MOP) and 3-carbethoxysoralen (3-CPs), MC s mutants showed higher sensitivity to photoadducts of 8-MOP than to (3-CPs). MC s mutants were also highly sensitive to a DNA cross-linking agent, cisplatin. Characterization of the sensitivity of mouse MC s mutants was analogous to that of Fanconi's anemia (FA)-derived cells. Low concentrations (10 ng/ml) of MMC induced chromosome aberration in a high incidence in mouse MC s cells, as well as in FA cells. The frequency of MMC-induced chromosome aberrations was normal in hybrid cells between normal human diploid somatic cells and mouse mutants and between FA cells and mouse wild cells, and hereditary deficiency became normal by hybrization. (Namekawa, K.)

  12. Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

    Science.gov (United States)

    Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

    2017-03-01

    Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

  13. PPARalpha/gamma expression and activity in mouse and human melanocytes and melanoma cells.

    Science.gov (United States)

    Eastham, Linda L; Mills, Caroline N; Niles, Richard M

    2008-06-01

    We examined the expression of PPARs and the effects of PPARalpha and PPARgamma agonists on growth of mouse and human melanocytes and melanoma cells. PPARalpha,beta, and PPARgamma mRNA qualitative expression in melan-a mouse melanocytes, B16 mouse melanoma, human melanocytes, and A375 and SK-mel28 human melanoma cells was determined by RT-PCR, while quantitative PPARalpha mRNA levels were determined by QuantiGene assay. PPARalpha and PPARgamma protein was assessed by Western blotting. The effect of natural and synthetic PPAR ligands on cell growth was determined by either hemocytometer counting or crystal violet assay. PPAR transcriptional activity was determined by a PPRE-reporter gene assay, while knockdown of PPARalpha expression was achieved by transient transfection of siRNA. Both mouse and human melanoma cells produced more PPARalpha and PPARgamma protein compared to melanocytes. PPARalpha mRNA levels were elevated in human melanoma cells, but not in mouse melanoma cells relative to melanocytes. Silencing of PPARalpha in human melanoma cells did not alter cell proliferation or morphology. PPARgamma-selective agonists inhibited the growth of both mouse and human melanoma cells, while PPARalpha-selective agonists had limited effects. Increased expression of PPARalpha in melanoma relative to melanocytes may be a common occurrence, however its biologic significance remains to be determined. PPARgamma agonists may be useful for arresting the growth of some melanomas.

  14. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    Energy Technology Data Exchange (ETDEWEB)

    Webb, Carol F., E-mail: carol-webb@omrf.org [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK (United States); Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Ratliff, Michelle L., E-mail: michelle-ratliff@omrf.org [Immunobiology and Cancer Research, Oklahoma Medical Research Foundation, Oklahoma City, OK (United States); Powell, Rebecca, E-mail: rebeccapowell@gmail.com [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Wirsig-Wiechmann, Celeste R., E-mail: celeste-wirsig@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Lakiza, Olga, E-mail: olga-lakiza@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States); Obara, Tomoko, E-mail: tomoko-obara@ouhsc.edu [Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK (United States)

    2015-08-07

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights: • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development.

  15. A developmentally plastic adult mouse kidney cell line spontaneously generates multiple adult kidney structures

    International Nuclear Information System (INIS)

    Webb, Carol F.; Ratliff, Michelle L.; Powell, Rebecca; Wirsig-Wiechmann, Celeste R.; Lakiza, Olga; Obara, Tomoko

    2015-01-01

    Despite exciting new possibilities for regenerative therapy posed by the ability to induce pluripotent stem cells, recapitulation of three-dimensional kidneys for repair or replacement has not been possible. ARID3a-deficient mouse tissues generated multipotent, developmentally plastic cells. Therefore, we assessed the adult mouse ARID3a−/− kidney cell line, KKPS5, which expresses renal progenitor surface markers as an alternative cell source for modeling kidney development. Remarkably, these cells spontaneously developed into multicellular nephron-like structures in vitro, and engrafted into immunocompromised medaka mesonephros, where they formed mouse nephron structures. These data implicate KKPS5 cells as a new model system for studying kidney development. - Highlights: • An ARID3a-deficient mouse kidney cell line expresses multiple progenitor markers. • This cell line spontaneously forms multiple nephron-like structures in vitro. • This cell line formed mouse kidney structures in immunocompromised medaka fish kidneys. • Our data identify a novel model system for studying kidney development

  16. Oocyte-like cells induced from mouse spermatogonial stem cells.

    Science.gov (United States)

    Wang, Lu; Cao, Jinping; Ji, Ping; Zhang, Di; Ma, Lianghong; Dym, Martin; Yu, Zhuo; Feng, Lixin

    2012-08-06

    During normal development primordial germ cells (PGCs) derived from the epiblast are the precursors of spermatogonia and oogonia. In culture, PGCs can be induced to dedifferentiate to pluripotent embryonic germ (EG) cells in the presence of various growth factors. Several recent studies have now demonstrated that spermatogonial stem cells (SSCs) can also revert back to pluripotency as embryonic stem (ES)-like cells under certain culture conditions. However, the potential dedifferentiation of SSCs into PGCs or the potential generation of oocytes from SSCs has not been demonstrated before. We report that mouse male SSCs can be converted into oocyte-like cells in culture. These SSCs-derived oocytes (SSC-Oocs) were similar in size to normal mouse mature oocytes. They expressed oocyte-specific markers and gave rise to embryos through parthenogenesis. Interestingly, the Y- and X-linked testis-specific genes in these SSC-Oocs were significantly down-regulated or turned off, while oocyte-specific X-linked genes were activated. The gene expression profile appeared to switch to that of the oocyte across the X chromosome. Furthermore, these oocyte-like cells lost paternal imprinting but acquired maternal imprinting. Our data demonstrate that SSCs might maintain the potential to be reprogrammed into oocytes with corresponding epigenetic reversals. This study provides not only further evidence for the remarkable plasticity of SSCs but also a potential system for dissecting molecular and epigenetic regulations in germ cell fate determination and imprinting establishment during gametogenesis.

  17. Oocyte-like cells induced from mouse spermatogonial stem cells

    Directory of Open Access Journals (Sweden)

    Wang Lu

    2012-08-01

    Full Text Available Abstract Background During normal development primordial germ cells (PGCs derived from the epiblast are the precursors of spermatogonia and oogonia. In culture, PGCs can be induced to dedifferentiate to pluripotent embryonic germ (EG cells in the presence of various growth factors. Several recent studies have now demonstrated that spermatogonial stem cells (SSCs can also revert back to pluripotency as embryonic stem (ES-like cells under certain culture conditions. However, the potential dedifferentiation of SSCs into PGCs or the potential generation of oocytes from SSCs has not been demonstrated before. Results We report that mouse male SSCs can be converted into oocyte-like cells in culture. These SSCs-derived oocytes (SSC-Oocs were similar in size to normal mouse mature oocytes. They expressed oocyte-specific markers and gave rise to embryos through parthenogenesis. Interestingly, the Y- and X-linked testis-specific genes in these SSC-Oocs were significantly down-regulated or turned off, while oocyte-specific X-linked genes were activated. The gene expression profile appeared to switch to that of the oocyte across the X chromosome. Furthermore, these oocyte-like cells lost paternal imprinting but acquired maternal imprinting. Conclusions Our data demonstrate that SSCs might maintain the potential to be reprogrammed into oocytes with corresponding epigenetic reversals. This study provides not only further evidence for the remarkable plasticity of SSCs but also a potential system for dissecting molecular and epigenetic regulations in germ cell fate determination and imprinting establishment during gametogenesis.

  18. Expression of GABAergic receptors in mouse taste receptor cells.

    Directory of Open Access Journals (Sweden)

    Margaret R Starostik

    Full Text Available BACKGROUND: Multiple excitatory neurotransmitters have been identified in the mammalian taste transduction, with few studies focused on inhibitory neurotransmitters. Since the synthetic enzyme glutamate decarboxylase (GAD for gamma-aminobutyric acid (GABA is expressed in a subset of mouse taste cells, we hypothesized that other components of the GABA signaling pathway are likely expressed in this system. GABA signaling is initiated by the activation of either ionotropic receptors (GABA(A and GABA(C or metabotropic receptors (GABA(B while it is terminated by the re-uptake of GABA through transporters (GATs. METHODOLOGY/PRINCIPAL FINDINGS: Using reverse transcriptase-PCR (RT-PCR analysis, we investigated the expression of different GABA signaling molecules in the mouse taste system. Taste receptor cells (TRCs in the circumvallate papillae express multiple subunits of the GABA(A and GABA(B receptors as well as multiple GATs. Immunocytochemical analyses examined the distribution of the GABA machinery in the circumvallate papillae. Both GABA(A-and GABA(B- immunoreactivity were detected in the peripheral taste receptor cells. We also used transgenic mice that express green fluorescent protein (GFP in either the Type II taste cells, which can respond to bitter, sweet or umami taste stimuli, or in the Type III GAD67 expressing taste cells. Thus, we were able to identify that GABAergic receptors are expressed in some Type II and Type III taste cells. Mouse GAT4 labeling was concentrated in the cells surrounding the taste buds with a few positively labeled TRCs at the margins of the taste buds. CONCLUSIONS/SIGNIFICANCE: The presence of GABAergic receptors localized on Type II and Type III taste cells suggests that GABA is likely modulating evoked taste responses in the mouse taste bud.

  19. Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts

    Science.gov (United States)

    Krais, Annette M.; Mühlbauer, Karl-Rudolf; Kucab, Jill E.; Chinbuah, Helena; Cornelius, Michael G.; Wei, Quan-Xiang; Hollstein, Monica; Phillips, David H.; Arlt, Volker M.; Schmeiser, Heinz H.

    2015-01-01

    We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by 32P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 μM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells. PMID:25230394

  20. Two pore channel 2 differentially modulates neural differentiation of mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Zhe-Hao Zhang

    Full Text Available Nicotinic acid adenine dinucleotide phosphate (NAADP is an endogenous Ca(2+ mobilizing nucleotide presented in various species. NAADP mobilizes Ca(2+ from acidic organelles through two pore channel 2 (TPC2 in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.

  1. Recovery of deficient homologous recombination in Brca2-depleted mouse cells by wild-type Rad51 expression.

    Science.gov (United States)

    Lee, Shauna A; Roques, Céline; Magwood, Alissa C; Masson, Jean-Yves; Baker, Mark D

    2009-02-01

    The BRCA2 tumor suppressor is important in maintaining genomic stability. BRCA2 is proposed to control the availability, cellular localization and DNA binding activity of the central homologous recombination protein, RAD51, with loss of BRCA2 resulting in defective homologous recombination. Nevertheless, the roles of BRCA2 in regulating RAD51 and how other proteins implicated in RAD51 regulation, such as RAD52 and RAD54 function relative to BRCA2 is not known. In this study, we tested whether defective homologous recombination in Brca2-depleted mouse hybridoma cells could be rectified by expression of mouse Rad51 or the Rad51-interacting mouse proteins, Rad52 and Rad54. In the Brca2-depleted cells, defective homologous recombination can be restored by over-expression of wild-type mouse Rad51, but not mouse Rad52 or Rad54. Correction of the homologous recombination defect requires Rad51 ATPase activity. A sizeable fraction ( approximately 50%) of over-expressed wild-type Rad51 is nuclear localized. The restoration of homologous recombination in the presence of a low (i.e., non-functional) level of Brca2 by wild-type Rad51 over-expression is unexpected. We suggest that Rad51 may access the nuclear compartment in a Brca2-independent manner and when Rad51 is over-expressed, the normal requirement for Brca2 control over Rad51 function in homologous recombination is dispensable. Our studies support loss of Rad51 function as a critical underlying factor in the homologous recombination defect in the Brca2-depleted cells.

  2. Development and function of human innate immune cells in a humanized mouse model.

    Science.gov (United States)

    Rongvaux, Anthony; Willinger, Tim; Martinek, Jan; Strowig, Till; Gearty, Sofia V; Teichmann, Lino L; Saito, Yasuyuki; Marches, Florentina; Halene, Stephanie; Palucka, A Karolina; Manz, Markus G; Flavell, Richard A

    2014-04-01

    Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34(+) progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

  3. Radiation response of spermatogonial stem cells in the mouse

    International Nuclear Information System (INIS)

    Bootsma, A.L.

    1978-01-01

    Spermatogonial stem cells are able to repopulate the testis by forming clones that elongate along the walls of the seminiferous tubules depleted of spermatogenetic cells as a result of an irradiation. The surviving number of stem cells after irradiation was estimated by determining the fraction of repopulated tubules in cross-sections of the testis 11 weeks after irradiation. This fraction, called the 'repopulation index', is assumed to be directly proportional to the number of surviving stem cells. The response of spermatogonial stem cells in the CBA mouse to 1-MeV fission neutrons was investigated. Radioresistant, colony forming stem cells in the mouse testis move into a much more radiosensitive phase of their cell cycle shortly after irradiation. This is demonstrated in publication II in experiments in which total doses of 300 rad of neutrons and 1200 rad of X-rays were split into two equal fractions. The radiation response of spermatogonial stem cells in the mouse which survived various doses of fission neutrons 24 hours before was studied in publication III. Twenty four hours after a dose of 150 rad of fission neutrons all first-dose survivors have moved from a radioresistant (D 0 89+-4 rad in this study) towards a radiosensitive phase of their cell cycle. Spermatogonial stem cells which survive a neutron dose of 150 rad all belong to a radioresistant stem cell population in the seminiferous epithelium. The data in publication IV show that during the first 26 days after a dose of 150 rad of neutrons the stem cell population first increases and then slowly decreases its radiosensitivity, to stay fixed at a relatively high level. (Auth.)

  4. Isolation of Kupffer Cells and Hepatocytes from a Single Mouse Liver

    DEFF Research Database (Denmark)

    Aparicio-Vergara, Marcela; Tencerova, Michaela; Morgantini, Cecilia

    2017-01-01

    Liver perfusion is a common technique used to isolate parenchymal and non-parenchymal liver cells for in vitro experiments. This method allows hepatic cells to be separated based on their size and weight, by centrifugation using a density gradient. To date, other methods allow the isolation of only...... one viable hepatic cellular fraction from a single mouse; either parenchymal (hepatocytes) or non-parenchymal cells (i.e., Kupffer cells or hepatic stellate cells). Here, we describe a method to isolate both hepatocytes and Kupffer cells from a single mouse liver, thereby providing the unique...... advantage of studying different liver cell types that have been isolated from the same organism....

  5. DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

    International Nuclear Information System (INIS)

    Yaki, T.

    1982-01-01

    DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

  6. Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model.

    Science.gov (United States)

    Swift, Brenna E; Williams, Brent A; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A; Viswanathan, Sowmya; Martinez-Lopez, Joaquin; Keating, Armand

    2012-07-01

    Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89-99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma

  7. Endothelial Cells Control Pancreatic Cell Fate at Defined Stages through EGFL7 Signaling

    Directory of Open Access Journals (Sweden)

    Der-I Kao

    2015-02-01

    Full Text Available Although endothelial cells have been shown to affect mouse pancreatic development, their precise function in human development remains unclear. Using a coculture system containing human embryonic stem cell (hESC-derived progenitors and endothelial cells, we found that endothelial cells play a stage-dependent role in pancreatic development, in which they maintain pancreatic progenitor (PP self-renewal and impair further differentiation into hormone-expressing cells. The mechanistic studies suggest that the endothelial cells act through the secretion of EGFL7. Consistently, endothelial overexpression of EGFL7 in vivo using a transgenic mouse model resulted in an increase of PP proliferation rate and a decrease of differentiation toward endocrine cells. These studies not only identified the role of EGFL7 as the molecular handle involved in the crosstalk between endothelium and pancreatic epithelium, but also provide a paradigm for using hESC stepwise differentiation to dissect the stage-dependent roles of signals controlling organogenesis.

  8. Morphometric studies with attached mouse C3H/10T 1/2 cells

    International Nuclear Information System (INIS)

    Geard, C.R.; Harding, T.

    1981-01-01

    Studies of in vitro transformation using the Syrian hamster embryo cell system and the mouse C3H/10T 1/2 cell system form an integral part of this laboratory's activities. As part of the studies with the mouse cell line we have monitored the behavior of these cells in culture in order to ascertain those variables which might influence the expression of transformation. The study of transformed cells versus normal cells could lead to insight into an earlier definition of transformation that the clonal morphological change currently in use. This present report details the changes in cellular morphology with time in culture of normal mouse C3H/10T 1/2 cells from early passages (9 to 13) and x-ray transformed cells which have been maintained in culture for three years

  9. Sertoli cells maintain Leydig cell number and peritubular myoid cell activity in the adult mouse testis.

    Directory of Open Access Journals (Sweden)

    Diane Rebourcet

    Full Text Available The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.

  10. Metformin prevents methylglyoxal-induced apoptosis of mouse Schwann cells

    International Nuclear Information System (INIS)

    Ota, Kimiko; Nakamura, Jiro; Li, Weiguo; Kozakae, Mika; Watarai, Atsuko; Nakamura, Nobuhisa; Yasuda, Yutaka; Nakashima, Eirtaro; Naruse, Keiko; Watabe, Kazuhiko; Kato, Koichi; Oiso, Yutaka; Hamada, Yoji

    2007-01-01

    Methylglyoxal (MG) is involved in the pathogenesis of diabetic complications via the formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS). To clarify whether the antidiabetic drug metformin prevents Schwann cell damage induced by MG, we cultured mouse Schwann cells in the presence of MG and metformin. Cell apoptosis was evaluated using Hoechst 33342 nuclear staining, caspase-3 activity, and c-Jun-N-terminal kinase (JNK) phosphorylation. Intracellular ROS formation was determined by flow cytometry, and AMP-activated kinase (AMPK) phosphorylation was also examined. MG treatment resulted in blunted cell proliferation, an increase in the number of apoptotic cells, and the activation of caspase-3 and JNK along with enhanced intracellular ROS formation. All of these changes were significantly inhibited by metformin. No significant activation of AMPK by MG or metformin was observed. Taken together, metformin likely prevents MG-induced apoptotic signals in mouse Schwann cells by inhibiting the formation of AGEs and ROS

  11. Analysis of glycoprotein E-selectin ligANDs on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells

    KAUST Repository

    Merzaban, Jasmeen S.

    2011-06-09

    Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34+ cells) display greater E-selectin binding than those obtained from mouse (lin-/Sca-1+/c-kit+ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34+ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved byWestern blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ∼ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand- 1 glycoform "CLA," and CD43. Human, but not mouse, cells displayed a prominent ∼ 100-kDa band, exclusively comprising the CD44 glycoform "HCELL."E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL\\'s contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures. © 2011 by The American Society of Hematology.

  12. Human amniotic epithelial cell feeder layers maintain mouse embryonic stem cell pluripotency via epigenetic regulation of the c-Myc promoter.

    Science.gov (United States)

    Liu, Te; Cheng, Weiwei; Liu, Tianjin; Guo, Lihe; Huang, Qin; Jiang, Lizhen; Du, Xiling; Xu, Fuhui; Liu, Zhixue; Lai, Dongmei

    2010-02-01

    Mouse embryonic stem cells (ESCs) are typically cultured on a feeder layer of mouse embryonic fibroblasts (MEFs), with leukemia inhibitory factor (LIF) added to maintain them in an undifferentiated state. We have previously shown that human amniotic epithelial cells (hAECs) can be used as feeder cells to maintain mouse ESC pluripotency, but the mechanism for this is unknown. In the present study, we found that CpG islands 5' of the c-Myc gene remain hypomethylated in mouse ESCs cultured on hAECs. In addition, levels of acetylation of histone H3 and trimethylation of histone H3K4 in the c-Myc gene promoter were higher in ES cells cultured on hAECs than those in ES cells cultured on MEFs. These data suggested that hAECs can alter mouse ESC gene expression via epigenetic modification of c-Myc, providing a possible mechanism for the hAEC-induced maintenance of ESCs in an undifferentiated state.

  13. A human-like senescence-associated secretory phenotype is conserved in mouse cells dependent on physiological oxygen.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Coppé

    2010-02-01

    Full Text Available Cellular senescence irreversibly arrests cell proliferation in response to oncogenic stimuli. Human cells develop a senescence-associated secretory phenotype (SASP, which increases the secretion of cytokines and other factors that alter the behavior of neighboring cells. We show here that "senescent" mouse fibroblasts, which arrested growth after repeated passage under standard culture conditions (20% oxygen, do not express a human-like SASP, and differ from similarly cultured human cells in other respects. However, when cultured in physiological (3% oxygen and induced to senesce by radiation, mouse cells more closely resemble human cells, including expression of a robust SASP. We describe two new aspects of the human and mouse SASPs. First, cells from both species upregulated the expression and secretion of several matrix metalloproteinases, which comprise a conserved genomic cluster. Second, for both species, the ability to promote the growth of premalignant epithelial cells was due primarily to the conserved SASP factor CXCL-1/KC/GRO-alpha. Further, mouse fibroblasts made senescent in 3%, but not 20%, oxygen promoted epithelial tumorigenesis in mouse xenographs. Our findings underscore critical mouse-human differences in oxygen sensitivity, identify conditions to use mouse cells to model human cellular senescence, and reveal novel conserved features of the SASP.

  14. Characterization of Bovine 5′-flanking Region during Differentiation of Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Hye-Jeong Jang

    2015-12-01

    Full Text Available Embryonic stem cells (ESCs have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (−420/+181 bovine NANOG 5′-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (−420/+181 promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

  15. In vitro toxicity assay of cisplatin on mouse acute lymphoblastic leukaemia and spermatogonial stem cells.

    Science.gov (United States)

    Shabani, R; Ashtari, K; Behnam, B; Izadyar, F; Asgari, H; Asghari Jafarabadi, M; Ashjari, M; Asadi, E; Koruji, M

    2016-06-01

    Testicular cancer is the most common cancer affecting men in reproductive age, and cisplatin is one of the major helpful chemotherapeutic agents for treatment of this cancer. In addition, exposure of testes cancer cells to cisplatin could potentially eliminate tumour cells from germ cells in patients. The aim of this study was to evaluate the effect of cisplatin on viability of mouse acute lymphoblastic leukaemia cell line (EL-4) and neonatal mouse spermatogonial cells in vitro. In this study, the isolated spermatogonial stem cells (SSC) and EL-4 were divided into six groups including control (received medium), sham (received DMSO in medium) and experimental groups which received different doses of cisplatin (0.5, 5, 10 and 15 μg ml(-1) ). Cells viability was evaluated with MTT assay. The identity of the cultured cells was confirmed by the expression of specific markers. Our finding showed that viability of both SSC and EL-4 cells was reduced with the dose of 15 μg/ml when compared to the control group (P ≤ 0.05). Also, the differences between the IC50 in doses 10 and 15 μg/ml at different time were significant (P ≤ 0.05). The number of TUNEL-positive cells was increased, and the BAX and caspase-3 expressions were upregulated in EL4 cells for group that received an effective dose of cisplatin). In conclusion, despite the dramatic effects of cisplatin on both cells, spermatogonial stem cells could form colony in culture. © 2015 Blackwell Verlag GmbH.

  16. Resveratrol protects mouse embryonic stem cells from ionizing radiation by accelerating recovery from DNA strand breakage.

    Science.gov (United States)

    Denissova, Natalia G; Nasello, Cara M; Yeung, Percy L; Tischfield, Jay A; Brenneman, Mark A

    2012-01-01

    Resveratrol has elicited many provocative anticancer effects in laboratory animals and cultured cells, including reduced levels of oxidative DNA damage, inhibition of tumor initiation and progression and induction of apoptosis in tumor cells. Use of resveratrol as a cancer-preventive agent in humans will require that its anticancer effects not be accompanied by damage to normal tissue stem or progenitor cells. In mouse embryonic stem cells (mESC) or early mouse embryos exposed to ethanol, resveratrol has been shown to suppress apoptosis and promote survival. However, in cells exposed to genotoxic stress, survival may come at the expense of genome stability. To learn whether resveratrol can protect stem cells from DNA damage and to study its effects on genomic integrity, we exposed mESC pretreated with resveratrol to ionizing radiation (IR). Forty-eight hours pretreatment with a comparatively low concentration of resveratrol (10 μM) improved survival of mESC >2-fold after exposure to 5 Gy of X-rays. Cells pretreated with resveratrol sustained the same levels of reactive oxygen species and DNA strand breakage after IR as mock-treated controls, but repaired DNA damage more rapidly and resumed cell division sooner. Frequencies of IR-induced mutation at a chromosomal reporter locus were not increased in cells pretreated with resveratrol as compared with controls, indicating that resveratrol can improve viability in mESC after DNA damage without compromising genomic integrity.

  17. Contribution of Mouse Embryonic Stem Cells and Induced Pluripotent Stem Cells to Chimeras through Injection and Coculture of Embryos

    OpenAIRE

    Guo, Jitong; Wu, Baojiang; Li, Shuyu; Bao, Siqin; Zhao, Lixia; Hu, Shuxiang; Sun, Wei; Su, Jie; Dai, Yanfeng; Li, Xihe

    2014-01-01

    Blastocyst injection and morula aggregation are commonly used to evaluate stem cell pluripotency based on chimeric contribution of the stem cells. To assess the protocols for generating chimeras from stem cells, 8-cell mouse embryos were either injected or cocultured with mouse embryonic stem cells and induced pluripotent stem cells, respectively. Although a significantly higher chimera rate resulted from blastocyst injection, the highest germline contribution resulted from injection of 8-cel...

  18. Targeting Phosphoinositide 3-Kinase γ in Airway Smooth Muscle Cells to Suppress Interleukin-13-Induced Mouse Airway Hyperresponsiveness

    Science.gov (United States)

    Jiang, Haihong; Xie, Yan; Abel, Peter W.; Toews, Myron L.; Townley, Robert G.; Casale, Thomas B.

    2012-01-01

    We recently reported that phosphoinositide 3-kinase γ (PI3Kγ) directly regulates airway smooth muscle (ASM) contraction by modulating Ca2+ oscillations. Because ASM contraction plays a critical role in airway hyperresponsiveness (AHR) of asthma, the aim of the present study was to determine whether targeting PI3Kγ in ASM cells could suppress AHR in vitro and in vivo. Intranasal administration into mice of interleukin-13 (IL-13; 10 μg per mouse), a key pathophysiologic cytokine in asthma, induced AHR after 48 h, as assessed by invasive tracheostomy. Intranasal administration of a broad-spectrum PI3K inhibitor or a PI3Kγ-specific inhibitor 1 h before AHR assessment attenuated IL-13 effects. Airway responsiveness to bronchoconstrictor agonists was also examined in precision-cut mouse lung slices pretreated without or with IL-13 for 24 h. Acetylcholine and serotonin dose-response curves indicated that IL-13-treated lung slices had a 40 to 50% larger maximal airway constriction compared with controls. Furthermore, acetylcholine induced a larger initial Ca2+ transient and increased Ca2+ oscillations in IL-13-treated primary mouse ASM cells compared with control cells, correlating with increased cell contraction. As expected, PI3Kγ inhibitor treatment attenuated IL-13-augmented airway contractility of lung slices and ASM cell contraction. In both control and IL-13-treated ASM cells, small interfering RNA-mediated knockdown of PI3Kγ by 70% only reduced the initial Ca2+ transient by 20 to 30% but markedly attenuated Ca2+ oscillations and contractility of ASM cells by 50 to 60%. This report is the first to demonstrate that PI3Kγ in ASM cells is important for IL-13-induced AHR and that acute treatment with a PI3Kγ inhibitor can ameliorate AHR in a murine model of asthma. PMID:22543031

  19. Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines

    OpenAIRE

    Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

    2016-01-01

    Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However,...

  20. Nuclear hormone receptor expression in mouse kidney and renal cell lines.

    Directory of Open Access Journals (Sweden)

    Daisuke Ogawa

    Full Text Available Nuclear hormone receptors (NHRs are transcription factors that regulate carbohydrate and lipid metabolism, immune responses, and inflammation. Although several NHRs, including peroxisome proliferator-activated receptor-γ (PPARγ and PPARα, demonstrate a renoprotective effect in the context of diabetic nephropathy (DN, the expression and role of other NHRs in the kidney are still unrecognized. To investigate potential roles of NHRs in the biology of the kidney, we used quantitative real-time polymerase chain reaction to profile the expression of all 49 members of the mouse NHR superfamily in mouse kidney tissue (C57BL/6 and db/m, and cell lines of mesangial (MES13, podocyte (MPC, proximal tubular epithelial (mProx24 and collecting duct (mIMCD3 origins in both normal and high-glucose conditions. In C57BL/6 mouse kidney cells, hepatocyte nuclear factor 4α, chicken ovalbumin upstream promoter transcription factor II (COUP-TFII and COUP-TFIII were highly expressed. During hyperglycemia, the expression of the NHR 4A subgroup including neuron-derived clone 77 (Nur77, nuclear receptor-related factor 1, and neuron-derived orphan receptor 1 significantly increased in diabetic C57BL/6 and db/db mice. In renal cell lines, PPARδ was highly expressed in mesangial and proximal tubular epithelial cells, while COUP-TFs were highly expressed in podocytes, proximal tubular epithelial cells, and collecting duct cells. High-glucose conditions increased the expression of Nur77 in mesangial and collecting duct cells, and liver x receptor α in podocytes. These data demonstrate NHR expression in mouse kidney cells and cultured renal cell lines and suggest potential therapeutic targets in the kidney for the treatment of DN.

  1. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Studies have shown that embryonic stem (ES) cells can be successfully differentiated into liver cells, which offer the potential unlimited cell source for a variety of end-stage liver disease. In our study, in order to induce mouse ES cells to differentiate into hepatocyte-like cells under chemically defined conditions, ES cells ...

  2. A novel podoplanin-GFPCre mouse strain for gene deletion in lymphatic endothelial cells.

    Science.gov (United States)

    Gil, Hyea Jin; Ma, Wanshu; Oliver, Guillermo

    2018-04-01

    The lymphatic vascular system is a one-direction network of thin-walled capillaries and larger vessels covered by a continuous layer of endothelial cells responsible for maintaining fluid homeostasis. Some of the main functions of the lymphatic vasculature are to drain fluid from the extracellular spaces and return it back to the blood circulation, lipid absorption from the intestinal tract, and transport of immune cells to lymphoid organs. A number of genes controlling the development of the mammalian lymphatic vasculature have been identified in the last few years, and their functional roles started to be characterized using gene inactivation approaches in mice. Unfortunately, only few mouse Cre strains relatively specific for lymphatic endothelial cells (LECs) are currently available. In this article, we report the generation of a novel Podoplanin (Pdpn) GFPCre transgenic mouse strain using its 5' regulatory region. Pdpn encodes a transmembrane mucin-type O-glycoprotein that is expressed on the surface of embryonic and postnatal LECs, in addition to few other cell types. Our detailed characterization of this novel strain indicates that it will be a valuable additional genetic tool for the analysis of gene function in LECs. © 2018 Wiley Periodicals, Inc.

  3. Radiosensitization of mouse spermatogenic stem cells by Ro-07-0582

    International Nuclear Information System (INIS)

    Suzuki, N.; Withers, R.; Hunter, N.

    1977-01-01

    The hypoxic character of the spermatogenic stem cells of the mouse testis was investigated by measuring the effect on radiosensitivity of treatment with the hypoxic cell radiosensitizer, Ro-07-0582 or hyperbaric oxygen (30 psi). The D 0 values obtained were 181 (161-207) rad for irradiation alone, 140 (133-148) rad for irradiation after treatment with Ro-07-0582, and about 100 rad for irradiation in the presence of hyperbaric oxygen. Ro-07-0582 alone was slightly cytotoxic. The results demonstrate that mouse spermatogenic stem cells are radiosensitized by Ro-07-0582 or hyperbaric oxygen and are not as well oxygenated as other normal tissues

  4. Mouse mannose-binding lectin-A and ficolin-A inhibit lipopolysaccharide-mediated pro-inflammatory responses on mast cells

    DEFF Research Database (Denmark)

    Ma, Ying Jie; Kang, Hee Jung; Kim, Ji Yeon

    2013-01-01

    It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response...... cytokine production by LPS-mediated TLR4 in mBMMCs appears to be down-regulated, indicating that mouse MBL and ficolin may have an inhibitory function toward mouse TLR4-mediated excessive inflammation on the mast cells.......It is unknown how soluble pattern-recognition receptors in blood, such as mannose-binding lectin (MBL) and ficolins, modulate mast cell-mediated inflammatory responses. We investigate how mouse MBL-A or ficolin-A regulate mouse bone marrow-derived mast cells (mBMMCs)-derived inflammatory response...

  5. Genetic control of the radiosensitivity of lymphoid cells for antibody-forming ability in CXS series of recombinant inbred mouse strains

    International Nuclear Information System (INIS)

    Okumoto, M.; Mori, N.; Nishikawa, R.; Imai, S.; Hilgers, J.; Takamori, Y.; Yagasaki, O.

    1992-01-01

    Incidence of radiation-induced lymphomas differs remarkably among various mouse strains. BALB/cHeA (C) mice are highly susceptible to radiation induction of lymphomas, while STS/A (S) mice are resistant. Thus, the induction of the disease is controlled by some genetic factors. To examine an involvement of radiosensitivity of lymphoid cells in lymphomagenesis, we have compared genetic control of the radiosensitivity for antibody-forming ability with that of lymphoma development in BALB/cHeA, STS/A, (CXS)F 1 hybrids and CXS series of recombinant inbred strains. Decrease of number of splenic plaque-forming cell (PFC) in Jerne's method by 3 Gy of X-irradiation for BALB/cHeA mice was larger than that for STS/A mice by more than one order of magnitude. (CXS)F 1 hybrid mice showed small number of decrease of PFC similar to STS/A mice suggesting that phenotype of radioresistance was dominant over sensitivity. The best concordance between genetic markers and radiosensitivities of antibody-forming ability in recombinant inbred strains was observed in a region containing Igh locus on chromosome 12. The results show that one locus controlling the radioresistance of lymphoid cells for antibody-forming ability might exist in the region containing Igh locus, and that this region clearly differ from a region with Ifa locus on chromosome 4 which regulate the susceptibility to radiation-induced lymphomagenesis. (author)

  6. Stepwise development of MAIT cells in mouse and human.

    Directory of Open Access Journals (Sweden)

    Emmanuel Martin

    2009-03-01

    Full Text Available Mucosal-associated invariant T (MAIT cells display two evolutionarily conserved features: an invariant T cell receptor (TCRalpha (iTCRalpha chain and restriction by the nonpolymorphic class Ib major histocompatibility complex (MHC molecule, MHC-related molecule 1 (MR1. MR1 expression on thymus epithelial cells is not necessary for MAIT cell development but their accumulation in the gut requires MR1 expressing B cells and commensal flora. MAIT cell development is poorly known, as these cells have not been found in the thymus so far. Herein, complementary human and mouse experiments using an anti-humanValpha7.2 antibody and MAIT cell-specific iTCRalpha and TCRbeta transgenic mice in different genetic backgrounds show that MAIT cell development is a stepwise process, with an intra-thymic selection followed by peripheral expansion. Mouse MAIT cells are selected in an MR1-dependent manner both in fetal thymic organ culture and in double iTCRalpha and TCRbeta transgenic RAG knockout mice. In the latter mice, MAIT cells do not expand in the periphery unless B cells are added back by adoptive transfer, showing that B cells are not required for the initial thymic selection step but for the peripheral accumulation. In humans, contrary to natural killer T (NKT cells, MAIT cells display a naïve phenotype in the thymus as well as in cord blood where they are in low numbers. After birth, MAIT cells acquire a memory phenotype and expand dramatically, up to 1%-4% of blood T cells. Finally, in contrast with NKT cells, human MAIT cell development is independent of the molecular adaptor SAP. Interestingly, mouse MAIT cells display a naïve phenotype and do not express the ZBTB16 transcription factor, which, in contrast, is expressed by NKT cells and the memory human MAIT cells found in the periphery after birth. In conclusion, MAIT cells are selected by MR1 in the thymus on a non-B non-T hematopoietic cell, and acquire a memory phenotype and expand in the

  7. Apoptosis of mouse hippocampal cells induced by Taenia crassiceps metacestode factor.

    Science.gov (United States)

    Zepeda, N; Solano, S; Copitin, N; Chávez, J L; Fernández, A M; García, F; Tato, P; Molinari, J L

    2017-03-01

    Seizures, headache, depression and neurological deficits are the signs and symptoms most frequently reported in human neurocysticercosis. However, the cause of the associated learning and memory deficits is unknown. Here, we used Taenia crassiceps infection in mice as a model of human cysticercosis. The effects of T. crassiceps metacestode infection or T. crassiceps metacestode factor (MF) treatment on mouse hippocampal cells were studied; control mice were included. At 45 days after infection or treatment of the mice with MF, all mice were anaesthetized and perfused transcardially with saline followed by phosphate-buffered 10% formalin. Then the brains were carefully removed. Coronal sections stained using several techniques were analysed. Extensive and significant apoptosis was found in the experimental animals, mainly in the dentate gyrus, CA1, CA2, CA3 and neighbouring regions, in comparison with the apparently intact cells from control mice (P < 0.01). These results suggest that neurological deficits, especially the learning and memory deficits, may be generated by extensive apoptosis of hippocampal cells.

  8. Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

    Directory of Open Access Journals (Sweden)

    Ivanna Ihnatovych

    Full Text Available Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C. Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate- cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

  9. 35S induced dominant lethals in male germ cells of mouse

    International Nuclear Information System (INIS)

    Satyanarayana Reddy, K.; Reddy, P.D.; Reddi, O.S.

    1977-01-01

    (CBA female x C 3 H/He male) F 1 males born to 35 S (20 μCi) treated animals during major organogenesis period were tested for dominant lethal mutations at maturity. The pre-implantation loss showed an increase from 6.88% in the control to 10.92% in 35 S treated animals. Similarly the post-implantation loss has increased from 3.96% (control) to 7.40%. As a result of the increased pre- and post-losses the total loss showed a significant increase (17.51%) in F 1 males born to 35 S treated animals when compared to controls (10.57%). Thus the results clearly show that 35 S is mutagenic in male germ cells of mouse. (author)

  10. Allergen and Epitope Targets of Mouse-Specific T Cell Responses in Allergy and Asthma

    Directory of Open Access Journals (Sweden)

    Véronique Schulten

    2018-02-01

    Full Text Available Mouse allergy has become increasingly common, mainly affecting laboratory workers and inner-city households. To date, only one major allergen, namely Mus m 1, has been described. We sought to identify T cell targets in mouse allergic patients. PBMC from allergic donors were expanded with either murine urine or epithelial extract and subsequently screened for cytokine production (IL-5 and IFNγ in response to overlapping peptides spanning the entire Mus m 1 sequence, peptides from various Mus m 1 isoforms [major urinary proteins (MUPs], peptides from mouse orthologs of known allergens from other mammalian species and peptides from proteins identified by immunoproteomic analysis of IgE/IgG immunoblots of mouse urine and epithelial extracts. This approach let to the identification of 106 non-redundant T cell epitopes derived from 35 antigens. Three major T cell-activating regions were defined in Mus m 1 alone. Moreover, our data show that immunodominant epitopes were largely shared between Mus m 1 and other MUPs even from different species, suggesting that sequence conservation in different allergens is a determinant for immunodominance. We further identified several novel mouse T cell antigens based on their homology to known mammalian allergens. Analysis of cohort-specific T cell responses revealed that rhinitis and asthmatic patients recognized different epitope repertoires. Epitopes defined herein can be formulated into an epitope “megapool” used to diagnose mouse allergy and study mouse-specific T cell responses directly ex vivo. This analysis of T cell epitopes provides a good basis for future studies to increase our understanding of the immunopathology associated with MO-allergy and asthma.

  11. Hypothalamic stem cells control ageing speed partly through exosomal miRNAs.

    Science.gov (United States)

    Zhang, Yalin; Kim, Min Soo; Jia, Baosen; Yan, Jingqi; Zuniga-Hertz, Juan Pablo; Han, Cheng; Cai, Dongsheng

    2017-08-03

    It has been proposed that the hypothalamus helps to control ageing, but the mechanisms responsible remain unclear. Here we develop several mouse models in which hypothalamic stem/progenitor cells that co-express Sox2 and Bmi1 are ablated, as we observed that ageing in mice started with a substantial loss of these hypothalamic cells. Each mouse model consistently displayed acceleration of ageing-like physiological changes or a shortened lifespan. Conversely, ageing retardation and lifespan extension were achieved in mid-aged mice that were locally implanted with healthy hypothalamic stem/progenitor cells that had been genetically engineered to survive in the ageing-related hypothalamic inflammatory microenvironment. Mechanistically, hypothalamic stem/progenitor cells contributed greatly to exosomal microRNAs (miRNAs) in the cerebrospinal fluid, and these exosomal miRNAs declined during ageing, whereas central treatment with healthy hypothalamic stem/progenitor cell-secreted exosomes led to the slowing of ageing. In conclusion, ageing speed is substantially controlled by hypothalamic stem cells, partially through the release of exosomal miRNAs.

  12. Mouse mammary tumor virus uses mouse but not human transferrin receptor 1 to reach a low pH compartment and infect cells

    International Nuclear Information System (INIS)

    Wang Enxiu; Obeng-Adjei, Nyamekye; Ying Qihua; Meertens, Laurent; Dragic, Tanya; Davey, Robert A.; Ross, Susan R.

    2008-01-01

    Mouse mammary tumor virus (MMTV) is a pH-dependent virus that uses mouse transferrin receptor 1 (TfR1) for entry into cells. Previous studies demonstrated that MMTV could induce pH 5-dependent fusion-from-with of mouse cells. Here we show that the MMTV envelope-mediated cell-cell fusion requires both the entry receptor and low pH (pH 5). Although expression of the MMTV envelope and TfR1 was sufficient to mediate low pH-dependent syncytia formation, virus infection required trafficking to a low pH compartment; infection was independent of cathepsin-mediated proteolysis. Human TfR1 did not support virus infection, although envelope-mediated syncytia formation occurred with human cells after pH 5 treatment and this fusion depended on TfR1 expression. However, although the MMTV envelope bound human TfR1, virus was only internalized and trafficked to a low pH compartment in cells expressing mouse TfR1. Thus, while human TfR1 supported cell-cell fusion, because it was not internalized when bound to MMTV, it did not function as an entry receptor. Our data suggest that MMTV uses TfR1 for all steps of entry: cell attachment, induction of the conformational changes in Env required for membrane fusion and internalization to an appropriate acidic compartment

  13. Endogenous superoxide dismutase and catalase activities and radiation resistance in mouse cell lines

    International Nuclear Information System (INIS)

    Davy, C.A.; Tesfay, Z.; Jones, J.; Rosenberg, R.C.; McCarthy, C.; Ostrand-Rosenberg, S.

    1988-01-01

    The relationship between the endogenous cytoplasmic levels of the enzymes superoxide dismutase and catalase and the inhibition of cell proliferation by γ-radiation has been studied in 11 mouse cell lines. The resistance of these mouse cell lines to radiation was found to vary by over 25-fold. No correlation was found between the cytoplasmic level of CuZn-superoxide dismutase or catalase and the resistance to radiation as measured by extrapolation number (EN), quasi-threshold dose (Dsub(q)), or Dsub(o). None of the cell lines had detectable cytoplasmic Mn-superoxide dismutase. The apparent Ksub(i) of potassium cyanide for mouse CuZn-superoxide dismutase was determined (Ksub(i) = 6.5 μmol dm -3 ). (author)

  14. Interaction of X-irradiated mouse cells in heterokaryons

    International Nuclear Information System (INIS)

    Hofmanova, J.; Spurna, V.

    1985-01-01

    The frequency of heterokaryon formation and the ability of DNA synthesis in the system of mouse X-irradiated L fibroblasts and non-irradiated or irradiated LS/BL lymphosarcoma cells were studied. The frequency of heterokaryons after fusion of one or both irradiated parental cells was 3 to 6 times higher than in the non-irradiated cell cultures. In these heterokaryons we found 1.5 to 3 times more nuclei of irradiated L cells capable of DNA synthesis than in the population of non-fused irradiated cells. (author)

  15. Identification and characterization of adult mouse meniscus stem/progenitor cells.

    Science.gov (United States)

    Gamer, Laura W; Shi, Rui Rui; Gendelman, Ashira; Mathewson, Dylan; Gamer, Jackson; Rosen, Vicki

    Meniscal damage is a common problem that accelerates the onset of knee osteoarthritis. Stem cell-based tissue engineering treatment approaches have shown promise in preserving meniscal tissue and restoring meniscal function. The purpose of our study was to identify meniscus-derived stem/progenitor cells (MSPCs) from mouse, a model system that allows for in vivo analysis of the mechanisms underlying meniscal injury and healing. MSPCs were isolated from murine menisci grown in explant culture and characterized for stem cell properties. Flow cytometry was used to detect the presence of surface antigens related to stem cells, and qRT-PCR was used to examine the gene expression profile of MSPCs. Major proteins associated with MSPCs were localized in the adult mouse knee using immunohistochemistry. Our data show that MSPCs have universal stem cell-like properties including clonogenicity and multi-potentiality. MSPCs expressed the mesenchymal stem cell markers CD44, Sca-1, CD90, and CD73 and when cultured had elevated levels of biglycan and collagen type I, important extracellular matrix components of adult meniscus. MSPC also expressed significant levels of Lox and Igf-1, genes associated with the embryonic meniscus. Localization studies showed staining for these same proteins in the superficial and outer zones of the adult mouse meniscus, regions thought to harbor endogenous repair cells. MSPCs represent a novel resident stem cell population in the murine meniscus. Analysis of MSPCs in mice will allow for a greater understanding of the cell biology of the meniscus, essential information for enhancing therapeutic strategies for treating knee joint injury and disease.

  16. Mouse neuroblastoma cell based model and the effect of epileptic events on calcium oscillations and neural spikes

    Science.gov (United States)

    Kim, Suhwan; Baek, Juyeong; Jung, Unsang; Lee, Sangwon; Jung, Woonggyu; Kim, Jeehyun; Kang, Shinwon

    2013-05-01

    Recently, Mouse neuroblastoma cells are considered as an attractive model for the study of human neurological and prion diseases, and intensively used as a model system in different areas. Among those areas, differentiation of neuro2a (N2A) cells, receptor mediated ion current, and glutamate induced physiological response are actively investigated. The reason for the interest to mouse neuroblastoma N2A cells is that they have a fast growing rate than other cells in neural origin with a few another advantages. This study evaluated the calcium oscillations and neural spikes recording of mouse neuroblastoma N2A cells in an epileptic condition. Based on our observation of neural spikes in mouse N2A cell with our proposed imaging modality, we report that mouse neuroblastoma N2A cells can be an important model related to epileptic activity studies. It is concluded that the mouse neuroblastoma N2A cells produce the epileptic spikes in vitro in the same way as produced by the neurons or the astrocytes. This evidence advocates the increased and strong level of neurotransmitters release by enhancement in free calcium using the 4-aminopyridine which causes the mouse neuroblastoma N2A cells to produce the epileptic spikes and calcium oscillation.

  17. Mouse cloning and somatic cell reprogramming using electrofused blastomeres.

    Science.gov (United States)

    Riaz, Amjad; Zhao, Xiaoyang; Dai, Xiangpeng; Li, Wei; Liu, Lei; Wan, Haifeng; Yu, Yang; Wang, Liu; Zhou, Qi

    2011-05-01

    Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.

  18. Intrinsic Contribution of Perforin to NK-Cell Homeostasis during Mouse Cytomegalovirus Infection

    Directory of Open Access Journals (Sweden)

    Maja eArapovic

    2016-04-01

    Full Text Available In addition to their role as effector cells in virus control, natural killer (NK cells have an immunoregulatory function in shaping the antiviral T-cell response. This function is further pronounced in perforin-deficient mice that show the enhanced NK-cell proliferation and cytokine secretion upon mouse cytomegalovirus (MCMV infection. Here we confirmed that stronger activation and maturation of NK cells in perforin-deficient mice correlates with higher MCMV load. To further characterize the immunoregulatory potential of perforin, we compared the response of NK cells that express or do not express perforin using bone-marrow chimeras. Our results demonstrated that the enhanced proliferation and maturation of NK cells in MCMV-infected bone-marrow chimeras is an intrinsic property of perforin-deficient NK cells. Thus, in addition to confirming that NK-cell proliferation is virus load dependent, our data extend this notion demonstrating that perforin plays an intrinsic role as a feedback mechanism in regulation of NK-cell proliferation during viral infections.

  19. Programmed Cell Death, Proliferating Cell Nuclear Antigen and p53 Expression in Mouse Colon Mucosa during Diet-Induced Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Mauro Risio

    2000-01-01

    Full Text Available Western‐style diets (WDs trigger and sustain the early phases of tumorigenesis in mouse colon, and when continued throughout the life span lead to the development of dysplastic crypts. In order to evaluate the roles both of cell proliferation and programmed cell death (PCD in WD‐induced tumorigenesis, immunohistochemical detection of proliferating nuclear antigen (PCNA, in situ end labeling (TUNEL of DNA breaks, and p53 protein were carried out in mouse colonic mucosa during prolonged feeding of two WDs. PCNA Labeling Index of colonic crypts was significantly higher in WD‐treated animals than in controls only at the beginning of the nutritional study, the gap rapidly bridged by increased cell proliferation spontaneously occurring in the colonic mucosa during aging. A transient early homeostatic activation of PCD at the base of the crypt also was observed in WD groups. No changes in PCD were seen in the upper third of the crypt or in surface epithelium throughout the study, indicating that PCD in that colonic crypt segment produces a constant flux of cell loss, uninfluenced by homeostatic fluctuations. A major finding was an irreversible, progressive, age‐related decline of PCD at the crypt base in both control and treated animals that occurred during the second half of the rodents  life span. p53 protein was not immunohistochemically detected, suggesting that neither overexpression of wild‐type nor mutated forms of the protein are involved in the above mentioned changes.

  20. 3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency

    International Nuclear Information System (INIS)

    Dayton, E.T.; Pharr, P.; Ogawa, M.; Serafin, W.E.; Austen, K.F.; Levi-Schaffer, F.; Stevens, R.L.

    1988-01-01

    As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated interleukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. The authors now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content ∼ 50-fold and their carboxypeptidase. A content ∼ 100-fold, and augment ∼ their biosynthesis of proteoglycans bearing 35 S-labeled haparin relative to 35 S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment

  1. Effect of Short-Term Hypergravity Treatment on Mouse 2-Cell Embryo Development

    Science.gov (United States)

    Ning, Li-Na; Lei, Xiao-Hua; Cao, Yu-Jing; Zhang, Yun-Fang; Cao, Zhong-Hong; Chen, Qi; Duan, En-Kui

    2015-11-01

    Though there are numerous biological experiments, which have been performed in a space environment, to study the physiological effect of space travel on living organisms, while the potential effect of weightlessness or short-term hypergravity on the reproductive system in most species, particularly in mammalian is still controversial and unclear. In our previous study, we investigated the effect of space microgravity on the development of mouse 4-cell embryos by using Chinese SJ-8. .Unexpectedly, we did not get any developed embryo during the space-flight. Considering that the process of space experiment is quite different from most experiments done on earth in several aspects such as, the vibration and short-term hypergravity during the rock launching and landing. Thus we want to know whether the short-term hypergravity produced by the launch process affect the early embryo development in mice, and howthe early embryos respond to the hypergravity. In present study, we are mimicking the short-term hypergravity during launch by using a centrifuge to investigate its influence on the development of early embryo (2-cell) in mice. We also examined the actin filament distribution in 2-cell embryos by immunostaining to test their potential capacity of development under short-term hypergravity exposure. Our results showed that most 2-cell embryos in the hypergravity exposure groups developed into blastocysts with normal morphology after 72h cultured in vitro, and there is no obvious difference in the development rate of blastocyst formation compared to the control. Moreover, there were no statistically significant differences in birth rates after oviduct transfer of 2-cell mouse embryos exposed on short-term hypergravity compared with 1 g condition. In addition, the well-organized actin distribution appeared in 2-cell embryos after exposed on hypergravity and also in the subsequent developmental blastocysts. Taken together, our data shows that short-term exposure in

  2. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  3. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    International Nuclear Information System (INIS)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-01-01

    Highlights: • LPA 5 inhibits the cell growth and motile activities of 3T3 cells. • LPA 5 suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA 5 on the cell motile activities inhibited by LPA 1 in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA 5 in 3T3 cells. • LPA signaling via LPA 5 acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA 1 –LPA 6 ) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA 1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA 5 in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA 1 and LPA 5 on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA 5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA 1

  4. Novel insights into embryonic stem cell self-renewal revealed through comparative human and mouse systems biology networks.

    Science.gov (United States)

    Dowell, Karen G; Simons, Allen K; Bai, Hao; Kell, Braden; Wang, Zack Z; Yun, Kyuson; Hibbs, Matthew A

    2014-05-01

    Embryonic stem cells (ESCs), characterized by their ability to both self-renew and differentiate into multiple cell lineages, are a powerful model for biomedical research and developmental biology. Human and mouse ESCs share many features, yet have distinctive aspects, including fundamental differences in the signaling pathways and cell cycle controls that support self-renewal. Here, we explore the molecular basis of human ESC self-renewal using Bayesian network machine learning to integrate cell-type-specific, high-throughput data for gene function discovery. We integrated high-throughput ESC data from 83 human studies (~1.8 million data points collected under 1,100 conditions) and 62 mouse studies (~2.4 million data points collected under 1,085 conditions) into separate human and mouse predictive networks focused on ESC self-renewal to analyze shared and distinct functional relationships among protein-coding gene orthologs. Computational evaluations show that these networks are highly accurate, literature validation confirms their biological relevance, and reverse transcriptase polymerase chain reaction (RT-PCR) validation supports our predictions. Our results reflect the importance of key regulatory genes known to be strongly associated with self-renewal and pluripotency in both species (e.g., POU5F1, SOX2, and NANOG), identify metabolic differences between species (e.g., threonine metabolism), clarify differences between human and mouse ESC developmental signaling pathways (e.g., leukemia inhibitory factor (LIF)-activated JAK/STAT in mouse; NODAL/ACTIVIN-A-activated fibroblast growth factor in human), and reveal many novel genes and pathways predicted to be functionally associated with self-renewal in each species. These interactive networks are available online at www.StemSight.org for stem cell researchers to develop new hypotheses, discover potential mechanisms involving sparsely annotated genes, and prioritize genes of interest for experimental validation

  5. Further characterization of protein kinase C in mouse mast cells

    International Nuclear Information System (INIS)

    White, J.R.; Ishizaka, T.

    1986-01-01

    Bridging of cell-bound IgE antibody molecules on colony stimulating factor dependent mouse mast cell line (PT-18) cells by multivalent antigen induces the mobilization and uptake of Ca 2+ monitored by Quin-2 and the production of diacylglycerol. Exposure of the sensitized cells to antigen also induces a substantial increase in protein kinase C (PKC) activity in the plasma membrane (340 units to 1375 units: 1 unit = 1 pmol of 32 P incorporated into Histone H-1/min/10 7 cells), within 30 seconds. There is also an increase in 3 H phorbol-12, 13-dibutyrate ( 3 H-PDB) binding which parallels the increase in PKC activity both in kinetics and antigen dose dependency. Determination of K/sub m/ and V/sub max/ for PKC revealed no difference between the cytosolic and membranous forms of PKC. Partial purification of PKC from the membrane of sensitized mast cells which had been labeled with 32 P and stimulated with DNP-HSA revealed a protein of 80-84,000 molecular weight, which migrated on polyacrylamide gel electrophoresis just above an authentic standard of PKC purified from rat brain. Treatment of the PKC from mouse mast cell membrane with alkaline phosphatase resulted in a reduction of phosphorylating activity and bindability of 3 H-PDB. In conclusion, the authors speculate that activation of mouse mast cells by cross-linking IgE results in the phosphorylation of a silent-pool of PKC converting it from an inactive state to an activated form

  6. Generation of stratified squamous epithelial progenitor cells from mouse induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Satoru Yoshida

    Full Text Available BACKGROUND: Application of induced pluripotent stem (iPS cells in regenerative medicine will bypass ethical issues associated with use of embryonic stem cells. In addition, patient-specific IPS cells can be useful to elucidate the pathophysiology of genetic disorders, drug screening, and tailor-made medicine. However, in order to apply iPS cells to mitotic tissue, induction of tissue stem cells that give rise to progeny of the target organ is required. METHODOLOGY/PRINCIPAL FINDINGS: We induced stratified epithelial cells from mouse iPS cells by co-culture with PA6 feeder cells (SDIA-method with use of BMP4. Clusters of cells positive for the differentiation markers KRT1 or KRT12 were observed in KRT14-positive colonies. We successfully cloned KRT14 and p63 double-positive stratified epithelial progenitor cells from iPS-derived epithelial cells, which formed stratified epithelial sheets consisting of five- to six-polarized epithelial cells in vitro. When these clonal cells were cultured on denuded mouse corneas, a robust stratified epithelial layer was observed with physiological cell polarity including high levels of E-cadherin, p63 and K15 expression in the basal layer and ZO-1 in the superficial layer, recapitulating the apico-basal polarity of the epithelium in vivo. CONCLUSIONS/SIGNIFICANCE: These results suggest that KRT14 and p63 double-positive epithelial progenitor cells can be cloned from iPS cells in order to produce polarized multilayer epithelial cell sheets.

  7. Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity.

    Science.gov (United States)

    Kawamoto, Kohei; Izumikawa, Masahiko; Beyer, Lisa A; Atkin, Graham M; Raphael, Yehoash

    2009-01-01

    Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be

  8. The common mouse protozoa Tritrichomonas muris alters mucosal T cell homeostasis and colitis susceptibility.

    Science.gov (United States)

    Escalante, Nichole K; Lemire, Paul; Cruz Tleugabulova, Mayra; Prescott, David; Mortha, Arthur; Streutker, Catherine J; Girardin, Stephen E; Philpott, Dana J; Mallevaey, Thierry

    2016-12-12

    The mammalian gastrointestinal tract hosts a diverse community of microbes including bacteria, fungi, protozoa, helminths, and viruses. Through coevolution, mammals and these microbes have developed a symbiosis that is sustained through the host's continuous sensing of microbial factors and the generation of a tolerant or pro-inflammatory response. While analyzing T cell-driven colitis in nonlittermate mouse strains, we serendipitously identified that a nongenetic transmissible factor dramatically increased disease susceptibility. We identified the protozoan Tritrichomonas muris as the disease-exacerbating element. Furthermore, experimental colonization with T. muris induced an elevated Th1 response in the cecum of naive wild-type mice and accelerated colitis in Rag1 -/- mice after T cell transfer. Overall, we describe a novel cross-kingdom interaction within the murine gut that alters immune cell homeostasis and disease susceptibility. This example of unpredicted microbial priming of the immune response highlights the importance of studying trans-kingdom interactions and serves as a stark reminder of the importance of using littermate controls in all mouse research. © 2016 Escalante et al.

  9. Cell lineage mapping of taste bud cells and keratinocytes in the mouse tongue and soft palate.

    Science.gov (United States)

    Okubo, Tadashi; Clark, Cheryl; Hogan, Brigid L M

    2009-02-01

    The epithelium of the mouse tongue and soft palate consists of at least three distinct epithelial cell populations: basal cells, keratinized cells organized into filiform and fungiform papillae, and taste receptor cells present in tight clusters known as taste buds in the fungiform and circumvallate papillae and soft palate. All three cell types develop from the simple epithelium of the embryonic tongue and palate, and are continually replaced in the adult by cell turnover. Previous studies using pulse-chase tritiated thymidine labeling in the adult mouse provided evidence for a high rate of cell turnover in the keratinocytes (5-7 days) and taste buds (10 days). However, little is known about the localization and phenotype of the long-term stem or progenitor cells that give rise to the mature taste bud cells and surrounding keratinocytes in these gustatory tissues. Here, we make use of a tamoxifen-inducible K14-CreER transgene and the ROSA26 LacZ reporter allele to lineage trace the mature keratinocytes and taste bud cells of the early postnatal and adult mouse tongue and soft palate. Our results support the hypothesis that both the pore keratinocytes and receptor cells of the taste bud are derived from a common K14(+)K5(+)Trp63(+)Sox2(+) population of bipotential progenitor cells located outside the taste bud. The results are also compatible with models in which the keratinocytes of the filiform and fungiform papillae are derived from basal progenitor cells localized at the base of these structures.

  10. Developmental immunolocalization of the Klotho protein in mouse kidney epithelial cells

    Directory of Open Access Journals (Sweden)

    J.H. Song

    2014-01-01

    Full Text Available A defect in Klotho gene expression in the mouse results in a syndrome that resembles rapid human aging. In this study, we investigated the detailed distribution and the time of the first appearance of Klotho in developing and adult mouse kidney. Kidneys from 16-(F16, 18-(F18 and 20-day-old (F20 fetuses, 1- (P1, 4- (P4, 7- (P7, 14- (P14, and 21-day-old (P21 pups and adults were processed for immunohistochemistry and immunoblot analyses. In the developing mouse kidney, Klotho immunoreactivity was initially observed in a few cells of the connecting tubules (CNT of 18-day-old fetus (F and in the medullary collecting duct (MCD and distal nephron of the F16 developing kidney. In F20, Klotho immunoreactivity was increased in CNT and additionally observed in the outer portion of MCD and tip of the renal papilla. During the first 3 weeks after birth, Klotho-positive cells gradually disappeared from the MCD due to apoptosis, but remained in the CNT and cortical collecting ducts (CCD. In the adult mouse, the Klotho protein was expressed only in a few cells of the CNT and CCD in cortical area. Also, Klotho immunoreactivity was observed in the aquaporin 2-positive CNT, CCD, and NaCl co-transporter-positive distal convoluted tubule (DCT cells and type B and nonA-nonB intercalated cells of CNT, DCT, and CCD. Collectively, our data indicate that immunolocalization of Klotho is closely correlated with proliferation in the intercalated cells of CNT and CCD from aging, and may be involved in the regulation of tubular proliferation.

  11. Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Ishizuka, Toshiaki, E-mail: tishizu@ndmc.ac.jp [Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama 359-8513 (Japan); Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro [Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama 359-8513 (Japan)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the DNA synthesis via induction of superoxide. Black-Right-Pointing-Pointer Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. Black-Right-Pointing-Pointer Angiotensin II enhanced differentiation into mesodermal progenitor cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT{sub 1}R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression

  12. Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

    International Nuclear Information System (INIS)

    Ishizuka, Toshiaki; Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro

    2012-01-01

    Highlights: ► Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. ► Angiotensin II may enhance the DNA synthesis via induction of superoxide. ► Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. ► Angiotensin II enhanced differentiation into mesodermal progenitor cells. ► Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT 1 R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression. Treatment with Ang II enhanced the phosphorylation of p38 MAPK in Col IV- exposed iPS cells. These results suggest that the stimulation

  13. Comparative action spectra for pyrimidine dimer formation in Cloudman S91 mouse melanoma and EMT6 mouse mammary carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Hill, H Z [New Jersey, Medical School, Newark (USA); Setlow, R B [Brookhaven National Lab., Upton, NY (USA)

    1982-05-01

    Pyrimidine dimer formation in melanotic mouse melanoma cells, Cloudman S91H-, and in mouse mammary carcinoma cells, EMT6, was compared as a function of wavelength by irradiating equal numbers of cells from the two cell lines simultaneously. More dimers were formed in EMT6 than in S91H- by light of wavelengths less than 289nm, while light of higher wavelengths caused equivalent dimer formation, as measured by the Micrococcus luteus UV-endonuclease assay. The cells of S91H- are lightly melanotic, yet shielding at lower wavelengths is considerable. It is speculated that melanin pigmentation arose by selection during an evolutionary period when UV-C light reaching the earth's surface was significantly greater than it is today.

  14. E-cadherin promotes incorporation of mouse epiblast stem cells into normal development.

    Directory of Open Access Journals (Sweden)

    Satoshi Ohtsuka

    Full Text Available Mouse epiblast stem cells (mEpiSCs are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.

  15. Cyclin G1 inhibits the proliferation of mouse endometrial stromal cell in decidualization

    Directory of Open Access Journals (Sweden)

    Xu Qian

    2017-01-01

    Full Text Available Uterine stromal cell decidualization is a dynamic physiological process in which cell proliferation, differentiation and apoptosis are orchestrated and occur in a temporal and cell-specific manner. This process is important for successful embryo implantation. Many cell-cycle regulators are involved in decidualization. The protein cyclin G1 is a unique regulator of the cell cycle with dual functions in cell proliferation. It was reported that cyclin G1 is expressed in mouse uterine stromal cells during the period of peri-implantation. To prove the function of cyclin G1 in mouse uterine stromal cells during this period, immunohistochemistry was used to stain mouse uterine tissues on days 4-8 of pregnancy. The results showed obvious spatial and temporal expression of cyclin G1 in uterine stromal cells, and that it is expressed in the cells of the primary decidual zone (PDZ on day 5 and secondary decidual zone (SDZ on days 6 and 7, when the stromal cells experienced active proliferation and differentiation was initiated. Applying the decidualization model of cultured primary stromal cells in vitro, we further revealed that the expression of cyclin G1 is associated with decidualization of stromal cells induced by medroxyprogesterone acetate (MPA and estradiol-17β (E2. RNA interference was used for the knockdown of cyclin G1 in the induced decidual cells. Flow cytometry analysis indicated that the proportion of cells in the S stage was increased, and decreased in the G2/M phase. Our study indicates that cyclin G1, as a negative regulator of the cell cycle, plays an important role in the process of decidualization in mouse uterine stromal cells by inhibiting cell-cycle progression.

  16. Poly (ADP-ribose) metabolism in alkylated mouse L5178Y cells

    International Nuclear Information System (INIS)

    Boyle, J.M.

    1985-01-01

    Poly ADP-ribosylation of two mouse lymphoma cell lines, L5178Y (LS) and the radiation and alkylating agent resistant derivative AII, was investigated by uptake of [ 3 H]NAD by permeabilised cells into acid-precipitable material that was sensitive to phosphodiesterase but insensitive to DNase and RNase. Basal activities in both lymphoma lines were 3-4-fold greater than in mouse L1210 leukaemia cells. However, total endogenous poly (ADP-R) polymerase activity in both L5178Y cell lines, stimulated by a large excess of DNase in the presence of Triton X-100, was less than half the activity in L1210 cells. Doses of N-methyl-N-nitrosourea (MNU) that produced 20-50% survival of colony-forming units increased poly (ADP-R) in both lymphoma lines by only 25% compared with 377% in L1210 cells when synthesis was measured immediately after a 30-min exposure of MNU. Concentrations of 3-aminobenzamide (3AB) above 2.5 mM inhibited colony-forming ability of lymphoma cells and equally inhibited uptake of [ 14 C]formate into protein, RNA and DNA indicating that 3AB behaves as a general metabolic poison. Non-toxic concentrations of 3AB potentiated cell killing by MNU to a similar degree in both lymphoma cell lines. In conclusion, the authors have found little evidence to support the hypothesis that the differential sensitivity of LS and AII is related to poly ADP-ribosylation. Compared with other mouse cells, L5178Y cells appear deficient in poly (ADP-R) polymerase and poly (ADP-R) glycohydrolase activities

  17. Hematopoietic stem cell-derived exosomes promote hematopoietic differentiation of mouse embryonic stem cells in vitro via inhibiting the miR126/Notch1 pathway.

    Science.gov (United States)

    Liao, Feng-Ling; Tan, Lin; Liu, Hua; Wang, Jin-Ju; Ma, Xiao-Tang; Zhao, Bin; Chen, Yanfang; Bihl, Ji; Yang, Yi; Chen, Ri-Ling

    2018-04-01

    Cell-derived exosomes (EXs) can modulate target cell differentiation via microRNAs (miRs) that they carried. Previous studies have shown that miR126 is highly expressed in hematopoietic stem cells (HSCs) and plays a role in hematopoiesis via modulating the Notch pathway that participates in progenitors' cell fate decisions. In this study we investigated whether HSC-derived EXs (HSC-EXs) could affect the differentiation of mouse embryonic stem cells (ESCs) into HSCs. We prepared HSC-EXs con , HSC-EXs sc and HSC-EXs miR126 from control HSCs and the HSCs transfected with scramble control or miR126 mimics, respectively. HSC-EXs were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis. We incubated the collected EXs with mouse ESCs over a 10-d differentiation induction period, during which HSC-EXs and a Notch pathway activator (Jagged1, 100 ng/mL) were added to the cultures every 3 d. After the 10-d differentiation period, the expression levels of miR126, SSEA1, CD117, Sca1, Notch1 and Hes1 in ESCs were assessed. The generated HSCs were validated by flow cytometry using antibodies against HSC markers (CD117, CD34 and Sca1). Our results revealed that: (1) transfection with miR126 mimics significantly increased miR126 levels in HSC-EXs miR126 . (2) HSC-EX co-culture promoted mouse ESCs differentiation into HSCs with the most prominent effect found in the HSC-EXs miR126 co-culture. (3) HSC differentiation was verified by reduced SSEA1 expression and increased CD117 and Sca1 expression. (4) All the effects caused by HSC-EXs were accompanied by significant reduction of Notch1 and Hes1 expression, thus inhibition of the Notch1/Hes1 pathway, whereas activation of Notch by Jagged1 abolished the effects of HSC-EXs miR126 . In conclusion, HSC-EXs promote hematopoietic differentiation of mouse ESCs in vitro by inhibiting the miR126/Notch1 pathway.

  18. Immunohistochemical localization of translationally controlled tumor protein in the mouse digestive system.

    Science.gov (United States)

    Sheverdin, Vadim; Jung, Jiwon; Lee, Kyunglim

    2013-09-01

    Translationally controlled tumor protein (TCTP) is a housekeeping protein, highly conserved among various species. It plays a major role in cell differentiation, growth, proliferation, apoptosis and carcinogenesis. Studies reported so far on TCTP expression in different digestive organs have not led to any understanding of the role of TCTP in digestion, so we localized TCTP in organs of the mouse digestive system employing immunohistochemical techniques. Translationally controlled tumor protein was found expressed in all organs studied: tongue, salivary glands, esophagus, stomach, small and large intestines, liver and pancreas. The expression of TCTP was found to be predominant in epithelia and neurons of myenteric nerve ganglia; high in serous glands (parotid, submandibular, gastric, intestinal crypts, pancreatic acini) and in neurons of myenteric nerve ganglia, and moderate to low in epithelia. In epithelia, expression of TCTP varied depending on its type and location. In enteric neurons, TCTP was predominantly expressed in the processes. Translationally controlled tumor protein expression in the liver followed porto-central gradient with higher expression in pericentral hepatocytes. In the pancreas, TCTP was expressed in both acini and islet cells. Our finding of nearly universal localization and expression of TCTP in mouse digestive organs points to the hitherto unrecognized functional importance of TCTP in the digestive system and suggests the need for further studies of the possible role of TCTP in the proliferation, secretion, absorption and neural regulation of the digestive process and its importance in the physiology and pathology of digestive process. © 2013 Anatomical Society.

  19. Mechanism of testosterone deficiency in the transgenic sickle cell mouse.

    Directory of Open Access Journals (Sweden)

    Biljana Musicki

    Full Text Available Testosterone deficiency is associated with sickle cell disease (SCD, but its underlying mechanism is not known. We investigated the possible occurrence and mechanism of testosterone deficiency in a mouse model of human SCD. Transgenic sickle male mice (Sickle exhibited decreased serum and intratesticular testosterone and increased luteinizing hormone (LH levels compared with wild type (WT mice, indicating primary hypogonadism in Sickle mice. LH-, dbcAMP-, and pregnenolone- (but not 22-hydroxycholesterol- stimulated testosterone production by Leydig cells isolated from the Sickle mouse testis was decreased compared to that of WT mice, implying defective Leydig cell steroidogenesis. There also was reduced protein expression of steroidogenic acute regulatory protein (STAR, but not cholesterol side-chain cleavage enzyme (P450scc, in the Sickle mouse testis. These data suggest that the capacity of P450scc to support testosterone production may be limited by the supply of cholesterol to the mitochondria in Sickle mice. The sickle mouse testis exhibited upregulated NADPH oxidase subunit gp91phox and increased oxidative stress, measured as 4-hydroxy-2-nonenal, and unchanged protein expression of an antioxidant glutathione peroxidase-1. Mice heterozygous for the human sickle globin (Hemi exhibited intermediate hypogonadal changes between those of WT and Sickle mice. These results demonstrate that testosterone deficiency occurs in Sickle mice, mimicking the human condition. The defects in the Leydig cell steroidogenic pathway in Sickle mice, mainly due to reduced availability of cholesterol for testosterone production, may be related to NADPH oxidase-derived oxidative stress. Our findings suggest that targeting testicular oxidative stress or steroidogenesis mechanisms in SCD offers a potential treatment for improving phenotypic changes associated with testosterone deficiency in this disease.

  20. Network reconstruction of the mouse secretory pathway applied on CHO cell transcriptome data

    DEFF Research Database (Denmark)

    Lund, Anne Mathilde; Kaas, Christian Schrøder; Brandl, Julian

    2017-01-01

    , counting 801 different components in mouse. By employing our mouse RECON to the CHO-K1 genome in a comparative genomic approach, we could reconstruct the protein secretory pathway of CHO cells counting 764 CHO components. This RECON furthermore facilitated the development of three alternative methods...... to study protein secretion through graphical visualizations of omics data. We have demonstrated the use of these methods to identify potential new and known targets for engineering improved growth and IgG production, as well as the general observation that CHO cells seem to have less strict transcriptional...... regulation of protein secretion than healthy mouse cells.  Conclusions: The RECON of the secretory pathway represents a strong tool for interpretation of data related to protein secretion as illustrated with transcriptomic data of Chinese Hamster Ovary (CHO) cells, the main platform for mammalian protein...

  1. Expression of aquaporin-7 and aquaporin-9 in tanycyte cells and choroid plexus during mouse estrus cycle.

    Science.gov (United States)

    Yaba, A; Sozen, B; Suzen, B; Demir, N

    2017-03-01

    Tanycytes are special ependymal cells located in the ventrolateral wall and floor of the third ventricle having processes extending nuclei that regulate reproductive functions and around of vessels in median eminance. The aquaporins (AQPs) are a family of transmembrane proteins that transport water and glycerol. AQP-7 and -9 are permeable to other small molecules as glycerol and therefore called aquaglyceroporins. In this study, we aimed to show localization of AQP-7 and -9 in epithelial cells of choroid plexus and tanycytes during female mouse estrus cycle. AQP-7 and -9 proteins were detected in α2 and β1 tanycytes in prœstrus stage. Interestingly, there is no staining in estrus stage in any type of tanycytes. We observed weak immunoreactivity in α1, α2 and β1 tanycyte cells in metestrus stage for AQP-7 and α1 for AQP-9 protein. AQP-7 and -9 showed intense immunoreactivity in α2, β1 and β2 tanycyte cells during diestrus stage. Consequently, AQP-7 and -9 showed differential staining pattern in different stages of mouse estrus cycle. In the light of our findings and other recent publications, we suggest that AQP-7 and -9-mediated glycerol transport in tanycyte cells might be under hormonal control to use glycerol as a potential energy substrate during mouse estrus cycle. Copyright © 2016. Published by Elsevier Masson SAS.

  2. Prox1 Inhibits Proliferation and Is Required for Differentiation of the Oligodendrocyte Cell Lineage in the Mouse.

    Directory of Open Access Journals (Sweden)

    Kentaro Kato

    Full Text Available Central nervous system injury induces a regenerative response in ensheathing glial cells comprising cell proliferation, spontaneous axonal remyelination, and limited functional recovery, but the molecular mechanisms are not fully understood. In Drosophila, this involves the genes prospero and Notch controlling the balance between glial proliferation and differentiation, and manipulating their levels in glia can switch the response to injury from prevention to promotion of repair. In the mouse, Notch1 maintains NG2 oligodendrocyte progenitor cells (OPCs in a progenitor state, but what factor may enable oligodendrocyte (OL differentiation and functional remyelination is not understood. Here, we asked whether the mammalian homologue of prospero, Prox1, is involved. Our data show that Prox1 is distributed in NG2+ OPCs and in OLs in primary cultured cells, and in the mouse spinal cord in vivo. siRNA prox1 knockdown in primary OPCs increased cell proliferation, increased NG2+ OPC cell number and decreased CC1+ OL number. Prox1 conditional knockout in the OL cell lineage in mice increased NG2+ OPC cell number, and decreased CC1+ OL number. Lysolecithin-induced demyelination injury caused a reduction in CC1+ OLs in homozygous Prox1-/- conditional knockout mice compared to controls. Remarkably, Prox1-/- conditional knockout mice had smaller lesions than controls. Altogether, these data show that Prox1 is required to inhibit OPC proliferation and for OL differentiation, and could be a relevant component of the regenerative glial response. Therapeutic uses of glia and stem cells to promote regeneration and repair after central nervous system injury would benefit from manipulating Prox1.

  3. Deriving multipotent stem cells from mouse spermatogonial stem cells: a new tool for developmental and clinical research

    NARCIS (Netherlands)

    de Rooij, Dirk G.; Mizrak, S. Canan

    2008-01-01

    In recent years, embryonic stem (ES) cell-like cells have been obtained from cultured mouse spermatogonial stem cells (SSCs). These advances have shown that SSCs can transition from being the stem cell-producing cells of spermatogenesis to being multipotent cells that can differentiate into

  4. Extract of mouse embryonic stem cells induces the expression of pluripotency genes in human adipose tissue-derived stem cells.

    Science.gov (United States)

    Salehi, Paria Motamen; Foroutan, Tahereh; Javeri, Arash; Taha, Masoumeh Fakhr

    2017-11-01

    In some previous studies, the extract of embryonic carcinoma cells (ECCs) and embryonic stem cells (ESCs) have been used to reprogram somatic cells to more dedifferentiated state. The aim of this study was to investigate the effect of mouse ESCs extract on the expression of some pluripotency markers in human adipose tissue-derived stem cells (ADSCs). Human ADSCs were isolated from subcutaneous abdominal adipose tissue and characterized by flow cytometric analysis for the expression of some mesenchymal stem cell markers and adipogenic and osteogenic differentiation. Frequent freeze-thaw technique was used to prepare cytoplasmic extract of ESCs. Plasma membranes of the ADSCs were reversibly permeabilized by streptolysin-O (SLO). Then the permeabilized ADSCs were incubated with the ESC extract and cultured in resealing medium. After reprogramming, the expression of some pluripotency genes was evaluated by RT-PCR and quantitative real-time PCR (qPCR) analyses. Third-passaged ADSCs showed a fibroblast-like morphology and expressed mesenchymal stem cell markers. They also showed adipogenic and osteogenic differentiation potential. QPCR analysis revealed a significant upregulation in the expression of some pluripotency genes including OCT4 , SOX2 , NANOG , REX1 and ESG1 in the reprogrammed ADSCs compared to the control group. These findings showed that mouse ESC extract can be used to induce reprogramming of human ADSCs. In fact, this method is applicable for reprogramming of human adult stem cells to a more pluripotent sate and may have a potential in regenerative medicine.

  5. Ulex europaeus 1 lectin targets microspheres to mouse Peyer's patch M-cells in vivo.

    Science.gov (United States)

    Foster, N; Clark, M A; Jepson, M A; Hirst, B H

    1998-03-01

    The interaction of latex microspheres with mouse Peyer's patch membranous M-cells was studied in a mouse gut loop model after the microspheres were coated with a variety of agents. Carboxylated microspheres (diameter 0.5 micron) were covalently coated with lectins Ulex europaeus 1, Concanavalin A, Euonymus europaeus and Bandeiraea simplicifolia 1 isolectin-B4, human immunoglobulin A or bovine serum albumin. Of the treatments examined, only Ulex europaeus (UEA1) resulted in significant selective binding of microspheres to M-cells. UEA1-coated microspheres bound to M-cells at a level 100-fold greater than BSA-coated microspheres, but binding to enterocytes was unaffected. Incubation of UEA1-coated microspheres with alpha-L-fucose reduced M-cell binding to a level comparable with BSA-coated microspheres. This indicated that targeting by UEA1 was via a carbohydrate receptor on the M-cell surface. Adherence of UEA1-coated microspheres to M-cells occurred within 10 min of inoculation into mouse gut loops and UEA1-coated microspheres were transported to 10 microns below the apical surface of M-cells within 60 min of inoculation. UEA1-coated microspheres also targeted mouse Peyer's patch M-cells after intragastric administration. These results demonstrated that altering the surface chemistry of carboxylated polystyrene microspheres increased M-cell targeting, suggesting a strategy to enhance delivery of vaccine antigens to the mucosal immune system.

  6. Tumour-cell killing by X-rays and immunity quantitated in a mouse model system

    International Nuclear Information System (INIS)

    Porteous, D.D.; Porteous, K.M.; Hughes, M.J.

    1979-01-01

    As part of an investigation of the interaction of X-rays and immune cytotoxicity in tumour control, an experimental mouse model system has been used in which quantitative anti-tumour immunity was raised in prospective recipients of tumour-cell suspensions exposed to varying doses of X-rays in vitro before injection. Findings reported here indicate that, whilst X-rays kill a proportion of cells, induced immunity deals with a fixed number dependent upon the immune status of the host, and that X-rays and anti-tumour immunity do not act synergistically in tumour-cell killing. The tumour used was the ascites sarcoma BP8. (author)

  7. Cell cycle evaluation of granulosa cells in the {gamma}-irradiated mouse ovarian follicles

    Energy Technology Data Exchange (ETDEWEB)

    KIm, Jin Kyu; Lee, Chang Joo; Lee, Young Keun [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of); Song, Kang Won; Yoon, Yong Dal [Hanyang Univ., Seoul (Korea, Republic of)

    1999-03-01

    This study was carried out to evaluate the biochemical and morphological effects of ionizing radiation on mouse ovarian follicles. Immature mice (ICR, 3 week-old) were irradiated with a dose of LD{sub 80(30)} at KAERI. The ovaries were collected after 6 hours, 12 hours, 1 day, and 2 days post irradiation. With the morphological basis of the histological staining with hematoxylin-eosin, immunohistochemical preparation using in situ 3'-end labeling was evaluated. Flow cytometric evaluation of DNA extracted from the whole ovary was performed. The percentage of A{sub 0} (subpopulation of cells with degraded DNA and with lower DNA fluorescence than G{sub 0}/G{sub 1} cells), apoptotic, cells in the cell cycle was significantly higher in the irradiated group than in the control group. The number of in situ 3'-end labeled follicles increased at 6 hours post irradiation. All the analyses represented that the ionizing radiation-induced follicular atresia was taken place via an apoptotic degeneration. Such a degeneration underwent very fast and acutely. Therefore, it is concluded that the radiation-induced follicular degeneration is, like the spontaneous atresia, mediated by an acute apoptosis of follicular granulosa cells. Flow cytometric evaluation of cell cycles can make the role for quantifying the atretic follicles and understanding the mechanism of the radiation-induced cell death.

  8. Role of Abcg2 During Mouse Embroyonic Stem Cell Diffferentiation

    Science.gov (United States)

    Role of Abcg2 During Mouse Embryonic Stem Cell Differentiation. Abcg2 is a multidrug resistance ATP-binding cassette (ABC) transporter whose activity may be considered a hallmark of stem cell plasticity. The role of Abcg2 during early embryogenesis, however, is unclear. Studies...

  9. Mouse neuroblastoma cell-based model and the effect of epileptic events on calcium oscillations and neural spikes

    Science.gov (United States)

    Kim, Suhwan; Jung, Unsang; Baek, Juyoung; Lee, Sangwon; Jung, Woonggyu; Kim, Jeehyun; Kang, Shinwon

    2013-01-01

    Recently, mouse neuroblastoma cells have been considered as an attractive model for the study of human neurological and prion diseases, and they have been intensively used as a model system in different areas. For example, the differentiation of neuro2a (N2A) cells, receptor-mediated ion current, and glutamate-induced physiological responses have been actively investigated with these cells. These mouse neuroblastoma N2A cells are of interest because they grow faster than other cells of neural origin and have a number of other advantages. The calcium oscillations and neural spikes of mouse neuroblastoma N2A cells in epileptic conditions are evaluated. Based on our observations of neural spikes in these cells with our proposed imaging modality, we reported that they can be an important model in epileptic activity studies. We concluded that mouse neuroblastoma N2A cells produce epileptic spikes in vitro in the same way as those produced by neurons or astrocytes. This evidence suggests that increased levels of neurotransmitter release due to the enhancement of free calcium from 4-aminopyridine causes the mouse neuroblastoma N2A cells to produce epileptic spikes and calcium oscillations.

  10. Direct contact with endoderm-like cells efficiently induces cardiac progenitors from mouse and human pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Hideki Uosaki

    Full Text Available RATIONALE: Pluripotent stem cell-derived cardiac progenitor cells (CPCs have emerged as a powerful tool to study cardiogenesis in vitro and a potential cell source for cardiac regenerative medicine. However, available methods to induce CPCs are not efficient or require high-cost cytokines with extensive optimization due to cell line variations. OBJECTIVE: Based on our in-vivo observation that early endodermal cells maintain contact with nascent pre-cardiac mesoderm, we hypothesized that direct physical contact with endoderm promotes induction of CPCs from pluripotent cells. METHOD AND RESULT: To test the hypothesis, we cocultured mouse embryonic stem (ES cells with the endodermal cell line End2 by co-aggregation or End2-conditioned medium. Co-aggregation resulted in strong induction of Flk1(+ PDGFRa(+ CPCs in a dose-dependent manner, but the conditioned medium did not, indicating that direct contact is necessary for this process. To determine if direct contact with End2 cells also promotes the induction of committed cardiac progenitors, we utilized several mouse ES and induced pluripotent (iPS cell lines expressing fluorescent proteins under regulation of the CPC lineage markers Nkx2.5 or Isl1. In agreement with earlier data, co-aggregation with End2 cells potently induces both Nkx2.5(+ and Isl1(+ CPCs, leading to a sheet of beating cardiomyocytes. Furthermore, co-aggregation with End2 cells greatly promotes the induction of KDR(+ PDGFRa(+ CPCs from human ES cells. CONCLUSIONS: Our co-aggregation method provides an efficient, simple and cost-effective way to induce CPCs from mouse and human pluripotent cells.

  11. Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells.

    Science.gov (United States)

    Lv, Lei; Zhang, Tianwei; Yi, Qiyi; Huang, Yun; Wang, Zheng; Hou, Heli; Zhang, Huan; Zheng, Wei; Hao, Qiaomei; Guo, Zongyou; Cooke, Howard J; Shi, Qinghua

    2012-08-01

    Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.

  12. EXAMINATION OF THE GERM CELL CHIMERA FORMING POTENTIAL OF MOUSE EMBRYONIC STEM CELLS

    Directory of Open Access Journals (Sweden)

    V.B. CÂRSTEA

    2007-05-01

    Full Text Available The aim of this study was to examine the factors, which influence the chimeraforming potential of mouse embryonic stem cells (ES cells. In our work, we examinethe chimera producing ability of R1 and R1/E mouse ES cell lines. We found that thepassage number affects chimera-forming capability of the ES cells. With theincreasing of the passage number, it could be getting less chimera animal, and onlythe R1/E ES cell line derived cells could contribute to the germ cells. At first, wecompared the marker of pluripotency using immunostaining and RT PCR, but wecould not find any difference between the R1 and R1/E cell in this way. Atchromosome analysis, we found, that the number of aneuploid cells, in R1 ES cellline, dramatically increased after 10 passages. We thought that the reason is thatduring the cell division Y chromosome could not arrange correctly between the twonewly derived progeny cells. To prove our conception, we made X and YchromosomeFISH analyses. We found, that the aneuploid R1 and R1/E ES cellscontain only one X and one Y chromosome, so not the loss of Y chromosome causethe problem at the germ cell formation. At last, we made the karyotypeanalysis of R1 and R1/E ES cells at different passages. The karyotype analysisdemonstrated that in the case of R1 ES cell line, the 41 and 42-chromosomecontaining cells hold trisomy. With the increasing of the passages number, thenumber of trisomy containing aneuploid cells increased. The aneuploid ES cells cancontribute to the different tissuses of chimera animals, but cannot form viable germcells.

  13. EXAMINATION OF THE GERM CELL CHIMERA FORMING POTENTIAL OF MOUSE EMBRYONIC STEM CELLS

    Directory of Open Access Journals (Sweden)

    CÂRSTEA V. B

    2007-01-01

    Full Text Available The aim of this study was to examine the factors, which influence the chimeraforming potential of mouse embryonic stem cells (ES cells. In our work, we examinethe chimera producing ability of R1 and R1/E mouse ES cell lines. We found that thepassage number affects chimera-forming capability of the ES cells. With theincreasing of the passage number, it could be getting less chimera animal, and onlythe R1/E ES cell line derived cells could contribute to the germ cells. At first, wecompared the marker of pluripotency using immunostaining and RT PCR, but wecould not find any difference between the R1 and R1/E cell in this way. Atchromosome analysis, we found, that the number of aneuploid cells, in R1 ES cellline, dramatically increased after 10 passages. We thought that the reason is thatduring the cell division Y chromosome could not arrange correctly between the twonewly derived progeny cells. To prove our conception, we made X and YchromosomeFISH analyses. We found, that the aneuploid R1 and R1/E ES cellscontain only one X and one Y chromosome, so not the loss of Y chromosome causethe problem at the germ cell formation. At last, we made the karyotypeanalysis of R1 and R1/E ES cells at different passages. The karyotype analysisdemonstrated that in the case of R1 ES cell line, the 41 and 42-chromosomecontaining cells hold trisomy. With the increasing of the passages number, thenumber of trisomy containing aneuploid cells increased. The aneuploid ES cells cancontribute to the different tissuses of chimera animals, but cannot form viable germcells.

  14. Generation of tooth-periodontium complex structures using high-odontogenic potential dental epithelium derived from mouse embryonic stem cells.

    Science.gov (United States)

    Zhang, Yancong; Li, Yongliang; Shi, Ruirui; Zhang, Siqi; Liu, Hao; Zheng, Yunfei; Li, Yan; Cai, Jinglei; Pei, Duanqing; Wei, Shicheng

    2017-06-08

    A number of studies have shown that tooth-like structures can be regenerated using induced pluripotent stem cells and mouse embryonic stem (mES) cells. However, few studies have reported the regeneration of tooth-periodontium complex structures, which are more suitable for clinical tooth transplantation. We established an optimized approach to induce high-odontogenic potential dental epithelium derived from mES cells by temporally controlling bone morphogenic protein 4 (BMP4) function and regenerated tooth-periodontium complex structures in vivo. First, immunofluorescence and quantitative reverse transcription-polymerase chain reaction were used to identify the watershed of skin and the oral ectoderm. LDN193189 was then used to inhibit the BMP4 receptor around the watershed, followed by the addition of exogenous BMP4 to promote BMP4 function. The generated dental epithelium was confirmed by western blot analysis and immunofluorescence. The generated epithelium was ultimately combined with embryonic day 14.5 mouse mesenchyme and transplanted into the renal capsules of nude mice. After 4 weeks, the tooth-periodontium complex structure was examined by micro-computed tomography (CT) and hematoxylin and eosin (H&E) staining. Our study found that the turning point of oral ectoderm differentiation occurred around day 3 after the embryoid body was transferred to a common culture plate. Ameloblastin-positive dental epithelial cells were detected following the temporal regulation of BMP4. Tooth-periodontium complex structures, which included teeth, a periodontal membrane, and alveolar bone, were formed when this epithelium was combined with mouse dental mesenchyme and transplanted into the renal capsules of nude mice. Micro-CT and H&E staining revealed that the generated tooth-periodontium complex structures shared a similar histological structure with normal mouse teeth. An optimized induction method was established to promote the differentiation of mES cells into dental

  15. Requirement of B-Raf, C-Raf, and A-Raf for the growth and survival of mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Guo, Wenjing; Hao, Baixia; Wang, Qian; Lu, Yingying; Yue, Jianbo

    2013-01-01

    Extracellular signal-regulated kinases (ERKs) have been implicated to be dispensable for self-renewal of mouse embryonic stem (ES) cells, and simultaneous inhibition of both ERK signaling and glycogen synthase kinase 3 (GSK3) not only allows mouse ES cells to self-renew independent of extracellular stimuli but also enables more efficient derivation of naïve ES cells from mouse and rat strains. Interestingly, some ERKs stay active in mouse ES cells which are maintained in regular medium containing leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP). Yet, the upstream signaling for ERK activation and their roles in mouse ES cells, other than promoting or priming differentiation, have not been determined. Here we found that mouse ES cells express three forms of Raf kinases, A-Raf, B-Raf, and C-Raf. Knocking-down each single Raf member failed to affect the sustained ERK activity, neither did A-Raf and B-Raf double knockdown or B-Raf and C-Raf double knockdown change it in ES cells. Interestingly, B-Raf and C-Raf double knockdown, not A-Raf and B-Raf knockdown, inhibited the maximal ERK activation induced by LIF, concomitant with the slower growth of ES cells. On the other hand, A-Raf, B-Raf, and C-Raf triple knockdown markedly inhibited both the maximal and sustained ERK activity in ES cells. Moreover, Raf triple knockdown, similar to the treatment of U-0126, an MEK inhibitor, significantly inhibited the survival and proliferation of ES cells, thereby compromising the colony propagation of mouse ES cells. In summary, our data demonstrate that all three Raf members are required for ERK activation in mouse ES cells and are involved in growth and survival of mouse ES cells. - Highlights: ●Mouse ES (mES) cells express all three Raf members, A-Raf, B-Raf, and C-Raf. ●Leukemia inhibitory factor (LIF) temporally activates ERKs in mES cells. ●B-Raf and C-Raf are required for LIF-induced maximal ERKs activity in mES cells. ●All Raf members are

  16. Requirement of B-Raf, C-Raf, and A-Raf for the growth and survival of mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Wenjing; Hao, Baixia; Wang, Qian; Lu, Yingying; Yue, Jianbo, E-mail: jbyue@me.com

    2013-11-01

    Extracellular signal-regulated kinases (ERKs) have been implicated to be dispensable for self-renewal of mouse embryonic stem (ES) cells, and simultaneous inhibition of both ERK signaling and glycogen synthase kinase 3 (GSK3) not only allows mouse ES cells to self-renew independent of extracellular stimuli but also enables more efficient derivation of naïve ES cells from mouse and rat strains. Interestingly, some ERKs stay active in mouse ES cells which are maintained in regular medium containing leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP). Yet, the upstream signaling for ERK activation and their roles in mouse ES cells, other than promoting or priming differentiation, have not been determined. Here we found that mouse ES cells express three forms of Raf kinases, A-Raf, B-Raf, and C-Raf. Knocking-down each single Raf member failed to affect the sustained ERK activity, neither did A-Raf and B-Raf double knockdown or B-Raf and C-Raf double knockdown change it in ES cells. Interestingly, B-Raf and C-Raf double knockdown, not A-Raf and B-Raf knockdown, inhibited the maximal ERK activation induced by LIF, concomitant with the slower growth of ES cells. On the other hand, A-Raf, B-Raf, and C-Raf triple knockdown markedly inhibited both the maximal and sustained ERK activity in ES cells. Moreover, Raf triple knockdown, similar to the treatment of U-0126, an MEK inhibitor, significantly inhibited the survival and proliferation of ES cells, thereby compromising the colony propagation of mouse ES cells. In summary, our data demonstrate that all three Raf members are required for ERK activation in mouse ES cells and are involved in growth and survival of mouse ES cells. - Highlights: ●Mouse ES (mES) cells express all three Raf members, A-Raf, B-Raf, and C-Raf. ●Leukemia inhibitory factor (LIF) temporally activates ERKs in mES cells. ●B-Raf and C-Raf are required for LIF-induced maximal ERKs activity in mES cells. ●All Raf members are

  17. Effects of cortisol on the primary response of mouse spleen cell cultures to heterologous erythrocytes

    International Nuclear Information System (INIS)

    Dracott, B.N.

    1974-01-01

    Cell viability and the production of direct PFC were studied in mouse spleen cell cultures after cortisol treatment in vivo or in vitro at various times relative to primary stimulation with SRBC in vitro. Cortisol treatment in vivo reduced spleen cell numbers by 88 percent after 48 hr, but cultures of the remaining cells produced as many PFC in vitro as did cultures of equal numbers of normal spleen cells. In normal spleen cell cultures incubated with cortisol for 4 hr prior to the addition of antigen, peak responses of PFC/culture and PFC/10 6 cells occurred 24 hr later than in controls and averaged, respectively, 27 and 141 percent of control values. Minimum viable cell numbers were observed in cortisol-treated cultures after 3 days; thereafter cell numbers gradually increased. These results were not significantly altered when cultures were treated simultaneously with cortisol and antigen. The response was not suppressed if the addition of antigen preceded that of cortisol by more than 4 hr. Suppression was also considerably reduced if fetal calf serum was used when preparing cells for culture

  18. Tumor cell-derived PDGF-B potentiates mouse mesenchymal stem cells-pericytes transition and recruitment through an interaction with NRP-1

    Directory of Open Access Journals (Sweden)

    Haque Inamul

    2010-08-01

    Full Text Available Abstract Background New blood vessel formation, or angiogenic switch, is an essential event in the development of solid tumors and their metastatic growth. Tumor blood vessel formation and remodeling is a complex and multi-step processes. The differentiation and recruitment of mural cells including vascular smooth muscle cells and pericytes are essential steps in tumor angiogenesis. However, the role of tumor cells in differentiation and recruitment of mural cells has not yet been fully elucidated. This study focuses on the role of human tumor cells in governing the differentiation of mouse mesenchymal stem cells (MSCs to pericytes and their recruitment in the tumor angiogenesis process. Results We show that C3H/10T1/2 mouse embryonic mesenchymal stem cells, under the influence of different tumor cell-derived conditioned media, differentiate into mature pericytes. These differentiated pericytes, in turn, are recruited to bind with capillary-like networks formed by endothelial cells on the matrigel under in vitro conditions and recruited to bind with blood vessels on gel-foam under in vivo conditions. The degree of recruitment of pericytes into in vitro neo-angiogenesis is tumor cell phenotype specific. Interestingly, invasive cells recruit less pericytes as compared to non-invasive cells. We identified tumor cell-secreted platelet-derived growth factor-B (PDGF-B as a crucial factor controlling the differentiation and recruitment processes through an interaction with neuropilin-1 (NRP-1 in mesenchymal stem cells. Conclusion These new insights into the roles of tumor cell-secreted PDGF-B-NRP-1 signaling in MSCs-fate determination may help to develop new antiangiogenic strategies to prevent the tumor growth and metastasis and result in more effective cancer therapies.

  19. Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling.

    Science.gov (United States)

    Ito, Takuji; Bai, Tao; Tanaka, Tetsuji; Yoshida, Kenji; Ueyama, Takashi; Miyajima, Masayasu; Negishi, Takayuki; Kawasaki, Takahiko; Takamatsu, Hyota; Kikutani, Hitoshi; Kumanogoh, Atsushi; Yukawa, Kazunori

    2015-02-01

    The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild‑type (WT) mice. Administration of β‑estradiol to infant Sema4D‑deficient (Sema4D‑/‑) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β‑estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin‑B1, was examined as well as the level of apoptosis in the vaginal epithelia of five‑week‑old WT and Sema4D‑/‑ mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin‑B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase‑3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five‑week‑old Sema4D‑/‑ mice compared with WT mice. The addition of recombinant Sema4D to Sema4D‑/‑ vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis‑inducing activity of Sema4D. The

  20. Semaphorin 4D induces vaginal epithelial cell apoptosis to control mouse postnatal vaginal tissue remodeling

    Science.gov (United States)

    ITO, TAKUJI; BAI, TAO; TANAKA, TETSUJI; YOSHIDA, KENJI; UEYAMA, TAKASHI; MIYAJIMA, MASAYASU; NEGISHI, TAKAYUKI; KAWASAKI, TAKAHIKO; TAKAMATSU, HYOTA; KIKUTANI, HITOSHI; KUMANOGOH, ATSUSHI; YUKAWA, KAZUNORI

    2015-01-01

    The opening of the mouse vaginal cavity to the skin is a postnatal tissue remodeling process that occurs at approximately five weeks of age for the completion of female genital tract maturation at puberty. The tissue remodeling process is primarily composed of a hormonally triggered apoptotic process predominantly occurring in the epithelium of the distal section of the vaginal cavity. However, the detailed mechanism underlying the apoptotic induction remains to be elucidated. In the present study, it was observed that the majority of BALB/c mice lacking the class 4 semaphorin, semaphorin 4D (Sema4D), developed imperforate vagina and hydrometrocolpos resulting in a perpetually unopened vaginal cavity regardless of a normal estrogen level comparable with that in wild-type (WT) mice. Administration of β-estradiol to infant Sema4D-deficient (Sema4D−/−) mice did not induce precocious vaginal opening, which was observed in WT mice subjected to the same β-estradiol administration, excluding the possibility that the closed vaginal phenotype was due to insufficient estrogen secretion at the time of vaginal opening. In order to assess the role of Sema4D in the postnatal vaginal tissue remodeling process, the expression of Sema4D and its receptor, plexin-B1, was examined as well as the level of apoptosis in the vaginal epithelia of five-week-old WT and Sema4D−/− mice. Immunohistochemical analyses confirmed the localization of Sema4D and plexin-B1 in the mouse vaginal epithelia. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunohistochemistry detecting activated caspase-3 revealed significantly fewer apoptotic cells in situ in the vaginal mucosa of five-week-old Sema4D−/− mice compared with WT mice. The addition of recombinant Sema4D to Sema4D−/− vaginal epithelial cells in culture significantly enhanced apoptosis of the vaginal epithelial cells, demonstrating the apoptosis-inducing activity of Sema4D. The experimental reduction of

  1. Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

    International Nuclear Information System (INIS)

    Kawahara, Takeshi

    2012-01-01

    Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine–aminopterin–thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed α, β, and γ subunits of high-affinity immunoglobulin E (IgE) receptor (FcεRI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-α, and cyclooxygenase 2, and production of prostaglandin D 2 and leukotriene C 4 in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-α expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on FcεRI- and TLR-mediated effector functions of mast cells.

  2. Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

    Energy Technology Data Exchange (ETDEWEB)

    Kawahara, Takeshi, E-mail: tkawafb@shinshu-u.ac.jp [Integrated Department of Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, Nagano (Japan)

    2012-11-01

    Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed {alpha}, {beta}, and {gamma} subunits of high-affinity immunoglobulin E (IgE) receptor (Fc{epsilon}RI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-{alpha}, and cyclooxygenase 2, and production of prostaglandin D{sub 2} and leukotriene C{sub 4} in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-{alpha} expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on Fc{epsilon}RI- and TLR-mediated effector functions of mast cells.

  3. Transcription of a novel mouse semaphorin gene, M-semaH, correlates with the metastatic ability of mouse tumor cell lines

    DEFF Research Database (Denmark)

    Christensen, C R; Klingelhöfer, Jörg; Tarabykina, S

    1998-01-01

    identified a novel member of the semaphorin/collapsin family in the two metastatic cell lines. We have named it M-semaH. Northern hybridization to a panel of tumor cell lines revealed transcripts in 12 of 12 metastatic cell lines but in only 2 of 6 nonmetastatic cells and none in immortalized mouse...

  4. Radiological protection effect on vanillin derivative VND3207 radiation-induced cytogenetic damage in mouse bone marrow cells

    International Nuclear Information System (INIS)

    Wang Chuangao; Wang Li; Zhou Pingkun; Wang Zhongwen; Hu Yongzhe; Jin Haiming; Zhang Xueqing; Chen Ying

    2010-01-01

    Objective: To study the protection of vanillin derivative VND3207 on the cytogenetic damage of mouse bone marrow cell induced by ionizing radiation. Methods: BALB/c mice were randomly divided into five groups: normal control group, 2 Gy dose irradiation group, and three groups of 2 Gy irradiation with VND3207 protection at doses of 10, 50 and 100 mg/kg, respectively. VND3207 was given by intragastric administration once a day for five days. Two hours after the last drug administration, the mice were irradiated with 2 Gy γ-rays. The changes of polychromatophilic erythroblasts micronuclei (MN), chromosome aberration (CA) and mitosis index (MI) of mouse bone marrow cells were observed at 24 and 48 h after irradiation. Results: Under the protection of VND3207 at the dosages 10, 50, 100 μmg/kg, the yields of poly-chromatophilic erythroblasts MN and CA of bone marrow cells were significantly decreased (t=2.36-4.26, P<0.05), and the marrow cells MI remained much higher level compared with the irradiated mice without drug protection (t=2.58, 2.01, P<0.05). The radiological protection effect was drug dose-dependent, and the administration of VND3207 at the dosage of 100 mg/kg resulted in reduction by 50 % and 65% in the yields of MN and CA, respectively. Conclusions: VND3207 had a good protection effect of on γ-ray induced cytogentic damage of mouse bone marrow cells. (authors)

  5. Cell membrane and cell junctions in differentiation of preimplanted mouse embryos.

    Science.gov (United States)

    Izquierdo, L; Fernández, S; López, T

    1976-12-01

    Cell membrane and cell junctions in differentiation of preimplanted mouse embryos, (membrana celular y uniones celulares en la diferenciación del embrión de ratón antes de la implantación). Arch. Biol. Med. Exper. 10: 130-134, 1976. The development of cell junctions that seal the peripheral blastomeres could be a decisive step in the differentiation of morulae into blastocysts. The appearance of these junctions is studied by electron microscopy of late morulae and initial blastocysts. Zonulae occludentes as well as impermeability to lanthanum emulsion precedes the appearance of the blastocel and hence might be considered as one of its necessary causes.

  6. Differentiation of Mouse Ovarian Stem Cells Toward Oocyte-Like Structure by Coculture with Granulosa Cells.

    Science.gov (United States)

    Parvari, Soraya; Yazdekhasti, Hossein; Rajabi, Zahra; Gerayeli Malek, Valliollah; Rastegar, Tayebeh; Abbasi, Mehdi

    2016-11-01

    An increasing body of evidence has confirmed existence and function of ovarian stem cells (OSCs). In this study, a novel approach on differentiation of OSCs into oocyte-like cells (OLCs) has been addressed. Recently, different methods have been recruited to isolate and describe aspects of OSCs, but newer and more convenient strategies in isolation are still growing. Herein, a morphology-based method was used to isolate OSCs. Cell suspension of mouse neonatal ovaries was cultured and formed colonies were harvested mechanically and cultivated on mouse embryonic fibroblasts. For differentiation induction, colonies transferred on inactive granulosa cells. Results showed that cells in colonies were positive for alkaline phosphatase activity and reverse transcription-polymerase chain reaction (RT-PCR) confirmed the pluripotency characteristics of cells. Immunofluorescence revealed a positive signal for OCT4, DAZL, MVH, and SSEA1 in colonies as well. Results of RT-PCR and immunofluorescence confirmed that some OLCs were generated within the germ stem cell (GSCs) colonies. The applicability of morphological selection for isolation of GSCs was verified. This method is easier and more economic than other techniques. Our results demonstrate that granulosa cells were effective in inducing the differentiation of OSCs into OLCs through direct cell-to-cell contacts.

  7. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    International Nuclear Information System (INIS)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-01-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10 6 receptors per cell. The cell line with the highest 125 I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10 6 receptors with a K/sub d/ of 10 -9 M. This level was not dependent on exposure to metals but could be increased further to 2 x 10 7 receptors per cell by addition of sodium butyrate to the culture medium. The α and β subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the α and β subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  8. Selective binding and transcytosis of Ulex europaeus 1 lectin by mouse Peyer's patch M-cells in vivo.

    Science.gov (United States)

    Clark, M A; Jepson, M A; Simmons, N L; Hirst, B H

    1995-12-01

    The in vivo interaction of the lectin Ulex europaeus agglutinin 1 with mouse Peyer's patch follicle-associated epithelial cells was studied in the mouse Peyer's patch gut loop model by immunofluorescence and electron microscopy. The lectin targets to mouse Peyer's patch M-cells and is rapidly endocytosed and transcytosed. These processes are accompanied by morphological changes in the M-cell microvilli and by redistribution of polymerised actin. The demonstration of selective binding and uptake of a lectin by intestinal M-cells in vivo suggests that M-cell-specific surface glycoconjugates might act as receptors for the selective adhesion/uptake of microorganisms.

  9. In utero bisphenol A exposure disrupts germ cell nest breakdown and reduces fertility with age in the mouse

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wei, E-mail: weiwang2@illinois.edu; Hafner, Katlyn S., E-mail: katlynhafner@gmail.com; Flaws, Jodi A., E-mail: jflaws@illinois.edu

    2014-04-15

    Bisphenol A (BPA) is a known reproductive toxicant in rodents. However, the effects of in utero BPA exposure on early ovarian development and the consequences of such exposure on female reproduction in later reproductive life are unclear. Thus, we determined the effects of in utero BPA exposure during a critical developmental window on germ cell nest breakdown, a process required for establishment of the finite primordial follicle pool, and on female reproduction. Pregnant FVB mice (F0) were orally dosed daily with tocopherol-striped corn oil (vehicle), diethylstilbestrol (DES; 0.05 μg/kg, positive control), or BPA (0.5, 20, and 50 μg/kg) from gestational day 11 until birth. Ovarian morphology and gene expression profiles then were examined in F1 female offspring on postnatal day (PND) 4 and estrous cyclicity was examined daily after weaning for 30 days. F1 females were also subjected to breeding studies with untreated males at three to nine months. The results indicate that BPA inhibits germ cell nest breakdown via altering expression of selected apoptotic factors. BPA also significantly advances the age of first estrus, shortens the time that the females remain in estrus, and increases the time that the females remain in metestrus and diestrus compared to controls. Further, F1 females exposed to low doses of BPA exhibit various fertility problems and have a significantly higher percentage of dead pups compared to controls. These results indicate that in utero exposure to low doses of BPA during a critical ovarian developmental window interferes with early ovarian development and reduces fertility with age. - Highlights: • In utero BPA exposure inhibits germ cell nest breakdown in female mouse offspring. • In utero BPA exposure alters expression of apoptosis regulators in the ovaries of mouse offspring. • In utero BPA exposure advances first estrus age and alters cyclicity in mouse offspring. • In utero BPA exposure causes various fertility problems in

  10. In utero bisphenol A exposure disrupts germ cell nest breakdown and reduces fertility with age in the mouse

    International Nuclear Information System (INIS)

    Wang, Wei; Hafner, Katlyn S.; Flaws, Jodi A.

    2014-01-01

    Bisphenol A (BPA) is a known reproductive toxicant in rodents. However, the effects of in utero BPA exposure on early ovarian development and the consequences of such exposure on female reproduction in later reproductive life are unclear. Thus, we determined the effects of in utero BPA exposure during a critical developmental window on germ cell nest breakdown, a process required for establishment of the finite primordial follicle pool, and on female reproduction. Pregnant FVB mice (F0) were orally dosed daily with tocopherol-striped corn oil (vehicle), diethylstilbestrol (DES; 0.05 μg/kg, positive control), or BPA (0.5, 20, and 50 μg/kg) from gestational day 11 until birth. Ovarian morphology and gene expression profiles then were examined in F1 female offspring on postnatal day (PND) 4 and estrous cyclicity was examined daily after weaning for 30 days. F1 females were also subjected to breeding studies with untreated males at three to nine months. The results indicate that BPA inhibits germ cell nest breakdown via altering expression of selected apoptotic factors. BPA also significantly advances the age of first estrus, shortens the time that the females remain in estrus, and increases the time that the females remain in metestrus and diestrus compared to controls. Further, F1 females exposed to low doses of BPA exhibit various fertility problems and have a significantly higher percentage of dead pups compared to controls. These results indicate that in utero exposure to low doses of BPA during a critical ovarian developmental window interferes with early ovarian development and reduces fertility with age. - Highlights: • In utero BPA exposure inhibits germ cell nest breakdown in female mouse offspring. • In utero BPA exposure alters expression of apoptosis regulators in the ovaries of mouse offspring. • In utero BPA exposure advances first estrus age and alters cyclicity in mouse offspring. • In utero BPA exposure causes various fertility problems in

  11. Common themes and cell type specific variations of higher order chromatin arrangements in the mouse

    Directory of Open Access Journals (Sweden)

    Cremer Thomas

    2005-12-01

    Full Text Available Abstract Background Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. Results All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. Conclusion Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation.

  12. Ras Signaling Regulates Stem Cells and Amelogenesis in the Mouse Incisor.

    Science.gov (United States)

    Zheng, X; Goodwin, A F; Tian, H; Jheon, A H; Klein, O D

    2017-11-01

    The role of Ras signaling during tooth development is poorly understood. Ras proteins-which are activated by many upstream pathways, including receptor tyrosine kinase cascades-signal through multiple effectors, such as the mitogen-activated protein kinase (MAPK) and PI3K pathways. Here, we utilized the mouse incisor as a model to study how the MAPK and PI3K pathways regulate dental epithelial stem cells and amelogenesis. The rodent incisor-which grows continuously throughout the life of the animal due to the presence of epithelial and mesenchymal stem cells-provides a model for the study of ectodermal organ renewal and regeneration. Utilizing models of Ras dysregulation as well as inhibitors of the MAPK and PI3K pathways, we found that MAPK and PI3K regulate dental epithelial stem cell activity, transit-amplifying cell proliferation, and enamel formation in the mouse incisor.

  13. The Effects of Imatinib Mesylate on Cellular Viability, Platelet Derived Growth Factor and Stem Cell Factor in Mouse Testicular Normal Leydig Cells.

    Science.gov (United States)

    Kheradmand, Fatemeh; Hashemnia, Seyyed Mohammad Reza; Valizadeh, Nasim; Roshan-Milani, Shiva

    2016-01-01

    Growth factors play an essential role in the development of tumor and normal cells like testicular leydig cells. Treatment of cancer with anti-cancer agents like imatinib mesylate may interfere with normal leydig cell activity, growth and fertility through failure in growth factors' production or their signaling pathways. The purpose of the study was to determine cellular viability and the levels of, platelet derived growth factor (PDGF) and stem cell factor (SCF) in normal mouse leydig cells exposed to imatinib, and addressing the effect of imatinib on fertility potential. The mouse TM3 leydig cells were treated with 0 (control), 2.5, 5, 10 and 20 μM imatinib for 2, 4 and 6 days. Each experiment was repeated three times (15 experiments in each day).The cellular viability and growth factors levels were assessed by MTT and ELISA methods, respectively. For statistical analysis, one-way ANOVA with Tukey's post hoc and Kruskal-Wallis test were performed. A p-value less than 0.05 was considered statistically significant. With increasing drug concentration, cellular viability decreased significantly (pcellular viability, PDGF and SCF levels. Imatinib may reduce fertility potential especially at higher concentrations in patients treated with this drug by decreasing cellular viability. The effect of imatinib on leydig cells is associated with PDGF stimulation. Of course future studies can be helpful in exploring the long term effects of this drug.

  14. Patch clamp study of mouse glomus cells using a whole carotid body.

    Science.gov (United States)

    Yamaguchi, Shigeki; Lande, Boris; Kitajima, Toshimitsu; Hori, Yuichi; Shirahata, Machiko

    2004-03-04

    Some electrophysiological characteristics of mouse glomus cells (DBA/2J strain) were investigated using an undissociated carotid body. The carotid body with major carotid arteries was placed in a recording chamber, and glomus cells were visualized with a water immersion lens combined with an infrared differential interference video camera. Patch clamp experiments revealed that voltage-gated outward current, but not inward current, was easily observed in glomus cells. Pharmacological experiments and the kinetics of the current suggest that outward current is via delayed rectifier, A type, and large conductance calcium-activated K channels. Furthermore, K current was reversibly attenuated by mild hypoxia. The results suggest electrophysiological similarities of glomus cells among the cat, the rat, and the DBA/2J mouse. The method appears useful for physiological experiments.

  15. Impact of 2-bromopropane on mouse embryonic stem cells and ...

    African Journals Online (AJOL)

    This study shows that 2-BP (5 to 10 μM) induces apoptotic processes in mouse embryonic stem cells (ESC-B5), but exerts no effects at treatment dosages below 5 μM. In ESC-B5 cells, 2-BP directly increased the content of reactive oxygen species (ROS), significantly increased the cytoplasmic free calcium and nitric oxide ...

  16. Improved cellular response of ion modified poly(lactic acid-co-glycolic acid) substrates for mouse fibroblast cells

    International Nuclear Information System (INIS)

    Adhikari, Ananta Raj; Geranpayeh, Tanya; Chu, Wei Kan; Otteson, Deborah C.

    2016-01-01

    In this report, the effects of argon (Ar) ion irradiation on poly(lactic acid-co-glycolic acid) (PLGA) substrates on biocompatibility were studied. PLGA scaffold substrates were prepared by spin coating glass surfaces with PLGA dissolved in anhydrous chloroform. Previously, we showed that surface modifications of PLGA films using ion irradiation modulate the inherent hydrophobicity of PLGA surface. Here we show that with increasing ion dose (1 × 10 12 to 1 × 10 14 ions/cm 2 ), hydrophobicity and surface roughness decreased. Biocompatibility for NIH3T3 mouse fibroblast cells was increased by argon irradiation of PLGA substrates. On unirradiated PLGA films, fibroblasts had a longer doubling time and cell densities were 52% lower than controls after 48 h in vitro. Argon irradiated PLGA substrates supported growth rates similar to control. Despite differences in cell cycle kinetics, there was no detectible cytotoxicity observed on any substrate. This demonstrates that argon ion irradiation can be used to tune the surface microstructure and generate substrates that are more compatible for the cell growth and proliferation. - Highlights: • Argon irradiation modifies surface chemistry and increases hydrophilicity of poly(lactic-glycolic) acid (PLGA) films. • Both native and irradiated PLGA films were not cytotoxic for mouse fibroblasts. • Fibroblast proliferation increased on PLGA substrates modified with higher doses of Argon irradiation. • Surface modification with Argon irradiation increases biocompatibility of PLGA films.

  17. Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER.

    Science.gov (United States)

    Dodds, E; Dunckley, M G; Naujoks, K; Michaelis, U; Dickson, G

    1998-04-01

    Cationic lipid-DNA complexes (lipoplexes) have been widely used as gene transfer vectors which avoid the adverse immunogenicity and potential for viraemia of viral vectors. With the long-term aim of gene transfer into skeletal muscle in vivo, we describe a direct in vitro comparison of two commercially available cationic lipid formulations, Lipofectamine and DOSPER. Optimisation of transfection was performed in the C2C12 mouse muscle cell line, before further studies in primary mouse myoblasts and C2C12 myotubes. Reporter gene constructs expressing either E. coli beta-galactosidase or green fluorescent protein (GFP) were used in order to evaluate transfection efficiency by histochemical staining or FACS analysis, respectively. Both lipid formulations were able to promote efficient, reproducible gene transfer in C2C12 cells, and to transfect primary mouse myoblast cultures successfully. However, DOSPER exhibited the important advantage of being able to transfect cells in the presence of serum of both bovine and murine origin. This feature allowed increased cell survival during in vitro transfections, and may be advantageous for direct in vivo gene transfer efficacy.

  18. Caffeine Abolishes the Ultraviolet-Induced REV3 Translesion Replication Pathway in Mouse Cells

    Directory of Open Access Journals (Sweden)

    Kouichi Yamada

    2011-11-01

    Full Text Available When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s, which insert nucleotide(s opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF. In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells, and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3−/− p53−/−, UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors.

  19. Mesenchymal and embryonic characteristics of stem cells obtained from mouse dental pulp

    DEFF Research Database (Denmark)

    Guimarães, Elisalva Teixeira; Cruz, Gabriela Silva; de Jesus, Alan Araújo

    2011-01-01

    abnormalities was evaluated by G banding. RESULTS: The mouse dental pulp stem cells (mDPSC) were highly proliferative, plastic-adherent, and exhibited a polymorphic morphology predominantly with stellate or fusiform shapes. The presence of cell clusters was observed in cultures of mDPSC. Some cells were...

  20. Mouse ES cells have a potential to differentiate into odontoblast-like cells using hanging drop method.

    Science.gov (United States)

    Kawai, R; Ozeki, N; Yamaguchi, H; Tanaka, T; Nakata, K; Mogi, M; Nakamura, H

    2014-05-01

    We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVβ3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Miyoshi, Ko, E-mail: miyoshi@cc.okayama-u.ac.jp [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan); Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato [Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikatacho, Okayama 700-8558 (Japan)

    2009-10-30

    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li{sub 2}CO{sub 3} were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  2. Lithium treatment elongates primary cilia in the mouse brain and in cultured cells

    International Nuclear Information System (INIS)

    Miyoshi, Ko; Kasahara, Kyosuke; Miyazaki, Ikuko; Asanuma, Masato

    2009-01-01

    The molecular mechanisms underlying the therapeutic effects of lithium, a first-line antimanic mood stabilizer, have not yet been fully elucidated. Treatment of the algae Chlamydomonas reinhardtii with lithium has been shown to induce elongation of their flagella, which are analogous structures to vertebrate cilia. In the mouse brain, adenylyl cyclase 3 (AC3) and certain neuropeptide receptors colocalize to the primary cilium of neuronal cells, suggesting a chemosensory function for the primary cilium in the nervous system. Here we show that lithium treatment elongates primary cilia in the mouse brain and in cultured cells. Brain sections from mice chronically fed with Li 2 CO 3 were subjected to immunofluorescence study. Primary cilia carrying both AC3 and the receptor for melanin-concentrating hormone (MCH) were elongated in the dorsal striatum and nucleus accumbens of lithium-fed mice, as compared to those of control animals. Moreover, lithium-treated NIH3T3 cells and cultured striatal neurons exhibited elongation of the primary cilia. The present results provide initial evidence that a psychotropic agent can affect ciliary length in the central nervous system, and furthermore suggest that lithium exerts its therapeutic effects via the upregulation of cilia-mediated MCH sensing. These findings thus contribute novel insights into the pathophysiology of bipolar mood disorder and other psychiatric diseases.

  3. Hypersensitivity of mouse NEIL1-knockdown cells to hydrogen peroxide during S phase

    International Nuclear Information System (INIS)

    Yamamoto, Ryohei; Ohshiro, Yukari; Shimotani, Tatsuhiko; Yamamoto, Mizuki; Matsuyama, Satoshi; Ide, Hiroshi; Kubo, Kihei

    2014-01-01

    Oxidative base damage occurs spontaneously due to reactive oxygen species generated as byproducts of respiration and other pathological processes in mammalian cells. Many oxidized bases are mutagenic and/or toxic, and most are repaired through the base excision repair pathway. Human endonuclease VIII-like protein 1 (hNEIL1) is thought to play an important role during the S phase of the cell cycle by removing oxidized bases in DNA replication fork-like (bubble) structures, and the protein level of hNEIL1 is increased in S phase. Compared with hNEIL1, there is relatively little information on the properties of the mouse ortholog mNEIL1. Since mouse cell nuclei lack endonuclease III-like protein (NTH) activity, in contrast to human cell nuclei, mNEIL1 is a major DNA glycosylase for repair of oxidized pyrimidines in mouse nuclei. In this study, we made mNEIL1-knockdown cells using an shRNA expression vector and examined the cell cycle-related variation in hydrogen peroxide (H 2 O 2 ) sensitivity. Hypersensitivity to H 2 O 2 caused by mNEIL1 knockdown was more significant in S phase than in G1 phase, suggesting that mNEIL1 has an important role during S phase, similarly to hNEIL1

  4. Transplantation of Adipose Derived Stromal Cells into the Developing Mouse Eye

    International Nuclear Information System (INIS)

    Yu, Song-Hee; Jang, Yu-Jin; Lee, Eun-Shil; Hwang, Dong-Youn; Jeon, Chang-Jin

    2010-01-01

    Adipose derived stromal cells (ADSCs) were transplanted into a developing mouse eye to investigate the influence of a developing host micro environment on integration and differentiation. Green fluorescent protein-expressing ADSCs were transplanted by intraocular injections. The age of the mouse was in the range of 1 to 10 days postnatal (PN). Survival dates ranged from 7 to 28 post transplantation (DPT), at which time immunohistochemistry was performed. The transplanted ADSCs displayed some morphological differentiations in the host eye. Some cells expressed microtubule associated protein 2 (marker for mature neuron), or glial fibrillary acid protein (marker for glial cell). In addition, some cells integrated into the ganglion cell layer. The integration and differentiation of the transplanted ADSCs in the 5 and 10 PN 7 DPT were better than in the host eye the other age ranges. This study was aimed at demonstrating how the age of host micro environment would influence the differentiation and integration of the transplanted ADSCs. However, it was found that the integration and differentiation into the developing retina were very limited when compared with other stem cells, such as murine brain progenitor cell

  5. Overproduction of S-adenosylmethionine decarboxylase in ethylglyoxal-bis(guanylhydrazone)-resistant mouse FM3A cells.

    Science.gov (United States)

    Suzuki, T; Sadakata, Y; Kashiwagi, K; Hoshino, K; Kakinuma, Y; Shirahata, A; Igarashi, K

    1993-07-15

    A variant cell line, termed SAM-1, which overproduced S-adenosylmethionine decarboxylase (AdoMetDC), was isolated by treatment of mouse FM3A cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequent incubation with ethylglyoxal bis(guanylhydrazone), an inhibitor of the enzyme. The cells were resistant to ethylglyoxal bis(guanylhydrazone), and showed AdoMetDC activity approximately five-times higher than control cells. The rate of AdoMetDC synthesis and the amount of AdoMetDC existing in SAM-1 cells were about five-times those in control cells. The amount of AdoMetDC mRNA existing in SAM-1 cells was five-times more than that in control cells. The amount of 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine, an irreversible inhibitor of AdoMetDC, necessary to inhibit cell growth was also five-times more in SAM-1 cells than in control cells. However, the following were the same in both SAM-1 and control cells; the amount of genomic DNA for AdoMetDC, the size and nucleotide sequence of 5' untranslated region of AdoMetDC mRNA, the deduced amino acid sequence (334 residues) from the nucleotide sequence of AdoMetDC cDNA and the degradation rate (t1/2 = about 4 h) of AdoMetDC. In addition, AdoMetDC mRNA in control cells was slightly more stable than that in SAM-1 cells. The results indicate that the overproduction of AdoMetDC in SAM-1 cells was caused by the increase of AdoMetDC mRNA. The variant cell line is convenient for studying the regulation of AdoMetDC and the physiological function of polyamines.

  6. Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells.

    Science.gov (United States)

    Deb, Tushar B; Coticchia, Christine M; Dickson, Robert B

    2004-09-10

    c-Myc-overexpressing mammary epithelial cells are proapoptotic; their survival is strongly promoted by epidermal growth factor (EGF). We now demonstrate that EGF-induced Akt activation and survival in transgenic mouse mammary tumor virus-c-Myc mouse mammary carcinoma cells are both calcium/calmodulin-dependent. Akt activation is abolished by the phospholipase C-gamma inhibitor U-73122, by the intracellular calcium chelator BAPTA-AM, and by the specific calmodulin antagonist W-7. These results implicate calcium/calmodulin in the activation of Akt in these cells. In addition, Akt activation by serum and insulin is also inhibited by W-7. EGF-induced and calcium/calmodulin-mediated Akt activation occurs in both tumorigenic and non-tumorigenic mouse and human mammary epithelial cells, independent of their overexpression of c-Myc. These results imply that calcium/calmodulin may be a common regulator of Akt activation, irrespective of upstream receptor activator, mammalian species, and transformation status in mammary epithelial cells. However, only c-Myc-overexpressing mouse mammary carcinoma cells (but not normal mouse mammary epithelial cells) undergo apoptosis in the presence of the calmodulin antagonist W-7, indicating the vital selective role of calmodulin for survival of these cells. Calcium/calmodulin-regulated Akt activation is mediated directly by neither calmodulin kinases nor phosphatidylinositol 3-kinase (PI-3 kinase). Pharmacological inhibitors of calmodulin kinase kinase and calmodulin kinases II and III do not inhibit EGF-induced Akt activation, and calmodulin antagonist W-7 does not inhibit phosphotyrosine-associated PI-3 kinase activation. Akt is, however, co-immunoprecipitated with calmodulin in an EGF-dependent manner, which is inhibited by calmodulin antagonist W-7. We conclude that calmodulin may serve a vital regulatory function to direct the localization of Akt to the plasma membrane for its activation by PI-3 kinase.

  7. Species-specific differences in peroxisome proliferation, catalase, and SOD2 upregulation as well as toxicity in human, mouse, and rat hepatoma cells induced by the explosive and environmental pollutant 2,4,6-trinitrotoluene.

    Science.gov (United States)

    Naumenko, Ekaterina Anatolevna; Ahlemeyer, Barbara; Baumgart-Vogt, Eveline

    2017-03-01

    2,4,6-Trinitrotoluene (TNT) has been widely used as an explosive substance and its toxicity is still of interest as it persisted in polluted areas. TNT is metabolized in hepatocytes which are prone to its toxicity. Since analysis of the human liver or hepatocytes is restricted due to ethical reasons, we investigated the effects of TNT on cell viability, reactive oxygen species (ROS) production, peroxisome proliferation, and antioxidative enzymes in human (HepG2), mouse (Hepa 1-6), and rat (H4IIEC3) hepatoma cell lines. Under control conditions, hepatoma cells of all three species were highly comparable exhibiting identical proliferation rates and distribution of their cell cycle phases. However, we found strong differences in TNT toxicity with the lowest IC 50 values (highest cell death rate) for rat cells, whereas human and mouse cells were three to sevenfold less sensitive. Moreover, a strong decrease in cellular dehydrogenase activity (MTT assay) and increased ROS levels were noted. TNT caused peroxisome proliferation with rat hepatoma cells being most responsive followed by those from mouse and human. Under control conditions, rat cells contained fivefold higher peroxisomal catalase and mitochondrial SOD2 activities and a twofold higher capacity to reduce MTT than human and mouse cells. TNT treatment caused an increase in catalase and SOD2 mRNA and protein levels in human and mouse, but not in rat cells. Similarly, human and mouse cells upregulated SOD2 activity, whereas rat cells failed therein. We conclude that TNT induced oxidative stress, peroxisome proliferation and mitochondrial damage which are highest in rat cells rendering them most susceptible toward TNT. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 989-1006, 2017. © 2016 Wiley Periodicals, Inc.

  8. Chemical coding and chemosensory properties of cholinergic brush cells in the mouse gastrointestinal and biliary tract

    Directory of Open Access Journals (Sweden)

    Burkhard eSchütz

    2015-03-01

    Full Text Available The mouse gastro-intestinal and biliary tract mucosal epithelia harbor choline acetyltransferase (ChAT-positive brush cells with taste cell-like traits. With the aid of two transgenic mouse lines that express green fluorescent protein (EGFP under the control of the ChAT promoter (EGFPChAT and by using in situ hybridization and immunohistochemistry we found that EGFPChAT cells were clustered in the epithelium lining the gastric groove. EGFPChAT cells were numerous in the gall bladder and bile duct, and found scattered as solitary cells along the small and large intestine. While all EGFPChAT cells were also ChAT-positive, expression of the high-affinity choline transporter (ChT1 was never detected. Except for the proximal colon, EGFPChAT cells also lacked detectable expression of the vesicular acetylcholine transporter (VAChT. EGFPChAT cells were found to be separate from enteroendocrine cells, however they were all immunoreactive for cytokeratin 18 (CK18, transient receptor potential melastatin-like subtype 5 channel (TRPM5, and for cyclooxygenases 1 (COX1 and 2 (COX2. The ex vivo stimulation of colonic EGFPChAT cells with the bitter substance denatonium resulted in a strong increase in intracellular calcium, while in other epithelial cells such an increase was significantly weaker and also timely delayed. Subsequent stimulation with cycloheximide was ineffective in both cell populations. Given their chemical coding and chemosensory properties, EGFPChAT brush cells thus may have integrative functions and participate in induction of protective reflexes and inflammatory events by utilizing ACh and prostaglandins for paracrine signaling.

  9. VSVΔG/EBOV GP-induced innate protection enhances natural killer cell activity to increase survival in a lethal mouse adapted Ebola virus infection.

    Science.gov (United States)

    Williams, Kinola J N; Qiu, Xiangguo; Fernando, Lisa; Jones, Steven M; Alimonti, Judie B

    2015-02-01

    Members of the species Zaire ebolavirus cause severe hemorrhagic fever with up to a 90% mortality rate in humans. The VSVΔG/EBOV GP vaccine has provided 100% protection in the mouse, guinea pig, and nonhuman primate (NHP) models, and has also been utilized as a post-exposure therapeutic to protect mice, guinea pigs, and NHPs from a lethal challenge of Ebola virus (EBOV). EBOV infection causes rapid mortality in human and animal models, with death occurring as early as 6 days after infection, suggesting a vital role for the innate immune system to control the infection before cells of the adaptive immune system can assume control. Natural killer (NK) cells are the predominant cell of the innate immune response, which has been shown to expand with VSVΔG/EBOV GP treatment. In the current study, an in vivo mouse model of the VSVΔG/EBOV GP post-exposure treatment was used for a mouse adapted (MA)-EBOV infection, to determine the putative VSVΔG/EBOV GP-induced protective mechanism of NK cells. NK depletion studies demonstrated that mice with NK cells survive longer in a MA-EBOV infection, which is further enhanced with VSVΔG/EBOV GP treatment. NK cell mediated cytotoxicity and IFN-γ secretion was significantly higher with VSVΔG/EBOV GP treatment. Cell mediated cytotoxicity assays and perforin knockout mice experiments suggest that there are perforin-dependent and -independent mechanisms involved. Together, these data suggest that NK cells play an important role in VSVΔG/EBOV GP-induced protection of EBOV by increasing NK cytotoxicity, and IFN-γ secretion.

  10. Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells.

    Science.gov (United States)

    Zhang, Meixiang; Ngo, Justine; Pirozzi, Filomena; Sun, Ying-Pu; Wynshaw-Boris, Anthony

    2018-03-15

    Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development. We optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols. Single and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically

  11. Radiosensitivity of mouse germ cells

    International Nuclear Information System (INIS)

    Matsuda, Yoichi; Takeuchi, Toyoko; Maemori, Mamiko; Seki, Naohiko; Tobari, Izuo

    1991-01-01

    To estimate radiosensitivity of mouse germ cells the analysis of chromosome aberrations was performed at diakinesis-metaphase I of spermatocytes and first-cleavage metaphase of one-cell embryos after exposure to radiations at various stages of primary spermatocytes and spermatids. The result provided evidence that there are two major types of DNA damage in X-irradiated sperm : (1) short-lived DNA lesions ; the lesions are subject to repair inhibition by agents added in G 1 , and are converted into chromosome-type aberrations during G 1 , and (2) long-lived DNA lesions ; the lesions persist until S phase and repair of the lesions is inhibited by caffeine, hydroxyurea and arabinofuranosyl cytosine in G 2 . The characteristic of X-ray damage induced in spermiogenic stage and repair mechanism for the damage in the fertilized egg were discussed comparing with the results with two chemicals, methyl methanesulfonate (MMS) and mitomycin C (MMC). (J.P.N.)

  12. A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells

    Directory of Open Access Journals (Sweden)

    Reza Ebrahimzadeh-Vesal

    2014-08-01

    Conclusion: In this study, we demonstrated the in vitro generation of mouse embryonic stem cells to germ cells by using a backbone vector containing the fusion gene Stra8-EGFP. The Stra8 gene is a retinoic acid-responsive protein and is able to regulate meiotic initiation.

  13. Establishment of c-myc-immortalized Kupffer cell line from a C57BL/6 mouse strain

    Directory of Open Access Journals (Sweden)

    Hiroshi Kitani

    2014-01-01

    Full Text Available We recently demonstrated in several mammalian species, a novel procedure to obtain liver-macrophages (Kupffer cells in sufficient numbers and purity using a mixed primary culture of hepatocytes. In this study, we applied this method to the C57BL/6 mouse liver and established an immortalized Kupffer cell line from this mouse strain. The hepatocytes from the C57BL/6 adult mouse liver were isolated by a two-step collagenase perfusion method and cultured in T25 culture flasks. Similar to our previous studies, the mouse hepatocytes progressively changed their morphology into a fibroblastic appearance after a few days of culture. After 7–10 days of culture, Kupffer-like cells, which were contaminants in the hepatocyte fraction at the start of the culture, actively proliferated on the mixed fibroblastic cell sheet. At this stage, a retroviral vector containing the human c-myc oncogene and neomycin resistance gene was introduced into the mixed culture. Gentle shaking of the culture flask, followed by the transfer and brief incubation of the culture supernatant, resulted in a quick and selective adhesion of Kupffer cells to a plastic dish surface. After selection with G418 and cloning by limiting dilutions, a clonal cell line (KUP5 was established. KUP5 cells displayed typical macrophage morphology and were stably passaged at 4–5 days intervals for more than 5 months, with a population doubling time of 19 h. KUP5 cells are immunocytochemically positive for mouse macrophage markers, such as Mac-1, F4/80. KUP5 cells exhibited substantial phagocytosis of polystyrene microbeads and the release of inflammatory cytokines upon lipopolysaccharide stimulation. Taken together, KUP5 cells provide a useful means to study the function of Kupffer cells in vitro.

  14. Efficient Differentiation of Mouse Embryonic Stem Cells into Insulin-Producing Cells

    Directory of Open Access Journals (Sweden)

    Szu-Hsiu Liu

    2012-01-01

    Full Text Available Embryonic stem (ES cells are a potential source of a variety of differentiated cells for cell therapy, drug discovery, and toxicology screening. Here, we present an efficacy strategy for the differentiation of mouse ES cells into insulin-producing cells (IPCs by a two-step differentiation protocol comprising of (i the formation of definitive endoderm in monolayer culture by activin A, and (ii this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Differentiated cells can be obtained within approximately 7 days. The differentiation IPCs combined application of RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties. In our study, we demonstrated that IPCs produced pancreatic transcription factors, endocrine progenitor marker, definitive endoderm, pancreatic β-cell markers, and Langerhans α and δ cells. The IPCs released insulin in a manner that was dose dependent upon the amount of glucose added. These techniques may be able to be applied to human ES cells, which would have very important ramifications for treating human disease.

  15. Anti-tumor activity of high-dose EGFR tyrosine kinase inhibitor and sequential docetaxel in wild type EGFR non-small cell lung cancer cell nude mouse xenografts

    Science.gov (United States)

    Tang, Ning; Zhang, Qianqian; Fang, Shu; Han, Xiao; Wang, Zhehai

    2017-01-01

    Treatment of non-small-cell lung cancer (NSCLC) with wild-type epidermal growth factor receptor (EGFR) is still a challenge. This study explored antitumor activity of high-dose icotinib (an EGFR tyrosine kinase inhibitor) plus sequential docetaxel against wild-type EGFR NSCLC cells-generated nude mouse xenografts. Nude mice were subcutaneously injected with wild-type EGFR NSCLC A549 cells and divided into different groups for 3-week treatment. Tumor xenograft volumes were monitored and recorded, and at the end of experiments, tumor xenografts were removed for Western blot and immunohistochemical analyses. Compared to control groups (negative control, regular-dose icotinib [IcoR], high-dose icotinib [IcoH], and docetaxel [DTX]) and regular icotinib dose (60 mg/kg) with docetaxel, treatment of mice with a high-dose (1200 mg/kg) of icotinib plus sequential docetaxel for 3 weeks (IcoH-DTX) had an additive effect on suppression of tumor xenograft size and volume (P Icotinib-containing treatments markedly reduced phosphorylation of EGFR, mitogen activated protein kinase (MAPK), and protein kinase B (Akt), but only the high-dose icotinib-containing treatments showed an additive effect on CD34 inhibition (P icotinib plus docetaxel had a similar effect on mouse weight loss (a common way to measure adverse reactions in mice), compared to the other treatment combinations. The study indicate that the high dose of icotinib plus sequential docetaxel (IcoH-DTX) have an additive effect on suppressing the growth of wild-type EGFR NSCLC cell nude mouse xenografts, possibly through microvessel density reduction. Future clinical trials are needed to confirm the findings of this study. PMID:27852073

  16. β-catenin functions pleiotropically in differentiation and tumorigenesis in mouse embryo-derived stem cells.

    Directory of Open Access Journals (Sweden)

    Noriko Okumura

    Full Text Available The canonical Wnt/β-catenin signaling pathway plays a crucial role in the maintenance of the balance between proliferation and differentiation throughout embryogenesis and tissue homeostasis. β-Catenin, encoded by the Ctnnb1 gene, mediates an intracellular signaling cascade activated by Wnt. It also plays an important role in the maintenance of various types of stem cells including adult stem cells and cancer stem cells. However, it is unclear if β-catenin is required for the derivation of mouse embryo-derived stem cells. Here, we established β-catenin-deficient (β-cat(Δ/Δ mouse embryo-derived stem cells and showed that β-catenin is not essential for acquiring self-renewal potential in the derivation of mouse embryonic stem cells (ESCs. However, teratomas formed from embryo-derived β-cat(Δ/Δ ESCs were immature germ cell tumors without multilineage differentiated cell types. Re-expression of functional β-catenin eliminated their neoplastic, transformed phenotype and restored pluripotency, thereby rescuing the mutant ESCs. Our findings demonstrate that β-catenin has pleiotropic effects in ESCs; it is required for the differentiation of ESCs and prevents them from acquiring tumorigenic character. These results highlight β-catenin as the gatekeeper in differentiation and tumorigenesis in ESCs.

  17. A new immuno- dystrophin-deficient model, the NSG-mdx4Cv mouse, provides evidence for functional improvement following allogeneic satellite cell transplantation

    Science.gov (United States)

    Arpke, Robert W.; Darabi, Radbod; Mader, Tara L.; Zhang, Yu; Toyama, Akira; Lonetree, Cara-lin; Nash, Nardina; Lowe, Dawn A.; Perlingeiro, Rita C.R.; Kyba, Michael

    2013-01-01

    Transplantation of a myogenic cell population into an immunodeficient recipient is an excellent way of assessing the in vivo muscle-generating capacity of that cell population. To facilitate both allogeneic and xenogeneic transplantations of muscle-forming cells in mice we have developed a novel immunodeficient muscular dystrophy model, the NSG-mdx4Cv mouse. The IL2Rg mutation, which is linked to the Dmd gene on the X chromosome, simultaneously depletes NK cells and suppresses thymic lymphomas, issues that limit the utility of the SCID/mdx model. The NSG-mdx4Cv mouse presents a muscular dystrophy of similar severity to the conventional mdx mouse. We show that this animal supports robust engraftment of both pig and dog muscle mononuclear cells. The question of whether satellite cells prospectively isolated by flow cytometry can confer a functional benefit upon transplantation has been controversial. Using allogeneic Pax7-ZsGreen donors and NSG-mdx4Cv recipients, we demonstrate definitively that as few as 900 FACS-isolated satellite cells can provide functional regeneration in vivo, in the form of an increased mean maximal force-generation capacity in cell-transplanted muscles, compared to a sham-injected control group. These studies highlight the potency of satellite cells to improve muscle function, and the utility of the NSG-mdx4Cv model for studies on muscle regeneration and Duchenne muscular dystrophy therapy. PMID:23606600

  18. Mouse embryonic retina delivers information controlling cortical neurogenesis.

    Directory of Open Access Journals (Sweden)

    Ciro Bonetti

    2010-12-01

    Full Text Available The relative contribution of extrinsic and intrinsic mechanisms to cortical development is an intensely debated issue and an outstanding question in neurobiology. Currently, the emerging view is that interplay between intrinsic genetic mechanisms and extrinsic information shape different stages of cortical development. Yet, whereas the intrinsic program of early neocortical developmental events has been at least in part decoded, the exact nature and impact of extrinsic signaling are still elusive and controversial. We found that in the mouse developing visual system, acute pharmacological inhibition of spontaneous retinal activity (retinal waves-RWs during embryonic stages increase the rate of corticogenesis (cell cycle withdrawal. Furthermore, early perturbation of retinal spontaneous activity leads to changes of cortical layer structure at a later time point. These data suggest that mouse embryonic retina delivers long-distance information capable of modulating cell genesis in the developing visual cortex and that spontaneous activity is the candidate long-distance acting extrinsic cue mediating this process. In addition, these data may support spontaneous activity to be a general signal coordinating neurogenesis in other developing sensory pathways or areas of the central nervous system.

  19. Radioprotection of mouse CNS endothelial cells in vivo

    International Nuclear Information System (INIS)

    Lyubimova, N.; Coultas, P.; Martin, R.

    1996-01-01

    Full text: Radioprotection using the minor groove binding DNA ligand Hoechst 33342 has been demonstrated in vitro, and more recently in vivo, in mouse lung. Intravenous administration was used for the lung studies, and both endothelial and alveolar epithelial cells-showed good up-take. Radiation damage to the endothelial cell population has also been postulated as important in late developing radionecrosis of spinal cord and brain. Endothelial cell density in brain can be readily determined by a fluorescent-histochemical technique. Treatment with a monoamine oxidase inhibitor and subsequent injection with L-DOPA results in an accumulation of dopamine (DA) in CNS endothelial cells. DA is converted to a fluorophore by exposure to paraformaldehyde, and cell numbers assayed by fluorescence microscopy. Earlier studies used this technique to monitor post-irradiation changes in endothelial cell density in rodent brain and showed the loss, within 24 hours, of a sensitive subpopulation comprising about 15% of the endothelial cells. Ten minutes after intravenous injection of Hoechst 33342 (80mg/kg) the ligand is confined by its limited penetration to the endothelial cells in mouse brain. When we irradiated at this time, there was protection against early endothelial cell loss. Ablation of the sensitive subpopulation in unprotected mice takes place over a dose range of 1 to 3 Gy γ-rays, but doses between 12 to 20 Gy are required in the presence of ligand. This protection equates to a very high dose modification factor of about 7 and possibly reflects a suppression of apoptosis in the sensitive endothelial subpopulation. The extent to which there is enhanced survival in the endothelial population as a whole and how the observed protection affects late CNS necrosis development has yet to be determined. However present results clearly show potential for the use of DNA-binding radioprotectors with limited penetration for investigations into the relative significance of

  20. Cytogenetic effects of sildenafil citrate (Viagra) on SWR/J mouse bone marrow cells.

    Science.gov (United States)

    Abou-Tarboush, Faisal Mohamed; Abdel-Samad, Mohamed Fathy

    2010-10-01

    The present study was conducted to investigate the cytogenetic effects of sildenafil citrate in SWR/J mouse bone marrow cells. Thirty-six males and 36 females were used and divided into four groups. Each group contained 18 animals (9 males and 9 females), weighing 30-35 g. These animals were orally administered with a single dose of 13, 26 or 40 mg/kg sildenafil citrate solution. A control group received normal saline in an identical condition. The animals were sacrificed at 12, 24 or 48 h, after the treatment. Chromosome aberrations were investigated in 50 metaphases per animal. No significant differences in the percentages of mitotic indices or in the frequencies of chromosome aberrations were observed between treated male and female mice at any doses or at any time intervals used, therefore, data from the two sexes were pooled when analyzed statistically. No significant (p < 0.05) differences in the percentages of mitotic indices or in the frequencies of chromosome aberrations were observed between sildenafil citrate-treated groups and the control group at any doses or at any time intervals used. However, the percentages of centromeric adhesions increased significantly (p < 0.01) in treated groups as compared with the control group at all doses and at all time intervals used. In conclusion, the results of the present study suggest that sildenafil citrate does not have cytogenetic effects on mouse bone marrow cells, but the centromeric adhesions induced by this drug need further studies to confirm them and to investigate the possible mechanism(s) responsible for such effect.

  1. Protective Effects of Mouse Bone Marrow Mesenchymal Stem Cell Soup on Staurosporine Induced Cell Death in PC12 and U87 Cell Lines

    Directory of Open Access Journals (Sweden)

    Hossein Zhaleh

    2016-11-01

    Full Text Available Mouse bone marrow mesenchymal stem cells (mBMSCs soup is promising tool for the treatment of neurodegenerative diseases. mBMSCs soup is easily obtained and is capable of transplantation without rejection. We investigated the effects of mBMSC soup on staurosporine-induced cell death in PC12 and U87 cells lines. The percentage of cell viability, cell death, NO concentration, total neurite length (TNL and fraction of cell differentiation (f% were assessed. Viability assay showed that mBM soup (24 and 48h in time dependent were increased cell viability (p<0.05 and also cell death assay showed that cell death in time dependent were decreased, respectively (p<0.05. TNL and fraction of cell differentiation significantly were increased compared with treatment1 (p<0.05. Our data showed that mBM Soup protects cells, increases cell viability, suppresses cell death and improvement the neurite elongation. We concluded that Mouse bone marrow mesenchymal stem cell soup plays an important protective role in staurosporine-induced cell death in PC12 and U87 cell lines.

  2. Anti-cancer potential of MAPK pathway inhibition in paragangliomas-effect of different statins on mouse pheochromocytoma cells

    NARCIS (Netherlands)

    Fliedner, S.M.; Engel, T.G.P.; Lendvai, N.K.; Shankavaram, U.; Nolting, S.; Wesley, R.; Elkahloun, A.G.; Ungefroren, H.; Oldoerp, A.; Lampert, G.; Lehnert, H.; Timmers, H.J.; Pacak, K.

    2014-01-01

    To date, malignant pheochromocytomas and paragangliomas (PHEOs/PGLs) cannot be effectively cured and thus novel treatment strategies are urgently needed. Lovastatin has been shown to effectively induce apoptosis in mouse PHEO cells (MPC) and the more aggressive mouse tumor tissue-derived cells

  3. Immunocompromised and immunocompetent mouse models for head and neck squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Lei ZG

    2016-01-01

    Full Text Available Zhen-ge Lei,1,* Xiao-hua Ren,2,* Sha-sha Wang,3 Xin-hua Liang,3,4 Ya-ling Tang3,5 1Department of Oral and Maxillofacial Surgery, Stomatological Hospital Affiliated to Nanchang University, Nanchang, Jiangxi, 2Department of Stomatology, Sichuan Medical Science Academy and Sichuan Provincial People’s Hospital, 3State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, 4Department of Oral and Maxillofacial Surgery, West China College of Stomatology, Sichuan University, 5Department of Oral Pathology, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, People’s Republic of China *These authors contributed equally to this work Abstract: Mouse models can closely mimic human oral squamous epithelial carcinogenesis, greatly expand the in vivo research possibilities, and play a critical role in the development of diagnosis, monitoring, and treatment of head and neck squamous cell carcinoma. With the development of the recent research on the contribution of immunity/inflammation to cancer initiation and progression, mouse models have been divided into two categories, namely, immunocompromised and immunocompetent mouse models. And thus, this paper will review these two kinds of models applied in head and neck squamous cell carcinoma to provide a platform to understand the complicated histological, molecular, and genetic changes of oral squamous epithelial tumorigenesis. Keywords: head and neck squamous cell carcinoma, HNSCC, mouse models, immunocompromised models, immunocompetent models, transgenic models

  4. Post-irradiation regeneration of early B-lymphocyte precursor cells in mouse bone marrow

    International Nuclear Information System (INIS)

    Park, Y.-H.; Osmond, D.G.

    1989-01-01

    To examine the sequential development of early B-cell precursors in mouse bone marrow, B-lineage cells have been examined during a wave of post-irradiation regeneration. Cell phenotypes have been defined for (i) terminal deoxynucleotidyl transferase (TdT); (ii) B220 glycoprotein, (iii) μ heavy chains in the cytoplasm (cμ) and at the cell surface (sμ). Three populations of μ - cells (TdT + 14.8 - ; TdT + 14.8 + ; TdT - 14.8 + ) have been proposed to be early B-cell precursors which would give rise to cμ + sμ - pre-B cells and to sμ + B lymphocytes. The timing, cell-size shifts and progressive amplification of the waves of regeneration accord with a dynamic model in which the TdT + 14.8 - , TdT + 14.8 + and TdT - 14.8 + cells form three successive stages in B-cell differentiation before the expression of μ chains, presumptively including the stage of μ chain gene rearrangement. In addition, the results provide an experimental system for the enrichment of early B-cell precursors in mouse bone marrow. (author)

  5. CD19 CAR-targeted T cells induce long-term remission and B Cell Aplasia in an immunocompetent mouse model of B cell acute lymphoblastic leukemia.

    Directory of Open Access Journals (Sweden)

    Marco L Davila

    Full Text Available Although many adults with B cell acute lymphoblastic leukemia (B-ALL are induced into remission, most will relapse, underscoring the dire need for novel therapies for this disease. We developed murine CD19-specific chimeric antigen receptors (CARs and an immunocompetent mouse model of B-ALL that recapitulates the disease at genetic, cellular, and pathologic levels. Mouse T cells transduced with an all-murine CD3ζ/CD28-based CAR that is equivalent to the one being used in our clinical trials, eradicate B-ALL in mice and mediate long-term B cell aplasias. In this model, we find that increasing conditioning chemotherapy increases tumor eradication, B cell aplasia, and CAR-modified T cell persistence. Quantification of recipient B lineage cells allowed us to estimate an in vivo effector to endogenous target ratio for B cell aplasia maintenance. In mice exhibiting a dramatic B cell reduction we identified a small population of progenitor B cells in the bone marrow that may serve as a reservoir for long-term CAR-modified T cell stimulation. Lastly, we determine that infusion of CD8+ CAR-modified T cells alone is sufficient to maintain long-term B cell eradication. The mouse model we report here should prove valuable for investigating CAR-based and other therapies for adult B-ALL.

  6. Enhanced casein kinase II activity during mouse embryogenesis. Identification of a 110-kDa phosphoprotein as the major phosphorylation product in mouse embryos and Krebs II mouse ascites tumor cells

    DEFF Research Database (Denmark)

    Schneider, H R; Reichert, G H; Issinger, O G

    1986-01-01

    Mouse embryos at various stages of development were used to study the relationship of protein kinase activities with normal embryogenesis. Casein kinase II (CKII) activity in developing mouse embryos shows a 3-4-fold activity increase at day 12 of gestation. Together with the CKII activity...... mouse tumour cells also show an enhanced CKII activity. Here too, a 110-kDa phosphoprotein was the major phosphoryl acceptor. Partial proteolytic digestion shows that both proteins are identical. Other protein kinases tested (cAMP- and cGMP-dependent protein kinases) only show a basal level of enzyme...

  7. Dose-related estrogen effects on gene expression in fetal mouse prostate mesenchymal cells.

    Directory of Open Access Journals (Sweden)

    Julia A Taylor

    Full Text Available Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα and androgen receptors and convert stimuli from circulating estrogens and androgens into paracrine signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2 in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM induce genes involved in morphological tissue development and sterol biosynthesis but suppress genes involved in growth factor signaling. Genes involved in cell adhesion were enriched among both up-regulated and down-regulated genes. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM were enriched in the glycolytic pathway. At the highest dose (100 nM, E2 induced genes enriched for cell adhesion, steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF- β signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas secreted growth factors and cytokines might play significant roles when estrogen level is high.

  8. Enhancement of cell-cell contact by a nonmitogenic lectin increases blastogenic response and IL-2 release by mitogen-stimulated mouse thymocytes.

    Science.gov (United States)

    Favero, J; Marti, J; Dornand, J; Bonnafous, J C; Mani, J C

    1986-03-01

    We have examined the influence of peanut agglutinin (PNA), a lectin which agglutinates but does not stimulate mouse thymocytes, on the responsiveness of these cells to concanavalin A (Con A) or galactose oxidase stimulation. Binding low amounts of PNA on unseparated mouse thymocytes pretreated with neuraminidase highly enhances the mitogenic response and the level of interleukin 2 release in the culture medium upon Con A stimulation. We have shown that PNA present on the cell surface acts as a crosslinking agent which favors intercellular binding between accessory cells (macrophages) and thymocytes, leading through this enhanced cooperation by cell-cell contact to an enhanced blastogenic response.

  9. An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

    Science.gov (United States)

    Fisher, Cynthia L; Marks, Hendrik; Cho, Lily Ting-Yin; Andrews, Robert; Wormald, Sam; Carroll, Thomas; Iyer, Vivek; Tate, Peri; Rosen, Barry; Stunnenberg, Hendrik G; Fisher, Amanda G; Skarnes, William C

    2017-12-01

    Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Mouse adhalin

    DEFF Research Database (Denmark)

    Liu, L; Vachon, P H; Kuang, W

    1997-01-01

    . To analyze the biological roles of adhalin, we cloned the mouse adhalin cDNA, raised peptide-specific antibodies to its cytoplasmic domain, and examined its expression and localization in vivo and in vitro. The mouse adhalin sequence was 80% identical to that of human, rabbit, and hamster. Adhalin...... was specifically expressed in striated muscle cells and their immediate precursors, and absent in many other cell types. Adhalin expression in embryonic mouse muscle was coincident with primary myogenesis. Its expression was found to be up-regulated at mRNA and protein levels during myogenic differentiation...

  11. The effect of diamide on potassium transport and cellular morphology in mouse L-cells

    International Nuclear Information System (INIS)

    Szekely, J.G.; Sargent, M.D.; Copps, T.P.; Lobreau, A.U.

    1982-06-01

    The effects of diamide (diazenedicarboxylic acid bis (N,N'-dimethylamide)) on the transport of potassium in mouse L-cells have been investigated using 86 Rb + as a tracer. Active, ouabain-sensitive uptake is reduced after 0.4 to 0.6 mol/L diamide treatment. The size of reduction depends on the temperature and the presence of glucose in the medium. These results suggest that the elimination of reduced glutathione by diamide is the major factor controlling the level of K + transport in treated L-cells. In addition to decreasing active transport, diamide produces dramatic changes in cellular ultrastructure, probably through altered Na + /K + balance and its action on tubulin. Clear organelle-free regions appear surrounded by vacuoles and swollen mitkchondria regions. The clear areas of cytoplasm eventually pinch off from the cell

  12. Improved cellular response of ion modified poly(lactic acid-co-glycolic acid) substrates for mouse fibroblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Adhikari, Ananta Raj, E-mail: aa8381@gmail.com [Department of Sciences, Wentworth Institute of Technology, Boston MA 02115 (United States); Geranpayeh, Tanya [Department of Biology and Biochemistry, University of Houston, Houston, TX 77204 (United States); Chu, Wei Kan [Texas Center for Superconductivity, University of Houston, Houston, TX 77204 (United States); Department of Physics, University of Houston, Houston, TX 77204 (United States); Otteson, Deborah C. [Department of Biology and Biochemistry, University of Houston, Houston, TX 77204 (United States); Department of Basic and Vision Sciences, College of Optometry, University of Houston, Houston, TX 77204 (United States)

    2016-03-01

    In this report, the effects of argon (Ar) ion irradiation on poly(lactic acid-co-glycolic acid) (PLGA) substrates on biocompatibility were studied. PLGA scaffold substrates were prepared by spin coating glass surfaces with PLGA dissolved in anhydrous chloroform. Previously, we showed that surface modifications of PLGA films using ion irradiation modulate the inherent hydrophobicity of PLGA surface. Here we show that with increasing ion dose (1 × 10{sup 12} to 1 × 10{sup 14} ions/cm{sup 2}), hydrophobicity and surface roughness decreased. Biocompatibility for NIH3T3 mouse fibroblast cells was increased by argon irradiation of PLGA substrates. On unirradiated PLGA films, fibroblasts had a longer doubling time and cell densities were 52% lower than controls after 48 h in vitro. Argon irradiated PLGA substrates supported growth rates similar to control. Despite differences in cell cycle kinetics, there was no detectible cytotoxicity observed on any substrate. This demonstrates that argon ion irradiation can be used to tune the surface microstructure and generate substrates that are more compatible for the cell growth and proliferation. - Highlights: • Argon irradiation modifies surface chemistry and increases hydrophilicity of poly(lactic-glycolic) acid (PLGA) films. • Both native and irradiated PLGA films were not cytotoxic for mouse fibroblasts. • Fibroblast proliferation increased on PLGA substrates modified with higher doses of Argon irradiation. • Surface modification with Argon irradiation increases biocompatibility of PLGA films.

  13. Sildenafil promotes eNOS activation and inhibits NADPH oxidase in the transgenic sickle cell mouse penis.

    Science.gov (United States)

    Musicki, Biljana; Bivalacqua, Trinity J; Champion, Hunter C; Burnett, Arthur L

    2014-02-01

    Sickle cell disease (SCD)-associated vasculopathy in the penis is characterized by aberrant nitric oxide and phosphodiesterase (PDE) 5 signaling, and by increased oxidative stress. Preliminary clinical trials show that continuous treatment with PDE5 inhibitor sildenafil unassociated with sexual activity decreases priapic activity in patients with SCD. However, the mechanism of its vasculoprotective effect in the penis remains unclear. We evaluated whether continuous administration of PDE5 inhibitor sildenafil promotes eNOS function at posttranslational levels and decreases superoxide-producing enzyme NADPH oxidase activity in the sickle cell mouse penis. SCD transgenic mice were used as an animal model of SCD. WT mice served as controls. Mice received treatment with the PDE5 inhibitor sildenafil (100 mg/kg/day) or vehicle for 3 weeks. eNOS phosphorylation on Ser-1177 (positive regulatory site), eNOS interactions with heat-shock protein 90 (HSP90) (positive regulator), phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177), an NADPH oxidase catalytic subunit gp91(phox), and a marker of oxidative stress (4-hydroxy-2-nonenal [HNE]) were measured by Western blot. Effect of continuous sildenafil treatment on eNOS posttranslational activation, NADPH oxidase catalytic subunit, and oxidative stress in the penis of the sickle cell mouse. Continuous treatment with sildenafil reversed (P penis. Sildenafil treatment of WT mice did not affect any of these parameters. Our findings that sildenafil enhances eNOS activation and inhibits NADPH oxidase function in the sickle cell mouse penis offers a vasculoprotective molecular basis for the therapeutic effect of sildenafil in the penis in association with SCD. © 2013 International Society for Sexual Medicine.

  14. Global gene expression analysis in a mouse model for Norrie disease: late involvement of photoreceptor cells.

    Science.gov (United States)

    Lenzner, Steffen; Prietz, Sandra; Feil, Silke; Nuber, Ulrike A; Ropers, H-Hilger; Berger, Wolfgang

    2002-09-01

    Mutations in the NDP gene give rise to a variety of eye diseases, including classic Norrie disease (ND), X-linked exudative vitreoretinopathy (EVRX), retinal telangiectasis (Coats disease), and advanced retinopathy of prematurity (ROP). The gene product is a cystine-knot-containing extracellular signaling molecule of unknown function. In the current study, gene expression was determined in a mouse model of ND, to unravel disease-associated mechanisms at the molecular level. Gene transcription in the eyes of 2-year-old Ndp knockout mice was compared with that in the eyes of age-matched wild-type control animals, by means of cDNA subtraction and microarrays. Clones (n = 3072) from the cDNA subtraction libraries were spotted onto glass slides and hybridized with fluorescently labeled RNA-derived targets. More than 230 differentially expressed clones were sequenced, and their expression patterns were verified by virtual Northern blot analysis. Numerous gene transcripts that are absent or downregulated in the eye of Ndp knockout mice are photoreceptor cell specific. In younger Ndp knockout mice (up to 1 year old), however, all these transcripts were found to be expressed at normal levels. The identification of numerous photoreceptor cell-specific transcripts with a reduced expression in 2-year-old, but not in young, Ndp knockout mice indicates that normal gene expression in these light-sensitive cells of mutant mice is established and maintained over a long period and that rods and cones are affected relatively late in the mouse model of ND. Obviously, the absence of the Ndp gene product is not compatible with long-term survival of photoreceptor cells in the mouse.

  15. Effects of BIO on proliferation and chondrogenic differentiation of mouse marrow derived mesenchymal stem cells

    Directory of Open Access Journals (Sweden)

    Nasrin Fallah

    2013-06-01

    Full Text Available In vitroexpansion of mesenchymal stem cell (MSCs into large number is necessary fortheir application in cell-based treatment of articular cartilage defects. On the other hand,some studies have indicated that BIO (6-Bromoindirubin-3-Oxime possesses mitogeniceffects on cell culture. The objective of the present study was to examine the effect of BIO onin vitro expansion and chondrogenic differentiation of mouse marrow-derived MSCs. Theculture was established using bone marrow tissue obtained from 10 NMRI mice. MSC natureof the isolated cells was verified according to the minimal criteria proposed for MSC.Passaged-3 cells were seeded in 24-well culture plates and treated by 0.05, 0.01, 0.1, 1.0 and1.5 μM BIO forsevendays. The culture without BIO was taken as the control. At the end ofcultivation period, the cultures were examinedfor viable cell number which was then used tocalculate population doubling time (PDT. The BIO with higher proliferation-promoting effectwas investigated for its chondrogenic effect on MSC culture. There was significantly moreviable cells at the cultures treated by 0.1 μM BIO. At this culture the cells tended to doubletheir population in rapid rate (each 43.07 hr than the cells treated with the other BIOconcentrations (p< 0.05. Interestingly treatment of MSC chondrogenic culture with 0.1 μMBIO ledto the up-regulation of cartilage specific genes including aggrecan, collagen II andSox9. In conclusion BIO at 0.1 μM could enhance mouse MSC in vitro proliferation as well astheir chondrogenic differentiation. These findings would be of great importance for the fieldof regenerative medicine.

  16. Generation of electrophysiologically functional cardiomyocytes from mouse induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Hongran Wang

    2016-03-01

    Full Text Available Induced pluripotent stem (iPS cells can efficiently differentiate into the three germ layers similar to those formed by differentiated embryonic stem (ES cells. This provides a new source of cells in which to establish preclinical allogeneic transplantation models. Our iPS cells were generated from mouse embryonic fibroblasts (MEFs transfected with the Yamanaka factors, the four transcription factors (Oct4, Sox2, Klf4 and c-Myc, without antibiotic selection or MEF feeders. After the formation of embryoid bodies (EBs, iPS cells spontaneously differentiated into Flk1-positive cardiac progenitors and cardiomyocytes expressing cardiac-specific markers such as alpha sarcomeric actinin (α-actinin, cardiac alpha myosin heavy chain (α-MHC, cardiac troponin T (cTnT, and connexin 43 (CX43, as well as cardiac transcription factors Nk2 homebox 5 (Nkx2.5 and gata binding protein 4 (gata4. The electrophysiological activity of iPS cell-derived cardiomyocytes (iPS-CMs was detected in beating cell clusters with optical mapping and RH237 a voltage-sensitive dye, and in single contracting cells with patch-clamp technology. Incompletely differentiated iPS cells formed teratomas when transplanted into a severe combined immunodeficiency (SCID mouse model of myocardial infarction. Our results show that somatic cells can be reprogrammed into pluripotent stem cells, which in turn spontaneously differentiate into electrophysiologically functional mature cardiomyocytes expressing cardiac-specific makers, and that these cells can potentially be used to repair myocardial infarction (MI in the future.

  17. Chemical coding and chemosensory properties of cholinergic brush cells in the mouse gastrointestinal and biliary tract.

    Science.gov (United States)

    Schütz, Burkhard; Jurastow, Innokentij; Bader, Sandra; Ringer, Cornelia; von Engelhardt, Jakob; Chubanov, Vladimir; Gudermann, Thomas; Diener, Martin; Kummer, Wolfgang; Krasteva-Christ, Gabriela; Weihe, Eberhard

    2015-01-01

    The mouse gastro-intestinal and biliary tract mucosal epithelia harbor choline acetyltransferase (ChAT)-positive brush cells with taste cell-like traits. With the aid of two transgenic mouse lines that express green fluorescent protein (EGFP) under the control of the ChAT promoter (EGFP (ChAT) ) and by using in situ hybridization and immunohistochemistry we found that EGFP (ChAT) cells were clustered in the epithelium lining the gastric groove. EGFP (ChAT) cells were numerous in the gall bladder and bile duct, and found scattered as solitary cells along the small and large intestine. While all EGFP (ChAT) cells were also ChAT-positive, expression of the high-affinity choline transporter (ChT1) was never detected. Except for the proximal colon, EGFP (ChAT) cells also lacked detectable expression of the vesicular acetylcholine transporter (VAChT). EGFP (ChAT) cells were found to be separate from enteroendocrine cells, however they were all immunoreactive for cytokeratin 18 (CK18), transient receptor potential melastatin-like subtype 5 channel (TRPM5), and for cyclooxygenases 1 (COX1) and 2 (COX2). The ex vivo stimulation of colonic EGFP (ChAT) cells with the bitter substance denatonium resulted in a strong increase in intracellular calcium, while in other epithelial cells such an increase was significantly weaker and also timely delayed. Subsequent stimulation with cycloheximide was ineffective in both cell populations. Given their chemical coding and chemosensory properties, EGFP (ChAT) brush cells thus may have integrative functions and participate in induction of protective reflexes and inflammatory events by utilizing ACh and prostaglandins for paracrine signaling.

  18. Efficient derivation of functional hepatocytes from mouse induced pluripotent stem cells by a combination of cytokines and sodium butyrate

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qi; YANG Yang; ZHANG Jian; WANG Guo-ying; LIU Wei; QIU Dong-bo; HEI Zi-qing; YING Qi-long; CHEN Gui-hua

    2011-01-01

    Background Hepatocyte transplantation has been proposed as an alternative to whole-organ transplantation to support many forms of hepatic insufficiency.Unfortunately,the lack of donor livers makes it difficult to obtain enough viable human hepatocytes for hepatocyte-based therapies.Therefore,it is urgent to find new ways to provide ample hepatocytes.Induced pluripotent stem (iPS) cells,a breakthrough in stem cell research,may terminate these hinders for cell transplantation.For the promise of iPS cells to be realized in liver diseases,it is necessary to determine if and how efficient they can be differentiated into functional hepatocytes.Methods In this study,we directly compared the hepatic-differentiation capacity of mouse iPS cells and embryonic stem (ES) cells with three different induction approaches:conditions via embryonic body (EB) formation plus cytokines,conditions by combination of dimethyl sulfoxide and sodium butyrate and chemically defined,serum free monolayer conditions.Among these three induction conditions,more homogenous populations can be promoted under chemically defined,serum free conditions.The cells generated under these conditions exhibited hepatic functions in vitro,including glycogen storage,indocynine green (ICG) uptake and release as well as urea secretion.Although efficient hepatocytes differentiation from mouse iPS cells were observed,mouse iPS cells showed relatively lower hepatic induction efficiency compared with mouse ES cells.Results Mouse iPS cells would be efficiently differentiated into functional hepatocytes in vitro,which may be helpful in facilitating the development of hepatocytes for transplantation and for research on drug discovery.Conclusion We demonstrate that mouse iPS cells retain full potential for fetal liver development and describe procedures that facilitates the efficient generation of highly differentiated human hepatocyte-like cells from iPS cells in vitro.

  19. Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Winzi, Maria K.; Hyttel, Poul; Dale, Jacqueline Kim

    2011-01-01

    The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However......, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed...... the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells...

  20. Specific Tandem 3'UTR Patterns and Gene Expression Profiles in Mouse Thy1+ Germline Stem Cells.

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    Yan Huang

    Full Text Available A recently developed strategy of sequencing alternative polyadenylation (APA sites (SAPAS with second-generation sequencing technology can be used to explore complete genome-wide patterns of tandem APA sites and global gene expression profiles. spermatogonial stem cells (SSCs maintain long-term reproductive abilities in male mammals. The detailed mechanisms by which SSCs self-renew and generate mature spermatozoa are not clear. To understand the specific alternative polyadenylation pattern and global gene expression profile of male germline stem cells (GSCs, mainly referred to SSCs here, we isolated and purified mouse Thy1+ cells from testis by magnetic-activated cell sorting (MACS and then used the SAPAS method for analysis, using pluripotent embryonic stem cells (ESCs and differentiated mouse embryonic fibroblast cells (MEFs as controls. As a result, we obtained 99,944 poly(A sites, approximately 40% of which were newly detected in our experiments. These poly(A sites originated from three mouse cell types and covered 17,499 genes, including 831 long non-coding RNA (lncRNA genes. We observed that GSCs tend to have shorter 3'UTR lengths while MEFs tend towards longer 3'UTR lengths. We also identified 1337 genes that were highly expressed in GSCs, and these genes were highly consistent with the functional characteristics of GSCs. Our detailed bioinformatics analysis identified APA site-switching events at 3'UTRs and many new specifically expressed genes in GSCs, which we experimentally confirmed. Furthermore, qRT-PCR was performed to validate several events of the 334 genes with distal-to-proximal poly(A switch in GSCs. Consistently APA reporter assay confirmed the total 3'UTR shortening in GSCs compared to MEFs. We also analyzed the cis elements around the proximal poly(A site preferentially used in GSCs and found C-rich elements may contribute to this regulation. Overall, our results identified the expression level and polyadenylation site

  1. Specific Tandem 3'UTR Patterns and Gene Expression Profiles in Mouse Thy1+ Germline Stem Cells

    Science.gov (United States)

    Lin, Zhuoheng; Feng, Xuyang; Jiang, Xue; Songyang, Zhou; Huang, Junjiu

    2015-01-01

    A recently developed strategy of sequencing alternative polyadenylation (APA) sites (SAPAS) with second-generation sequencing technology can be used to explore complete genome-wide patterns of tandem APA sites and global gene expression profiles. spermatogonial stem cells (SSCs) maintain long-term reproductive abilities in male mammals. The detailed mechanisms by which SSCs self-renew and generate mature spermatozoa are not clear. To understand the specific alternative polyadenylation pattern and global gene expression profile of male germline stem cells (GSCs, mainly referred to SSCs here), we isolated and purified mouse Thy1+ cells from testis by magnetic-activated cell sorting (MACS) and then used the SAPAS method for analysis, using pluripotent embryonic stem cells (ESCs) and differentiated mouse embryonic fibroblast cells (MEFs) as controls. As a result, we obtained 99,944 poly(A) sites, approximately 40% of which were newly detected in our experiments. These poly(A) sites originated from three mouse cell types and covered 17,499 genes, including 831 long non-coding RNA (lncRNA) genes. We observed that GSCs tend to have shorter 3'UTR lengths while MEFs tend towards longer 3'UTR lengths. We also identified 1337 genes that were highly expressed in GSCs, and these genes were highly consistent with the functional characteristics of GSCs. Our detailed bioinformatics analysis identified APA site-switching events at 3'UTRs and many new specifically expressed genes in GSCs, which we experimentally confirmed. Furthermore, qRT-PCR was performed to validate several events of the 334 genes with distal-to-proximal poly(A) switch in GSCs. Consistently APA reporter assay confirmed the total 3'UTR shortening in GSCs compared to MEFs. We also analyzed the cis elements around the proximal poly(A) site preferentially used in GSCs and found C-rich elements may contribute to this regulation. Overall, our results identified the expression level and polyadenylation site profiles and

  2. Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Guedes, Ana M V; Henrique, Domingos; Abranches, Elsa

    2016-01-01

    Mouse Embryonic Stem cells (mESCs) show heterogeneous and dynamic expression of important pluripotency regulatory factors. Single-cell analysis has revealed the existence of cell-to-cell variability in the expression of individual genes in mESCs. Understanding how these heterogeneities are regulated and what their functional consequences are is crucial to obtain a more comprehensive view of the pluripotent state.In this chapter we describe how to analyze transcriptional heterogeneity by monitoring gene expression of Nanog, Oct4, and Sox2, using single-molecule RNA FISH in single mESCs grown in different cell culture medium. We describe in detail all the steps involved in the protocol, from RNA detection to image acquisition and processing, as well as exploratory data analysis.

  3. Development and Characterization of a Human and Mouse Intestinal Epithelial Cell Monolayer Platform

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    Kenji Kozuka

    2017-12-01

    Full Text Available Summary: We describe the development and characterization of a mouse and human epithelial cell monolayer platform of the small and large intestines, with a broad range of potential applications including the discovery and development of minimally systemic drug candidates. Culture conditions for each intestinal segment were optimized by correlating monolayer global gene expression with the corresponding tissue segment. The monolayers polarized, formed tight junctions, and contained a diversity of intestinal epithelial cell lineages. Ion transport phenotypes of monolayers from the proximal and distal colon and small intestine matched the known and unique physiology of these intestinal segments. The cultures secreted serotonin, GLP-1, and FGF19 and upregulated the epithelial sodium channel in response to known biologically active agents, suggesting intact secretory and absorptive functions. A screen of over 2,000 pharmacologically active compounds for inhibition of potassium ion transport in the mouse distal colon cultures led to the identification of a tool compound. : Siegel and colleagues describe their development of a human and mouse intestinal epithelial cell monolayer platform that maintains the cellular, molecular, and functional characteristics of tissue for each intestinal segment. They demonstrate the platform's application to drug discovery by screening a library of over 2,000 compounds to identify an inhibitor of potassium ion transport in the mouse distal colon. Keywords: intestinal epithelium, organoids, monolayer, colon, small intestine, phenotype screening assays, enteroid, colonoid

  4. Regulation of cAMP on the first mitotic cell cycle of mouse embryos.

    Science.gov (United States)

    Yu, Aiming; Zhang, Zhe; Bi, Qiang; Sun, Bingqi; Su, Wenhui; Guan, Yifu; Mu, Runqing; Miao, Changsheng; Zhang, Jie; Yu, Bingzhi

    2008-03-01

    Mitosis promoting factor (MPF) plays a central role during the first mitosis of mouse embryo. We demonstrated that MPF activity increased when one-cell stage mouse embryo initiated G2/M transition following the decrease of cyclic adenosine 3', 5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) activity. When cAMP and PKA activity increases again, MPF activity decreases and mouse embryo starts metaphase-anaphase transition. In the downstream of cAMP/PKA, there are some effectors such as polo-like kinase 1 (Plk1), Cdc25, Mos (mitogen-activated protein kinase kinase kinase), MEK (mitogen-activated protein kinase kinase), mitogen-activated protein kinase (MAPK), Wee1, anaphase-promoting complex (APC), and phosphoprotein phosphatase that are involved in the regulation of MPF activity. Here, we demonstrated that following activation of MPF, MAPK activity was steady, whereas Plk1 activity fluctuated during the first cell cycle. Plk1 activity was the highest at metaphase and decreased at metaphase-anaphase transition. Further, we established a mathematical model using Gepasi algorithm and the simulation was in agreement with the experimental data. Above all the evidences, we suggested that cAMP and PKA might be the upstream factors which were included in the regulation of the first cell cycle development of mouse embryo. Copyright 2007 Wiley-Liss, Inc.

  5. Isolation of an 18,000-dalton hypusine-containing protein from cultured mouse neuroblastoma cells

    International Nuclear Information System (INIS)

    Dou, Q.P.; Chen, K.Y.

    1987-01-01

    An 18,000-dalton protein can be metabolically labeled by [ 3 H]putrescine or spermidine in mammalian cells. The labeling is due to a post-translational conversion of a lysine residue to hypusine residue. Previous studies indicated that the labeling is growth-dependent and is greatly diminished in mouse neuroblastoma cells after differentiation. To further study the physiological functions of this protein in the differentiation of mouse neuroblastoma cells, they have developed a simple procedure to purify this protein from cultured NB-15 mouse neuroblastoma cells. The 4-steps procedure included a Cibacron-Blue column, an omega-diaminooctyl-agarose column, a Sephadex G-50 column, and a Mono Q column. The procedure resulted in a 500-fold purification and the preparation appeared to be homogenous as judged by SDS-PAGE. Peptide map analysis using V-8 protease digestion method indicated that the 18,000-dalton hypusine-containing protein from NB-15 cells was identical to eukaryotic initiation factor 4D isolated from rabbit reticulocytes. This purification scheme also enabled them to detect a very faintly labeled protein in NB-15 cells. This weakly labeled protein had an apparent molecular weight of 22,000-dalton and pI of 5.0

  6. Senescent mouse cells fail to overtly regulate the HIRA histone chaperone and do not form robust Senescence Associated Heterochromatin Foci

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    Enders Greg H

    2010-06-01

    Full Text Available Abstract Background Cellular senescence is a permanent growth arrest that occurs in response to cellular stressors, such as telomere shortening or activation of oncogenes. Although the process of senescence growth arrest is somewhat conserved between mouse and human cells, there are some critical differences in the molecular pathways of senescence between these two species. Recent studies in human fibroblasts have defined a cell signaling pathway that is initiated by repression of a specific Wnt ligand, Wnt2. This, in turn, activates a histone chaperone HIRA, and culminates in formation of specialized punctate domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF, that are enriched in the histone variant, macroH2A. SAHF are thought to repress expression of proliferation-promoting genes, thereby contributing to senescence-associated proliferation arrest. We asked whether this Wnt2-HIRA-SAHF pathway is conserved in mouse fibroblasts. Results We show that mouse embryo fibroblasts (MEFs and mouse skin fibroblasts, do not form robust punctate SAHF in response to an activated Ras oncogene or shortened telomeres. However, senescent MEFs do exhibit elevated levels of macroH2A staining throughout the nucleus as a whole. Consistent with their failure to fully activate the SAHF assembly pathway, the Wnt2-HIRA signaling axis is not overtly regulated between proliferating and senescent mouse cells. Conclusions In addition to the previously defined differences between mouse and human cells in the mechanisms and phenotypes associated with senescence, we conclude that senescent mouse and human fibroblasts also differ at the level of chromatin and the signaling pathways used to regulate chromatin. These differences between human and mouse senescence may contribute to the increased propensity of mouse fibroblasts (and perhaps other mouse cell types to become immortalized and transformed, compared to human cells.

  7. Non-invasive bioluminescence imaging to monitor the immunological control of a plasmablastic lymphoma-like B cell neoplasia after hematopoietic cell transplantation.

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    Martin Chopra

    Full Text Available To promote cancer research and to develop innovative therapies, refined pre-clinical mouse tumor models that mimic the actual disease in humans are of dire need. A number of neoplasms along the B cell lineage are commonly initiated by a translocation recombining c-myc with the immunoglobulin heavy-chain gene locus. The translocation is modeled in the C.129S1-Igha(tm1(MycJanz/J mouse which has been previously engineered to express c-myc under the control of the endogenous IgH promoter. This transgenic mouse exhibits B cell hyperplasia and develops diverse B cell tumors. We have isolated tumor cells from the spleen of a C.129S1-Igha(tm1(MycJanz/J mouse that spontaneously developed a plasmablastic lymphoma-like disease. These cells were cultured, transduced to express eGFP and firefly luciferase, and gave rise to a highly aggressive, transplantable B cell lymphoma cell line, termed IM380. This model bears several advantages over other models as it is genetically induced and mimics the translocation that is detectable in a number of human B cell lymphomas. The growth of the tumor cells, their dissemination, and response to treatment within immunocompetent hosts can be imaged non-invasively in vivo due to their expression of firefly luciferase. IM380 cells are radioresistant in vivo and mice with established tumors can be allogeneically transplanted to analyze graft-versus-tumor effects of transplanted T cells. Allogeneic hematopoietic stem cell transplantation of tumor-bearing mice results in prolonged survival. These traits make the IM380 model very valuable for the study of B cell lymphoma pathophysiology and for the development of innovative cancer therapies.

  8. Long term effects of lipopolysaccharide on satellite glial cells in mouse dorsal root ganglia

    Energy Technology Data Exchange (ETDEWEB)

    Blum, E. [Laboratory of Experimental Surgery, Hadassah-Hebrew University Medical Center, Mount Scopus, Jerusalem 91240 (Israel); Procacci, P.; Conte, V.; Sartori, P. [Dipartimento di Scienze Biomediche per la Salute, University of Milan, via Mangiagalli 14, I-20133 Milano (Italy); Hanani, M., E-mail: hananim@cc.huji.ac.il [Laboratory of Experimental Surgery, Hadassah-Hebrew University Medical Center, Mount Scopus, Jerusalem 91240 (Israel)

    2017-01-01

    Lipopolysaccharide (LPS) has been used extensively to study neuroinflammation, but usually its effects were examined acutely (24 h<). We have shown previously that a single intraperitoneal LPS injection activated satellite glial cells (SGCs) in mouse dorsal root ganglia (DRG) and altered several functional parameters in these cells for at least one week. Here we asked whether the LPS effects would persist for 1 month. We injected mice with a single LPS dose and tested pain behavior, assessed SGCs activation in DRG using glial fibrillary acidic protein (GFAP) immunostaining, and injected a fluorescent dye intracellularly to study intercellular coupling. Electron microscopy was used to quantitate changes in gap junctions. We found that at 30 days post-LPS the threshold to mechanical stimulation was lower than in controls. GFAP expression, as well as the magnitude of dye coupling among SGCs were greater than in controls. Electron microscopy analysis supported these results, showing a greater number of gap junctions and an abnormal growth of SGC processes. These changes were significant, but less prominent than at 7 days post-LPS. We conclude that a single LPS injection exerts long-term behavioral and cellular changes. The results are consistent with the idea that SGC activation contributes to hyperalgesia. - Highlights: • A single lipopolysaccharides injection activated glia in mouse dorsal root ganglia for 30 days. • This was accompanied by increased communications by gap junctions among glia and by hyperalgesia. • Glial activation and coupling may contribute to chronic pain.

  9. Generation of competent bone marrow-derived antigen presenting cells from the deer mouse (Peromyscus maniculatus

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    Farrell Regina M

    2004-09-01

    Full Text Available Abstract Background Human infections with Sin Nombre virus (SNV and related New World hantaviruses often lead to hantavirus cardiopulmonary syndrome (HCPS, a sometimes fatal illness. Lungs of patients who die from HCPS exhibit cytokine-producing mononuclear infiltrates and pronounced pulmonary inflammation. Deer mice (Peromyscus maniculatus are the principal natural hosts of SNV, in which the virus establishes life-long persistence without conspicuous pathology. Little is known about the mechanisms SNV employs to evade the immune response of deer mice, and experimental examination of this question has been difficult because of a lack of methodologies for examining such responses during infection. One such deficiency is our inability to characterize T cell responses because susceptible syngeneic deer mice are not available. Results To solve this problem, we have developed an in vitro method of expanding and generating competent antigen presenting cells (APC from deer mouse bone marrow using commercially-available house mouse (Mus musculus granulocyte-macrophage colony stimulating factor. These cells are capable of processing and presenting soluble protein to antigen-specific autologous helper T cells in vitro. Inclusion of antigen-specific deer mouse antibody augments T cell stimulation, presumably through Fc receptor-mediated endocytosis. Conclusions The use of these APC has allowed us to dramatically expand deer mouse helper T cells in culture and should permit extensive characterization of T cell epitopes. Considering the evolutionary divergence between deer mice and house mice, it is probable that this method will be useful to other investigators using unconventional models of rodent-borne diseases.

  10. Halofuginone suppresses growth of human uterine leiomyoma cells in a mouse xenograft model.

    Science.gov (United States)

    Koohestani, Faezeh; Qiang, Wenan; MacNeill, Amy L; Druschitz, Stacy A; Serna, Vanida A; Adur, Malavika; Kurita, Takeshi; Nowak, Romana A

    2016-07-01

    Does halofuginone (HF) inhibit the growth of human uterine leiomyoma cells in a mouse xenograft model? HF suppresses the growth of human uterine leiomyoma cells in a mouse xenograft model through inhibiting cell proliferation and inducing apoptosis. Uterine leiomyomas are the most common benign tumors of the female reproductive tract. HF can suppress the growth of human uterine leiomyoma cells in vitro. The mouse xenograft model reflects the characteristics of human leiomyomas. Primary leiomyoma smooth muscle cells from eight patients were xenografted under the renal capsule of adult, ovariectomized NOD-scid IL2Rγ(null) mice (NSG). Mice were treated with two different doses of HF or vehicle for 4 weeks with six to eight mice per group. Mouse body weight measurements and immunohistochemical analysis of body organs were carried out to assess the safety of HF treatment. Xenografted tumors were measured and analyzed for cellular and molecular changes induced by HF. Ovarian steroid hormone receptors were evaluated for possible modulation by HF. Treatment of mice carrying human UL xenografts with HF at 0.25 or 0.50 mg/kg body weight for 4 weeks resulted in a 35-40% (P leiomyoma cells in an in vivo model, HF was administered to mice whose tolerance and metabolism of the drug may differ from that in humans. Also, the longer term effects of HF treatment are yet unclear. The results of this study showing the effectiveness of HF in reducing UL tumor growth by interfering with the main cellular processes regulating cell proliferation and apoptosis are in agreement with previous studies on the effects of HF on other fibrotic diseases. HF can be considered as a candidate for reducing the size of leiomyomas, particularly prior to surgery. This project was funded by NIH PO1HD057877 and R01 HD064402. Authors report no competing interests. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights

  11. Radiation response of mouse lymphoid and myeloid cell lines. Pt. 1

    International Nuclear Information System (INIS)

    Radford, I.R.

    1994-01-01

    The sensitivity of 10 mouse lymphoid or myeloid cell lines to γ-ray- and DNA-associated 125 I-decay-induced clonogenic cell killing have been compared with their rate of loss of viability (membrane integrity) and with their putative cell type of origin. The increased sensitivity of haematopoietic cell lines to killing by DNA dsb may be related to their mode of death (apoptosis versus necrosis). Mode of cell death may thus be an important factor in determining the 'inherent radiosensitivity' of normal cells/tissues. Haematopoietic cell lines that undergo rapid interphase apoptotic death showed extreme sensitivity to DNA dsb. (author)

  12. Dose-dependent effects of ouabain on spiral ganglion neurons and Schwann cells in mouse cochlea.

    Science.gov (United States)

    Zhang, Zhi-Jian; Guan, Hong-Xia; Yang, Kun; Xiao, Bo-Kui; Liao, Hua; Jiang, Yang; Zhou, Tao; Hua, Qing-Quan

    2017-10-01

    This study aimed in fully investigating the toxicities of ouabain to mouse cochlea and the related cellular environment, and providing an optimal animal model system for cell transplantation in the treatment of auditory neuropathy (AN) and sensorineural hearing loss (SNHL). Different dosages of ouabain were applied to mouse round window. The auditory brainstem responses and distortion product otoacoustic emissions were used to evaluate the cochlear function. The immunohistochemical staining and cochlea surface preparation were performed to detect the spiral ganglion neurons (SGNs), Schwann cells and hair cells. Ouabain at the dosages of 0.5 mM, 1 mM and 3 mM selectively and permanently destroyed SGNs and their functions, while leaving the hair cells relatively intact. Ouabain at 3 mM resulted in the most severe SGNs loss and induced significant loss of Schwann cells started as early as 7 days and with further damages at 14 and 30 days after ouabain exposure. The application of ouabain to mouse round window induces damages of SGNs and Schwann cells in a dose- and time-dependent manner, this study established a reliable and accurate animal model system of AN and SNHL.

  13. Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses.

    Science.gov (United States)

    Simerly, Calvin; McFarland, Dave; Castro, Carlos; Lin, Chih-Cheng; Redinger, Carrie; Jacoby, Ethan; Mich-Basso, Jocelyn; Orwig, Kyle; Mills, Parker; Ahrens, Eric; Navara, Chris; Schatten, Gerald

    2011-07-01

    Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more naïve states in these inter-specific chimera assays will be an important future endeavor. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Ectopic expression of the calcium-binding protein parvalbumin in mouse liver endothelial cells

    DEFF Research Database (Denmark)

    Castillo, M B; Berchtold, M W; Rülicke, T

    1997-01-01

    To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical...... methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent...... vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased...

  15. [Relationship between sensitivity of tumor cells to chemotherapeutic agent in vivo and in vitro: experiment with mouse lymphoma cells].

    Science.gov (United States)

    Li, Chuan-gang; Li, Mo-lin; Shu, Xiao-hong; Jia, Yu-jie; Liu, Yong-ji; Li, Ming

    2007-06-12

    To study the relationship of the sensitivity of tumor cells to chemotherapeutic agent between in vivo and in vitro. Mouse lymphoma cells of the line E14 were cultured and melphalan resistant EL4 cell line (EL4/melphalan) was established by culturing EL4 cells with continuous low-concentration and intermittent gradually-increasing-concentration of melphalan in vitro. MTT assay was used to evaluate the drug sensitivity and the resistance index of the EL4/melphalan cells to melphalan was calculated. EL4/melphalan and EL4 cells of the concentration of 5 x 10(8)/L were inoculated separately into 20 C57BL/6 mice subcutaneously. 12 days later, the EL4 and EL4/melphalan tumor-bearing mice were randomly divided into 2 groups respectively, 5 mice in each group. Treatment groups were given 7.5 mg/kg melphalan intraperitoneally, and control groups were given the same volume of normal saline. The tumor size was observed every other day. Compared with the EL4 cells, the EL4/melphalan cells had no obvious changes morphologically. They could grow in RPMI 1640 medium containing 5 mg/ml melphalan. The resistance index was 2.87 against melphalan. After the treatment of melphalan of the dose 7.5 mg/kg, the tumor sizes of the treatment groups and control groups inoculated with both EL4 cells and the EL4/melphalan cells gradually decreased at the similar speed, and about one week later all tumors disappeared. However, the tumors of the control groups grew progressively and all the mice died at last. The chemotherapeutic effects of tumors in vivo have nothing to do with the effects of the chemotherapeutic agents on tumor cells in vitro. The tumor cells resistant to melphalan in vitro remain sensitive to the drug in vivo.

  16. A novel approach for amplification and purification of mouse oligodendrocyte progenitor cells

    Directory of Open Access Journals (Sweden)

    Junlin Yang

    2016-08-01

    Full Text Available Although transgenic and knockout mice are widely used to study the specification and differentiation of oligodendrocyte precursor cells (OPCs, mouse primary OPCs are difficult to be purified and maintained, and many in vitro studies have to resort to rat OPCs as substitutes. In this study, we reported that mouse O4 negative early-stage OPCs can be obtained by culturing cortical tissue blocks, and the simultaneous treatment of OPCs with PDGFaa, bFGF and EGF is the key for the propagation of mouse OPCs in culture. Epidermal growth factor (EGF was found to be a potent mitogen for OPCs and cooperate with Platelet Derived Growth Factor-AA (PDGFaa to extend cell division and inhibit their differentiation. EGF also collaborates with PDGFaa and basic fibroblast growth factor (bFGF to convert bipolar or tripolar OPCs to more vital fibroblast-like OPCs without compromising their oligodendrocyte differentiation potential. In addition, EGF promoted the survival and proliferation of glial progenitor cells (GPCs derived from primary OPC cultures, and a mixture of GPCs and OPCs can be obtained and propagated in the presence of EGF, bFGF and PDGFaa. Once EGF is withdrawn, GPC population decreased sharply and fibroblast-like OPCs changed into typical OPCs morphology, then homogeneous OPCs were obtained subsequently.

  17. Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells.

    Science.gov (United States)

    Winzi, Maria K; Hyttel, Poul; Dale, Jacqueline Kim; Serup, Palle

    2011-11-01

    The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures.

  18. Elimination of mouse tumor cells from neonate spermatogonial cells utilizing cisplatin-entrapped folic acid-conjugated poly(lactic-co-glycolic acid) nanoparticles in vitro.

    Science.gov (United States)

    Shabani, Ronak; Ashjari, Mohsen; Ashtari, Khadijeh; Izadyar, Fariborz; Behnam, Babak; Khoei, Samideh; Asghari-Jafarabadi, Mohamad; Koruji, Morteza

    2018-01-01

    Some male survivors of childhood cancer are suffering from azoospermia. In addition, spermatogonial stem cells (SSCs) are necessary for the improvement of spermatogenesis subsequent to exposure to cytotoxic agents such as cisplatin. The aim of this study was to evaluate the anticancer activity of cisplatin-loaded folic acid-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) on mouse malignant cell line (EL4) and SSCs in vitro. SSCs were co-cultured with mouse malignant cell line (EL4) cells and divided into four culture groups: 1) control (cells were co-cultured in the culture medium), 2) co-cultured cells were treated with cisplatin (10 μg/mL), 3) co-cultured cells were treated with cisplatin-loaded folic acid-conjugated PLGA NPs, and 4) co-cultures were treated with folic acid-conjugated PLGA for 48 hours. The NPs were prepared, characterized, and targeted with folate. In vitro release characteristics, loading efficiency, and scanning electron microscopy and transmission electron microscopy images were studied. Cancer cells were assayed after treatment using flow cytometry and TUNEL assay. The co-cultures of SSCs and EL4 cells were injected into seminiferous tubules of the testes after treating with cis-diaminedichloroplatinum/PLGA NPs. The mean diameter of PLGA NPs ranged between 150 and 250 nm. The number of TUNEL-positive cells increased, and the expression of Bax and caspase-3 were upregulated in EL4 cells in Group 4 compared with Group 2. There was no pathological tumor in testes after transplantation with treated co-cultured cells. The PLGA NPs appeared to act as a promising carrier for cisplatin administration, which was consistent with a higher activation of apoptosis than free drug.

  19. Reversibility of β-Cell-Specific Transcript Factors Expression by Long-Term Caloric Restriction in db/db Mouse

    Directory of Open Access Journals (Sweden)

    Chunjun Sheng

    2016-01-01

    Full Text Available Type 2 diabetes (T2D is characterized by β-cell dedifferentiation, but underlying mechanisms remain unclear. The purpose of the current study was to explore the mechanisms of β-cell dedifferentiation with and without long-term control of calorie intake. We used a diabetes mouse model (db/db to analyze the changes in the expression levels of β-cell-specific transcription factors (TFs and functional factors with long-term caloric restriction (CR. Our results showed that chronic euglycemia was maintained in the db/db mice with long-term CR intervention, and β-cell dedifferentiation was significantly reduced. The expression of Glut2, Pdx1, and Nkx6.1 was reversed, while MafA expression was significantly increased with long-term CR. GLP-1 pathway was reactivated with long-term CR. Our work showed that the course of β-cell dedifferentiation can intervene by long-term control of calorie intake. Key β-cell-specific TFs and functional factors play important roles in maintaining β-cell differentiation. Targeting these factors could optimize T2D therapies.

  20. 17β-Estradiol Promotes Schwann Cell Proliferation and Differentiation, Accelerating Early Remyelination in a Mouse Peripheral Nerve Injury Model

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    Yan Chen

    2016-01-01

    Full Text Available Estrogen induces oligodendrocyte remyelination in response to demyelination in the central nervous system. Our objective was to determine the effects of 17β-estradiol (E2 on Schwann cell function and peripheral nerve remyelination after injury. Adult male C57BL/6J mice were used to prepare the sciatic nerve transection injury model and were randomly categorized into control and E2 groups. To study myelination in vitro, dorsal root ganglion (DRG explant culture was prepared using 13.5-day-old mouse embryos. Primary Schwann cells were isolated from the sciatic nerves of 1- to 3-day-old Sprague–Dawley rats. Immunostaining for myelin basic protein (MBP expression and toluidine blue staining for myelin sheaths demonstrated that E2 treatment accelerates early remyelination in the “nerve bridge” region between the proximal and distal stumps of the transection injury site in the mouse sciatic nerve. The 5-bromo-2′-deoxyuridine incorporation assay revealed that E2 promotes Schwann cell proliferation in the bridge region and in the primary culture, which is blocked using AKT inhibitor MK2206. The in vitro myelination in the DRG explant culture determined showed that the MBP expression in the E2-treated group is higher than that in the control group. These results show that E2 promotes Schwann cell proliferation and myelination depending on AKT activation.

  1. 17β-Estradiol Promotes Schwann Cell Proliferation and Differentiation, Accelerating Early Remyelination in a Mouse Peripheral Nerve Injury Model

    Science.gov (United States)

    Chen, Yan; Guo, Wenjie; Li, Wenjuan; Cheng, Meng; Hu, Ying; Xu, Wenming

    2016-01-01

    Estrogen induces oligodendrocyte remyelination in response to demyelination in the central nervous system. Our objective was to determine the effects of 17β-estradiol (E2) on Schwann cell function and peripheral nerve remyelination after injury. Adult male C57BL/6J mice were used to prepare the sciatic nerve transection injury model and were randomly categorized into control and E2 groups. To study myelination in vitro, dorsal root ganglion (DRG) explant culture was prepared using 13.5-day-old mouse embryos. Primary Schwann cells were isolated from the sciatic nerves of 1- to 3-day-old Sprague–Dawley rats. Immunostaining for myelin basic protein (MBP) expression and toluidine blue staining for myelin sheaths demonstrated that E2 treatment accelerates early remyelination in the “nerve bridge” region between the proximal and distal stumps of the transection injury site in the mouse sciatic nerve. The 5-bromo-2′-deoxyuridine incorporation assay revealed that E2 promotes Schwann cell proliferation in the bridge region and in the primary culture, which is blocked using AKT inhibitor MK2206. The in vitro myelination in the DRG explant culture determined showed that the MBP expression in the E2-treated group is higher than that in the control group. These results show that E2 promotes Schwann cell proliferation and myelination depending on AKT activation. PMID:27872858

  2. Structural and functional development of rat and mouse gastric mucous cells in relation to their proliferative activity

    International Nuclear Information System (INIS)

    Wattel, W.

    1978-01-01

    An investigation has been carried out to find a relation between the differentiation and the mitotic activity of gastric mucous cells of the rat and the mouse. It is shown that the bulk mucous production is carried out by the older, non-proliferative, surface mucous cells that line the foveolae and the gastric surface. One experiment describes the renewal of mouse gastric mucous cells following fast neutron irradiation. (C.F.)

  3. Properties of mouse CD40: differential expression of CD40 epitopes on dendritic cells and epithelial cells

    NARCIS (Netherlands)

    van den Berg, T. K.; Hasbold, J.; Renardel de Lavalette, C.; Döpp, E. A.; Dijkstra, C. D.; Klaus, G. G.

    1996-01-01

    In this study we describe the tissue distribution of mouse CD40 using two monoclonal antibodies (mAb) against different epitopes of the molecule. In lymphoid tissues CD40 was expressed by B lymphocytes. Most B cells in typical B-cell compartments were CD40-positive, including germinal centre B

  4. Cell death induced on cell cultures and nude mouse skin by non-thermal, nanosecond-pulsed generated plasma.

    Directory of Open Access Journals (Sweden)

    Arnaud Duval

    Full Text Available Non-thermal plasmas are gaseous mixtures of molecules, radicals, and excited species with a small proportion of ions and energetic electrons. Non-thermal plasmas can be generated with any high electro-magnetic field. We studied here the pathological effects, and in particular cell death, induced by nanosecond-pulsed high voltage generated plasmas homogeneously applied on cell cultures and nude mouse skin. In vitro, Jurkat cells and HMEC exhibited apoptosis and necrosis, in dose-dependent manner. In vivo, on nude mouse skin, cell death occurred for doses above 113 J/cm(2 for the epidermis, 281 J/cm(2 for the dermis, and 394 J/cm(2 for the hypodermis. Using electron microscopy, we characterized apoptosis for low doses and necrosis for high doses. We demonstrated that these effects were not related to thermal, photonic or pH variations, and were due to the production of free radicals. The ability of cold plasmas to generate apoptosis on cells in suspension and, without any sensitizer, on precise skin areas, opens new fields of application in dermatology for extracorporeal blood cell treatment and the eradication of superficial skin lesions.

  5. Human mesenchymal stem cells towards non-alcoholic steatohepatitis in an immunodeficient mouse model

    International Nuclear Information System (INIS)

    Winkler, Sandra; Borkham-Kamphorst, Erawan; Stock, Peggy; Brückner, Sandra; Dollinger, Matthias; Weiskirchen, Ralf; Christ, Bruno

    2014-01-01

    Non-alcoholic steatohepatitis (NASH) is a frequent clinical picture characterised by hepatic inflammation, lipid accumulation and fibrosis. When untreated, NASH bears a high risk of developing liver cirrhosis and consecutive hepatocellular carcinoma requiring liver transplantation in its end-stage. However, donor organ scarcity has prompted the search for alternatives, of which hepatocyte or stem cell-derived hepatocyte transplantation are regarded auspicious options of treatment. Mesenchymal stem cells (MSC) are able to differentiate into hepatocyte-like cells and thus may represent an alternative cell source to primary hepatocytes. In addition these cells feature anti-inflammatory and pro-regenerative characteristics, which might favour liver recovery from NASH. The aim of this study was to investigate the potential benefit of hepatocyte-like cells derived from human bone marrow MSC in a mouse model of diet-induced NASH. Seven days post-transplant, human hepatocyte-like cells were found in the mouse liver parenchyma. Triglyceride depositions were lowered in the liver but restored to normal in the blood. Hepatic inflammation was attenuated as verified by decreased expression of the acute phase protein serum amyloid A, inflammation-associated markers (e.g. lipocalin 2), as well as the pro-inflammatory cytokine TNFα. Moreover, the proliferation of host hepatocytes that indicate the regenerative capacity in livers receiving cell transplants was enhanced. Transplantation of MSC-derived human hepatocyte-like cells corrects NASH in mice by restoring triglyceride depositions, reducing inflammation and augmenting the regenerative capacity of the liver. - Highlights: • First time to show NASH in an immune-deficient mouse model. • Human MSC attenuate NASH and improve lipid homeostasis. • MSC act anti-fibrotic and augment liver regeneration by stimulation of proliferation. • Pre-clinical assessment of human MSC for stem cell-based therapy of NASH

  6. Human mesenchymal stem cells towards non-alcoholic steatohepatitis in an immunodeficient mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Winkler, Sandra, E-mail: sandra.pelz@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Borkham-Kamphorst, Erawan, E-mail: ekamphorst@ukaachen.de [Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital Aachen, Pauwelsstraße 30, D-52074 Aachen (Germany); Stock, Peggy, E-mail: peggy.stock@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Brückner, Sandra, E-mail: sandra.brueckner@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Dollinger, Matthias, E-mail: matthias.dollinger@uniklinik-ulm.de [Department for Internal Medicine I, University Hospital Ulm, Albert-Einstein-Allee 23, D-89081 Ulm (Germany); Weiskirchen, Ralf, E-mail: rweiskirchen@ukaachen.de [Institute of Clinical Chemistry and Pathobiochemistry, RWTH University Hospital Aachen, Pauwelsstraße 30, D-52074 Aachen (Germany); Christ, Bruno, E-mail: bruno.christ@medizin.uni-leipzig.de [Applied Molecular Hepatology Laboratory, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, University Hospital Leipzig, Liebigstraße 21, D-04103 Leipzig (Germany); Translational Centre for Regenerative Medicine (TRM), University of Leipzig, Leipzig (Germany)

    2014-08-15

    Non-alcoholic steatohepatitis (NASH) is a frequent clinical picture characterised by hepatic inflammation, lipid accumulation and fibrosis. When untreated, NASH bears a high risk of developing liver cirrhosis and consecutive hepatocellular carcinoma requiring liver transplantation in its end-stage. However, donor organ scarcity has prompted the search for alternatives, of which hepatocyte or stem cell-derived hepatocyte transplantation are regarded auspicious options of treatment. Mesenchymal stem cells (MSC) are able to differentiate into hepatocyte-like cells and thus may represent an alternative cell source to primary hepatocytes. In addition these cells feature anti-inflammatory and pro-regenerative characteristics, which might favour liver recovery from NASH. The aim of this study was to investigate the potential benefit of hepatocyte-like cells derived from human bone marrow MSC in a mouse model of diet-induced NASH. Seven days post-transplant, human hepatocyte-like cells were found in the mouse liver parenchyma. Triglyceride depositions were lowered in the liver but restored to normal in the blood. Hepatic inflammation was attenuated as verified by decreased expression of the acute phase protein serum amyloid A, inflammation-associated markers (e.g. lipocalin 2), as well as the pro-inflammatory cytokine TNFα. Moreover, the proliferation of host hepatocytes that indicate the regenerative capacity in livers receiving cell transplants was enhanced. Transplantation of MSC-derived human hepatocyte-like cells corrects NASH in mice by restoring triglyceride depositions, reducing inflammation and augmenting the regenerative capacity of the liver. - Highlights: • First time to show NASH in an immune-deficient mouse model. • Human MSC attenuate NASH and improve lipid homeostasis. • MSC act anti-fibrotic and augment liver regeneration by stimulation of proliferation. • Pre-clinical assessment of human MSC for stem cell-based therapy of NASH.

  7. Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells.

    Science.gov (United States)

    Wang, Ruoxing; Guo, Yan-Lin

    2012-10-01

    Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. MicroRNA modulation induced by AICA ribonucleotide in J1 mouse ES cells.

    Directory of Open Access Journals (Sweden)

    Xiaoyan Shi

    Full Text Available ES cells can propagate indefinitely, maintain self-renewal, and differentiate into almost any cell type of the body. These properties make them valuable in the research of embryonic development, regenerative medicine, and organ transplantation. MicroRNAs (miRNAs are considered to have essential functions in the maintenance and differentiation of embryonic stem cells (ES cells. It was reported that, strong external stimuli, such as a transient low-pH and hypoxia stress, were conducive to the formation of induced pluripotent stem cells (iPS cells. AICA ribonucleotide (AICAR is an AMP-activated protein kinase activator, which can let cells in the state of energy stress. We have demonstrated that AICAR can maintain the pluripotency of J1 mouse ES cells through modulating protein expression in our previous research, but its effects on ES cell miRNA expression remain unknown. In this study, we conducted small RNA high-throughput sequencing to investigate AICAR influence on J1 mouse ES cells by comparing the miRNA expression patterns of the AICAR-treated cells and those without treatment. The result showed that AICAR can significantly modulate the expression of multiple miRNAs, including those have crucial functions in ES cell development. Some differentially expressed miRNAs were selected and confirmed by real-time PCR. For the differently expressed miRNAs identified, further study was conducted regarding the pluripotency and differentiation associated miRNAs with their targets. Moreover, miR-134 was significantly down-regulated after AICAR treatment, and this was suggested to be directly associated with the up-regulated pluripotency markers, Nanog and Sox2. Lastly, Myc was significantly down-regulated after AICAR treatment; therefore, we predicted miRNAs that may target Myc and identified that AICAR induced up-regulation of miR-34a, 34b, and 34c can repress Myc expression in J1 mouse ES cells. Taken together, our study provide a new mechanism for

  9. SAMHD1 restricts HIV-1 replication and regulates interferon production in mouse myeloid cells.

    Directory of Open Access Journals (Sweden)

    Ruonan Zhang

    Full Text Available SAMHD1 restricts the replication of HIV-1 and other retroviruses in human myeloid and resting CD4(+ T cells and that is counteracted in SIV and HIV-2 by the Vpx accessory protein. The protein is a phosphohydrolase that lowers the concentration of deoxynucleoside triphosphates (dNTP, blocking reverse transcription of the viral RNA genome. Polymorphisms in the gene encoding SAMHD1 are associated with Aicardi-Goutières Syndrome, a neurological disorder characterized by increased type-I interferon production. SAMHD1 is conserved in mammals but its role in restricting virus replication and controlling interferon production in non-primate species is not well understood. We show that SAMHD1 is catalytically active and expressed at high levels in mouse spleen, lymph nodes, thymus and lung. siRNA knock-down of SAMHD1 in bone marrow-derived macrophages increased their susceptibility to HIV-1 infection. shRNA knock-down of SAMHD1 in the murine monocytic cell-line RAW264.7 increased its susceptibility to HIV-1 and murine leukemia virus and increased the levels of the dNTP pool. In addition, SAMHD1 knock-down in RAW264.7 cells induced the production of type-I interferon and several interferon-stimulated genes, modeling the situation in Aicardi-Goutières Syndrome. Our findings suggest that the role of SAMHD1 in restricting viruses is conserved in the mouse. The RAW264.7 cell-line serves as a useful tool to study the antiviral and innate immune response functions of SAMHD1.

  10. Evaluation of Depigmenting Activity by 8-Hydroxydaidzein in Mouse B16 Melanoma Cells and Human Volunteers

    Directory of Open Access Journals (Sweden)

    Ching-Gong Lin

    2009-09-01

    Full Text Available In our previous study, 8-hydroxydaidzein (8-OHDe was demonstrated to be a potent and unique suicide substrate of mushroom tyrosinase. In this study, the compound was evaluated for in vitro cellular tyrosinase and melanogenesis inhibitory activities in mouse B16 melanoma cells and for in vivo skin-whitening activity in human volunteers. Tyrosinase activity and melanogenesis in the cell culture incubated with 10 µM of 8-OHDe were decreased to 20.1% and 51.8% of control, respectively, while no obvious cytotoxicity was observed in this concentration. In contrast, a standard tyrosinase inhibitor, kojic acid, showed 69.9% and 71.3% of control in cellular tyrosinase and melanogenesis activity, respectively, at a concentration as high as 100 µM. Hence, 8-OHDe exhibited more than an inhibitory effects on melanin production in B16 cells 10-fold stronger than kojic acid. In addition, when a cream containing 4% 8-OHDe was applied to human skin in an in vivo study, significant increases in the dL*-values were observed after three weeks. Moreover, the increase in the dL*-values after 8-week treatment with 4% 8-OHDe (from -0.57 to 1.94 is stronger than those of 2% 8-OHDe treatment (from 0.26 to 0.94 and 2% ascorbic acid-2-glucoside treatment (from 0.07 to 1.54. From the results of the study, it was concluded that 8-OHDe, the potent suicide substrate of mushroom tyrosinase, has depigmenting activities in both mouse melanoma cells and in human volunteers. Thus, the compound has significant potential for use in cosmetics as a skin-whitening ingredient.

  11. Photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses

    CSIR Research Space (South Africa)

    Thobakgale, Lebogang

    2017-01-01

    Full Text Available This presentation is about the photo-transfection of mouse embryonic stem cells with plasmid DNA using femtosecond laser pulses. It outlines the background on embryonic stem cells (ES) and phototransfection....

  12. Effects of Treatment with Bone Morphogenetic Protein 4 and Co-culture on Expression of Piwil2 Gene in Mouse Differentiated Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Mehdi Forouzandeh-Moghadam

    2009-01-01

    Full Text Available Background: Specific growth factors and feeder layers seem to have important roles in in vitroembryonic stem cells (ESCs differentiation. In this study,the effects of bone morphogenetic protein4 (BMP4 and mouse embryonic fibroblasts (MEFs co-culture system on germ cell differentiationfrom mouse ESCs were studied.Materials and Methods: Cell suspension was prepared from one-day-old embryoid body (EBand cultured for four days in DMEM medium containing 20% fetal bovine serum (FBS in thefollowing groups: simple culture (SC, simple culture with BMP4 (SCB, co-culture (CO-C andco-culture with BMP4 (CO-CB. Expression of piwi-like homolog 2 (Piwil2, the germ cell-specificgene, was evaluated in the different study groups by using quantitative real time polymerase chainreaction (RT-PCR. Testis was used as a positive control.Results: The maximum and minimum Piwil2 expression was observed in SC and SCB groups,respectively. A significant difference was observed in Piwil2 expression between SCB and otherstudy groups (p<0.05.Conclusion: The findings of this study showed that neither the addition of BMP4 in culture mediumnor the use of MEFs as a feeder layer have a positive effect on late germ cell induction from mouseESCs.

  13. Augmentation of Antitumor Immunity by Human and Mouse CAR T Cells Secreting IL-18

    Directory of Open Access Journals (Sweden)

    Biliang Hu

    2017-09-01

    Full Text Available The effects of transgenically encoded human and mouse IL-18 on T cell proliferation and its application in boosting chimeric antigen receptor (CAR T cells are presented. Robust enhancement of proliferation of IL-18-secreting human T cells occurred in a xenograft model, and this was dependent on TCR and IL-18R signaling. IL-18 augmented IFN-γ secretion and proliferation of T cells activated by the endogenous TCR. TCR-deficient, human IL-18-expressing CD19 CAR T cells exhibited enhanced proliferation and antitumor activity in the xenograft model. Antigen-propelled activation of cytokine helper ensemble (APACHE CAR T cells displayed inducible expression of IL-18 and enhanced antitumor immunity. In an intact mouse tumor model, CD19-IL-18 CAR T cells induced deeper B cell aplasia, significantly enhanced CAR T cell proliferation, and effectively augmented antitumor effects in mice with B16F10 melanoma. These findings point to a strategy to develop universal CAR T cells for patients with solid tumors.

  14. Culture conditions have an impact on the maturation of traceable, transplantable mouse embryonic stem cell-derived otic progenitor cells.

    Science.gov (United States)

    Abboud, Nesrine; Fontbonne, Arnaud; Watabe, Isabelle; Tonetto, Alain; Brezun, Jean Michel; Feron, François; Zine, Azel

    2017-09-01

    The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1-green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two-step short-term differentiation method involving an induction period of 5 days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF-β inhibitors and insulin-like growth factor IGF-1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5 days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin-injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons

  15. The effect of interferon-β on mouse neural progenitor cell survival and differentiation

    International Nuclear Information System (INIS)

    Hirsch, Marek; Knight, Julia; Tobita, Mari; Soltys, John; Panitch, Hillel; Mao-Draayer, Yang

    2009-01-01

    Interferon-β (IFN-β) is a mainstay therapy for relapse-remitting multiple sclerosis (MS). However, the direct effects of IFN-β on the central nervous system (CNS) are not well understood. To determine whether IFN-β has direct neuroprotective effects on CNS cells, we treated adult mouse neural progenitor cells (NPCs) in vitro with IFN-β and examined the effects on proliferation, apoptosis, and differentiation. We found that mouse NPCs express high levels of IFNα/β receptor (IFNAR). In response to IFN-β treatment, no effect was observed on differentiation or proliferation. However, IFN-β treated mouse NPCs demonstrated decreased apoptosis upon growth factor withdrawal. Pathway-specific polymerase chain reaction (PCR) arrays demonstrated that IFN-β treatment upregulated the STAT 1 and 2 signaling pathway, as well as GFRA2, NOD1, Caspases 1 and 12, and TNFSF10. These results suggest that IFN-β can directly affect NPC survival, possibly playing a neuroprotective role in the CNS by modulating neurotrophic factors.

  16. The effect of interferon-{beta} on mouse neural progenitor cell survival and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Hirsch, Marek [Neurology Department, University of Vermont College of Medicine, Burlington, VT (United States); Knight, Julia [Neuroscience Department, University of Vermont College of Medicine, Burlington, VT (United States); Tobita, Mari; Soltys, John; Panitch, Hillel [Neurology Department, University of Vermont College of Medicine, Burlington, VT (United States); Mao-Draayer, Yang, E-mail: yang.mao-draayer@vtmednet.org [Neurology Department, University of Vermont College of Medicine, Burlington, VT (United States)

    2009-10-16

    Interferon-{beta} (IFN-{beta}) is a mainstay therapy for relapse-remitting multiple sclerosis (MS). However, the direct effects of IFN-{beta} on the central nervous system (CNS) are not well understood. To determine whether IFN-{beta} has direct neuroprotective effects on CNS cells, we treated adult mouse neural progenitor cells (NPCs) in vitro with IFN-{beta} and examined the effects on proliferation, apoptosis, and differentiation. We found that mouse NPCs express high levels of IFN{alpha}/{beta} receptor (IFNAR). In response to IFN-{beta} treatment, no effect was observed on differentiation or proliferation. However, IFN-{beta} treated mouse NPCs demonstrated decreased apoptosis upon growth factor withdrawal. Pathway-specific polymerase chain reaction (PCR) arrays demonstrated that IFN-{beta} treatment upregulated the STAT 1 and 2 signaling pathway, as well as GFRA2, NOD1, Caspases 1 and 12, and TNFSF10. These results suggest that IFN-{beta} can directly affect NPC survival, possibly playing a neuroprotective role in the CNS by modulating neurotrophic factors.

  17. Age-dependent transition from cell-level to population-level control in murine intestinal homeostasis revealed by coalescence analysis.

    Directory of Open Access Journals (Sweden)

    Zheng Hu

    Full Text Available In multi-cellular organisms, tissue homeostasis is maintained by an exquisite balance between stem cell proliferation and differentiation. This equilibrium can be achieved either at the single cell level (a.k.a. cell asymmetry, where stem cells follow strict asymmetric divisions, or the population level (a.k.a. population asymmetry, where gains and losses in individual stem cell lineages are randomly distributed, but the net effect is homeostasis. In the mature mouse intestinal crypt, previous evidence has revealed a pattern of population asymmetry through predominantly symmetric divisions of stem cells. In this work, using population genetic theory together with previously published crypt single-cell data obtained at different mouse life stages, we reveal a strikingly dynamic pattern of stem cell homeostatic control. We find that single-cell asymmetric divisions are gradually replaced by stochastic population-level asymmetry as the mouse matures to adulthood. This lifelong process has important developmental and evolutionary implications in understanding how adult tissues maintain their homeostasis integrating the trade-off between intrinsic and extrinsic regulations.

  18. The phenotype of FancB-mutant mouse embryonic stem cells

    OpenAIRE

    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu, Lingchuan; Hasty, Paul

    2011-01-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslink...

  19. In vitro effects of triiodothyronine on gene expression in mouse trophoblast cells.

    Science.gov (United States)

    Silva, J F; Ocarino, N M; Serakides, R

    2015-01-01

    The objective of the present study was to evaluate the effects of different doses of T3 (10(-4) M, 10(-7) M, 10(-9) M) on the in vitro gene expression of Tpbp, Prl3b1, VEGF, PGF, PL-1, and INFy in mouse trophoblast cells by real-time RT-PCR. Doses of 10(-7) and 10(-9) M T3 increased the mRNA levels of Tpbp, Pl3b1, VEGF, PGF, INFy and PL-1. In contrast, the dose of 10(-4) M reduced the gene expression of PL-1 and VEGF. T3 affected the gene expression of differentiation, hormonal, immune and angiogenic factors in mouse trophoblast cells. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. A Functional Analysis on the Interspecies Interaction between Mouse LFA-1 and Human Intercellular Adhesion Molecule-1 at the Cell Level

    Directory of Open Access Journals (Sweden)

    David Núñez

    2017-12-01

    Full Text Available The interaction between intercellular adhesion molecules (ICAM and the integrin leukocyte function-associated antigen-1 (LFA-1 is crucial for the regulation of several physiological and pathophysiological processes like cell-mediated elimination of tumor or virus infected cells, cancer metastasis, or inflammatory and autoimmune processes. Using purified proteins it was reported a species restriction for the interaction of ICAM-1 and LFA-1, being mouse ICAM-1 able to interact with human LFA-1 but not human ICAM-1 with mouse LFA-1. However, in vivo results employing tumor cells transfected with human ICAM-1 suggest that functionally mouse LFA-1 can recognize human ICAM-1. In order to clarify the interspecies cross-reactivity of the ICAM-1/LFA-1 interaction, we have performed functional studies analyzing the ability of human soluble ICAM-1 and human/mouse LFA-1 derived peptides to inhibit cell aggregation and adhesion as well as cell-mediated cytotoxicity in both mouse and human systems. In parallel, the affinity of the interaction between mouse LFA-1-derived peptides and human ICAM-1 was determined by calorimetry assays. According to the results obtained, it seems that human ICAM-1 is able to interact with mouse LFA-1 on intact cells, which should be taking into account when using humanized mice and xenograft models for the study of immune-related processes.

  1. A Functional Analysis on the Interspecies Interaction between Mouse LFA-1 and Human Intercellular Adhesion Molecule-1 at the Cell Level.

    Science.gov (United States)

    Núñez, David; Comas, Laura; Lanuza, Pilar M; Sánchez-Martinez, Diego; Pérez-Hernández, Marta; Catalán, Elena; Domingo, María Pilar; Velázquez-Campoy, Adrián; Pardo, Julián; Gálvez, Eva M

    2017-01-01

    The interaction between intercellular adhesion molecules (ICAM) and the integrin leukocyte function-associated antigen-1 (LFA-1) is crucial for the regulation of several physiological and pathophysiological processes like cell-mediated elimination of tumor or virus infected cells, cancer metastasis, or inflammatory and autoimmune processes. Using purified proteins it was reported a species restriction for the interaction of ICAM-1 and LFA-1, being mouse ICAM-1 able to interact with human LFA-1 but not human ICAM-1 with mouse LFA-1. However, in vivo results employing tumor cells transfected with human ICAM-1 suggest that functionally mouse LFA-1 can recognize human ICAM-1. In order to clarify the interspecies cross-reactivity of the ICAM-1/LFA-1 interaction, we have performed functional studies analyzing the ability of human soluble ICAM-1 and human/mouse LFA-1 derived peptides to inhibit cell aggregation and adhesion as well as cell-mediated cytotoxicity in both mouse and human systems. In parallel, the affinity of the interaction between mouse LFA-1-derived peptides and human ICAM-1 was determined by calorimetry assays. According to the results obtained, it seems that human ICAM-1 is able to interact with mouse LFA-1 on intact cells, which should be taking into account when using humanized mice and xenograft models for the study of immune-related processes.

  2. NaCl responsive taste cells in the mouse fungiform taste buds.

    Science.gov (United States)

    Yoshida, R; Horio, N; Murata, Y; Yasumatsu, K; Shigemura, N; Ninomiya, Y

    2009-03-17

    Previous studies have demonstrated that rodents' chorda tympani (CT) nerve fibers responding to NaCl can be classified according to their sensitivities to the epithelial sodium channel (ENaC) blocker amiloride into two groups: amiloride-sensitive (AS) and -insensitive (AI). The AS fibers were shown to respond specifically to NaCl, whereas AI fibers broadly respond to various electrolytes, including NaCl. These data suggest that salt taste transduction in taste cells may be composed of at least two different systems; AS and AI ones. To further address this issue, we investigated the responses to NaCl, KCl and HCl and the amiloride sensitivity of mouse fungiform papilla taste bud cells which are innervated by the CT nerve. Comparable with the CT data, the results indicated that 56 NaCl-responsive cells tested were classified into two groups; 25 cells ( approximately 44%) narrowly responded to NaCl and their NaCl response were inhibited by amiloride (AS cells), whereas the remaining 31 cells ( approximately 56%) responded not only to NaCl, but to KCl and/or HCl and showed no amiloride inhibition of NaCl responses (AI cells). Amiloride applied to the basolateral side of taste cells had no effect on NaCl responses in the AS and AI cells. Single cell reverse transcription-polymerase chain reaction (RT-PCR) experiments indicated that ENaC subunit mRNA was expressed in a subset of AS cells. These findings suggest that the mouse fungiform taste bud is composed of AS and AI cells that can transmit taste information differently to their corresponding types of CT fibers, and apical ENaCs may be involved in the NaCl responses of AS cells.

  3. Insulin receptor internalization defect in an insulin-resistant mouse melanoma cell line

    International Nuclear Information System (INIS)

    Androlewicz, M.J.; Straus, D.S.; Brandenburg, D.F.

    1989-01-01

    Previous studies from this laboratory demonstrated that the PG19 mouse melanoma cell line does not exhibit a biological response to insulin, whereas melanoma x mouse embryo fibroblast hybrids do respond to insulin. To investigate the molecular basis of the insulin resistance of the PG19 melanoma cells, insulin receptors from the insulin-resistant melanoma cells and insulin-sensitive fibroblast x melanoma hybrid cells were analyzed by the technique of photoaffinity labeling using the photoprobe 125 I-NAPA-DP-insulin. Photolabeled insulin receptors from the two cell types have identical molecular weights as determined by SDS gel electrophoresis under reducing and nonreducing conditions, indicating that the receptors on the two cell lines are structurally similar. Insulin receptor internalization studies revealed that the hybrid cells internalize receptors to a high degree at 37 degree C, whereas the melanoma cells internalize receptors to a very low degree or not at all. The correlation between ability to internalize insulin receptors and sensitivity to insulin action in this system suggests that uptake of the insulin-receptor complex may be required for insulin action in these cells. Insulin receptors from the two cell lines autophosphorylate in a similar insulin-dependent manner both in vitro and in intact cells, indicating that insulin receptors on the melanoma and hybrid cells have functional tyrosine protein kinase activity. Therefore, the block in insulin action in the PG19 melanoma cells appears to reside at a step beyond insulin-stimulated receptor autophosphorylation

  4. [Activation of nucleolar organizers during in vitro cultivation of mouse R1 embryonic stem cells].

    Science.gov (United States)

    Kunafina, E R; Chaplina, M V; Filiasova, E I; Gibanova, N V; Khodarovich, Iu M; Larionov, O A; Zatsepina, O V

    2005-01-01

    We studies the activities of ribosomal genes (nucleolus forming regions of chromosomes) at successive stages of cultivation of the mouse R1 embryonic stem cells. The total number and number of active nucleolar organizers were estimated by means of in situ hybridization with mouse rDNA probes and argentophilic staining of nucleolus forming chromosomes regions from the 16th until the 32nd passages. The data we obtained suggest that the total number of nucleolar organizers per metaphase plate was constant (as a rule, eight), while the mean number of active nucleolar organizers progressively increased from the early (16th) to the late (32nd) passages: 5.2 +/- 0.4 versus 7.4 +/- 0.9 argentophilic organizers per cell. Cell heterogeneity by the number of active nucleolar organizers also increased during the late passages. Taken together, these data suggest activation of DNA transcription and synthesis of ribosomes during cultivation of mouse R1 embryonic stem cells. Based on the experimental and published data, it has been proposed that activation of ribosomal genes correlates in time with a decreased capacity of embryonic stem cells for pluripotent differentiation.

  5. CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

    International Nuclear Information System (INIS)

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

    2009-01-01

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1 C YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+) s evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

  6. Growth retardation of paramecium and mouse cells by shielding them from background radiation

    International Nuclear Information System (INIS)

    Kawanishi, Masanobu; Okuyama, Katsuyuki; Shiraishi, Kazunori; Matsuda, Yatsuka; Taniguchi, Ryoichi; Shiomi, Nobuyuki; Yonezawa, Morio; Yagi, Takashi

    2012-01-01

    In the 1970s and 1980s, Planel et al. reported that the growth of paramecia was decreased by shielding them from background radiation. In the 1990s, Takizawa et al. found that mouse cells displayed a decreased growth rate under shielded conditions. The purpose of the present study was to confirm that growth is impaired in organisms that have been shielded from background radiation. Radioprotection was produced with a shielding chamber surrounded by a 15 cm thick iron wall and a 10 cm thick paraffin wall that reduced the γ ray and neutron levels in the chamber to 2% and 25% of the background levels, respectively. Although the growth of Paramecium tetraurelia was not impaired by short-term radioprotection (around 10 days), which disagreed with the findings of Planel et al., decreased growth was observed after long-term (40-50 days) radiation shielding. When mouse lymphoma L5178Y cells were incubated inside or outside of the shielding chamber for 7 days, the number of cells present on the 6th and 7th days under the shielding conditions was significantly lower than that present under the non-shielding conditions. These inhibitory effects on cell growth were abrogated by the addition of a 137 Cs γ-ray source disk to the chamber. Furthermore, no growth retardation was observed in XRCC4-deficient mouse M10 cells, which display impaired DNA double strand break repair. (author)

  7. Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines

    DEFF Research Database (Denmark)

    Svarcova, Olga; Dinnyes, A.; Polgar, Z.

    2009-01-01

    displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both......Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer...... ofmouse embryonic fibroblast (MEF) and mouse HM1 emryonic stem cells (HM1), were processed for autoradiography following 3H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF...

  8. Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells.

    Science.gov (United States)

    Nakano, Yu; Iwanaga, Shinya; Mizumoto, Hiroshi; Kajiwara, Toshihisa

    2018-03-03

    Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for mouse embryonic stem (ES) cells by applying a hollow fiber (HF)/organoid culture method. Cylindrical organoids, which had the potential for further spontaneous differentiation, were established inside of hollow fibers. Using this method, we improved the proliferation rate of mouse ES cells to produce an increased HSC population and achieved around a 40-fold higher production volume of HSCs in HF culture than in conventional EB culture. Therefore, the HF/organoid culture method may be a new mass culture method to acquire pluripotent stem cell-derived HSCs.

  9. Genomic targets of Brachyury (T in differentiating mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Amanda L Evans

    Full Text Available The T-box transcription factor Brachyury (T is essential for formation of the posterior mesoderm and the notochord in vertebrate embryos. Work in the frog and the zebrafish has identified some direct genomic targets of Brachyury, but little is known about Brachyury targets in the mouse.Here we use chromatin immunoprecipitation and mouse promoter microarrays to identify targets of Brachyury in embryoid bodies formed from differentiating mouse ES cells. The targets we identify are enriched for sequence-specific DNA binding proteins and include components of signal transduction pathways that direct cell fate in the primitive streak and tailbud of the early embryo. Expression of some of these targets, such as Axin2, Fgf8 and Wnt3a, is down regulated in Brachyury mutant embryos and we demonstrate that they are also Brachyury targets in the human. Surprisingly, we do not observe enrichment of the canonical T-domain DNA binding sequence 5'-TCACACCT-3' in the vicinity of most Brachyury target genes. Rather, we have identified an (AC(n repeat sequence, which is conserved in the rat but not in human, zebrafish or Xenopus. We do not understand the significance of this sequence, but speculate that it enhances transcription factor binding in the regulatory regions of Brachyury target genes in rodents.Our work identifies the genomic targets of a key regulator of mesoderm formation in the early mouse embryo, thereby providing insights into the Brachyury-driven genetic regulatory network and allowing us to compare the function of Brachyury in different species.

  10. Radiation damage to mouse testis cells from [/sup 99m/Tc] pertechnetate

    International Nuclear Information System (INIS)

    Mian, T.A.; Suzuki, N.; Glenn, H.J.; Haynie, T.P.; Meistrich, M.L.

    1977-01-01

    The radiation dose and the biologic damage to mouse testis from intravenously administered [/sup 99m/Tc] pertechnetate were studied. The dose was measured for penetrating radiations from /sup 99m/Tc, using calibrated thermoluminescent dosimeters and calculations from the uptake of the nuclide in the testis, and was found to be 4.9 rads per mCi of 99 Tc. The biologic damage was measured by the decrease in the number of sperm heads in the testis, counted both by hemacytometer and by Coulter counter. In preliminary experiments using external gamma radiation from 137 Cs, the number of sperm heads reached a minimum 29 days after irradiation. Twenty-nine days after injection of 5.8 mCi of /sup 99m/Tc, which gives 28 rads to the testis, the number of sperm heads decreased to 70% of control. The biologic effect corresponds to that seen after 40 rads of gamma radiation from 137 Cs. The damage to mouse testis cells from internally administered /sup 99m/Tc as measured in an in vivo system appears to be at least as significant as that from external gamma irradiation, if not more so

  11. Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Yong, E-mail: yongzhao@uic.edu [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Guo, Chengshan; Hwang, David; Lin, Brian; Dingeldein, Michael; Mihailescu, Dan; Sam, Susan; Sidhwani, Seema [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Zhang, Yongkang [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Jain, Sumit [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Skidgel, Randal A. [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Prabhakar, Bellur S. [Department of Immunology and Microbiology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Mazzone, Theodore [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Holterman, Mark J. [Department of Surgery, University of Illinois at Chicago, Chicago, IL 60612 (United States)

    2010-09-03

    Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.

  12. Assessment of a 42 metal salts chemical library in mouse embryonic stem cells

    Science.gov (United States)

    The developmental effects of xenobiotics on differentiation can be profiled using mouse embryonic stem cells (mESCs). The adherent cell differentiation and cytotoxicity (ACDC) technique was used to evaluate a library of 42 metal and metaloid salts. Jl mESCs were allowed to prolif...

  13. TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain.

    Science.gov (United States)

    Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

    2014-05-15

    Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology.

  14. Toxicity of silver nanoparticles in mouse embryonic stem cells and chemical based reprogramming of somatic cells to sphere cells

    Science.gov (United States)

    Rajanahalli Krishnamurthy, Pavan

    Abstract 1: Silver nanoparticles (Ag Np's) have an interesting surface chemistry and unique plasmonic properties. They are used in a wide variety of applications ranging from consumer products like socks, medical dressing, computer chips and it is also shown to have antimicrobial, anti bacterial activity and wound healing. Ag Np toxicity studies have been limited to date which needs to be critically addressed due to its wide applications. Mouse embryonic stem (MES) cells represent a unique cell population with the ability to undergo both self renewal and differentiation. They exhibit very stringent and tightly regulated mechanisms to circumvent DNA damage and stress response. We used 10 nm coated (polysaccharide) and uncoated Ag Np's to test its toxic effects on MES cells. MES cells and embryoid bodies (EB's) were treated with two concentrations of Ag Np's: 5 microg/ml and 50 ug/ml and exposed for 24, 48 and 72 hours. Increased cell death, ROS production and loss of mitochondrial membrane potential and alkaline phosphatase (AP) occur in a time and a concentration dependant manner. Due to increased cell death, there is a progressive increase in Annexin V (apoptosis) and Propidium Iodide (PI) staining (necrosis). Oct4 and Nanog undergo ubiquitination and dephosphorylation post-translational modifications in MES cells thereby altering gene expression of pluripotency factors and differentiation of EB's into all the three embryonic germ layers with specific growth factors were also inhibited after Ag Np exposure. Flow cytometry analysis revealed Ag Np's treated cells had altered cell cycle phases correlating with altered self renewal capacity. Our results suggest that Ag Np's effect MES cell self renewal, pluripotency and differentiation and serves as a perfect model system for studying toxicity induced by engineered Ag Np's. Abstract 2: The reprogramming of fibroblasts to pluripotent stem cells and the direct conversion of fibroblasts to functional neurons has been

  15. Annexin A7 suppresses lymph node metastasis of hepatocarcinoma cells in a mouse model

    International Nuclear Information System (INIS)

    Jin, Yanling; Wang, Shaoqing; Chen, Wenjing; Zhang, Jun; Wang, Bo; Guan, Hongwei; Tang, Jianwu

    2013-01-01

    Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death in China. This study investigated the effects of Annexin A7 (ANXA7) on the inhibition of HCC lymph node metastasis in a mouse model. The stable knockup and knockdown of Annexin A7-expressing HCC cells using Annexin A7 cDNA and shRNA vectors, respectively, were injected into a mouse footpad to establish primary and metastatic tumors in mice. On the 14th, 21st, and 28th days after HCC cells inoculation, the mice were sacrificed for inspection of primary and secondary tumors and immunohistochemistry of Annexin A7 expression. The lymph node metastasis rate of the F ANXA7-control group was 77%, and the lymph node metastasis rate of the F ANXA7-down group was 100% (p < 0.05). In contrast, the lymph node metastasis rate of the P ANXA7-up group was 0% and that of the P ANXA7-control group was 36% (p < 0.05). Furthermore, immunohistochemistry experiments revealed that the subcellular localization of Annexin A7 protein in both primary and lymph node-metastasized tumors was mainly in the cytosol. In addition, the expression of the 47 kDa and 51 kDa isoforms of Annexin A7 protein changed during tumor progression. This study indicated that Annexin A7 expression was able to inhibit HCC lymph node metastasis, whereas knockdown of Annexin A7 expression significantly induced HCC metastasis to local lymph nodes

  16. Chromosome alterations in the X-ray-induced transformants of cultured mouse cell line

    International Nuclear Information System (INIS)

    Kodama, Seiji; Komatsu, Kenshi; Okumura, Yutaka; Sasaki, M.S.

    1989-01-01

    Mouse m5S cells were subjected to soft X-ray irradiation. Twenty-four transformants were separated as indicators of focus formation. Two clones, cl.4103 and cl.6310, were chosen for the analysis of chromosome alterations in transformants. A parent strain, m5S/1M, served as the control. Anchorage independence (AG) was not detected in the control strain, irrespective of culture conditions and population doubling number (PDN). In the case of transformants, the frequency of AG was increased with increasing PDN for cl.4103, and was constant for cl.6310, irrespective of PDN. Karyotype of m5S/1M was 42, X, -Y, +der (6) t(6;13), t(8;8), +8, +15. In addition, -13, der(10) and -der(6)t(6;13), der(5), +mar occurred as karyotype alterations for cl.4103 and cl. 6310, respectively. The present experiment indicated that chromosome alterations secondary to primary alterations occur in a high frequency in the transforming process of X-ray irradiated cells, and that the secondary chromosome alterations result in selective proliferation of transformed clones. (Namekawa, K)

  17. Differentiation/Purification Protocol for Retinal Pigment Epithelium from Mouse Induced Pluripotent Stem Cells as a Research Tool.

    Directory of Open Access Journals (Sweden)

    Yuko Iwasaki

    Full Text Available To establish a novel protocol for differentiation of retinal pigment epithelium (RPE with high purity from mouse induced pluripotent stem cells (iPSC.Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated.We obtained iPS-RPE at high purity (approximately 98%. The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation.We successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE.

  18. Giant renin secretory granules in beige mouse renal afferent arterioles

    DEFF Research Database (Denmark)

    Jensen, B L; Rasch, Ruth; Nyengaard, Jens Randel

    1997-01-01

    The mutant beige mouse (C57BL/6 bg) has a disease characterised by abnormally enlarged cytoplasmic granules in a variety of cells. With the purpose of establishing a suitable cellular model for studying renin secretion, the present study was undertaken to compare renin granule morphology in beige...... (average granular volume 0.681 microm3), whereas 1-2 large granules were present per cell in beige mice. The volume of afferent arteriole that contained secretory granules was lower in the beige mice. We conclude that the beige mouse synthesizes, stores and releases active renin. Renin secretory granules...... in beige mice are grossly enlarged with 1-2 granules per juxtaglomerular cell. Compared with control mice, a similar amount of total renin granule volume per afferent arteriole is contained in a smaller part of beige mouse afferent arteriole. Granular cells from beige mice could therefore be a valuable...

  19. Dual Innervation of Neonatal Merkel Cells in Mouse Touch Domes

    Science.gov (United States)

    Luo, Wenqin

    2014-01-01

    Merkel cell-neurite complexes are specialized mechanosensory end organs that mediate discriminative touch sensation. It is well established that type I slowly adapting (SAI) mechanoreceptors, which express neural filament heavy chain (NFH), innervate Merkel cells. It was previously shown that neurotrophic factor NT3 and its receptor TrkC play crucial roles in controlling touch dome Merkel cell innervation of NFH+ fibers. In addition, nerve fibers expressing another neurotrophic tyrosine receptor kinase (NTRK), Ret, innervate touch dome Merkel cells as well. However, the relationship between afferents responsive to NT3/TrkC signaling and those expressing Ret is unclear. It is also controversial if these Ret+ fibers belong to the early or late Ret+ DRG neurons, which are defined based on the co-expression and developmental dependence of TrkA. To address these questions, we genetically traced Ret+ and TrkC+ fibers and analyzed their developmental dependence on TrkA. We found that Merkel cells in neonatal mouse touch domes receive innervation of two types of fibers: one group is Ret+, while the other subset expresses TrkC and NFH. In addition, Ret+ fibers depend on TrkA for their survival and normal innervation whereas NFH+ Merkel cell innervating fibers are almost unaltered in TrkA mutant mice, supporting that Ret+ and NFH+/TrkC+ afferents are two distinct groups. Ret signaling, on the other hand, plays a minor role for the innervation of neonatal touch domes. In contrast, Merkel cells in the glabrous skin are mainly contacted by NFH+/TrkC+ afferents. Taken together, our results suggest that neonatal Merkel cells around hair follicles receive dual innervation while Merkel cells in the glabrous skin are mainly innervated by only SAI mechanoreceptors. In addition, our results suggest that neonatal Ret+ Merkel cell innervating fibers most likely belong to the late but not early Ret+ DRG neurons. PMID:24637732

  20. Establishment and characterization of a hypocatalasemic mouse cell strain

    International Nuclear Information System (INIS)

    Utsumi, Hiroshi; Tano, Keizo; Hashimoto, Mitsumasa W.; Kodama, Seiji; Watanabe, Hiromitsu

    1998-01-01

    Contact-inhibited catalase-deficient fibroblast cell strain has been established from the homozygous hypocatalasemic C3H/Cs b mutant mouse. This cell strain has low level of catalase enzyme activity and has normal level of enzyme activities of both glutathione peroxidase and superoxide dismutase. Catalase-deficient C3H/Cs b mutant cell strain is markedly more sensitive to the toxicity of hydrogen peroxide compared to wild-type C3H/Cs a cell strain. In addition, mutant cell strain is sensitive to X-rays and near-UV compared to wild-type cell strain, but shows the same sensitivities to topoisomerase II inhibitors, adriamycin and 4'-(9-acridinylamino) methanesulfon-m-anisidide (m-AMSA), and the DNA cross-linking agents, cis-diamminedichloroplatinum (II) (cis-Pt) and trans-diamminedichloroplatinum (II) (trans-Pt). These cell strains will be of use in the study of the roles which catalase plays in the intracellular prevention of DNA damage induced by oxidative stress. (author)

  1. Basement membrane components secreted by mouse yolk sac carcinoma cell lines

    DEFF Research Database (Denmark)

    Damjanov, A; Wewer, U M; Tuma, B

    1990-01-01

    Three new cell lines (NE, ME, LRD) were cloned from mouse-embryo-derived teratocarcinomas and characterized on the basis of developmental, ultrastructural, and cytochemical criteria as nullipotent embryonal carcinoma (EC), pure parietal yolk sac (PYS) carcinoma and mixed parieto-visceral yolk sac...

  2. Adoptively transferred human lung tumor specific cytotoxic T cells can control autologous tumor growth and shape tumor phenotype in a SCID mouse xenograft model

    Directory of Open Access Journals (Sweden)

    Ferrone Soldano

    2007-06-01

    Full Text Available Abstract Background The anti-tumor efficacy of human immune effector cells, such as cytolytic T lymphocytes (CTLs, has been difficult to study in lung cancer patients in the clinical setting. Improved experimental models for the study of lung tumor-immune cell interaction as well as for evaluating the efficacy of adoptive transfer of immune effector cells are needed. Methods To address questions related to the in vivo interaction of human lung tumor cells and immune effector cells, we obtained an HLA class I + lung tumor cell line from a fresh surgical specimen, and using the infiltrating immune cells, isolated and characterized tumor antigen-specific, CD8+ CTLs. We then established a SCID mouse-human tumor xenograft model with the tumor cell line and used it to study the function of the autologous CTLs provided via adoptive transfer. Results The tumor antigen specific CTLs isolated from the tumor were found to have an activated memory phenotype and able to kill tumor cells in an antigen specific manner in vitro. Additionally, the tumor antigen-specific CTLs were fully capable of homing to and killing autologous tumors in vivo, and expressing IFN-γ, each in an antigen-dependent manner. A single injection of these CTLs was able to provide significant but temporary control of the growth of autologous tumors in vivo without the need for IL-2. The timing of injection of CTLs played an essential role in the outcome of tumor growth control. Moreover, immunohistochemical analysis of surviving tumor cells following CTL treatment indicated that the surviving tumor cells expressed reduced MHC class I antigens on their surface. Conclusion These studies confirm and extend previous studies and provide additional information regarding the characteristics of CTLs which can be found within a patient's tumor. Moreover, the in vivo model described here provides a unique window for observing events that may also occur in patients undergoing adoptive cellular

  3. Regulation of mouse retroelement MuERV-L/MERVL expression by REX1 and epigenetic control of stem cell potency.

    Directory of Open Access Journals (Sweden)

    Jon eSchoorlemmer

    2014-02-01

    Full Text Available About half of mammalian genomes is occupied by DNA sequences that originatefrom transposable elements. Retrotransposons can modulate gene expression indifferent ways and, particularly retrotransposon-derived LTRs, profoundly shapeexpression of both surrounding and distant genomic loci. This is especially important inpreimplantation development, during which extensive reprogramming of the genometakes place and cells pass through totipotent and pluripotent states. At this stage, themain mechanisms responsible for retrotransposon silencing i.e. DNA methylation, isinoperative. A particular retrotransposon called muERV-L/MERVL is expressed duringpreimplantation stages and contributes to the plasticity of mouse embryonic stem cells.This review will focus on the role of MERVL-derived sequences as controllingelements of gene expression specific for preimplantation development, two-cell stagespecific gene expression and stem cell pluripotency, the epigenetic mechanisms thatcontrol their expression, and the contributions of the pluripotency marker REX1 and therelated YY1 family of transcription factors to this regulation process.

  4. Experimental photoallergic contact dermatitis: a mouse model

    International Nuclear Information System (INIS)

    Maguire, H.C. Jr.; Kaidbey, K.

    1982-01-01

    We have induced photoallergic contact dermatitis in mice to 3,3',4',5 tetrachlorosalicylanilide (TCSA), chlorpromazine and 6-methylcoumarin. These compounds are known to produce photoallergic contact dermatitis in humans. The photoallergic contact dermatitis reaction in the mouse is immunologically specific viz. mice photosensitized to TCSA react, by photochallenge, to that compound and not to chlorpromazine, and conversely. The reaction requires UVA at both sensitization and challenge. It appears to be T-cell mediated in that it can be passively transferred to syngeneic mice by lymph node cells from actively sensitized mice, the histology of the reactions resembles that of classic allergic contact dermatitis in mice, challenge reactions are seen at 24 but not at 4 hr, and photoallergic contact dermatitis can be induced in B-cell deficient mice. The availability of a mouse model for the study of photo-ACD will facilitate the identification of pertinent control mechanisms and may aid in the management of the disease. It is likely that a bioassay for photoallergens of humans can be based on this mouse model

  5. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    Energy Technology Data Exchange (ETDEWEB)

    Huimin, Zhou [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Li, Jia [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Shujing, Wang [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Hongmei, Wang [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Department of Medical Microbiology and Parasitology, School of Medicine, Liaodong College, Dandong 118000 (China); Haiying, Chu [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Yichuan, Hu [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Jun, Cao [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China); Jianing, Zhang [Department of Biochemistry, Institute of Glycobiology, Dalian Medical University, Dalian 116027 (China)

    2006-06-23

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression.

  6. Divergent expression and roles for caveolin-1 in mouse hepatocarcinoma cell lines with varying invasive ability

    International Nuclear Information System (INIS)

    Zhou Huimin; Jia Li; Wang Shujing; Wang Hongmei; Chu Haiying; Hu Yichuan; Cao Jun; Zhang Jianing

    2006-01-01

    Caveolin-1 is the major component protein of caveolae and associated with a lot of cellular events such as endocytosis, cholesterol homeostasis, signal transduction, and tumorigenesis. The majority of results suggest that caveolin-1 might not only act as a tumor suppressor gene but also a promoting metastasis gene. In this study, the divergent expression and roles of caveolin-1 were investigated in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have high, low, and no metastatic potential in the lymph nodes, as compared with normal mouse liver cell line IAR-20. The results showed that expression of caveolin-1 mRNA and protein along with the amount of caveolae number in Hca-F cells was higher than that in Hca-P cells, but was not detectable in Hepa1-6 cells. When caveolin-1 expression in Hca-F cells was down-regulated by RNAi approach, Hca-F cells proliferation rate in vitro declined and the expression of lymphangiogenic factor VEGFA in Hca-F decreased as well. Furthermore, in vivo implantation assay indicated that reduction of caveolin-1 expression in Hca-F prevented the lymphatic metastasis tumor burden of Hca-F cells in 615 mice. These results suggest that caveolin-1 facilities the lymphatic metastasis ability of mouse hepatocarcinoma cells via regulation tumor cell growth and VEGFA expression

  7. SPDEF is required for mouse pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production.

    Science.gov (United States)

    Chen, Gang; Korfhagen, Thomas R; Xu, Yan; Kitzmiller, Joseph; Wert, Susan E; Maeda, Yutaka; Gregorieff, Alexander; Clevers, Hans; Whitsett, Jeffrey A

    2009-10-01

    Various acute and chronic inflammatory stimuli increase the number and activity of pulmonary mucus-producing goblet cells, and goblet cell hyperplasia and excess mucus production are central to the pathogenesis of chronic pulmonary diseases. However, little is known about the transcriptional programs that regulate goblet cell differentiation. Here, we show that SAM-pointed domain-containing Ets-like factor (SPDEF) controls a transcriptional program critical for pulmonary goblet cell differentiation in mice. Initial cell-lineage-tracing analysis identified nonciliated secretory epithelial cells, known as Clara cells, as the progenitors of goblet cells induced by pulmonary allergen exposure in vivo. Furthermore, in vivo expression of SPDEF in Clara cells caused rapid and reversible goblet cell differentiation in the absence of cell proliferation. This was associated with enhanced expression of genes regulating goblet cell differentiation and protein glycosylation, including forkhead box A3 (Foxa3), anterior gradient 2 (Agr2), and glucosaminyl (N-acetyl) transferase 3, mucin type (Gcnt3). Consistent with these findings, levels of SPDEF and FOXA3 were increased in mouse goblet cells after sensitization with pulmonary allergen, and the proteins were colocalized in goblet cells lining the airways of patients with chronic lung diseases. Deletion of the mouse Spdef gene resulted in the absence of goblet cells in tracheal/laryngeal submucosal glands and in the conducting airway epithelium after pulmonary allergen exposure in vivo. These data show that SPDEF plays a critical role in regulating a transcriptional network mediating the goblet cell differentiation and mucus hyperproduction associated with chronic pulmonary disorders.

  8. Intervention of oxygen-control ability to radiation sensitivity, cell aging and cell transformation

    International Nuclear Information System (INIS)

    Yoshii, Hanako; Watanabe, Masami

    2009-01-01

    Oxygen is essential for life, and cells have therefore developed numerous adaptive responses to oxygen change. Here, we examined the difference in oxygen-control functions of human (HE), mouse (ME), and Syrian hamster embryo (SHE) cells cultured under different oxygen conditions (0.5%, 2% and 20%), and also examined whether oxygen tensions contributed to cellular lifespan and transformation. HE cells had their replicative lifespan slightly extended under hypoxic (0.5% and 2% oxygen) conditions, but were not immortalized under any of the oxygen concentrations. On the other hand, although ME cells cultured under 20% oxygen tension decreased their proliferation potency temporarily at early stage, all rodent cells were immortalized and acquired anchorage-independency, regardless of oxygen tension. These results suggest that cellular oxygen control function is related to sensitivities cellular immortalization and transformation. To understand intervention of oxygen control ability on cellular immortalization and transformation, we examined the intracellular oxidative level, mitochondria functions and radiation sensitivity. Intracellular oxidative levels of hypoxically cultured rodent cells were significantly enhanced. Mitochondrial membrane potential was altered depend on oxygen tensions, but the change was not parallel to mitochondria number in rodent cells. ME cells were particularly sensitive to oxygen change, and showed a clear oxygen effect on the X-ray survival. However, there was no difference in frequency of radiation-induced micronuclei between HE and ME cells. These results suggest that the response to oxygen change differs markedly in HE and rodent cells. (author)

  9. Femtosecond laser assisted photo-transfection and differentiation of mouse embryonic stem cells

    Science.gov (United States)

    Thobakgale, Lebogang; Manoto, Sello; Ombinda Lemboumba, Satuurnin; Maaza, Malik; Mthunzi-Kufa, Patience

    2018-02-01

    In tissue engineering research, stem cells have been used as starting material in the synthesis of mammalian cells for the treatment of various cell based diseases. This is done by manipulating the DNA content of the cells to induce a specific effect such as increased proliferation or developing a new cell type through the process of differentiation. Such controlled gene expression of stem cells is achieved by the method of transfection, where exogenous plasmid deoxyribonucleic acid (pDNA) is inserted into a stem cell using chemical, viral or physical methods. In this research, we used femtosecond (fs) laser pulses from a home-build microscope system to perforate the cellular membrane and allow entry of selected pDNA to alter the behaviour of mouse embryonic stem cells (mESCs). In one set of experiments, we induce fluorescence on mESCs using green fluorescence protein plasmid (pGFP) while in other tests; differentiation of mESCs into endoderm cells is performed using Sox-17 plasmid DNA (pSox-17). Primitive endoderm formation was thereafter confirmed using polymerase chain reactions (PCR) and the Sox-17 primer. Cell viability studies using adenosine triphosphate were also conducted. From the data, it was concluded that the photo-transfection method is biocompatible since it was able to induce fluorescence in mESCs. Secondly, it was confirmed that Sox-17 was photo-transfected successfully using 6 μW laser power, 128 fs pulses and 1kHz pulse repetition rate.

  10. A transcriptome-wide screen for mRNAs enriched in fetal Leydig cells: CRHR1 agonism stimulates rat and mouse fetal testis steroidogenesis.

    Directory of Open Access Journals (Sweden)

    Erin N McDowell

    Full Text Available Fetal testis steroidogenesis plays an important role in the reproductive development of the male fetus. While regulators of certain aspects of steroidogenesis are known, the initial driver of steroidogenesis in the human and rodent fetal testis is unclear. Through comparative analysis of rodent fetal testis microarray datasets, 54 candidate fetal Leydig cell-specific genes were identified. Fetal mouse testis interstitial expression of a subset of these genes with unknown expression (Crhr1, Gramd1b, Itih5, Vgll3, and Vsnl1 was verified by whole-mount in situ hybridization. Among the candidate fetal Leydig cell-specific factors, three receptors (CRHR1, PRLR, and PROKR2 were tested for a steroidogenic function using ex vivo fetal testes treated with receptor agonists (CRH, PRL, and PROK2. While PRL and PROK2 had no effect, CRH, at low (approximately 1 to 10 nM concentration, increased expression of the steroidogenic genes Cyp11a1, Cyp17a1, Scarb1, and Star in GD15 mouse and GD17 rat testes, and in conjunction, testosterone production was increased. Exposure of GD15 fetal mouse testis to a specific CRHR1 antagonist blunted the CRH-induced steroidogenic gene expression and testosterone responses. Similar to ex vivo rodent fetal testes, ≥ 10 nM CRH exposure of MA-10 Leydig cells increased steroidogenic pathway mRNA and progesterone levels, showing CRH can enhance steroidogenesis by directly targeting Leydig cells. Crh mRNA expression was observed in rodent fetal hypothalamus, and CRH peptide was detected in rodent amniotic fluid. Together, these data provide a resource for discovering factors controlling fetal Leydig cell biology and suggest that CRHR1 activation by CRH stimulates rat and mouse fetal Leydig cell steroidogenesis in vivo.

  11. Sex-reversed somatic cell cloning in the mouse.

    Science.gov (United States)

    Inoue, Kimiko; Ogonuki, Narumi; Mekada, Kazuyuki; Yoshiki, Atsushi; Sado, Takashi; Ogura, Atsuo

    2009-10-01

    Somatic cell nuclear transfer has many potential applications in the fields of basic and applied sciences. However, it has a disadvantage that can never be overcome technically-the inflexibility of the sex of the offspring. Here, we report an accidental birth of a female mouse following nuclear transfer using an immature Sertoli cell. We produced a batch of 27 clones in a nuclear transfer experiment using Sertoli cells collected from neonatal male mice. Among them, one pup was female. This "male-derived female" clone grew into a normal adult and produced offspring by natural mating with a littermate. Chromosomal analysis revealed that the female clone had a 39,X karyotype, indicating that the Y chromosome had been deleted in the donor cell or at some early step during nuclear transfer. This finding suggests the possibility of resuming sexual reproduction after a single male is cloned, which should be especially useful for reviving extinct or endangered species.

  12. Identifying Candidate Reprogramming Genes in Mouse Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Gao, Fang; Li, Jingyu; Zhang, Heng; Yang, Xu; An, Tiezhu

    2017-08-01

    Factor-based induced reprogramming approaches have tremendous potential for human regenerative medicine, but the efficiencies of these approaches are still low. In this study, we analyzed the global transcriptional profiles of mouse induced pluripotent stem cells (miPSCs) and mouse embryonic stem cells (mESCs) from seven different labs and present here the first successful clustering according to cell type, not by lab of origin. We identified 2131 different expression genes (DEs) as candidate pluripotency-associated genes by comparing mESCs/miPSCs with somatic cells and 720 DEs between miPSCs and mESCs. Interestingly, there was a significant overlap between the two DE sets. Therefore, we defined the overlap DEs as "consensus DEs" including 313 miPSC-specific genes expressed at a higher level in miPSCs versus mESCs and 184 mESC-specific genes in total and reasoned that these may contribute to the differences in pluripotency between mESCs and miPSCs. A classification of "consensus DEs" according to their different expression levels between somatic cells and mESCs/miPSCs shows that 86% of the miPSC-specific genes are more highly expressed in somatic cells, while 73% of mESC-specific genes are highly expressed in mESCs/miPSCs, indicating that the miPSCs have not efficiently silenced the expression pattern of the somatic cells from which they are derived and failed to completely induce the genes with high expression levels in mESCs. We further revealed a strong correlation between oocyte-enriched factors and insufficiently induced mESC-specific genes and identified 11 hub genes via network analysis. In light of these findings, we postulated that these key hub genes might not only drive somatic cell nuclear transfer (SCNT) reprogramming but also augment the efficiency and quality of miPSC reprogramming.

  13. T cell progenitors in the mouse fetal liver

    International Nuclear Information System (INIS)

    Rabinowich, H.; Umiel, T.; Globerson, A.

    1983-01-01

    Fourteen-day mouse fetal liver was found to contain cells capable of giving rise to T as well as B cell functions. The experimental system consisted of congenic C3H/DiSn and (C3H/DiSn X C3H.SW)F1 lethally irradiated (900 R) mice reconstituted with C3H/DiSn fetal liver or bone marrow cells. Assays included thyroid allograft rejection as well as in vitro measurement of reactivity to phytohemagglutinin (PHA) and concanavalin A (Con A) and in a mixed lymphocyte culture (MLC) system in spleen, lymph node, and thymus cells. The fetal liver chimeras were found to become as capable as the bone marrow chimeras in responding in these various assays. The T cell responses lagged behind the responses to the B cell mitogens dextran sulfate (DXS) and lipopolysaccharide (LPS) (30 days after reconstitution, as compared with 14 days for DXS and 21 for LPS). The reacting cells were of the donor genotype, as revealed after treatment with C3H/DiSn (H-2k) anti-C3H.SW (H-2b) congenic sera. T cell responses were not manifest in thymectomized (TX) chimeras. Hence, the liver seems to contain cells capable of developing into T cell lineages in a thymus-dependent process

  14. The perivascular phagocyte of the mouse pineal gland: An antigen-presenting cell

    DEFF Research Database (Denmark)

    Møller, Morten; Rath, Martin F; Klein, David C

    2006-01-01

    The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal g...... for MHC class II protein and for CD68, a marker of monocytes/phagocytes. This study verifies that perivascular phagocytes with antigen-presenting properties are present in the mouse pineal gland....

  15. Delivery of Bone Marrow-Derived Mesenchymal Stem Cells Improves Tear Production in a Mouse Model of Sjögren’s Syndrome

    Directory of Open Access Journals (Sweden)

    Hema S. Aluri

    2017-01-01

    Full Text Available The purpose of the present study was to test the potential of mouse bone marrow-derived mesenchymal stem cells (BD-MSCs in improving tear production in a mouse model of Sjögren’s syndrome dry eye and to investigate the underlying mechanisms involved. NOD mice (n=20 were randomized to receive i.p. injection of sterile phosphate buffered saline (PBS, control or murine BD-MSCs (1 × 106 cells. Tears production was measured at baseline and once a week after treatment using phenol red impregnated threads. Cathepsin S activity in the tears was measured at the end of treatment. After 4 weeks, animals were sacrificed and the lacrimal glands were excised and processed for histopathology, immunohistochemistry, and RNA analysis. Following BD-MSC injection, tears production increased over time when compared to both baseline and PBS injected mice. Although the number of lymphocytic foci in the lacrimal glands of treated animals did not change, the size of the foci decreased by 40.5% when compared to control animals. The mRNA level of the water channel aquaporin 5 was significantly increased following delivery of BD-MSCs. We conclude that treatment with BD-MSCs increases tear production in the NOD mouse model of Sjögren’s syndrome. This is likely due to decreased inflammation and increased expression of aquaporin 5.

  16. Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

    Science.gov (United States)

    de Almeida-Leite, Camila Megale; Arantes, Rosa Maria Esteves

    2010-12-15

    Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. Proliferation assay of mouse embryonic stem (ES) cells exposed to atmospheric-pressure plasmas at room temperature

    International Nuclear Information System (INIS)

    Miura, Taichi; Hirano, Kazumi; Ogura, Chika; Ikeguchi, Masamichi; Seki, Atsushi; Nishihara, Shoko; Ando, Ayumi; Kanazawa, Tatsuya; Hamaguchi, Satoshi

    2014-01-01

    Proliferation assays of mouse embryonic stem (ES) cells have been performed with cell culture media exposed to atmospheric-pressure plasmas (APPs), which generate reactive species in the media at room temperature. It is found that serum in cell culture media functions as a scavenger of highly reactive species and tends to protect cells in the media against cellular damage. On the other hand, if serum is not present in a cell culture medium when it is exposed to APP, the medium becomes cytotoxic and cannot be detoxified by serum added afterwards. Plasma-induced cytotoxic media hinder proliferation of mouse ES cells and may even cause cell death. It is also shown by nuclear magnetic resonance spectroscopy that organic compounds in cell culture media are in general not significantly modified by plasma exposure. These results indicate that if there is no serum in media when they are exposed to APPs, highly reactive species (such as OH radicals) generated in the media by the APP exposure are immediately converted to less reactive species (such as H 2 O 2 ), which can no longer readily react with serum that is added to the medium after plasma exposure. This study has clearly shown that it is these less reactive species, rather than highly reactive species, that make the medium cytotoxic to mouse ES cells. (paper)

  18. Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells.

    OpenAIRE

    Van Schravendijk, C F; Hooghe-Peters, E L; De Meyts, P; Pipeleers, D G

    1984-01-01

    The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insu...

  19. Comparison between the radiosensitivity of human, mouse and chicken fibroblast-like cells using short-term endpoints

    International Nuclear Information System (INIS)

    Diatloff-Zito, C.; Loria, E.; Maciera-Coelho, A.; Deschavanne, P.J.; Malaise, E.P.

    1981-01-01

    A comparative study has been made of the radiosensitivity of fibroblastic cell lines from three different animal species: human, mouse and chicken. Endpoints reflecting short term responses were utilized: colony forming ability (CFA), DNA single strand break (SSB) repair and repair of potentially lethal damage (PLD). Regardless of the criterion employed, the response to radiation varies from one species to another. According to survival curves, chicken cells appear to be more radioresistant than those of human and mouse. SSB repair is apparently absent in murine cells, partial in chicken cells and complete in human cells. This lack of correlation between survival curves and SSB repair demonstrates that survival of irradiated cells does not depend only (or at all) on the repair of SSB. The repair of PLD is much more efficient in human and chicken cells than in murine cells. (author)

  20. Inhibition of Cell Division and DNA Replication Impair Mouse-Naïve Pluripotency Exit.

    Science.gov (United States)

    Waisman, Ariel; Vazquez Echegaray, Camila; Solari, Claudia; Cosentino, María Soledad; Martyn, Iain; Deglincerti, Alessia; Ozair, Mohammad Zeeshan; Ruzo, Albert; Barañao, Lino; Miriuka, Santiago; Brivanlou, Ali; Guberman, Alejandra

    2017-09-01

    The cell cycle has gained attention as a key determinant for cell fate decisions, but the contribution of DNA replication and mitosis in stem cell differentiation has not been extensively studied. To understand if these processes act as "windows of opportunity" for changes in cell identity, we established synchronized cultures of mouse embryonic stem cells as they exit the ground state of pluripotency. We show that initial transcriptional changes in this transition do not require passage through mitosis and that conversion to primed pluripotency is linked to lineage priming in the G1 phase. Importantly, we demonstrate that impairment of DNA replication severely blocks transcriptional switch to primed pluripotency, even in the absence of p53 activity induced by the DNA damage response. Our data suggest an important role for DNA replication during mouse embryonic stem cell differentiation, which could shed light on why pluripotent cells are only receptive to differentiation signals during G1, that is, before the S phase. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Adrenergic factors involved in the control of crypt cell proliferation in jejunum and descending colon of mouse.

    Science.gov (United States)

    Kennedy, M F; Tutton, P J; Barkla, D H

    1983-01-01

    The mitotic rates in the crypts of Lieberkühn of the proximal jejunum and descending colon of mouse, following different treatments, were measured using a stathmokinetic technique. Regression coefficients, representing mitotic rates, were then calculated by the method of least squares. Treatment with adrenaline, isoprenaline, phenylephrine, phentolamine, and yohimbine all resulted in decreased mitotic rate of jejunal and colonic crypt cells. Chemical sympathectomy and cryosympathectomy had a similar effect, and chemical sympathectomy was followed by a supersensitivity to clonidine. Intraperitoneal injection of metaraminol, clonidine, propranolol, prazosin, labetolol and simultaneous injection of propranolol and adrenaline all resulted in an increased rate of crypt cell proliferation in both jejunum and colon. A significant increase in mitotic rate was observed in both tissues at night. The amplitude of this diurnal variation was decreased in both jejunum and colon following chemical sympathectomy. In addition, the amplitude of this variation in jejunum was decreased after treatment with yohimbine or phentolamine. The results of the study suggest that the sympathetic nervous system stimulates epithelial cell proliferation in both the small and large intestine and that this effect is mediated by an alpha 2-adrenoceptor. By contrast, stimulation of alpha 1- and beta-adrenoceptors is inhibitory to cell proliferation in these tissues.

  2. Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells.

    Science.gov (United States)

    van den Brink, Susanne C; Baillie-Johnson, Peter; Balayo, Tina; Hadjantonakis, Anna-Katerina; Nowotschin, Sonja; Turner, David A; Martinez Arias, Alfonso

    2014-11-01

    Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call 'gastruloids'. © 2014. Published by The Company of Biologists Ltd.

  3. Spontaneous pyrogen production by mouse histiocytic and myelomonocytic tumor cell lines in vitro.

    Science.gov (United States)

    Bodel, P

    1978-05-01

    Tumor-associated fever occurs commonly in acute leukemias and lymphomas. We investigated the capacity for in vitro production of pyrogen by three mouse histiocytic lymphoma cell lines (J-774, PU5-1.8, p 388 D1), one myelomonoyctic line (WEHI-3), and tow lymphoma-derived lines, RAW-8 and R-8. Pyrogen was released spontaneously into the culture medium during growth by all cell lines with macrophage or myeloid characteristics including lysozyme production; R-8 cells, of presumed B-lymphocyte origin, did not produce pyrogen. When injected into mice, the pyrogens gave fever curves typical of endogenous pyrogen, were inactived by heating to 56 degrees C and by pronase digestion, and appeared to be secreted continuously by viable cells. Two pyrogenic molecular species produced by H-774 cells were identified by Sephadex filtration, one of mol wt approximately equal to 30,000, and the other greater than or equal to 60,000. By contrast, three carcinoma cell lines of human origin and SV-40 3T3 mouse fibroblasts did not produce pyrogen in vitro. These results suggest that some malignant cells derived from phagocytic cells of bone marrow origin retain their capacity for pyrogen production, and may spontaneously secrete pyrogen during growth.

  4. Proliferation of mouse endometrial stromal cells in culture is highly sensitive to lysophosphatidic acid signaling

    International Nuclear Information System (INIS)

    Aikawa, Shizu; Kano, Kuniyuki; Inoue, Asuka; Aoki, Junken

    2017-01-01

    Endometrial stromal cells (ESCs) proliferate rapidly both in vivo and in vitro. Here we show that proliferation of ESCs in vitro is strongly dependent on lysophosphatidic acid (LPA) signaling. LPA is produced by autotaxin (ATX) and induces various kinds of cellular processes including migration, proliferation and inhibition of cell death possibly through six G protein-coupled receptors (LPA 1-6 ). We found that ESCs proliferated rapidly in vitro in an autocrine manner and that the proliferation was prominently suppressed by either an ATX inhibitor (ONO-8430506) or an LPA 1/3 antagonist (Ki16425). Among the cells lines tested, mouse ESCs were the most sensitive to these inhibitors. Proliferation of ESCs isolated from either LPA 1 - or LPA 3 -deficient mice was comparable to proliferation of ESCs isolated from control mice. An LPA receptor antagonist (AM095), which was revealed to be a dual LPA 1 /LPA 3 antagonist, also suppressed the proliferation of ESCs. The present results show that LPA signaling has a critical role in the proliferation of ESCs, and that this role is possibly mediated redundantly by LPA 1 and LPA 3 . - Highlights: • Uterine endometrial stromal cells (ESCs) proliferate rapidly both in vivo and in vitro. • ESCs proliferated in vitro in an autocrine fashion. • Proliferation of mouse ESCs was prominently suppressed by inhibitors of lysophosphatidic acid (LPA) signaling. • LPA receptors, LPA 1 and LPA 3 , had redundant role in supporting the proliferation of ESCs.

  5. Quantitative transcriptional profiling of ATDC5 mouse progenitor cells during chondrogenesis

    DEFF Research Database (Denmark)

    Chen, Li; Fink, Trine; Zhang, Xiao-Yan

    2005-01-01

    During the differentiation of a mouse chondroprogenitor cell line, ATDC5, an analysis of the transcription cartilage-related genes was carried out using real-time RT-PCR in a semiquantitative fashion. A total number of 104 genes both previously linked to chondrogenesis and hitherto not associated...

  6. Titanium dioxide nanoparticle-induced cytotoxicity and the underlying mechanism in mouse myocardial cells

    Science.gov (United States)

    Zhou, Yingjun; Hong, Fashui; Wang, Ling

    2017-11-01

    Exposure to fine particulate matter (PM) is known to cause cardiovascular disease. While extensive research has focused on the risk of atmospheric PM to public health, particularly heart disease, limited studies to date have attempted to clarify the molecular mechanisms underlying myocardial cell damage caused by exposure to titanium dioxide nanoparticles (TiO2 NPs). Data from the current investigation showed that TiO2 NPs are deposited in myocardial mitochondria via the blood circulation accompanied by obvious ultrastructural changes and impairment of mitochondrial structure and function in mouse myocardial cells, including reduction in mitochondrial membrane potential and ATP production, aggravation of oxidative stress along with increased levels of reactive oxygen species, malondialdehyde and protein carbonyl, and decreased glutathione content and enzymatic activities, including superoxide dismutase and glutathione peroxidase. Furthermore, TiO2 NPs induced a significant decrease in the activities of complex I, complex II, complex III, complex IV, succinate dehydrogenase, NADH oxidase, Ca2+-ATPase, Na+/K+-ATPase, and Ca2+/Mg2+-ATPase, and upregulation of cytokine expression (including cytochrome c, caspase-3, and p-JNK) in mitochondria-mediated apoptosis while downregulating Bcl-2 expression in mouse myocardial cells. Our results collectively indicate that chronic exposure to TiO2 NPs induces damage in mitochondrial structure and function as well as mitochondria-mediated apoptosis in mouse myocardial cells, which may be closely associated with heart disease in animals and humans.

  7. A novel method of mouse ex utero transplantation of hepatic progenitor cells into the fetal liver

    International Nuclear Information System (INIS)

    Shikanai, Mima; Asahina, Kinji; Iseki, Sachiko; Teramoto, Kenichi; Nishida, Tomohiro; Shimizu-Saito, Keiko; Ota, Masato; Eto, Kazuhiro; Teraoka, Hirobumi

    2009-01-01

    Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or α-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.

  8. Expression of Podoplanin in the Mouse Tooth Germ and Apical Bud Cells

    Science.gov (United States)

    Sawa, Yoshihiko; Iwasawa, Kana; Ishikawa, Hiroyuki

    2008-01-01

    This study was designed to investigate the distribution of cells expressing podoplanin in the mouse tooth bud. Podoplanin expression was detected in enamel epithelia of the cervical loop at cell-cell contacts strongly, and weakly on the loosely aggregated stellate reticulum in the center and the neighboring stratum intermedium. Odontoblasts exhibited intense podoplanin expression at the junction with predentin while no expression was detected in the enamel organ containing ameloblasts. These results suggest that proliferating inner and outer enamel epithelia express podoplanin but that the expression is suppressed in the differentiated epithelia containing ameloblasts. On the other hand the podoplanin expression occurs in the differentiating odontoblasts and the expression is sustained in differentiated odontoblasts, indicating that odontoblasts have the strong ability to express podoplanin. In cultured apical bud cells podoplanin was detected at cell-cell contacts. In real-time PCR analysis the amount of podoplanin mRNA of the apical buds was 2-fold compared with the amount of kidney used as a positive control. These findings indicate that apical bud cells have the strong ability to express the podoplanin gene. Podoplanin is a mucin-type glycoprotein negatively charged by extensive O-glycosylation and a high content of sialic acid, which expresses the adhesive property. The podoplanin may contribute to form odontoblastic fiber or function as the anchorage to the tooth development and in proliferating epithelial cells of cervical loop and apical bud. PMID:18989465

  9. Chondroitin Sulfate Is Indispensable for Pluripotency and Differentiation of Mouse Embryonic Stem Cells

    Science.gov (United States)

    Izumikawa, Tomomi; Sato, Ban; Kitagawa, Hiroshi

    2014-01-01

    Chondroitin sulfate (CS) proteoglycans are present on the surfaces of virtually all cells and in the extracellular matrix and are required for cytokinesis at early developmental stages. Studies have shown that heparan sulfate (HS) is essential for maintaining mouse embryonic stem cells (ESCs) that are primed for differentiation, whereas the function of CS has not yet been elucidated. To clarify the role of CS, we generated glucuronyltransferase-I-knockout ESCs lacking CS. We found that CS was required to maintain the pluripotency of ESCs and promoted initial ESC commitment to differentiation compared with HS. In addition, CS-A and CS-E polysaccharides, but not CS-C polysaccharides, bound to E-cadherin and enhanced ESC differentiation. Multiple-lineage differentiation was inhibited in chondroitinase ABC-digested wild-type ESCs. Collectively, these results suggest that CS is a novel determinant in controlling the functional integrity of ESCs via binding to E-cadherin.

  10. Pax6- and Six3-mediated induction of lens cell fate in mouse and human ES cells.

    Directory of Open Access Journals (Sweden)

    Raymond M Anchan

    Full Text Available Embryonic stem (ES cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.

  11. Repair and mutation induction in mouse germ cells: a summary and some thoughts

    International Nuclear Information System (INIS)

    Russell, L.B.

    1979-01-01

    The various lines of evidence for repair of premutational damage in mouse germ cells are reviewed with the implications for future experiment planning. Relation between mutagenicity and carcinogenicity are discussed

  12. Tracing notochord-derived cells using a Noto-cre mouse: implications for intervertebral disc development.

    Science.gov (United States)

    McCann, Matthew R; Tamplin, Owen J; Rossant, Janet; Séguin, Cheryle A

    2012-01-01

    Back pain related to intervertebral disc degeneration is the most common musculoskeletal problem, with a lifetime prevalence of 82%. The lack of effective treatment for this widespread problem is directly related to our limited understanding of disc development, maintenance and degeneration. The aim of this study was to determine the developmental origins of nucleus pulposus cells within the intervertebral disc using a novel notochord-specific Cre mouse. To trace the fate of notochordal cells within the intervertebral disc, we derived a notochord-specific Cre mouse line by targeting the homeobox gene Noto. Expression of this gene is restricted to the node and the posterior notochord during gastrulation [embryonic day 7.5 (E7.5)-E12.5]. The Noto-cre mice were crossed with a conditional lacZ reporter for visualization of notochord fate in whole-mount embryos. We performed lineage-tracing experiments to examine the contribution of the notochord to spinal development from E12.5 through to skeletally mature mice (9 months). Fate mapping studies demonstrated that, following elongation and formation of the primitive axial skeleton, the notochord gives rise to the nucleus pulposus in fully formed intervertebral discs. Cellular localization of β-galactosidase (encoded by lacZ) and cytokeratin-8 demonstrated that both notochordal cells and chondrocyte-like nucleus pulposus cells are derived from the embryonic notochord. These studies establish conclusively that notochordal cells act as embryonic precursors to all cells found within the nucleus pulposus of the mature intervertebral disc. This suggests that notochordal cells might serve as tissue-specific progenitor cells within the disc and establishes the Noto-cre mouse as a unique tool to interrogate the contribution of notochordal cells to both intervertebral disc development and disc degeneration.

  13. Inactivation of STAT3 Signaling Impairs Hair Cell Differentiation in the Developing Mouse Cochlea.

    Science.gov (United States)

    Chen, Qianqian; Quan, Yizhou; Wang, Naitao; Xie, Chengying; Ji, Zhongzhong; He, Hao; Chai, Renjie; Li, Huawei; Yin, Shankai; Chin, Y Eugene; Wei, Xunbin; Gao, Wei-Qiang

    2017-07-11

    Although STAT3 signaling is demonstrated to regulate sensory cell differentiation and regeneration in the zebrafish, its exact role is still unclear in mammalian cochleae. Here, we report that STAT3 and its activated form are specifically expressed in hair cells during mouse cochlear development. Importantly, conditional cochlear deletion of Stat3 leads to an inhibition on hair cell differentiation in mice in vivo and in vitro. By cell fate analysis, inactivation of STAT3 signaling shifts the cell division modes from asymmetric to symmetric divisions from supporting cells. Moreover, inhibition of Notch signaling stimulates STAT3 phosphorylation, and inactivation of STAT3 signaling attenuates production of supernumerary hair cells induced by a Notch pathway inhibitor. Our findings highlight an important role of the STAT3 signaling during mouse cochlear hair cell differentiation and may have clinical implications for the recovery of hair cell loss-induced hearing impairment. Copyright © 2017 International Society for Stem Cell Research. Published by Elsevier Inc. All rights reserved.

  14. Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors

    DEFF Research Database (Denmark)

    Rothová, Michaela; Hölzenspies, Jurriaan J; Livigni, Alessandra

    2016-01-01

    Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive...... endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut...

  15. An inducible knockout mouse to model the cell-autonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias

    Science.gov (United States)

    Mirantes, Cristina; Eritja, Núria; Dosil, Maria Alba; Santacana, Maria; Pallares, Judit; Gatius, Sónia; Bergadà, Laura; Maiques, Oscar; Matias-Guiu, Xavier; Dolcet, Xavier

    2013-01-01

    SUMMARY PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ERT under the control of a chicken actin promoter, we have generated a tamoxifen-inducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors. PMID:23471917

  16. Comparative analysis of TCDD-induced AhR-mediated gene expression in human, mouse and rat primary B cells

    Energy Technology Data Exchange (ETDEWEB)

    Kovalova, Natalia, E-mail: kovalova@msu.edu [Department of Pharmacology and Toxicology, Michigan State University, Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States); Nault, Rance, E-mail: naultran@msu.edu [Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States); Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Crawford, Robert, E-mail: crawfo28@msu.edu [Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States); Zacharewski, Timothy R., E-mail: tzachare@msu.edu [Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States); Department of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Kaminski, Norbert E., E-mail: kamins11@msu.edu [Department of Pharmacology and Toxicology, Michigan State University, Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States)

    2017-02-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant that activates the aryl hydrocarbon receptor (AhR) resulting in altered gene expression. In vivo, in vitro, and ex vivo studies have demonstrated that B cells are directly impaired by TCDD, and are a sensitive target as evidenced by suppression of antibody responses. The window of sensitivity to TCDD-induced suppression of IgM secretion among mouse, rat and human B cells is similar. Specifically, TCDD must be present within the initial 12 h post B cell stimulation, indicating that TCDD disrupts early signaling network(s) necessary for B lymphocyte activation and differentiation. Therefore, we hypothesized that TCDD treatment across three different species (mouse, rat and human) triggers a conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Time course studies identified TCDD-elicited differential expression of 515 human, 2371 mouse and 712 rat orthologous genes over the 24-h period. 28 orthologs were differentially expressed in response to TCDD in all three species. Overrepresented pathways enriched in all three species included cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton and pathways in cancer. Differentially expressed genes functionally associated with cell-cell signaling in humans, immune response in mice, and oxidation reduction in rats. Overall, these results suggest that despite the conservation of the AhR and its signaling mechanism, TCDD elicits species-specific gene expression changes. - Highlights: • Kovalova TAAP Highlights Nov. 2016 • RNA-Seq identified TCDD-induced gene expression in PWM-activated primary B cells. • TCDD elicited differential expression of 515 human, 2371 mouse and 712

  17. Cell of Origin and Cancer Stem Cells in Tumor Suppressor Mouse Models of Glioblastoma.

    Science.gov (United States)

    Alcantara Llaguno, Sheila R; Xie, Xuanhua; Parada, Luis F

    2016-01-01

    The cellular origins and the mechanisms of progression, maintenance of tumorigenicity, and therapeutic resistance are central questions in the glioblastoma multiforme (GBM) field. Using tumor suppressor mouse models, our group recently reported two independent populations of adult GBM-initiating central nervous system progenitors. We found different functional and molecular subtypes depending on the tumor-initiating cell lineage, indicating that the cell of origin is a driver of GBM subtype diversity. Using an in vivo model, we also showed that GBM cancer stem cells (CSCs) or glioma stem cells (GSCs) contribute to resistance to chemotherapeutic agents and that genetic ablation of GSCs leads to a delay in tumor progression. These studies are consistent with the cell of origin and CSCs as critical regulators of the pathogenesis of GBM. © 2016 Alcantara Llaguno et al; Published by Cold Spring Harbor Laboratory Press.

  18. An increase in immature β-cells lacking Glut2 precedes the expansion of β-cell mass in the pregnant mouse.

    Directory of Open Access Journals (Sweden)

    Christine A Beamish

    Full Text Available A compensatory increase in β-cell mass occurs during pregnancy to counter the associated insulin resistance, and a failure in adaptation is thought to contribute to gestational diabetes. Insulin-expressing but glucose-transporter-2-low (Ins+Glut2LO progenitor cells are present in mouse and human pancreas, being predominantly located in extra-islet β-cell clusters, and contribute to the regeneration of the endocrine pancreas following induced ablation. We therefore sought to investigate the contribution of Ins+Glut2LO cells to β-cell mass expansion during pregnancy. Female C57Bl/6 mice were time mated and pancreata were collected at gestational days (GD 6, 9, 12, 15, and 18, and postpartum D7 (n = 4/time-point and compared to control (non-pregnant animals. Beta cell mass, location, proliferation (Ki67+, and proportion of Ins+Glut2LO cells were measured using immunohistochemistry and bright field or confocal microscopy. Beta cell mass tripled by GD18 and β-cell proliferation peaked at GD12 in islets (≥6 β-cells and small β-cell clusters (1-5 β-cells. The proportion and fraction of Ins+Glut2LO cells undergoing proliferation increased significantly at GD9 in both islets and clusters, preceding the increase in β-cell mass and proliferation, and their proliferation within clusters persisted until GD15. The overall number of clusters increased significantly at GD9. Quantitative PCR showed a significant increase in Pdx1 presence at GD9 vs. GD18 or control pancreas, and Pdx1 was visualized by immunohistochemistry within both Ins+Glut2LO and Ins+Glut2HI cells within clusters. These results indicate that Ins+Glut2LO cells are likely to contribute to β-cell mass expansion during pregnancy.

  19. A novel and effective cancer immunotherapy mouse model using antigen-specific B cells selected in vitro.

    Directory of Open Access Journals (Sweden)

    Tatsuya Moutai

    Full Text Available Immunotherapies such as adoptive transfer of T cells or natural killer cells, or monoclonal antibody (MoAb treatment have recently been recognized as effective means to treat cancer patients. However, adoptive transfer of B cells or plasma cells producing tumor-specific antibodies has not been applied as a therapy because long-term culture and selective expansion of antigen-specific B cells has been technically very difficult. Here, we describe a novel cancer immunotherapy that uses B-cell adoptive transfer. We demonstrate that germinal-center-like B cells (iGB cells induced in vitro from mouse naïve B cells become plasma cells and produce IgG antibodies for more than a month in the bone marrow of non-irradiated recipient mice. When transferred into mice, iGB cells producing antibody against a surrogate tumor antigen suppressed lung metastasis and growth of mouse melanoma cells expressing the same antigen and prolonged survival of the recipients. In addition, we have developed a novel culture system called FAIS to selectively expand antigen-specific iGB cells utilizing the fact that iGB cells are sensitive to Fas-induced cell death unless their antigen receptors are ligated by membrane-bound antigens. The selected iGB cells efficiently suppressed lung metastasis of melanoma cells in the adoptive immunotherapy model. As human blood B cells can be propagated as iGB cells using culture conditions similar to the mouse iGB cell cultures, our data suggest that it will be possible to treat cancer-bearing patients by the adoptive transfer of cancer-antigen-specific iGB cells selected in vitro. This new adoptive immunotherapy should be an alternative to the laborious development of MoAb drugs against cancers for which no effective treatments currently exist.

  20. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Gao, Xiugong; Sprando, Robert L.; Yourick, Jeffrey J.

    2015-01-01

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds

  1. Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Xiugong, E-mail: xiugong.gao@fda.hhs.gov; Sprando, Robert L.; Yourick, Jeffrey J.

    2015-08-15

    Developmental toxicity testing has traditionally relied on animal models which are costly, time consuming, and require the sacrifice of large numbers of animals. In addition, there are significant disparities between human beings and animals in their responses to chemicals. Thalidomide is a species-specific developmental toxicant that causes severe limb malformations in humans but not in mice. Here, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on differentiation of mouse embryonic stem cells (mESCs). C57BL/6 mESCs were allowed to differentiate spontaneously and RNA was collected at 24, 48, and 72 h after exposure to 0.25 mM thalidomide. Global gene expression analysis using microarrays revealed hundreds of differentially expressed genes upon thalidomide exposure that were enriched in gene ontology (GO) terms and canonical pathways associated with embryonic development and differentiation. In addition, many genes were found to be involved in small GTPases-mediated signal transduction, heart development, and inflammatory responses, which coincide with clinical evidences and may represent critical embryotoxicities of thalidomide. These results demonstrate that transcriptomics in combination with mouse embryonic stem cell differentiation is a promising alternative model for developmental toxicity assessment. - Highlights: • Studied genomic changes in mouse embryonic stem cells upon thalidomide exposure • Identified gene expression changes that may represent thalidomide embryotoxicity • The toxicogenomic changes coincide well with known thalidomide clinical outcomes. • The mouse embryonic stem cell model is suitable for developmental toxicity testing. • The model has the potential for high-throughput screening of a multitude of compounds.

  2. Inactivation of STAT3 Signaling Impairs Hair Cell Differentiation in the Developing Mouse Cochlea

    Directory of Open Access Journals (Sweden)

    Qianqian Chen

    2017-07-01

    Full Text Available Although STAT3 signaling is demonstrated to regulate sensory cell differentiation and regeneration in the zebrafish, its exact role is still unclear in mammalian cochleae. Here, we report that STAT3 and its activated form are specifically expressed in hair cells during mouse cochlear development. Importantly, conditional cochlear deletion of Stat3 leads to an inhibition on hair cell differentiation in mice in vivo and in vitro. By cell fate analysis, inactivation of STAT3 signaling shifts the cell division modes from asymmetric to symmetric divisions from supporting cells. Moreover, inhibition of Notch signaling stimulates STAT3 phosphorylation, and inactivation of STAT3 signaling attenuates production of supernumerary hair cells induced by a Notch pathway inhibitor. Our findings highlight an important role of the STAT3 signaling during mouse cochlear hair cell differentiation and may have clinical implications for the recovery of hair cell loss-induced hearing impairment.

  3. Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ruoxing [Department of Biological Sciences, The University of Southern Mississippi, 118 College Drive 5018, Hattiesburg, MS 39406 (United States); Guo, Yan-Lin, E-mail: yanlin.guo@usm.edu [Department of Biological Sciences, The University of Southern Mississippi, 118 College Drive 5018, Hattiesburg, MS 39406 (United States)

    2012-10-01

    Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs. -- Highlights: Black-Right-Pointing-Pointer Inhibition of Cdks slows down mESCs proliferation. Black-Right-Pointing-Pointer mESCs display remarkable recovery capacity from short-term cell cycle interruption. Black-Right-Pointing-Pointer Short-term cell cycle interruption does not compromise mESC self-renewal. Black

  4. Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Wang, Ruoxing; Guo, Yan-Lin

    2012-01-01

    Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs. -- Highlights: ► Inhibition of Cdks slows down mESCs proliferation. ► mESCs display remarkable recovery capacity from short-term cell cycle interruption. ► Short-term cell cycle interruption does not compromise mESC self-renewal. ► Oct4 and Nanog are up-regulated via de novo synthesis by cell cycle interruption.

  5. Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks.

    Science.gov (United States)

    Tichy, Elisia D; Pillai, Resmi; Deng, Li; Liang, Li; Tischfield, Jay; Schwemberger, Sandy J; Babcock, George F; Stambrook, Peter J

    2010-11-01

    Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells. Of the 2 major pathways that repair double-strand breaks, error-prone nonhomologous end joining (NHEJ) predominated in mouse embryonic fibroblasts, whereas the high fidelity homologous recombinational repair (HRR) predominated in ES cells. Microhomology-mediated end joining, an emerging repair pathway, persisted at low levels in all cell types examined. The levels of proteins involved in HRR and microhomology-mediated end joining were highly elevated in ES cells compared with mouse embryonic fibroblasts, whereas those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV protein was also low in ES cells. Attempts to increase the abundance of DNA Ligase IV protein by overexpression or inhibition of its degradation, and thereby elevate NHEJ in ES cells, were unsuccessful. When ES cells were induced to differentiate, however, the level of DNA Ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than NHEJ may lend ES cells an additional layer of genomic protection and that the limited levels of DNA Ligase IV may account for the low level of NHEJ activity.

  6. Fetal progenitor cell transplantation treats methylmalonic aciduria in a mouse model

    International Nuclear Information System (INIS)

    Buck, Nicole E.; Pennell, Samuel D.; Wood, Leonie R.; Pitt, James J.; Allen, Katrina J.; Peters, Heidi L.

    2012-01-01

    Highlights: ► Fetal cells were transplanted into a methylmalonic acid mouse model. ► Cell engraftment was detected in liver, spleen and bone marrow. ► Biochemical disease correction was measured in blood samples. ► A double dose of 5 million cells (1 week apart) proved more effective. ► Higher levels of engraftment may be required for greater disease correction. -- Abstract: Methylmalonic aciduria is a rare disorder caused by an inborn error of organic acid metabolism. Current treatment options are limited and generally focus on disease management. We aimed to investigate the use of fetal progenitor cells to treat this disorder using a mouse model with an intermediate form of methylmalonic aciduria. Fetal liver cells were isolated from healthy fetuses at embryonic day 15–17 and intravenously transplanted into sub-lethally irradiated mice. Liver donor cell engraftment was determined by PCR. Disease correction was monitored by urine and blood methylmalonic acid concentration and weight change. Initial studies indicated that pre-transplantation sub-lethal irradiation followed by transplantation with 5 million cells were suitable. We found that a double dose of 5 million cells (1 week apart) provided a more effective treatment. Donor cell liver engraftment of up to 5% was measured. Disease correction, as defined by a decrease in blood methylmalonic acid concentration, was effected in methylmalonic acid mice transplanted with a double dose of cells and who showed donor cell liver engraftment. Mean plasma methylmalonic acid concentration decreased from 810 ± 156 (sham transplanted) to 338 ± 157 μmol/L (double dose of 5 million cells) while mean blood C3 carnitine concentration decreased from 20.5 ± 4 (sham transplanted) to 5.3 ± 1.9 μmol/L (double dose of 5 million cells). In conclusion, higher levels of engraftment may be required for greater disease correction; however these studies show promising results for cell transplantation biochemical

  7. Fetal progenitor cell transplantation treats methylmalonic aciduria in a mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Buck, Nicole E., E-mail: nicole.buck@mcri.edu.au [Metabolic Research, Murdoch Childrens Research Institute, The University of Melbourne, Department of Paediatrics, Royal Children' s Hospital, Flemington Road, Parkville, VIC 3052 (Australia); Pennell, Samuel D.; Wood, Leonie R. [Metabolic Research, Murdoch Childrens Research Institute, The University of Melbourne, Department of Paediatrics, Royal Children' s Hospital, Flemington Road, Parkville, VIC 3052 (Australia); Pitt, James J. [Victorian Clinical Genetics Services, Murdoch Childrens Research Institute, Royal Children' s Hospital, Parkville (Australia); Allen, Katrina J. [Gastro and Food Allergy, Murdoch Childrens Research Institute, Parkville (Australia); Peters, Heidi L. [Metabolic Research, Murdoch Childrens Research Institute, The University of Melbourne, Department of Paediatrics, Royal Children' s Hospital, Flemington Road, Parkville, VIC 3052 (Australia)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer Fetal cells were transplanted into a methylmalonic acid mouse model. Black-Right-Pointing-Pointer Cell engraftment was detected in liver, spleen and bone marrow. Black-Right-Pointing-Pointer Biochemical disease correction was measured in blood samples. Black-Right-Pointing-Pointer A double dose of 5 million cells (1 week apart) proved more effective. Black-Right-Pointing-Pointer Higher levels of engraftment may be required for greater disease correction. -- Abstract: Methylmalonic aciduria is a rare disorder caused by an inborn error of organic acid metabolism. Current treatment options are limited and generally focus on disease management. We aimed to investigate the use of fetal progenitor cells to treat this disorder using a mouse model with an intermediate form of methylmalonic aciduria. Fetal liver cells were isolated from healthy fetuses at embryonic day 15-17 and intravenously transplanted into sub-lethally irradiated mice. Liver donor cell engraftment was determined by PCR. Disease correction was monitored by urine and blood methylmalonic acid concentration and weight change. Initial studies indicated that pre-transplantation sub-lethal irradiation followed by transplantation with 5 million cells were suitable. We found that a double dose of 5 million cells (1 week apart) provided a more effective treatment. Donor cell liver engraftment of up to 5% was measured. Disease correction, as defined by a decrease in blood methylmalonic acid concentration, was effected in methylmalonic acid mice transplanted with a double dose of cells and who showed donor cell liver engraftment. Mean plasma methylmalonic acid concentration decreased from 810 {+-} 156 (sham transplanted) to 338 {+-} 157 {mu}mol/L (double dose of 5 million cells) while mean blood C3 carnitine concentration decreased from 20.5 {+-} 4 (sham transplanted) to 5.3 {+-} 1.9 {mu}mol/L (double dose of 5 million cells). In conclusion, higher levels of engraftment may

  8. Piperlongumine inhibits LMP1/MYC-dependent mouse B-lymphoma cells

    International Nuclear Information System (INIS)

    Han, Seong-Su; Tompkins, Van S.; Son, Dong-Ju; Kamberos, Natalie L.; Stunz, Laura L.; Halwani, Ahmad; Bishop, Gail A.; Janz, Siegfried

    2013-01-01

    Highlights: •Mouse model of human Burkitt lymphoma revealed cancer inhibition by PL. •Treatment with PL led to apoptosis of malignant but not normal B cells. •PL inhibited LMP1–NF-κB–Myc-dependent target genes including p21-encoding Cdkn1a. •PL holds promise for new interventions approaches to hematologic malignancies. -- Abstract: Piperlongumine (PL), isolated from the fruit of Long pepper, Piper longum, is a cancer-inhibiting compound that selectively kills tumor cells while sparing their normal counterparts. Here we evaluated the efficacy with which PL suppresses malignant B cells derived from a newly developed, double-transgenic mouse model of human endemic Burkitt lymphoma (BL), designated mCD40-LMP1/iMyc Eμ . PL inhibited tumor cell proliferation in a concentration-dependent manner and induced apoptosis of neoplastic but not normal B cells. Treatment with PL resulted in downregulation of EBV-encoded LMP1, cellular Myc, constitutive NF-κB activity, and a host of LMP1-Myc-NF-κB-regulated target genes including Aurka, Bcat1, Bub1b, Ccnb1, Chek1, Fancd2, Tfrc and Xrcc6. Of note, p21 Cip1 -encoding Cdkn1a was suppressed independent of changes in Trp53 mRNA levels and p53 DNA-binding activity. Considering the central role of the LMP1–NF-κB–Myc axis in B-lineage neoplasia, these findings further our understanding of the mechanisms by which PL inhibits B-lymphoma and provide a preclinical rationale for the inclusion of PL in new interventions in blood cancers

  9. Piperlongumine inhibits LMP1/MYC-dependent mouse B-lymphoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Han, Seong-Su; Tompkins, Van S. [Department of Pathology, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Son, Dong-Ju [Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA (United States); Kamberos, Natalie L. [Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Stunz, Laura L. [Deparment of Microbiology, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Iowa City VAMC, Iowa City, IA (United States); Halwani, Ahmad [Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Bishop, Gail A. [Deparment of Microbiology, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA (United States); Iowa City VAMC, Iowa City, IA (United States); Janz, Siegfried, E-mail: siegfried-janz@uiowa.edu [Department of Pathology, University of Iowa Carver College of Medicine, Iowa City, IA (United States)

    2013-07-12

    Highlights: •Mouse model of human Burkitt lymphoma revealed cancer inhibition by PL. •Treatment with PL led to apoptosis of malignant but not normal B cells. •PL inhibited LMP1–NF-κB–Myc-dependent target genes including p21-encoding Cdkn1a. •PL holds promise for new interventions approaches to hematologic malignancies. -- Abstract: Piperlongumine (PL), isolated from the fruit of Long pepper, Piper longum, is a cancer-inhibiting compound that selectively kills tumor cells while sparing their normal counterparts. Here we evaluated the efficacy with which PL suppresses malignant B cells derived from a newly developed, double-transgenic mouse model of human endemic Burkitt lymphoma (BL), designated mCD40-LMP1/iMyc{sup Eμ}. PL inhibited tumor cell proliferation in a concentration-dependent manner and induced apoptosis of neoplastic but not normal B cells. Treatment with PL resulted in downregulation of EBV-encoded LMP1, cellular Myc, constitutive NF-κB activity, and a host of LMP1-Myc-NF-κB-regulated target genes including Aurka, Bcat1, Bub1b, Ccnb1, Chek1, Fancd2, Tfrc and Xrcc6. Of note, p21{sup Cip1}-encoding Cdkn1a was suppressed independent of changes in Trp53 mRNA levels and p53 DNA-binding activity. Considering the central role of the LMP1–NF-κB–Myc axis in B-lineage neoplasia, these findings further our understanding of the mechanisms by which PL inhibits B-lymphoma and provide a preclinical rationale for the inclusion of PL in new interventions in blood cancers.

  10. COUP-TFII controls mouse pancreatic β-cell mass through GLP-1-β-catenin signaling pathways.

    Directory of Open Access Journals (Sweden)

    Marie Boutant

    Full Text Available The control of the functional pancreatic β-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how β-cell mass is determined.Conditional chicken ovalbumin upstream promoter transcription factor II (COUP-TFII-deficient mice were generated and crossed with mice expressing Cre under the control of pancreatic duodenal homeobox 1 (pdx1 gene promoter. Ablation of COUP-TFII in pancreas resulted in glucose intolerance. Beta-cell number was reduced at 1 day and 3 weeks postnatal. Together with a reduced number of insulin-containing cells in the ductal epithelium and normal β-cell proliferation and apoptosis, this suggests decreased β-cell differentiation in the neonatal period. By testing islets isolated from these mice and cultured β-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces the expression of the β-catenin gene and its target genes such as cyclin D1 and axin 2. Moreover, induction of these genes by glucagon-like peptide 1 (GLP-1 via β-catenin was impaired in absence of COUP-TFII. The expression of two other target genes of GLP-1 signaling, GLP-1R and PDX-1 was significantly lower in mutant islets compared to control islets, possibly contributing to reduced β-cell mass. Finally, we demonstrated that COUP-TFII expression was activated by the Wnt signaling-associated transcription factor TCF7L2 (T-cell factor 7-like 2 in human islets and rat β-cells providing a feedback loop.Our findings show that COUP-TFII is a novel component of the GLP-1 signaling cascade that increases β-cell number during the neonatal period. COUP-TFII is required for GLP-1 activation of the β-catenin-dependent pathway and its expression is under the control of TCF7L2.

  11. Cultivation of mouse mammary tumor cells derived from DD/Tbr, 3

    International Nuclear Information System (INIS)

    Iwai, Mineko; Iwai, Yoshiaki; Takamori, Yasuhiko; Okumoto, Masaaki; Nishikawa, Ryosuke

    1981-01-01

    The factors affecting production of MuMTV by DD-762 cells, an established cell line from a spontaneous mammary tumor in a DD/Tbr mouse, were examined. When the cells were seeded and cultures medium were refreshed at every 3 - 4 day intervals without passage of cells, virus production began after exponential pase of cell growth and attained to peaks at every 10 - 12 days intervals up to approximately 60 days after seeding. MuMTV production was dependent on cell seeding density. Seeding at higher cell density, virus release occurred earlier. Maximum amount of MuMTV was observed with the medium containing 10 μg INS, 5 μg DXM and 10% FCS. The RDDP activities in the culture fluid were rapidly inactivated by incubation at 37 0 C. (author)

  12. GATA-1 directly regulates Nanog in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wen-Zhong; Ai, Zhi-Ying [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Wang, Zhi-Wei [School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027 (China); Chen, Lin-Lin [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Guo, Ze-Kun, E-mail: gzknwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Zhang, Yong, E-mail: zylabnwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China)

    2015-09-25

    Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression. - Highlights: • The Nanog proximal promoter conceives functional element for GATA-1. • GATA-1 occupies the Nanog proximal promoter in vitro and in vivo. • GATA-1 transcriptionally suppresses Nanog.

  13. A Transgenic Tri-Modality Reporter Mouse

    OpenAIRE

    Yan, Xinrui; Ray, Pritha; Paulmurugan, Ramasamy; Tong, Ricky; Gong, Yongquan; Sathirachinda, Ataya; Wu, Joseph C.; Gambhir, Sanjiv S.

    2013-01-01

    Transgenic mouse with a stably integrated reporter gene(s) can be a valuable resource for obtaining uniformly labeled stem cells, tissues, and organs for various applications. We have generated a transgenic mouse model that ubiquitously expresses a tri-fusion reporter gene (fluc2-tdTomato-ttk) driven by a constitutive chicken β-actin promoter. This "Tri-Modality Reporter Mouse" system allows one to isolate most cells from this donor mouse and image them for bioluminescent (fluc2), fluorescent...

  14. Dual Function of Wnt Signaling during Neuronal Differentiation of Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Hanjun Kim

    2015-01-01

    Full Text Available Activation of Wnt signaling enhances self-renewal of mouse embryonic and neural stem/progenitor cells. In contrast, undifferentiated ES cells show a very low level of endogenous Wnt signaling, and ectopic activation of Wnt signaling has been shown to block neuronal differentiation. Therefore, it remains unclear whether or not endogenous Wnt/β-catenin signaling is necessary for self-renewal or neuronal differentiation of ES cells. To investigate this, we examined the expression profiles of Wnt signaling components. Expression levels of Wnts known to induce β-catenin were very low in undifferentiated ES cells. Stable ES cell lines which can monitor endogenous activity of Wnt/β-catenin signaling suggest that Wnt signaling was very low in undifferentiated ES cells, whereas it increased during embryonic body formation or neuronal differentiation. Interestingly, application of small molecules which can positively (BIO, GSK3β inhibitor or negatively (IWR-1-endo, Axin stabilizer control Wnt/β-catenin signaling suggests that activation of that signaling at different time periods had differential effects on neuronal differentiation of 46C ES cells. Further, ChIP analysis suggested that β-catenin/TCF1 complex directly regulated the expression of Sox1 during neuronal differentiation. Overall, our data suggest that Wnt/β-catenin signaling plays differential roles at different time points of neuronal differentiation.

  15. Dual effects of fluoxetine on mouse early embryonic development

    International Nuclear Information System (INIS)

    Kim, Chang-Woon; Choe, Changyong; Kim, Eun-Jin; Lee, Jae-Ik; Yoon, Sook-Young; Cho, Young-Woo; Han, Sunkyu; Tak, Hyun-Min; Han, Jaehee; Kang, Dawon

    2012-01-01

    Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50 μM) for different durations. When late 2-cells were incubated with 5 μM fluoxetine for 6 h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5 μM) over 24 h showed a reduction in blastocyst formation. The addition of fluoxetine (5 μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K + channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ∼ 30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating. Highlights: ► Short-term exposure of 2-cells to fluoxetine enhances mouse blastocyst formation. ► The enhancive effect of fluoxetine is resulted from CaMKII activation

  16. Dual effects of fluoxetine on mouse early embryonic development

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chang-Woon [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of); Department of Obstetrics and Gynecology, Samsung Changwon Hospital, Sungkyunkwan University, Changwon 630-723 (Korea, Republic of); Choe, Changyong [National Institute of Animal Science, RDA, Cheonan 330-801 (Korea, Republic of); Kim, Eun-Jin [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of); Lee, Jae-Ik [Department of Obstetrics and Gynecology, Gyeongsang National University Hospital, Jinju 660-702 (Korea, Republic of); Yoon, Sook-Young [Fertility Center of CHA Gangnam Medical Center, CHA University, Seoul 135-081 (Korea, Republic of); Cho, Young-Woo; Han, Sunkyu; Tak, Hyun-Min; Han, Jaehee [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of); Kang, Dawon, E-mail: dawon@gnu.ac.kr [Department of Physiology and Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751 (Korea, Republic of)

    2012-11-15

    Fluoxetine, a selective serotonin reuptake inhibitor, regulates a variety of physiological processes, such as cell proliferation and apoptosis, in mammalian cells. Little is known about the role of fluoxetine in early embryonic development. This study was undertaken to investigate the effect of fluoxetine during mouse early embryonic development. Late two-cell stage embryos (2-cells) were cultured in the presence of various concentrations of fluoxetine (1 to 50 μM) for different durations. When late 2-cells were incubated with 5 μM fluoxetine for 6 h, the percentage that developed into blastocysts increased compared to the control value. However, late 2-cells exposed to fluoxetine (5 μM) over 24 h showed a reduction in blastocyst formation. The addition of fluoxetine (5 μM) together with KN93 or KN62 (calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitors) failed to increase blastocyst formation. Fluoxetine treatment inhibited TREK-1 and TREK-2, members of the two-pore domain K{sup +} channel family expressed in mouse embryos, activities, indicating that fluoxetine-induced membrane depolarization in late 2-cells might have resulted from TREK inhibition. In addition, long-term exposure to fluoxetine altered the TREK mRNA expression levels. Furthermore, injection of siRNA targeting TREKs significantly decreased blastocyst formation by ∼ 30% compared to injection of scrambled siRNA. Long-term exposure of fluoxetine had no effect on blastocyst formation of TREK deficient embryos. These results indicate that low-dose and short-term exposures of late 2-cells to fluoxetine probably increase blastocyst formation through activation of CaMKII-dependent signal transduction pathways, whereas long-term exposure decreases mouse early embryonic development through inhibition of TREK channel gating. Highlights: ► Short-term exposure of 2-cells to fluoxetine enhances mouse blastocyst formation. ► The enhancive effect of fluoxetine is resulted from Ca

  17. [PRODUCT OF THE BMI1--A KEY COMPONENT OF POLYCOMB--POSITIVELY REGULATES ADIPOCYTE DIFFERENTIATION OF MOUSE MESENCHYMAL STEM CELLS].

    Science.gov (United States)

    Petrov, N S; Vereschagina, N A; Sushilova, E N; Kropotov, A V; Miheeva, N F; Popov, B V

    2016-01-01

    Bmil is a key component of Polycomb (PcG), which in mammals controls the basic functions of mammalian somatic stem cells (SSC) such as self-renewal and differentiation. Bmi1 supports SSC via transcriptional suppression of genes associated with cell cycle and differentiation. The most studied target genes of Bmi1 are the genes of Ink4 locus, CdkI p16(Ink4a) and p1(Arf), suppression of which due to activating mutations of the BMI1 results in formation of cancer stem cells (CSC) and carcinomas in various tissues. In contrast, inactivation of BMI1 results in cell cycle arrest and cell senescence. Although clinical phenomena of hypo- and hyperactivation of BMI1 are well known, its targets and mechanisms of regulation of tissue specific SSC are still obscure. The goal of this study was to evaluate the regulatory role of BMI1 in adipocyte differentiation (AD) of mouse mesenchymal stem cells (MSC). Induction of AD in mouse MSC of the C3H10T1/2 cell line was associated with an increase in the expression levels of BMI1, the genes of pRb family (RB, p130) and demethylase UTX, but not methyltransferase EZH2, whose products regulate the methylation levels of H3K27. It was observed earlier that H3K27me3 may play the role of the epigenetic switch by promoting AD of human MSC via activating expression of the PPARγ2, the master gene of AD (Hemming et al., 2014). Here we show that inactivation of BMI1 using specific siRNA slows and decreases the levels of AD, but does not abolish it. This is associated with a complete inhibition of the expression of adipogenic marker genes--PPARγ2, ADIPOQ and a decrease in the expression of RB, p130, but not UTX. The results obtained give evidence that the epigenetic mechanism regulating AD differentiation in mouse and human MSC is different.

  18. Development of an in vitro culture method for stepwise differentiation of mouse embryonic stem cells and induced pluripotent stem cells into mature osteoclasts.

    Science.gov (United States)

    Nishikawa, Keizo; Iwamoto, Yoriko; Ishii, Masaru

    2014-05-01

    The development of methods for differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs) into functional cells have helped to analyze the mechanism regulating cellular processes and to explore cell-based assays for drug discovery. Although several reports have demonstrated methods for differentiation of mouse ESCs into osteoclast-like cells, it remains unclear whether these methods are applicable for differentiation of iPSCs to osteoclasts. In this study, we developed a simple method for stepwise differentiation of mouse ESCs and iPSCs into bone-resorbing osteoclasts based upon a monoculture approach consisting of three steps. First, based on conventional hanging-drop methods, embryoid bodies (EBs) were produced from mouse ESCs or iPSCs. Second, EBs were cultured in medium supplemented with macrophage colony-stimulating factor (M-CSF), and differentiated to osteoclast precursors, which expressed CD11b. Finally, ESC- or iPSC-derived osteoclast precursors stimulated with receptor activator of nuclear factor-B ligand (RANKL) and M-CSF formed large multinucleated osteoclast-like cells that expressed tartrate-resistant acid phosphatase and were capable of bone resorption. Molecular analysis showed that the expression of osteoclast marker genes such as Nfatc1, Ctsk, and Acp5 are increased in a RANKL-dependent manner. Thus, our procedure is simple and easy and would be helpful for stem cell-based bone research.

  19. Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks.

    Science.gov (United States)

    Bañuelos, C A; Banáth, J P; MacPhail, S H; Zhao, J; Eaves, C A; O'Connor, M D; Lansdorp, P M; Olive, P L

    2008-09-01

    Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PK(cs) appears to be responsible since mES cells expressed strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX(-/-) mES cells also expressed 6 times more DNA-PK(cs) than wild-type mES cells. Similar results were obtained for ATM(-/-) mES cells. Differentiation of mES cells led to an increase in DNA-PK(cs), an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PK(cs). These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage.

  20. Differentiate or Die: 3-Bromopyruvate and Pluripotency in Mouse Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Ana Sofia Rodrigues

    Full Text Available Pluripotent embryonic stem cells grown under standard conditions (ESC have a markedly glycolytic profile, which is shared with many different types of cancer cells. Thus, some therapeutic strategies suggest that pharmacologically shifting cancer cells towards an oxidative phenotype, using glycolysis inhibitors, may reduce cancer aggressiveness. Given the metabolic parallels between cancer and stemness would chemotherapeutical agents have an effect on pluripotency, and could a strategy involving these agents be envisioned to modulate stem cell fate in an accessible manner? In this manuscript we attempted to determine the effects of 3-bromopyruvate (3BrP in pluripotency. Although it has other intracellular targets, this compound is a potent inhibitor of glycolysis enzymes thought to be important to maintain a glycolytic profile. The goal was also to determine if we could contribute towards a pharmacologically accessible metabolic strategy to influence cell differentiation.Mouse embryonic stem cells (mESC grown under standard pluripotency conditions (in the presence of Leukemia Inducing Factor- LIF were treated with 3BrP. As a positive control for differentiation other mESCs were grown without LIF. Overall our results demonstrate that 3BrP negatively affects pluripotency, forcing cells to become less glycolytic and with more active mitochondria. These changes in metabolism are correlated with increased differentiation, even under pluripotency conditions (i.e. in the presence of LIF. However, 3BrP also significantly impaired cell function, and may have other roles besides affecting the metabolic profile of mESCs.Treatment of mESCs with 3BrP triggered a metabolic switch and loss of pluripotency, even in the presence of LIF. Interestingly, the positive control for differentiation allowed for a distinction between 3BrP effects and changes associated with spontaneous differentiation/loss of pluripotency in the absence of LIF. Additionally, there was a

  1. Differentiate or Die: 3-Bromopyruvate and Pluripotency in Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Rodrigues, Ana Sofia; Pereira, Sandro L; Correia, Marcelo; Gomes, Andreia; Perestrelo, Tânia; Ramalho-Santos, João

    2015-01-01

    Pluripotent embryonic stem cells grown under standard conditions (ESC) have a markedly glycolytic profile, which is shared with many different types of cancer cells. Thus, some therapeutic strategies suggest that pharmacologically shifting cancer cells towards an oxidative phenotype, using glycolysis inhibitors, may reduce cancer aggressiveness. Given the metabolic parallels between cancer and stemness would chemotherapeutical agents have an effect on pluripotency, and could a strategy involving these agents be envisioned to modulate stem cell fate in an accessible manner? In this manuscript we attempted to determine the effects of 3-bromopyruvate (3BrP) in pluripotency. Although it has other intracellular targets, this compound is a potent inhibitor of glycolysis enzymes thought to be important to maintain a glycolytic profile. The goal was also to determine if we could contribute towards a pharmacologically accessible metabolic strategy to influence cell differentiation. Mouse embryonic stem cells (mESC) grown under standard pluripotency conditions (in the presence of Leukemia Inducing Factor- LIF) were treated with 3BrP. As a positive control for differentiation other mESCs were grown without LIF. Overall our results demonstrate that 3BrP negatively affects pluripotency, forcing cells to become less glycolytic and with more active mitochondria. These changes in metabolism are correlated with increased differentiation, even under pluripotency conditions (i.e. in the presence of LIF). However, 3BrP also significantly impaired cell function, and may have other roles besides affecting the metabolic profile of mESCs. Treatment of mESCs with 3BrP triggered a metabolic switch and loss of pluripotency, even in the presence of LIF. Interestingly, the positive control for differentiation allowed for a distinction between 3BrP effects and changes associated with spontaneous differentiation/loss of pluripotency in the absence of LIF. Additionally, there was a slight

  2. Cell adhesion molecules expression pattern indicates that somatic cells arbitrate gonadal sex of differentiating bipotential fetal mouse gonad.

    Science.gov (United States)

    Piprek, Rafal P; Kolasa, Michal; Podkowa, Dagmara; Kloc, Malgorzata; Kubiak, Jacek Z

    2017-10-01

    Unlike other organ anlagens, the primordial gonad is sexually bipotential in all animals. In mouse, the bipotential gonad differentiates into testis or ovary depending on the genetic sex (XY or XX) of the fetus. During gonad development cells segregate, depending on genetic sex, into distinct compartments: testis cords and interstitium form in XY gonad, and germ cell cysts and stroma in XX gonad. However, our knowledge of mechanisms governing gonadal sex differentiation remains very vague. Because it is known that adhesion molecules (CAMs) play a key role in organogenesis, we suspected that diversified expression of CAMs should also play a crucial role in gonad development. Using microarray analysis we identified 129 CAMs and factors regulating cell adhesion during sexual differentiation of mouse gonad. To identify genes expressed differentially in three cell lines in XY and XX gonads: i) supporting (Sertoli or follicular cells), ii) interstitial or stromal cells, and iii) germ cells, we used transgenic mice expressing EGFP reporter gene and FACS cell sorting. Although a large number of CAMs expressed ubiquitously, expression of certain genes was cell line- and genetic sex-specific. The sets of CAMs differentially expressed in supporting versus interstitial/stromal cells may be responsible for segregation of these two cell lines during gonadal development. There was also a significant difference in CAMs expression pattern between XY supporting (Sertoli) and XX supporting (follicular) cells but not between XY and XX germ cells. This indicates that differential CAMs expression pattern in the somatic cells but not in the germ line arbitrates structural organization of gonadal anlagen into testis or ovary. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Matrix stiffness and oxigen tension modulate epigenetic conversion of mouse dermal fibroblasts into insulin producing cells.

    Directory of Open Access Journals (Sweden)

    Alessandro Zenobi

    2017-05-01

    Full Text Available In vivo, cells are surrounded by a three-dimensional (3-D organization of supporting matrix, neighboring cells and a gradient of chemical and mechanical signals (Antoni, et al., 2015. However, the present understanding of many biological processes is mainly based on two-dimensional (2-D systems that typically provides a static environment. In the present study, we tested two different 3-D culture systems and apply them to the epigenetic conversion of mouse dermal fibroblasts into insulin producing-cells (Pennarossa, et al., 2013; Brevini, et al., 2015, combining also the use of two oxygen tensions. In particular, cells were differentiated using the Polytetrafluoroethylene micro-bioreactor (PTFE and the Polyacrylamide (PAA gels with different stiffness (1 kPa; 4 kPa, maintained either in the standard 20% or in the more physiological 5% oxygen tensions. Standard differentiation performed on plastic substrates was assessed as a control. Cell morphology (Fig.1A, insulin expression and release were analyzed to evaluate the role of both stiffness and oxygen tension in the process. The results obtained showed that 1 kPa PAA gel and PTFE system induced a significantly higher insulin expression and release than plastic and 4 kPa PAA gel, especially in low oxygen condition (Fig.1B. Furthermore, comparing the efficiency of the two systems tested, 1 kPa PAA gel ensured a higher insulin transcription than PTFE (Fig.1C. Recent studies show the direct influence of substrates on lineage commitment and cell differentiation (Engler, et al., 2006; Evans, et al., 2009. The evidence here presented confirm that the use of an appropriate stiffness (similar to the pancreatic tissue, combined with a physiological oxygen tension, promote β-cell differentiation, with beneficial effects on cell functional activity and insulin release. The present results highlight the importance of 3-D cell rearrangement and oxigen tension to promote in vitro epigenetic conversion of

  4. p38 MAPK pathway is essential for self-renewal of mouse male germline stem cells (mGSCs).

    Science.gov (United States)

    Niu, Zhiwei; Mu, Hailong; Zhu, Haijing; Wu, Jiang; Hua, Jinlian

    2017-02-01

    Male germline stem cells (mGSCs), also called spermatogonial stem cells (SSCs), constantly generate spermatozoa in male animals. A number of preliminary studies on mechanisms of mGSC self-renewal have previously been conducted, revealing that several factors are involved in this regulated process. The p38 MAPK pathway is widely conserved in multiple cell types in vivo, and plays an important role in cell proliferation, differentiation, inflammation and apoptosis. However, its role in self-renewal of mGSCs has not hitherto been determined. Here, the mouse mGSCs were cultured and their identity was verified by semi-RT-PCR, alkaline phosphatase (AP) staining and immunofluorescence staining. Then, the p38 MAPK pathway was blocked by p38 MAPK-specific inhibitor SB202190. mGSC self-renewal ability was then analysed by observation of morphology, cell number, cell growth analysis, TUNEL incorporation assay and cell cycle analysis. Results showed that mouse mGSC self-renewal ability was significantly inhibited by SB202190. This study showed for the first time that the p38 MAPK pathway plays a key role in maintaining self-renewal capacity of mouse mGSCs, which offers a new self-renewal pathway for these cells and contributes to overall knowledge of the mechanisms of mGSC self-renewal. © 2016 John Wiley & Sons Ltd.

  5. Radiation response of mouse lymphoid and myeloid cell lines. Pt. 3

    International Nuclear Information System (INIS)

    Radford, I.R.; Murphy, T.K.

    1994-01-01

    The authors have examined the timing of γ-irradiation-induced death in relation to cell cycle progression using a panel of mouse lymphoid or myeloid cell lines. Death was found to occur immediately after irradiation ('rapid interphase' death), or after arrest in G 2 phase ('delayed interphase' death), or following one or more mitoses ('mitotic/delayed mitotic' death). In part II of this series of papers the authors demonstrated the occurrence of radiation-induced apoptosis in all these cell lines. Several of the cell lines showed different timing of death dependent upon the radiation dose used. These differences in the timing of radiation-induced death are shown to be useful indicators of the relative radiosensitivity of haematopoietic cell lines. (author)

  6. Differential effects of fractionated X irradiation on mouse spermatogonial stem cells

    NARCIS (Netherlands)

    van der Meer, Y.; Huiskamp, R.; Davids, J. A.; de rooij, D. G.

    1993-01-01

    The response of spermatogonial stem cells to fractionated X irradiation was studied in the various stages of the spermatogenic cycle of the CBA mouse. Fractionated doses of 2 + 2, 1 + 3, and 3 + 1 Gy with a 24-h interval between the doses were compared with a single dose of 4 Gy. The numbers of

  7. [Octanol preconditioning alleviates mouse cardiomyocyte swelling induced by simulated ischemia/reperfusion challenge in vitro].

    Science.gov (United States)

    Luo, Yukun; Fang, Jun; Fan, Lin; Lin, Chaogui; Chen, Zhaoyang; Chen, Lianglong

    2012-10-01

    To investigate the role of connexin 43-formed hemichannels in cell volume regulation induced by simulated ischemia/reperfusion (SI/R). Mouse cardiomyocytes isolated on a Langendorff apparatus with enzyme solution were aliquoted into control, SI/R and SI/R +octanol groups. Calcein-AM was used to stain the cells and the cell volume was measured with confocal microscope by stack scanning. Trypan blue was used to measure the cell viability after the treatments. Calcein-AM staining and cofocal microscopy yielded stable and reproducible results for cell volume measurement. Mouse cardiomyocytes subjected to simulated SI/R showed obvious cell swelling as compared with the control cells [(126∓6)% vs 100%, Poctanol preconditioning significantly attenuated the cell swelling [(113∓6)%, Poctanol preconditioning obviously reduced the viability of the cells with SI/R challenge [(31∓2)%, Poctanol can alleviate the cell swelling to enhance the viability of the cardiomyocytes following SI/R.

  8. Efficient and simple production of insulin-producing cells from embryonal carcinoma stem cells using mouse neonate pancreas extract, as a natural inducer.

    Directory of Open Access Journals (Sweden)

    Marzieh Ebrahimie

    Full Text Available An attractive approach to replace the destroyed insulin-producing cells (IPCs is the generation of functional β cells from stem cells. Embryonal carcinoma (EC stem cells are pluripotent cells which can differentiate into all cell types. The present study was carried out to establish a simple nonselective inductive culture system for generation of IPCs from P19 EC cells by 1-2 weeks old mouse pancreas extract (MPE. Since, mouse pancreatic islets undergo further remodeling and maturation for 2-3 weeks after birth, we hypothesized that the mouse neonatal MPE contains essential factors to induce in vitro differentiation of pancreatic lineages. Pluripotency of P19 cells were first confirmed by expression analysis of stem cell markers, Oct3/4, Sox-2 and Nanog. In order to induce differentiation, the cells were cultured in a medium supplemented by different concentrations of MPE (50, 100, 200 and 300 µg/ml. The results showed that P19 cells could differentiate into IPCs and form dithizone-positive cell clusters. The generated P19-derived IPCs were immunoreactive to proinsulin, insulin and insulin receptor beta. The expression of pancreatic β cell genes including, PDX-1, INS1 and INS2 were also confirmed. The peak response at the 100 µg/ml MPE used for investigation of EP300 and CREB1 gene expression. When stimulated with glucose, these cells synthesized and secreted insulin. Network analysis of the key transcription factors (PDX-1, EP300, CREB1 during the generation of IPCs resulted in introduction of novel regulatory candidates such as MIR17, and VEZF1 transcription factors, as well as MORN1, DKFZp761P0212, and WAC proteins. Altogether, we demonstrated the possibility of generating IPCs from undifferentiated EC cells, with the characteristics of pancreatic β cells. The derivation of pancreatic cells from EC cells which are ES cell siblings would provide a valuable experimental tool in study of pancreatic development and function as well as rapid

  9. Gradual regeneration of mouse testicular stem cells after exposure to ionizing radiation

    International Nuclear Information System (INIS)

    Meistrich, M.L.; Hunter, N.R.; Suzuki, N.; Trostle, P.K.; Withers, H.R.

    1978-01-01

    The regeneration of mouse testicular stem cells during 60 weeks after exposure to 600 or 1200 rad of γ radiation was examined. Restoration of spermatogenesis depended on stem cell survival, regeneration, and differentiation. Several assays were employed to measure the number of stem cells and their ability to repopulate the seminiferous epithelium as follows. Assay 1: The percentage of repopulated tubular cross sections was determined histologically at various times after irradiation. Assay 2: Mice were irradiated and, after given time intervals to allow for regeneration of stem cell numbers, a second dose was given. The percentage of repopulated tubular cross sections was determined 5 weeks later. Assay 3: The ability of the stem cells to produce spermatocytes and spermatids was assayed by the levels of the germ cell specific isoenzyme, LDH-X. Assay 4: The ability of the stem cells to produce sperm was assayed by the number of sperm heads in the testes. In addition, the ability of the stem cells to produce functional spermatozoa was measured by the fertility of the animals. The results obtained were as follows. All assays demonstrated that gradual regeneration of stem cell number occurred simultaneously with repopulation of the seminiferous epithelium by differentiating cells derived from stem cells. The regeneration kinetics of stem cells followed an exponential increase approaching a dose-dependent plateau below the level prior to irradiation. The doubling time for stem cells during the exponential portion was about 2 weeks. The regeneration of stem cell number after depletion by irradiation was gradual and incomplete, and only partially restored spermatogenesis. Correlation of regeneration with fertility data demonstrated that fertility was reestablished when sperm production returned to about 15% of control levels

  10. Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

    Science.gov (United States)

    Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

    2015-01-01

    Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. Results GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Conclusions Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed

  11. Retrovirus-mediated gene transfer of a human c-fos cDNA into mouse bone marrow stromal cells.

    Science.gov (United States)

    Roux, P; Verrier, B; Klein, B; Niccolino, M; Marty, L; Alexandre, C; Piechaczyk, M

    1991-11-01

    A cDNA encoding a complete human c-fos protein was isolated and inserted into two different murine MoMuLV-derived recombinant retroviruses allowing expression of c-fos protein in different cell types. One c-fos-expressing retrovirus, chosen for its ability to express high levels of proteins in fibroblast-like cells, was shown to potentiate long-term cultures of mouse bone marrow stromal cells in vitro and therefore constitutes a potential tool for immortalizing such cells. Moreover, when tested in an in vitro differentiation assay, stromal cells constitutively expressing c-fos favor the granulocyte differentiation of hematopoietic precursors. Interestingly, retroviruses expressing v-src and v-abl oncogenes, included as controls in our experiments, do not produce any detectable effects, whereas those expressing polyoma virus middle T antigen facilitate long-term growth in vitro of stromal cells that favor the macrophage differentiation pathway of bone marrow stem cells. Our observation supports the idea that constitutive expression of some oncogenes, including c-fos and polyoma virus middle T antigen, may influence cytokine production by bone marrow stromal cells.

  12. Analysis of X-ray induced cell-cycle perturbations in mouse osteosarcoma cells: a two-signal cell-cycle model

    International Nuclear Information System (INIS)

    Meeteren, A. van; Wijk, R. van; Stap, J.; Deys, B.F.

    1984-01-01

    The effects of X-irradiation on mouse osteosarcoma cells have been studied by time-lapse cinematography and the resulting pedigrees have been analysed statistically. It is shown that the irradiation treatment causes three types of cell kinetic lesions: cell death (disintegration), cell sterilization (failure to divide) and proliferation delay. The first two lesions are the most important with regard to survival of the irradiated cell in a clonal assay. Of these two lesions, sterilization appears to be highly correlated for sister cells, while this is not true for cell disintegration. This indicates that cell survival in a clonal assay may be a function of the ratio of the incidences of these two types of lesions. The X-ray-induced proliferation delay was studied in terms of intermitotic time distributions, mother-daughter correlation and sibling correlation in relation to the current cell-cycle phase at the time of treatment. This analysis shows that the effects of irradiation on these cell-cycle characteristics is highly cell-cycle-dependent. A qualitative model to account for the observations is presented. (author)

  13. Analysis of X-ray induced cell-cycle perturbations in mouse osteosarcoma cells: a two-signal cell-cycle model

    Energy Technology Data Exchange (ETDEWEB)

    Meeteren, A van; Wijk, R van [Rijksuniversiteit Utrecht (Netherlands); Stap, J; Deys, B F [Amsterdam Univ. (Netherlands)

    1984-03-01

    The effects of X-irradiation on mouse osteosarcoma cells have been studied by time-lapse cinematography and the resulting pedigrees have been analysed statistically. It is shown that the irradiation treatment causes three types of cell kinetic lesions: cell death (disintegration), cell sterilization (failure to divide) and proliferation delay. The first two lesions are the most important with regard to survival of the irradiated cell in a clonal assay. Of these two lesions, sterilization appears to be highly correlated for sister cells, while this is not true for cell disintegration. This indicates that cell survival in a clonal assay may be a function of the ratio of the incidences of these two types of lesions. The X-ray-induced proliferation delay was studied in terms of intermitotic time distributions, mother-daughter correlation and sibling correlation in relation to the current cell-cycle phase at the time of treatment. This analysis shows that the effects of irradiation on these cell-cycle characteristics is highly cell-cycle-dependent. A qualitative model to account for the observations is presented.

  14. Single cell analysis facilitates staging of Blimp1-dependent primordial germ cells derived from mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    John J Vincent

    Full Text Available The cell intrinsic programming that regulates mammalian primordial germ cell (PGC development in the pre-gonadal stage is challenging to investigate. To overcome this we created a transgene-free method for generating PGCs in vitro (iPGCs from mouse embryonic stem cells (ESCs. Using labeling for SSEA1 and cKit, two cell surface molecules used previously to isolate presumptive iPGCs, we show that not all SSEA1+/cKit+ double positive cells exhibit a PGC identity. Instead, we determined that selecting for cKit(bright cells within the SSEA1+ fraction significantly enriches for the putative iPGC population. Single cell analysis comparing SSEA1+/cKit(bright iPGCs to ESCs and embryonic PGCs demonstrates that 97% of single iPGCs co-express PGC signature genes Blimp1, Stella, Dnd1, Prdm14 and Dazl at similar levels to e9.5-10.5 PGCs, whereas 90% of single mouse ESC do not co-express PGC signature genes. For the 10% of ESCs that co-express PGC signature genes, the levels are significantly lower than iPGCs. Microarray analysis shows that iPGCs are transcriptionally distinct from ESCs and repress gene ontology groups associated with mesoderm and heart development. At the level of chromatin, iPGCs contain 5-methyl cytosine bases in their DNA at imprinted and non-imprinted loci, and are enriched in histone H3 lysine 27 trimethylation, yet do not have detectable levels of Mvh protein, consistent with a Blimp1-positive pre-gonadal PGC identity. In order to determine whether iPGC formation is dependent upon Blimp1, we generated Blimp1 null ESCs and found that loss of Blimp1 significantly depletes SSEA1/cKit(bright iPGCs. Taken together, the generation of Blimp1-positive iPGCs from ESCs constitutes a robust model for examining cell-intrinsic regulation of PGCs during the Blimp1-positive stage of development.

  15. Cre Fused with RVG Peptide Mediates Targeted Genome Editing in Mouse Brain Cells In Vivo.

    Science.gov (United States)

    Zou, Zhiyuan; Sun, Zhaolin; Li, Pan; Feng, Tao; Wu, Sen

    2016-12-14

    Cell penetrating peptides (CPPs) are short peptides that can pass through cell membranes. CPPs can facilitate the cellular entry of proteins, macromolecules, nanoparticles and drugs. RVG peptide (RVG hereinafter) is a 29-amino-acid CPP derived from a rabies virus glycoprotein that can cross the blood-brain barrier (BBB) and enter brain cells. However, whether RVG can be used for genome editing in the brain has not been reported. In this work, we combined RVG with Cre recombinase for bacterial expression. The purified RVG-Cre protein cut plasmids in vitro and traversed cell membranes in cultured Neuro2a cells. By tail vein-injecting RVG-Cre into Cre reporter mouse lines mTmG and Rosa26 lacZ , we demonstrated that RVG-Cre could target brain cells and achieve targeted somatic genome editing in adult mice. This direct delivery of the gene-editing enzyme protein into mouse brains with RVG is much safer than plasmid- or viral-based methods, holding promise for further applications in the treatment of various brain diseases.

  16. Induction of Functional 3D Ciliary Epithelium-Like Structure From Mouse Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Kinoshita, Hirofumi; Suzuma, Kiyoshi; Kaneko, Jun; Mandai, Michiko; Kitaoka, Takashi; Takahashi, Masayo

    2016-01-01

    To generate ciliary epithelium (CE) from mouse induced pluripotent stem (iPS) cells. Recently, a protocol for self-organizing optic cup morphogenesis in three-dimensional culture was reported, and it was suggested that ocular tissue derived from neural ectoderm could be differentiated. We demonstrated that a CE-like double-layered structure could be induced in simple culture by using a modified Eiraku differentiation protocol. Differentiation of a CE-like double-layered structure could be promoted by glycogen synthase kinase 3β (GSK-3β) inhibitor. Connexin43 and aquaporin1 were expressed in both thin layers, and induced CE-like cells expressed ciliary marker genes, such as cyclinD2, zic1, tgfb2, aldh1a3, wfdc1, otx1, BMP4, and BMP7. Increases in cytoplasmic and nuclear β-catenin in aggregates of the CE-like double-layered structure were confirmed by Western blot analysis. In addition, tankyrase inhibitor prevented the induction of the CE-like double-layered structure by GSK-3β inhibitor. Dye movement from pigmented cells to nonpigmented cells in the mouse iPS cell-derived CE-like structure was observed in a fluid movement experiment, consistent with the physiological function of CE in vivo. We could differentiate CE from mouse iPS cells in the present study. In the future, we hope that this CE-like complex will become useful as a graft for transplantation therapy in pathologic ocular hypotension due to CE dysfunction, and as a screening tool for the development of drugs for diseases associated with CE function.

  17. Knockdown of XBP1 by RNAi in Mouse Granulosa Cells Promotes Apoptosis, Inhibits Cell Cycle, and Decreases Estradiol Synthesis

    Directory of Open Access Journals (Sweden)

    Nan Wang

    2017-05-01

    Full Text Available Granulosa cells are crucial for follicular growth, development, and follicular atresia. X-box binding protein 1 (XBP1, a basic region-leucine zipper protein, is widely involved in cell differentiation, proliferation, apoptosis, cellular stress response, and other signaling pathways. In this study, RNA interference, flow cytometry, western blot, real-time PCR, Cell Counting Kit (CCK8, and ELISA were used to investigate the effect of XBP1 on steroidogenesis, apoptosis, cell cycle, and proliferation of mouse granulosa cells. ELISA analysis showed that XBP1 depletion significantly decreased the concentrations of estradiol (E2. Additionally, the expression of estrogen synthesis enzyme Cyp19a1 was sharply downregulated. Moreover, flow cytometry showed that knockdown of XBP1 increased the apoptosis rate and arrests the cell cycle in S-phase in granulosa cells (GCs. Further study confirmed these results. The expression of CCAAT-enhancer-binding protein homologous protein (CHOP, cysteinyl aspartate specific proteases-3 (caspase-3, cleaved caspase-3, and Cyclin E was upregulated, while that of Bcl-2, Cyclin A1, and Cyclin B1 was downregulated. Simultaneously, CCK8 analysis indicated that XBP1 disruption inhibited cell proliferation. In addition, XBP1 knockdown also alters the expression of Has2 and Ptgs2, two essential genes for folliculogenesis. Collectively, these data reveal a novel critical role of XBP1 in folliculogenesis by regulating the cell cycle, apoptosis, and steroid synthesis of mouse granulosa cells.

  18. Changes of mitochondrial structure, ATPase and Ca2+ concentration in spermatogenic cells of mouse testes induced by low dose radiation

    International Nuclear Information System (INIS)

    Wang Zhicheng; Liu Shuchun; Li Pengwu; Kang Shunai; Liang Shuo; Zhao Gang; Gong Shouliang

    2009-01-01

    Objective: To observe the ultrastructure, ATPase activity and Ca 2+ concentration ([Ca 2+ ]i) of mitochondria in the sperematogenic cells of mouse testes 3-24 h after low dose radiation with 0.025-0.200 Gy X-rays, and illuminate the effects of mitochondrion structure and relative biological function on apoptosis. Methods: The ultrastructure changes of mitochondria in the spermatogenic cells were observed with transmission electron microscope; the ATPase activity was measured with protein enzymic method; [Ca 2+ ]i was measured indirectly by flow cytometry with Fluo-3 probes. Results: The mitochondria swelled and vacuolizated, and their cristae were broken in the spermatogonia and spermatocytes 12 h after irradiation, and their nuclei were karyopyknosis, the acrosomal vesicle structure was ambiguity, the membrane structure was unclear, and the mitochondria in spermatids were vacuolization. The activities of Na + -K + -ATPase in mouse testis tissue 12 h after irradiated with 0.025-0.200 Gy decreased compared with those with 0 Gy, the Na + -K + -ATPase activities of the cells irradiated with 0.05-0.200 Gy decreased significantly compared with those with 0 Gy (P 2+ -ATPase of the cells irradiated with 0.025-0.200 Gy decreased significantly compared with those with 0 Gy (P 2+ ]i in mouse testis spermatogenic cells had similar dose-response relationship, [Ca 2+ ]i after irradiated with 0.075 Gy decreased compared with those with 0 Gy (P + -K + -ATPase in mouse testis tissues decreased obviously compared with those at 0 h (P 2+ -ATPase in mouse testis tissues increased slightly at 3 h, then decreased at 6-24 h compared with those at 0 h (P 2+ ]i in mouse testis spermatogenic cells had similar time course-response relationship, [Ca 2+ ]i at 12 h decreased significantly compared with at 0 h (P 2+ ]i induced by low dose radiation. (authors)

  19. Pleiotropy of Glycogen Synthase Kinase-3 Inhibition by CHIR99021 Promotes Self-Renewal of Embryonic Stem Cells from Refractory Mouse Strains

    Science.gov (United States)

    Ye, Shoudong; Tan, Li; Yang, Rongqing; Fang, Bo; Qu, Su; Schulze, Eric N.; Song, Houyan; Ying, Qilong; Li, Ping

    2012-01-01

    Background Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. β-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains. Methodology/Principal Findings We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of β-catenin and c-Myc protein levels. Stabilized β-catenin promoted ES self-renewal through two mechanisms. First, β-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor–independent manner. Second, β-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. β-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice. Conclusions/Significance Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools. PMID:22540008

  20. Ginsenoside Re Promotes Osteoblast Differentiation in Mouse Osteoblast Precursor MC3T3-E1 Cells and a Zebrafish Model

    Directory of Open Access Journals (Sweden)

    Hye-Min Kim

    2016-12-01

    Full Text Available Bone homeostasis is tightly regulated to balance bone formation and bone resorption. Many anabolic drugs are used as bone-targeted therapeutic agents for the promotion of osteoblast-mediated bone formation or inhibition of osteoclast-mediated bone resorption. Previous studies showed that ginsenoside Re has the effect of the suppression of osteoclast differentiation in mouse bone-marrow derived macrophages and zebrafish. Herein, we investigated whether ginsenoside Re affects osteoblast differentiation and mineralization in in vitro and in vivo models. Mouse osteoblast precursor MC3T3-E1 cells were used to investigate cell viability, alkaline phosphatase (ALP activity, and mineralization. In addition, we examined osteoblastic signaling pathways. Ginsenoside Re affected ALP activity without cytotoxicity, and we also observed the stimulation of osteoblast differentiation through the activation of osteoblast markers including runt-related transcription factor 2, type 1 collagen, ALP, and osteocalcin in MC3T3-E1 cells. Moreover, Alizarin red S staining indicated that ginsenoside Re increased osteoblast mineralization in MC3T3-E1 cells and zebrafish scales compared to controls. These results suggest that ginsenoside Re promotes osteoblast differentiation as well as inhibits osteoclast differentiation, and it could be a potential therapeutic agent for bone diseases.

  1. Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

    OpenAIRE

    Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

    2012-01-01

    Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomita...

  2. Immunohistochemical analysis of Clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis

    International Nuclear Information System (INIS)

    Hicks, Sarah M.; Vassallo, Jeffrey D.; Dieter, Matthew Z.; Lewis, Cindy L.; Whiteley, Laurence O.; Fix, Andrew S.; Lehman-McKeeman, Lois D.

    2003-01-01

    Immunohistochemical methods have been widely used to determine the histogenesis of spontaneous and chemically-induced mouse lung tumors. Typically, antigens for either alveolar Type II cells or bronchiolar epithelial Clara cells are studied. In the present work, the morphological and immunohistochemical phenotype of a transgenic mouse designed to develop lung tumors arising from Clara cells was evaluated. In this model, Clara cell-specific transformation is accomplished by directed expression of the SV40 large T antigen (TAg) under the mouse Clara cell secretory protein (CC10) promoter. In heterozygous mice, early lesions at 1 month of age consisted of hyperplastic bronchiolar epithelial cells. These progressed to adenoma by 2 months as proliferating epithelium extended into adjacent alveolar spaces. By 4 months, a large portion of the lung parenchyma was composed of tumor masses. Expression of constitutive CC10 was diminished in transgenic animals at all time points. Only the occasional cell or segment of the bronchiolar epithelium stained positively for CC10 by immunohistochemistry, and all tumors were found to be uniformly negative for staining. These results were corroborated by Western blotting, where CC10 was readily detectable in whole lung homogenate from nontransgenic animals, but not detected in lung from transgenic animals at any time point. Tumors were also examined for expression of surfactant apoprotein C (SPC), an alveolar Type II cell-specific marker, and found to be uniformly negative for staining. These results indicate that, in this transgenic model, expression of CC10, which is widely used to determine whether lung tumors arise from Clara cells, was reduced and subsequently lost during Clara cell tumor progression

  3. Mouse Hobit is a homolog of the transcriptional repressor Blimp-1 that regulates NKT cell effector differentiation

    NARCIS (Netherlands)

    van Gisbergen, Klaas P. J. M.; Kragten, Natasja A. M.; Hertoghs, Kirsten M. L.; Wensveen, Felix M.; Jonjic, Stipan; Hamann, Jörg; Nolte, Martijn A.; van Lier, Rene A. W.

    2012-01-01

    The transcriptional repressor Blimp-1 mediates the terminal differentiation of many cell types, including T cells. Here we identified Hobit (Znf683) as a previously unrecognized homolog of Blimp-1 that was specifically expressed in mouse natural killer T cells (NKT cells). Through studies of

  4. Systematic identification of cis-regulatory sequences active in mouse and human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Marica Grskovic

    2007-08-01

    Full Text Available Understanding the transcriptional regulation of pluripotent cells is of fundamental interest and will greatly inform efforts aimed at directing differentiation of embryonic stem (ES cells or reprogramming somatic cells. We first analyzed the transcriptional profiles of mouse ES cells and primordial germ cells and identified genes upregulated in pluripotent cells both in vitro and in vivo. These genes are enriched for roles in transcription, chromatin remodeling, cell cycle, and DNA repair. We developed a novel computational algorithm, CompMoby, which combines analyses of sequences both aligned and non-aligned between different genomes with a probabilistic segmentation model to systematically predict short DNA motifs that regulate gene expression. CompMoby was used to identify conserved overrepresented motifs in genes upregulated in pluripotent cells. We show that the motifs are preferentially active in undifferentiated mouse ES and embryonic germ cells in a sequence-specific manner, and that they can act as enhancers in the context of an endogenous promoter. Importantly, the activity of the motifs is conserved in human ES cells. We further show that the transcription factor NF-Y specifically binds to one of the motifs, is differentially expressed during ES cell differentiation, and is required for ES cell proliferation. This study provides novel insights into the transcriptional regulatory networks of pluripotent cells. Our results suggest that this systematic approach can be broadly applied to understanding transcriptional networks in mammalian species.

  5. Intrinsic bursting of AII amacrine cells underlies oscillations in the rd1 mouse retina.

    Science.gov (United States)

    Choi, Hannah; Zhang, Lei; Cembrowski, Mark S; Sabottke, Carl F; Markowitz, Alexander L; Butts, Daniel A; Kath, William L; Singer, Joshua H; Riecke, Hermann

    2014-09-15

    In many forms of retinal degeneration, photoreceptors die but inner retinal circuits remain intact. In the rd1 mouse, an established model for blinding retinal diseases, spontaneous activity in the coupled network of AII amacrine and ON cone bipolar cells leads to rhythmic bursting of ganglion cells. Since such activity could impair retinal and/or cortical responses to restored photoreceptor function, understanding its nature is important for developing treatments of retinal pathologies. Here we analyzed a compartmental model of the wild-type mouse AII amacrine cell to predict that the cell's intrinsic membrane properties, specifically, interacting fast Na and slow, M-type K conductances, would allow its membrane potential to oscillate when light-evoked excitatory synaptic inputs were withdrawn following photoreceptor degeneration. We tested and confirmed this hypothesis experimentally by recording from AIIs in a slice preparation of rd1 retina. Additionally, recordings from ganglion cells in a whole mount preparation of rd1 retina demonstrated that activity in AIIs was propagated unchanged to elicit bursts of action potentials in ganglion cells. We conclude that oscillations are not an emergent property of a degenerated retinal network. Rather, they arise largely from the intrinsic properties of a single retinal interneuron, the AII amacrine cell. Copyright © 2014 the American Physiological Society.

  6. Recovery of aging-related size increase of skin epithelial cells: in vivo mouse and in vitro human study.

    Directory of Open Access Journals (Sweden)

    Igor Sokolov

    Full Text Available The size increase of skin epithelial cells during aging is well-known. Here we demonstrate that treatment of aging cells with cytochalasin B substantially decreases cell size. This decrease was demonstrated on a mouse model and on human skin cells in vitro. Six nude mice were treated by topical application of cytochalasin B on skin of the dorsal left midsection for 140 days (the right side served as control for placebo treatment. An average decrease in cell size of 56±16% resulted. A reduction of cell size was also observed on primary human skin epithelial cells of different in vitro age (passages from 1 to 8. A cell strain obtained from a pool of 6 human subjects was treated with cytochalasin B in vitro for 12 hours. We observed a decrease in cell size that became statistically significant and reached 20-40% for cells of older passage (6-8 passages whereas no substantial change was observed for younger cells. These results may be important for understanding the aging processes, and for cosmetic treatment of aging skin.

  7. Human atopic dermatitis skin-derived T cells can induce a reaction in mouse keratinocytes in vivo

    DEFF Research Database (Denmark)

    Martel, Britta C; Blom, Lars; Dyring-Andersen, Beatrice

    2015-01-01

    . In comparison, blood -derived in vitro differentiated Th2 cells only induced a weak response in a few of the mice. Thus, we conclude that human AD skin-derived T cells can induce a reaction in mouse skin through induction of a proliferative response in the mouse keratinocytes. This article is protected......In atopic dermatitis (AD), the inflammatory response between skin infiltrating T cells and keratinocytes is fundamental to the development of chronic lesional eczema. The aim of this study was to investigate whether skin-derived T cells from AD patients could induce an inflammatory response in mice...... through keratinocyte activation and consequently cause development of eczematous lesions. Punch biopsies of lesional skin from AD patients were used to establish skin-derived T cell cultures and which were transferred into NOD.Cg-Prkd(scid) Il2rg(tm1Sug) /JicTac (NOG) mice. We found that subcutaneous...

  8. Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process

    Science.gov (United States)

    Ghosal, Abhisek; Sekar, Thillai V.

    2014-01-01

    Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na+-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na+-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS. PMID:24904078

  9. [Gefitineb inhibits the growth and induces the apoptosis of mouse I-10 Leydig testicular cancer cells in vitro].

    Science.gov (United States)

    Ji, Jie; Tong, Xu-hui; Zhang, Xin-yu; Gao, Qin; Li, Bei-bei; Wu, Xiao-xiang

    2015-09-01

    To observe the inhibitory effect of gefitineb on the proliferation and its inducing effect on the apoptosis of mouse I-10 Leydig testicular cancer cells in vitro. We treated I-10 Leydig testicular cancer cells of mice with gefitineb at 0, 1.25, 2.5, 5, 10, 20, and 40 µmol/L. Then we determined the inhibitory effect of gefitineb on the growth of the cells by MTT, detected their early and late apoptosis by Annexin V-FITC/propidium iodide double staining and Hoechst 33258 nuclear staining, respectively, and observed the expressions of apoptosis-related proteins Bcl-2, Bax and caspase 3/9 by Western blot. Compared with the blank control group, gefitineb significantly inhibited the proliferation of the I-10 cells at 10 and 20 µmol/L (P testicular cancer cells of mice and induce their apoptosis via the mitochondria-mediated apoptosis signaling pathway.

  10. Preclinical study of mouse pluripotent parthenogenetic embryonic stem cell derivatives for the construction of tissue-engineered skin equivalent.

    Science.gov (United States)

    Rao, Yang; Cui, Jihong; Yin, Lu; Liu, Wei; Liu, Wenguang; Sun, Mei; Yan, Xingrong; Wang, Ling; Chen, Fulin

    2016-10-22

    Embryonic stem cell (ESC) derivatives hold great promise for the construction of tissue-engineered skin equivalents (TESE). However, harvesting of ESCs destroys viable embryos and may lead to political and ethical concerns over their application. In the current study, we directed mouse parthenogenetic embryonic stem cells (pESCs) to differentiate into fibroblasts, constructed TESE, and evaluated its function in vivo. The stemness marker expression and the pluripotent differentiation ability of pESCs were tested. After embryoid body (EB) formation and adherence culture, mesenchymal stem cells (MSCs) were enriched and directed to differentiate into fibroblastic lineage. Characteristics of derived fibroblasts were assessed by quantitative real-time PCR and ELISA. Functional ability of the constructed TESE was tested by a mouse skin defects repair model. Mouse pESCs expressed stemness marker and could form teratoma containing three germ layers. MSCs could be enriched from outgrowths of EBs and directed to differentiate into fibroblastic lineage. These cells express a high level of growth factors including FGF, EGF, VEGF, TGF, PDGF, and IGF1, similar to those of ESC-derived fibroblasts and mouse fibroblasts. Seeded into collagen gels, the fibroblasts derived from pESCs could form TESE. Mouse skin defects could be successfully repaired 15 days after transplantation of TESE constructed by fibroblasts derived from pESCs. pESCs could be induced to differentiate into fibroblastic lineage, which could be applied to the construction of TESE and skin defect repair. Particularly, pESC derivatives avoid the limitations of political and ethical concerns, and provide a promising source for regenerative medicine.

  11. In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells.

    Science.gov (United States)

    Ishikura, Yukiko; Yabuta, Yukihiro; Ohta, Hiroshi; Hayashi, Katsuhiko; Nakamura, Tomonori; Okamoto, Ikuhiro; Yamamoto, Takuya; Kurimoto, Kazuki; Shirane, Kenjiro; Sasaki, Hiroyuki; Saitou, Mitinori

    2016-12-06

    The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. B cell-targeted therapy with anti-CD20 monoclonal antibody in a mouse model of Graves' hyperthyroidism.

    Science.gov (United States)

    Ueki, I; Abiru, N; Kobayashi, M; Nakahara, M; Ichikawa, T; Eguchi, K; Nagayama, Y

    2011-03-01

    Graves' disease is a B cell-mediated and T cell-dependent autoimmune disease of the thyroid which is characterized by overproduction of thyroid hormones and thyroid enlargement by agonistic anti-thyrotrophin receptor (TSHR) autoantibody. In addition to antibody secretion, B cells have recently been recognized to function as antigen-presenting/immune-modulatory cells. The present study was designed to evaluate the efficacy of B cell depletion by anti-mouse (m) CD20 monoclonal antibody (mAb) on Graves' hyperthyroidism in a mouse model involving repeated injection of adenovirus expressing TSHR A-subunit (Ad-TSHR289). We observe that a single injection of 250 µg/mouse anti-mCD20 mAb eliminated B cells efficiently from the periphery and spleen and to a lesser extent from the peritoneum for more than 3 weeks. B cell depletion before immunization suppressed an increase in serum immunoglobulin (Ig)G levels, TSHR-specific splenocyte secretion of interferon (IFN)-γ, anti-TSHR antibody production and development of hyperthyroidism. B cell depletion 2 weeks after the first immunization, a time-point at which T cells were primed but antibody production was not observed, was still effective at inhibiting antibody production and disease development without inhibiting splenocyte secretion of IFN-γ. By contrast, B cell depletion in hyperthyroid mice was therapeutically ineffective. Together, these data demonstrate that B cells are critical not only as antibody-producing cells but also as antigen-presenting/immune-modulatory cells in the early phase of the induction of experimental Graves' hyperthyroidism and, although therapeutically less effective, B cell depletion is highly efficient for preventing disease development. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.

  13. B cell-targeted therapy with anti-CD20 monoclonal antibody in a mouse model of Graves' hyperthyroidism

    Science.gov (United States)

    Ueki, I; Abiru, N; Kobayashi, M; Nakahara, M; Ichikawa, T; Eguchi, K; Nagayama, Y

    2011-01-01

    Graves' disease is a B cell-mediated and T cell-dependent autoimmune disease of the thyroid which is characterized by overproduction of thyroid hormones and thyroid enlargement by agonistic anti-thyrotrophin receptor (TSHR) autoantibody. In addition to antibody secretion, B cells have recently been recognized to function as antigen-presenting/immune-modulatory cells. The present study was designed to evaluate the efficacy of B cell depletion by anti-mouse (m) CD20 monoclonal antibody (mAb) on Graves' hyperthyroidism in a mouse model involving repeated injection of adenovirus expressing TSHR A-subunit (Ad-TSHR289). We observe that a single injection of 250 µg/mouse anti-mCD20 mAb eliminated B cells efficiently from the periphery and spleen and to a lesser extent from the peritoneum for more than 3 weeks. B cell depletion before immunization suppressed an increase in serum immunoglobulin (Ig)G levels, TSHR-specific splenocyte secretion of interferon (IFN)-γ, anti-TSHR antibody production and development of hyperthyroidism. B cell depletion 2 weeks after the first immunization, a time-point at which T cells were primed but antibody production was not observed, was still effective at inhibiting antibody production and disease development without inhibiting splenocyte secretion of IFN-γ. By contrast, B cell depletion in hyperthyroid mice was therapeutically ineffective. Together, these data demonstrate that B cells are critical not only as antibody-producing cells but also as antigen-presenting/immune-modulatory cells in the early phase of the induction of experimental Graves' hyperthyroidism and, although therapeutically less effective, B cell depletion is highly efficient for preventing disease development. PMID:21235532

  14. Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs

    Science.gov (United States)

    Schatten, Heide; Schatten, Gerald; Balczon, Ron; Simerly, Calvin; Mazia, Daniel

    1986-01-01

    The behavior of centrosomes during the stages of fertilization and cell division in mouse oocytes and in sea urchin eggs was monitored in an immunofluorescence microscope, using autoimmune centrosomal antiserum derived from a patient with scleroderma to label the centrosomal material. These observations showed that centrosomes reproduce during the interphase and aggregate and separate during cell mitosis. Results supported the hypothesis of Mazia (1984), who proposed that centrosomes are 'flexible bodies'. It was also found that, while the sea urchin centrosomes are paternally inherited as was initially proposed by Bovery (1904), the mouse centrosomes are of maternal origin.

  15. Single cell analysis of caspase-3 in apoptotic and non-apoptotic cells during mouse limb development

    Czech Academy of Sciences Publication Activity Database

    Adamová, Eva; Klepárník, Karel; Matalová, E.

    2014-01-01

    Roč. 3, - (2014), PP58 ISSN 2052-1219. [European Calcified Tissue Society Congress /41./. 17.05.2014-20.05.2014, Praha] R&D Projects: GA ČR GAP206/11/2377; GA ČR(CZ) GA14-28254S Institutional support: RVO:68081715 Keywords : single cell analysis * caspase-3 * mouse limb development Subject RIV: CB - Analytical Chemistry, Separation

  16. Cell cycle-dependent expression of Dub3, Nanog and the p160 family of nuclear receptor coactivators (NCoAs in mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Siem van der Laan

    Full Text Available Pluripotency of embryonic stem cells (ESC is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb. Esrrb contributes to the relaxation of the G1 to S-phase (G1/S checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation.

  17. Cell cycle-dependent expression of Dub3, Nanog and the p160 family of nuclear receptor coactivators (NCoAs) in mouse embryonic stem cells.

    Science.gov (United States)

    van der Laan, Siem; Golfetto, Eleonora; Vanacker, Jean-Marc; Maiorano, Domenico

    2014-01-01

    Pluripotency of embryonic stem cells (ESC) is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb). Esrrb contributes to the relaxation of the G1 to S-phase (G1/S) checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs) displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation.

  18. Novel technique to measure total IgM and IgG in vitro haemolysin production by mouse spleen cells, using /sup 51/Cr-labelled sheep red blood cells

    Energy Technology Data Exchange (ETDEWEB)

    Liske, R [Hoffmann-La Roche (F.) and Co., Basel (Switzerland)

    1980-06-12

    A quantitative method for measuring in vitro production of IgM and IgG haemolysis is described. Immune mouse spleen cells, /sup 51/Cr-labelled sheep red blood cells, guinea pig complement and -where applicable- rabbit anti-mouse gammaglobulin serum are incubated in the fluid phase at 37/sup 0/C, and the degree of chromium release measured in the supernatent. The assay gives reproducible results which compare well with the numbers of plaque-forming cells obtained in the conventional plaque-forming assay.

  19. Cell interactions in concanavalin A activated cation flux and DNA synthesis of mouse lymphocytes

    DEFF Research Database (Denmark)

    Owens, T; Kaplan, J G

    1980-01-01

    Co-culture at constant cell density of nude mouse spleen cells (by themselves unresponsive to the T-cell mitogen concanavalin A (Con A)), with congenic T-enriched lymphocyte suspensions and Con A caused anomalously high activation of K+ transport (measured by 86Rb uptake) and of incorporation...... cells. Attempts to demonstrate a diffusible factor in the supernatants of stimulated T cells were unsuccessful. The measured interaction is sufficient to explain our previous paradoxical findings that enrichment of T cells as measured by membrane markers did not cause a corresponding enrichment...

  20. A neonatal mouse spinal cord injury model for assessing post-injury adaptive plasticity and human stem cell integration.

    Directory of Open Access Journals (Sweden)

    Jean-Luc Boulland

    Full Text Available Despite limited regeneration capacity, partial injuries to the adult mammalian spinal cord can elicit variable degrees of functional recovery, mediated at least in part by reorganization of neuronal circuitry. Underlying mechanisms are believed to include synaptic plasticity and collateral sprouting of spared axons. Because plasticity is higher in young animals, we developed a spinal cord compression (SCC injury model in the neonatal mouse to gain insight into the potential for reorganization during early life. The model provides a platform for high-throughput assessment of functional synaptic connectivity that is also suitable for testing the functional integration of human stem and progenitor cell-derived neurons being considered for clinical cell replacement strategies. SCC was generated at T9-T11 and functional recovery was assessed using an integrated approach including video kinematics, histology, tract tracing, electrophysiology, and high-throughput optical recording of descending inputs to identified spinal neurons. Dramatic degeneration of axons and synaptic contacts was evident within 24 hours of SCC, and loss of neurons in the injured segment was evident for at least a month thereafter. Initial hindlimb paralysis was paralleled by a loss of descending inputs to lumbar motoneurons. Within 4 days of SCC and progressively thereafter, hindlimb motility began to be restored and descending inputs reappeared, but with examples of atypical synaptic connections indicating a reorganization of circuitry. One to two weeks after SCC, hindlimb motility approached sham control levels, and weight-bearing locomotion was virtually indistinguishable in SCC and sham control mice. Genetically labeled human fetal neural progenitor cells injected into the injured spinal cord survived for at least a month, integrated into the host tissue and began to differentiate morphologically. This integrative neonatal mouse model provides opportunities to explore early

  1. Diffusion chamber culture of mouse bone marrow cells, (1)

    International Nuclear Information System (INIS)

    Sigeta, Chiharu; Tanaka, Kimio; Kawakami, Masahito; Takahashi, Hiroshi; Ohkita, Takeshi

    1980-01-01

    Mouse bone marrow cells were cultured in diffusion chambers (DC) implanted in the peritoneal cavity of host mice. Host mice were subjected to (1) irradiation ( 60 Co 800 rad) and/or (2) phenylhydrazine induced anemia and then receiving irradiation ( 60 Co 600 rad). After culture periods of 3-7 days, the total number of cells in DC was increased. A marked increase in DC is due to the proliferation of granulocyte series. When host mice were subjected to anemia and irradiation, the start of cell proliferation in DC was delay about two days. On the whole, anemia and irradiation host reduced a little cell growth in DC. The number of immature granulocytes grown in DC in irradiated hosts or anemia and irradiated hosts increased and reached a plateu at day 5. During the plateu period, the proportions between immature and mature granulocytes in DC were kept constantly. The number of macrophages showed a two-phase increasing. Erythroid cells and lymphocytes rapidly disappeared from the chambers during 3 days. The number of erythroid cells was not significantly influenced even in anemia and irradiation hosts. (author)

  2. Nuclear Reprogramming in Mouse Primordial Germ Cells: Epigenetic Contribution

    Directory of Open Access Journals (Sweden)

    Massimo De Felici

    2011-01-01

    Full Text Available The unique capability of germ cells to give rise to a new organism, allowing the transmission of primary genetic information from generation to generation, depends on their epigenetic reprogramming ability and underlying genomic totipotency. Recent studies have shown that genome-wide epigenetic modifications, referred to as “epigenetic reprogramming”, occur during the development of the gamete precursors termed primordial germ cells (PGCs in the embryo. This reprogramming is likely to be critical for the germ line development itself and necessary to erase the parental imprinting and setting the base for totipotency intrinsic to this cell lineage. The status of genome acquired during reprogramming and the associated expression of key pluripotency genes render PGCs susceptible to transform into pluripotent stem cells. This may occur in vivo under still undefined condition, and it is likely at the origin of the formation of germ cell tumors. The phenomenon appears to be reproduced under partly defined in vitro culture conditions, when PGCs are transformed into embryonic germ (EG cells. In the present paper, I will try to summarize the contribution that epigenetic modifications give to nuclear reprogramming in mouse PGCs.

  3. Sox17-Mediated XEN Cell Conversion Identifies Dynamic Networks Controlling Cell-Fate Decisions in Embryo-Derived Stem Cells

    Directory of Open Access Journals (Sweden)

    Angela C.H. McDonald

    2014-10-01

    Full Text Available Little is known about the gene regulatory networks (GRNs distinguishing extraembryonic endoderm (ExEn stem (XEN cells from those that maintain the extensively characterized embryonic stem cell (ESC. An intriguing network candidate is Sox17, an essential transcription factor for XEN derivation and self-renewal. Here, we show that forced Sox17 expression drives ESCs toward ExEn, generating XEN cells that contribute to ExEn when placed back into early mouse embryos. Transient Sox17 expression is sufficient to drive this fate change during which time cells transit through distinct intermediate states prior to the generation of functional XEN-like cells. To orchestrate this conversion process, Sox17 acts in autoregulatory and feedforward network motifs, regulating dynamic GRNs directing cell fate. Sox17-mediated XEN conversion helps to explain the regulation of cell-fate changes and reveals GRNs regulating lineage decisions in the mouse embryo.

  4. Development of mPMab-1, a Mouse-Rat Chimeric Antibody Against Mouse Podoplanin.

    Science.gov (United States)

    Yamada, Shinji; Kaneko, Mika K; Nakamura, Takuro; Ichii, Osamu; Konnai, Satoru; Kato, Yukinari

    2017-04-01

    Podoplanin (PDPN), the ligand of C-type lectin-like receptor-2, is used as a lymphatic endothelial marker. We previously established clone PMab-1 of rat IgG 2a as a specific monoclonal antibody (mAb) against mouse PDPN. PMab-1 is also very sensitive in immunohistochemical analysis; however, rat mAbs seem to be unfavorable for pathologists because anti-mouse IgG and anti-rabbit IgG are usually used as secondary antibodies in commercially available kits for immunohistochemical analysis. In this study, we develop a mouse-rat chimeric antibody, mPMab-1 of mouse IgG 2a , which was derived from rat PMab-1 mAb. Immunohistochemical analysis shows that mPMab-1 detects podocytes of the kidney, lymphatic endothelial cells of the colon, and type I alveolar cells of the lung. Importantly, mPMab-1 is more sensitive than PMab-1. This conversion strategy from rat mAb to mouse mAb could be applicable to other mAbs.

  5. The toxicity of silver and silica nanoparticles in comparable human and mouse cell lines

    DEFF Research Database (Denmark)

    Foldbjerg, Rasmus; Beer, Christiane; Sutherland, Duncan S

    The toxicity of silica (SiO2) and PVP-coated silver (Ag) nanoparticles (NPs) was investigated in two pairs of human or mouse cell lines originating from lung epithelium (A549 and ASB-XIV) and macrophages (THP-1 and J744A.1). Both NPs were characterized in H2O and cell media and demonstrated to be...

  6. Micronuclei induced by municipal landfill leachate in mouse bone marrow cells in vivo

    International Nuclear Information System (INIS)

    Li Guangke; Sang Nan; Zhao Youcai

    2004-01-01

    The induction of micronuclei (MN) in polychromatic erythrocytes (PCE) of mouse bone marrow by municipal landfill leachate was studied in vivo. Results showed that mouse exposure via drinking water containing various concentrations of leachate caused a significant increase of MN frequencies in a concentration (Chemical oxygen demand measured with potassium dichromate oxidation, COD Cr )-dependent manner. MN induction in female and male mice was different at higher concentrations. This implies that leachate is a genotoxic agent in mammalian cells and that exposure to leachate in an aquatic environment may pose a potential genotoxic risk to human beings

  7. Cell lines generated from a chronic lymphocytic leukemia mouse model exhibit constitutive Btk and Akt signaling

    NARCIS (Netherlands)

    Singh, Simar Pal; Pillai, Saravanan Y.; de Bruijn, Marjolein J. W.; Stadhouders, Ralph; Corneth, Odilia B. J.; van den Ham, Henk Jan; Muggen, Alice; van Ijcken, Wilfred; Slinger, Erik; Kuil, Annemieke; Spaargaren, Marcel; Kater, Arnon P.; Langerak, Anton W.; Hendriks, Rudi W.

    2017-01-01

    Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature CD5(+) B cells in blood. Spontaneous apoptosis of CLL cells in vitro has hampered in-depth investigation of CLL pathogenesis. Here we describe the generation of three monoclonal mouse cell lines, EMC2, EMC4 and EMC6,

  8. Modification of nanocellulose by poly-lysine can inhibit the effect of fumonisin B1 on mouse liver cells.

    Science.gov (United States)

    Jebali, Ali; Yasini Ardakani, Seyed Ali; Shahdadi, Hossein; Balal Zadeh, Mohammad Hossein; Hekmatimoghaddam, Seyedhossein

    2015-02-01

    Fumonisin B1 is an important mycotoxin, mainly produced by Fusarium verticillioides. It has toxic effects on liver, brain, and kidney cells. The first aim of this study was to synthesize nanocellulose modified with poly-lysine (NMPL), and the second aim was to evaluate the adsorption of fumonisin B1 by NMPL. As third aim, the function of mouse liver cells was investigated after exposure to fumonisin B1, and fumonisin B1+ NMPL. In this study, NMPL was prepared using cross-linker, and then incubated with fumonisin B1 at controlled conditions. After incubation, the adsorption and release of fumonisin B1 were evaluated in each condition. Next, mouse liver cells were separately exposed to fumonisin B1, NMPL, and (fumonisin B1+NMPL). Then, the level of aniline aminotransferase (ALT) and aspartate aminotransferase (AST) was evaluated. It was found that both adsorption and release of fumonisin B1 were not affected by temperature and incubation time, but affected by pH and concentration of NMPL. Also, this study showed NMPL could adsorb fumonisin B1 in different foodstuffs. Importantly, although the levels of ALT and AST were increased when the cells were treated with fumonisin B1 alone, they were not affected when exposed to NMPL or (fumonisin B1+NMPL). The authors suggest that NMPL is a good adsorbent to remove and inhibit fumonisin B1. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Cocaine- and amphetamine-regulated transcript promotes the differentiation of mouse bone marrow-derived mesenchymal stem cells into neural cells

    OpenAIRE

    Jin Jiali; Chen Zhibin; Zhang Meijuan; Huang Danqing; Liu Zhuo; Huang Siyuan; Zhang Zhuo; Wang Zhongyuan; Chen Lei; Chen Ling; Xu Yun

    2011-01-01

    Abstract Background Neural tissue has limited potential to self-renew after neurological damage. Cell therapy using BM-MSCs (bone marrow mesenchymal stromal cells) seems like a promising approach for the treatment of neurological diseases. However, the neural differentiation of stem cells influenced by massive factors and interactions is not well studied at present. Results In this work, we isolated and identified MSCs from mouse bone marrow. Co-cultured with CART (0.4 nM) for six days, BM-MS...

  10. Effect of low dose X-ray irradiation on apoptosis in spermatogenic cells of mouse testes

    International Nuclear Information System (INIS)

    Liu Guangwei; Liu Shuchun; Lu Zhe; Gong Shouliang

    2003-01-01

    To study the effects of low dose radiation (LDR) with different doses of X-rays on the apoptosis in spermatogenic cells of male Kunming mouse testes. The time-effect and dose-effect of apoptosis in the different stages of spermatogenic cell cycles of mouse testis after LDR with different doses of X-rays were studied with light microscope using the methods of TdT-mediated dUTP nick end labeling (TUNEL) and HE staining. The apoptosis of spermatogenic cells induced by LDR had a remarkable regularity in cell types. When the dose was 0.025 Gy, spermatogonium apoptosis was taken as main. With the dose increase of irradiation (0.025-0.2 Gy), spermatocytes also showed an apoptotic change, but the apoptotic rate of spermatogonia was significantly higher than that of spermatocytes. Moreover, the apoptosis of spermatids and spermatozoa scarcely occurred after irradiation with low dose. The apoptosis of spermatogenic cells induced by LDR has a regular change, which provides a further experimental evidence for the mechanism study of hormesis by LDR

  11. Hypoxia-response plasmid vector producing bcl-2 shRNA enhances the apoptotic cell death of mouse rectum carcinoma.

    Science.gov (United States)

    Fujioka, Takashi; Matsunaga, Naoya; Okazaki, Hiroyuki; Koyanagi, Satoru; Ohdo, Shigehiro

    2010-01-01

    Hypoxia-induced gene expression frequently occurs in malignant solid tumors because they often have hypoxic areas in which circulation is compromised due to structurally disorganized blood vessels. Hypoxia-response elements (HREs) are responsible for activating gene transcription in response to hypoxia. In this study, we constructed a hypoxia-response plasmid vector producing short hairpin RNA (shRNA) against B-cell leukemia/lymphoma-2 (bcl-2), an anti-apoptotic factor. The hypoxia-response promoter was made by inserting tandem repeats of HREs upstream of cytomegalovirus (CMV) promoter (HRE-CMV). HRE-CMV shbcl-2 vector consisted of bcl-2 shRNA under the control of HRE-CMV promoter. In hypoxic mouse rectum carcinoma cells (colon-26), the production of bcl-2 shRNA driven by HRE-CMV promoter was approximately 2-fold greater than that driven by CMV promoter. A single intratumoral (i.t.) injection of 40 microg HRE-CMV shbcl-2 to colon-26 tumor-bearing mice caused apoptotic cell death, and repetitive treatment with HRE-CMV shbcl-2 (40 microg/mouse, i.t.) also significantly suppressed the growth of colon-26 tumor cells implanted in mice. Apoptotic and anti-tumor effects were not observed in tumor-bearing mice treated with CMV shbcl-2. These results reveal the ability of HRE-CMV shbcl-2 vector to suppress the expression of bcl-2 in hypoxic tumor cells and suggest the usefulness of our constructed hypoxia-response plasmid vector to treat malignant tumors. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10054FP].

  12. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    Science.gov (United States)

    Krivokharchenko, Alexander; Karmenyan, Artashes; Sarkisov, Oleg; Bader, Michael; Chiou, Arthur; Shakhbazyan, Avetik

    2012-01-01

    Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

  13. CAR-mediated repression of Foxo1 transcriptional activity regulates the cell cycle inhibitor p21 in mouse livers

    International Nuclear Information System (INIS)

    Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O.

    2014-01-01

    Highlights: • CAR activation decreased the level of Foxo1 in mouse livers. • CAR activation decreased the level of p21 in mouse livers. • CAR activation inhibited Foxo1 transcriptional activity in mouse livers. - Abstract: 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments

  14. Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

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    Yamano, Noriko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Kimura, Tohru, E-mail: tkimura@patho.med.osaka-u.ac.jp [Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Watanabe-Kushima, Shoko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Shinohara, Takashi [Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501 (Japan); Nakano, Toru, E-mail: tnakano@patho.med.osaka-u.ac.jp [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)

    2010-02-12

    Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.

  15. ProSAAS-derived peptides are differentially processed and sorted in mouse brain and AtT-20 cells.

    Directory of Open Access Journals (Sweden)

    Jonathan H Wardman

    Full Text Available ProSAAS is the precursor for some of the most abundant peptides found in mouse brain and other tissues, including peptides named SAAS, PEN, and LEN. Both SAAS and LEN are found in big and little forms due to differential processing. Initial processing of proSAAS is mediated by furin (and/or furin-like enzymes and carboxypeptidase D, while the smaller forms are generated by secretory granule prohormone convertases and carboxypeptidase E. In mouse hypothalamus, PEN and big LEN colocalize with neuropeptide Y. In the present study, little LEN and SAAS were detected in mouse hypothalamus but not in cell bodies of neuropeptide Y-expressing neurons. PEN and big LEN show substantial colocalization in hypothalamus, but big LEN and little LEN do not. An antiserum to SAAS that detects both big and little forms of this peptide did not show substantial colocalization with PEN or big LEN. To further study this, the AtT-20 cells mouse pituitary corticotrophic cell line was transfected with rat proSAAS and the distribution of peptides examined. As found in mouse hypothalamus, only some of the proSAAS-derived peptides colocalized with each other in AtT-20 cells. The two sites within proSAAS that are known to be efficiently cleaved by furin were altered by site-directed mutagenesis to convert the P4 Arg into Lys; this change converts the sequences from furin consensus sites into prohormone convertase consensus sites. Upon expression of the mutated form of proSAAS in AtT-20 cells, there was significantly more colocalization of proSAAS-derived peptides PEN and SAAS. Taken together, these results indicate that proSAAS is initially cleaved in the Golgi or trans-Golgi network by furin and/or furin-like enzymes and the resulting fragments are sorted into distinct vesicles and further processed by additional enzymes into the mature peptides.

  16. Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis.

    Science.gov (United States)

    Musicki, Biljana; Liu, Tongyun; Sezen, Sena F; Burnett, Arthur L

    2012-08-01

    Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Relative to hemi mice, SCD increased (Ppenis. Apocynin treatment of sickle mice reversed (P0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis. © 2012 International Society for Sexual Medicine.

  17. The Effect of Combination of Radiation with 5-Fluorouracil on Mouse Jejunal Crypt Cells

    International Nuclear Information System (INIS)

    Huh, Seung Jae; Park, Charn Il

    1985-01-01

    The interaction of radiation and 5-Fluorouracil (5-FU) on mouse jejunal crypt cells was studied using the microcolony survival assay. 150mg/kg of 5-FU was injected intraperitoneally 15 minutes before irradiation and 6 hours after irradiation. Jejunal crypt cells of mouse survived more when 5-FU was given 15 minutes before irradiation than giving it 6 hours after irradiation. The mean lethal doses (Do) of each of irradiation alone group, 5-FU injection group of 15 minutes preceding irradiation, and 5-FU injection group of 6 hours post irradiation were 135, 135, and 114 rad respectively. The dose effect factor (DEF) of each of 5-FU injection groups of 15 minutes preceding irradiation and of 6 hours post irradiation were 1.13 and 1.27

  18. Human spermatogonial stem cells display limited proliferation in vitro under mouse spermatogonial stem cell culture conditions.

    Science.gov (United States)

    Medrano, Jose V; Rombaut, Charlotte; Simon, Carlos; Pellicer, Antonio; Goossens, Ellen

    2016-11-01

    To study the ability of human spermatogonial stem cells (hSSCs) to proliferate in vitro under mouse spermatogonial stem cell (mSSC) culture conditions. Experimental basic science study. Reproductive biology laboratory. Cryopreserved testicular tissue with normal spermatogenesis obtained from three donors subjected to orchiectomy due to a prostate cancer treatment. Testicular cells used to create in vitro cell cultures corresponding to the following groups: [1] unsorted human testicular cells, [2] differentially plated human testicular cells, and [3] cells enriched with major histocompatibility complex class 1 (HLA - )/epithelial cell surface antigen (EPCAM + ) in coculture with inactivated testicular feeders from the same patient. Analyses and characterization including immunocytochemistry and quantitative reverse-transcription polymerase chain reaction for somatic and germ cell markers, testosterone and inhibin B quantification, and TUNEL assay. Putative hSSCs appeared in singlets, doublets, or small groups of up to four cells in vitro only when testicular cells were cultured in StemPro-34 medium supplemented with glial cell line-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF). Fluorescence-activated cell sorting with HLA - /EPCAM + resulted in an enrichment of 27% VASA + /UTF1 + hSSCs, compared to 13% in unsorted controls. Coculture of sorted cells with inactivated testicular feeders gave rise to an average density of 112 hSSCs/cm 2 after 2 weeks in vitro compared with unsorted cells (61 hSSCs/cm 2 ) and differentially plated cells (49 hSSCS/cm 2 ). However, putative hSSCs rarely stained positive for the proliferation marker Ki67, and their presence was reduced to the point of almost disappearing after 4 weeks in vitro. We found that hSSCs show limited proliferation in vitro under mSSC culture conditions. Coculture of HLA - /EPCAM + sorted cells with testicular

  19. Posttranslational inactivation of endothelial nitric oxide synthase in the transgenic sickle cell mouse penis

    Science.gov (United States)

    Musicki, Biljana; Champion, Hunter C.; Hsu, Lewis L.; Bivalacqua, Trinity J.; Burnett, Arthur L.

    2017-01-01

    INTRODUCTION Sickle cell disease (SCD)-associated priapism is characterized by endothelial nitric oxide synthase (eNOS) dysfunction in the penis. However, the mechanism of decreased eNOS function/activation in the penis in association with SCD is not known. AIMS Our hypothesis in the present study was that eNOS is functionally inactivated in the SCD penis in association with impairments in eNOS posttranslational phosphorylation and the enzyme’s interactions with its regulatory proteins. METHODS Sickle cell transgenic (sickle) mice were used as an animal model of SCD. Wild type (WT) mice served as controls. Penes were excised at baseline for molecular studies. eNOS phosphorylation on Ser-1177 (positive regulatory site) and Thr-495 (negative regulatory site), total eNOS, and phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177) expressions, and eNOS interactions with heat shock protein 90 (HSP90) and caveolin-1 were measured by Western blot. Constitutive NOS catalytic activity was measured by conversion of L-[14C]arginine-to-L-[14C]citrulline in the presence of calcium. MAIN OUTCOME MEASURES Molecular mechanisms of eNOS dysfunction in the sickle mouse penis. RESULTS eNOS phosphorylated on Ser-1177, an active portion of eNOS, was decreased in the sickle mouse penis compared to WT penis. eNOS interaction with its positive protein regulator HSP90, but not with its negative protein regulator caveolin-1, and phosphorylated AKT expression, as well as constitutive NOS activity, were also decreased in the sickle mouse penis compared to WT penis. eNOS phosphorylated on Thr-495, total eNOS, HSP90, and caveolin-1 protein expressions in the penis were not affected by SCD. CONCLUSION These findings provide a molecular basis for chronically reduced eNOS function in the penis by SCD, which involves decreased eNOS phosphorylation on Ser-1177 and decreased eNOS-HSP90 interaction. PMID:21143412

  20. Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells.

    Science.gov (United States)

    Hayashi, Katsuhiko; Saitou, Mitinori

    2013-08-01

    Oogenesis is an integrated process through which an egg acquires the potential for totipotency, a fundamental condition for creating new individuals. Reconstitution of oogenesis in a culture that generates eggs with proper function from pluripotent stem cells (PSCs) is therefore one of the key goals in basic biology as well as in reproductive medicine. Here we describe a stepwise protocol for the generation of eggs from mouse PSCs, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs are first induced into primordial germ cell-like cells (PGCLCs) that are in turn aggregated with somatic cells of female embryonic gonads, the precursors for adult ovaries. Induction of PGCLCs followed by aggregation with the somatic cells takes up to 8 d. The aggregations are then transplanted under the ovarian bursa, in which PGCLCs grow into germinal vesicle (GV) oocytes in ∼1 month. The PGCLC-derived GV oocytes can be matured into eggs in 1 d by in vitro maturation (IVM), and they can be fertilized with spermatozoa by in vitro fertilization (IVF) to obtain healthy and fertile offspring. This method provides an initial step toward reconstitution of the entire process of oogenesis in vitro.

  1. The cellular cancer resistance of the SR/CR mouse

    DEFF Research Database (Denmark)

    Koch, Janne; Hau, Jann; Jensen, Henrik Elvang

    2012-01-01

    The SR/CR mouse phenotype, first described in 1999 in BALB/c and later bred into C57BL/6 mice, is resistant to cancer formation following high doses of cancer cells administered intraperitoneally. The tumor cell targeting and destruction mechanisms have not been identified. By fluorescence-activa...... controls. Importantly, this differentially regulated immune response of SR/CR mice could not be found in response to challenge with the lymphoma cell line EL-4....

  2. Antitumor effect of cordycepin (3'-deoxyadenosine) on mouse melanoma and lung carcinoma cells involves adenosine A3 receptor stimulation.

    Science.gov (United States)

    Nakamura, Kazuki; Yoshikawa, Noriko; Yamaguchi, Yu; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru

    2006-01-01

    An attempt was made to elucidate the molecular targetfor the antitumor effects of cordycepin (3'-deoxyadenosine) using non-selective and selective adenosine A1, A2a, A2b and A3 receptor agonists and antagonists. Although adenosine and 2'-deoxyadenosine (up to 100 microM) had no effect, cordycepin showed remarkable inhibitory effects on the growth curves of B16-BL6 mouse melanoma (IC50= 39 microM) and mouse Lewis lung carcinoma (IC50 = 48 microM) cell lines in vitro. Among the adenosine receptor agonists and antagonists used (up to 100 microM), only 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), a selective adenosine A3 receptor agonist, notably inhibited the growth of both mouse tumor cell lines (B16-BL6; IC50 = 5 microM, LLC; 14 microM). In addition, the tumor growth inhibitory effect of cordycepin was antagonized by 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191), a selective adenosine A3 receptor antagonist. These results suggest that cordycepin exerts inhibitory effects on the growth of mouse melanoma and lung carcinoma cells by stimulating adenosine A3 receptors on tumor cells.

  3. Ibrutinib suppresses alloantibody responses in a mouse model of allosensitization.

    Science.gov (United States)

    Kim, Irene; Wu, Gordon; Chai, Ning-Ning; Klein, Andrew S; Jordan, Stanley

    2017-12-01

    Ibrutinib is a Bruton's tyrosine Kinase (BTK) antagonist that inhibits B cell receptor (BCR) signaling. Complete BTK deficiency is associated with absence of B-cells. Ibrutinb is currently approved by FDA for treatment of B-cell malignancies, including Waldenström macroglobulinaemia. We recently carried out studies to determine if ibrutinib could modify alloantibody responses. A mouse model of allogenic sensitization using a C57BL/6 mouse as the recipient of a skin allograft from an HLA-A2 transgenic mouse was utilized to examine the effects of ibrutinib on alloantibody responses and B cell effector functions. Donor-specific antibody (DSA) levels were measured in a flow-cytometric antibody binding assay. Splenic T and B cell subsets and plasma cells were analyzed in flow cytometry. Control mice developed peak levels of DSA IgM at day 14 PTx while the ibrutinib treated mice had significantly lower levels of DSA IgM (p=0.0047). Control mice developed HLA.A2-specific IgG antibodies at day 14 (230±60 MFI) and reached peak levels at day 21 (426±61 MFI). In contrast, mice in the treatment group had low levels of HLA.A2-specific IgG at day 14 (109±59 MFI, p=0.004) and day 21 (241±86 MFI, p=0.003). FACS analysis found a reduction of B220 + or CD19 + B cell population (pibrutinib attenuated recall DSA IgG responses to re-sensitization (pIbrutinib is effective in suppressing alloantibody responses through blocking BTK-mediated BCR signaling, leading to reduction of B cells and short-lived plasma cells in the spleens. Use of ibrutinib may provide benefits to HLA-sensitized transplant patients for alloantibody suppression. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Planar cell polarity enables posterior localization of nodal cilia and left-right axis determination during mouse and Xenopus embryogenesis.

    Directory of Open Access Journals (Sweden)

    Dragana Antic

    2010-02-01

    Full Text Available Left-right asymmetry in vertebrates is initiated in an early embryonic structure called the ventral node in human and mouse, and the gastrocoel roof plate (GRP in the frog. Within these structures, each epithelial cell bears a single motile cilium, and the concerted beating of these cilia produces a leftward fluid flow that is required to initiate left-right asymmetric gene expression. The leftward fluid flow is thought to result from the posterior tilt of the cilia, which protrude from near the posterior portion of each cell's apical surface. The cells, therefore, display a morphological planar polarization. Planar cell polarity (PCP is manifested as the coordinated, polarized orientation of cells within epithelial sheets, or as directional cell migration and intercalation during convergent extension. A set of evolutionarily conserved proteins regulates PCP. Here, we provide evidence that vertebrate PCP proteins regulate planar polarity in the mouse ventral node and in the Xenopus gastrocoel roof plate. Asymmetric anterior localization of VANGL1 and PRICKLE2 (PK2 in mouse ventral node cells indicates that these cells are planar polarized by a conserved molecular mechanism. A weakly penetrant Vangl1 mutant phenotype suggests that compromised Vangl1 function may be associated with left-right laterality defects. Stronger functional evidence comes from the Xenopus GRP, where we show that perturbation of VANGL2 protein function disrupts the posterior localization of motile cilia that is required for leftward fluid flow, and causes aberrant expression of the left side-specific gene Nodal. The observation of anterior-posterior PCP in the mouse and in Xenopus embryonic organizers reflects a strong evolutionary conservation of this mechanism that is important for body plan determination.

  5. Wound Healing Activity of Extracts and Formulations of Aloe vera, Henna, Adiantum capillus-veneris, and Myrrh on Mouse Dermal Fibroblast Cells.

    Science.gov (United States)

    Negahdari, Samira; Galehdari, Hamid; Kesmati, Mahnaz; Rezaie, Anahita; Shariati, Gholamreza

    2017-01-01

    Among the most important factors in wound healing pathways are transforming growth factor beta1 and vascular endothelial growth factor. Fibroblasts are the main cell in all phases wound closure. In this study, the extracts of plant materials such as Adiantum capillus-veneris , Commiphora molmol , Aloe vera , and henna and one mixture of them were used to treatment of normal mouse skin fibroblasts. Cytotoxic effects of each extract and their mixture were assessed on mouse skin fibroblasts cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. We performed migration assays to assess migration properties of mouse skin fibroblasts cells in response to the extracts. Changes in the gene expression of the Tgf β1 and Vegf-A genes were monitored by real-time polymerase chain reaction. A. capillus-veneris , C. molmol and henna extract improved the expression of Tgfβ1 gene. All used extracts upregulated the expression of Vegf-A gene and promoted the migration of mouse fibroblast cells in vitro . The present study demonstrated that the mentioned herbal extracts might be effective in wound healing, through the improvement in the migration of fibroblast cells and regulating the gene expression of Tgfβ1 and Vegf-A genes in fibroblast cells treated with extracts.

  6. Mechanical control of notochord morphogenesis by extra-embryonic tissues in mouse embryos.

    Science.gov (United States)

    Imuta, Yu; Koyama, Hiroshi; Shi, Dongbo; Eiraku, Mototsugu; Fujimori, Toshihiko; Sasaki, Hiroshi

    2014-05-01

    Mammalian embryos develop in coordination with extraembryonic tissues, which support embryonic development by implanting embryos into the uterus, supplying nutrition, providing a confined niche, and also providing patterning signals to embryos. Here, we show that in mouse embryos, the expansion of the amniotic cavity (AC), which is formed between embryonic and extraembryonic tissues, provides the mechanical forces required for a type of morphogenetic movement of the notochord known as convergent extension (CE) in which the cells converge to the midline and the tissue elongates along the antero-posterior (AP) axis. The notochord is stretched along the AP axis, and the expansion of the AC is required for CE. Both mathematical modeling and physical simulation showed that a rectangular morphology of the early notochord caused the application of anisotropic force along the AP axis to the notochord through the isotropic expansion of the AC. AC expansion acts upstream of planar cell polarity (PCP) signaling, which regulates CE movement. Our results highlight the importance of extraembryonic tissues as a source of the forces that control the morphogenesis of embryos. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  7. Effect of diphenyl ether herbicides and oxadiazon on porphyrin biosynthesis in mouse liver, rat primary hepatocyte culture and HepG2 cells.

    Science.gov (United States)

    Krijt, J; van Holsteijn, I; Hassing, I; Vokurka, M; Blaauboer, B J

    1993-01-01

    The effects of the herbicides fomesafen, oxyfluorfen, oxadiazon and fluazifop-butyl on porphyrin accumulation in mouse liver, rat primary hepatocyte culture and HepG2 cells were investigated. Ten days of herbicide feeding (0.25% in the diet) increased the liver porphyrins in male C57B1/6J mice from 1.4 +/- 0.6 to 4.8 +/- 2.1 (fomesafen) 16.9 +2- 2.9 (oxyfluorfen) and 25.9 +/- 3.1 (oxadiazon) nmol/g wet weight, respectively. Fluazifop-butyl had no effect on liver porphyrin metabolism. Fomesafen, oxyfluorfen and oxadiazon increased the cellular porphyrin content of rat hepatocytes after 24 h of incubation (control, 3.2 pmol/mg protein, fomesafen, oxyfluorfen and oxadiazon at 0.125 mM concentration 51.5, 54.3 and 44.0 pmol/mg protein, respectively). The porphyrin content of HepG2 cells increased from 1.6 to 18.2, 10.6 and 9.2 pmol/mg protein after 24 h incubation with the three herbicides. Fluazifop-butyl increased hepatic cytochrome P450 levels and ethoxy- and pentoxyresorufin O-dealkylase (EROD and PROD) activity, oxyfluorfen increased PROD activity. Peroxisomal palmitoyl CoA oxidation increased after fomesafen and fluazifop treatment to about 500% of control values both in mouse liver and rat hepatocytes. Both rat hepatocytes and HepG2 cells can be used as a test system for the porphyrogenic potential of photobleaching herbicides.

  8. Mouse androgenetic embryonic stem cells differentiated to multiple cell lineages in three embryonic germ layers in vitro.

    Science.gov (United States)

    Teramura, Takeshi; Onodera, Yuta; Murakami, Hideki; Ito, Syunsuke; Mihara, Toshihiro; Takehara, Toshiyuki; Kato, Hiromi; Mitani, Tasuku; Anzai, Masayuki; Matsumoto, Kazuya; Saeki, Kazuhiro; Fukuda, Kanji; Sagawa, Norimasa; Osoi, Yoshihiko

    2009-06-01

    The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible tissues for transplantation.

  9. Pre-crisis mouse cells show strain-specific covariation in the amount of 54-kilodalton phosphoprotein and in susceptibility to transformation by simian virus 40.

    Science.gov (United States)

    Chen, S; Blanck, G; Pollack, R E

    1983-09-01

    We have used several inbred mouse strains to examine the role of the 54-kilodalton (kDa) cellular phosphoprotein in transformation by the papovavirus simian virus 40. We have measured the endogenous 54-kDa phosphoprotein in cells obtained from these inbred mouse strains. To study the effect of passage, cell cultures were measured for amount of the 54-kDa phosphoprotein at the 2nd and 12th passages. In the absence of any transforming agent, the amount of endogenous 54-kDa phosphoprotein in early pre-crisis mouse cells varied in a strain-specific way. Transformation frequency varied coordinately with endogenous 54-kDa expression. Mouse strains whose cells produced a high level of endogenous 54-kDa phosphoprotein on passage did not further increase its expression after simian virus 40 transformation.

  10. Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice.

    Directory of Open Access Journals (Sweden)

    Budd A Tucker

    2011-04-01

    Full Text Available This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs, could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.

  11. Genome editing in mouse spermatogonial stem/progenitor cells using engineered nucleases.

    Directory of Open Access Journals (Sweden)

    Danielle A Fanslow

    Full Text Available Editing the genome to create specific sequence modifications is a powerful way to study gene function and promises future applicability to gene therapy. Creation of precise modifications requires homologous recombination, a very rare event in most cell types that can be stimulated by introducing a double strand break near the target sequence. One method to create a double strand break in a particular sequence is with a custom designed nuclease. We used engineered nucleases to stimulate homologous recombination to correct a mutant gene in mouse "GS" (germline stem cells, testicular derived cell cultures containing spermatogonial stem cells and progenitor cells. We demonstrated that gene-corrected cells maintained several properties of spermatogonial stem/progenitor cells including the ability to colonize following testicular transplantation. This proof of concept for genome editing in GS cells impacts both cell therapy and basic research given the potential for GS cells to be propagated in vitro, contribute to the germline in vivo following testicular transplantation or become reprogrammed to pluripotency in vitro.

  12. p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Moscatelli, Ilana; Pierantozzi, Enrico; Camaioni, Antonella; Siracusa, Gregorio [Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome (Italy); Campagnolo, Luisa, E-mail: campagno@med.uniroma2.it [Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome (Italy)

    2009-11-01

    Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75{sup NTR}), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75{sup NTR} and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75{sup NTR} and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75{sup NTR}/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75{sup NTR} or TrkA. Interestingly, immunoreactivity to anti-p75{sup NTR} antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75{sup NTR}, when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75{sup NTR} is turned on.

  13. p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Moscatelli, Ilana; Pierantozzi, Enrico; Camaioni, Antonella; Siracusa, Gregorio; Campagnolo, Luisa

    2009-01-01

    Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75 NTR ), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75 NTR and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75 NTR and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75 NTR /TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75 NTR or TrkA. Interestingly, immunoreactivity to anti-p75 NTR antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75 NTR , when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75 NTR is turned on.

  14. Recombinant Escherichia coli Trx-JZTX-III represses the proliferation of mouse hepatocellular carcinoma cells through induction of cell cycle arrest.

    Science.gov (United States)

    Sun, Mei-Na; Zhao, Xue-Jiao; Zhao, Han-Dong; Zhang, Wei-Guang; Li, Feng-Lan; Chen, Ming-Zi; Li, Hui; Li, Guangchao

    2013-06-01

    The aim of the present study was to investigate the effects of recombinant Escherichia coli (E. coli) Trx-jingzhaotoxin (JZTX)-III on cell growth in the mouse hepatocellular carcinoma (HCC) cell line Hepa1-6. The JZTX-III gene sequence was synthesized and cloned into the pET-32a(+) vector to construct the recombinant fusion protein Trx-JZTX-III, which was subsequently purified. Hepa1-6 cells were treated with 0 to 1,000-µg/ml concentrations of Trx-JZTX-III; this was demonstrated to affect cell viability, as determined by the 3-(4,5-dimethylthiazol‑2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. The expression of the proliferating cell nuclear antigen (PCNA) protein was investigated using western blot analysis. A colony formation assay was used to determine Hepa1-6 cell proliferation, and the migration ability of cells was determined using a wound‑healing assay. Additionally, flow cytometry was employed to observe changes in the cell cycle. The MTT assay and quantification of PCNA expression indicated that recombinant E. coli Trx-JZTX-III significantly repressed the proliferation of Hepa1-6 cells. Colony formation and the migration of malignant cells was inhibited following treatment with recombinant E. coli Trx-JZTX-III. Flow cytometry showed that recombinant E. coli Trx-JZTX-III induced G0/G1 cell cycle arrest. In conclusion, recombinant E. coli Trx-JZTX-III functions as a tumor suppressor drug in mouse HCC and its underlying mechanism may involve the induction of G0/G1 cell cycle arrest.

  15. Single-Cell Landscape of Transcriptional Heterogeneity and Cell Fate Decisions during Mouse Early Gastrulation

    Directory of Open Access Journals (Sweden)

    Hisham Mohammed

    2017-08-01

    Full Text Available The mouse inner cell mass (ICM segregates into the epiblast and primitive endoderm (PrE lineages coincident with implantation of the embryo. The epiblast subsequently undergoes considerable expansion of cell numbers prior to gastrulation. To investigate underlying regulatory principles, we performed systematic single-cell RNA sequencing (seq of conceptuses from E3.5 to E6.5. The epiblast shows reactivation and subsequent inactivation of the X chromosome, with Zfp57 expression associated with reactivation and inactivation together with other candidate regulators. At E6.5, the transition from epiblast to primitive streak is linked with decreased expression of polycomb subunits, suggesting a key regulatory role. Notably, our analyses suggest elevated transcriptional noise at E3.5 and within the non-committed epiblast at E6.5, coinciding with exit from pluripotency. By contrast, E6.5 primitive streak cells became highly synchronized and exhibit a shortened G1 cell-cycle phase, consistent with accelerated proliferation. Our study systematically charts transcriptional noise and uncovers molecular processes associated with early lineage decisions.

  16. The survival of cultured mouse cerebellar granule cells is not dependent on elevated potassium-ion concentration

    DEFF Research Database (Denmark)

    Mogensen, Helle Smidt; Hack, N; Balázs, R

    1994-01-01

    The effects of K(+)-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from d...

  17. Isolation of sphere-forming stem cells from the mouse inner ear.

    Science.gov (United States)

    Oshima, Kazuo; Senn, Pascal; Heller, Stefan

    2009-01-01

    The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies.

  18. Differential subnetwork of chemokines/cytokines in human, mouse, and rat brain cells after oxygen-glucose deprivation.

    Science.gov (United States)

    Du, Yang; Deng, Wenjun; Wang, Zixing; Ning, MingMing; Zhang, Wei; Zhou, Yiming; Lo, Eng H; Xing, Changhong

    2017-04-01

    Mice and rats are the most commonly used animals for preclinical stroke studies, but it is unclear whether targets and mechanisms are always the same across different species. Here, we mapped the baseline expression of a chemokine/cytokine subnetwork and compared responses after oxygen-glucose deprivation in primary neurons, astrocytes, and microglia from mouse, rat, and human. Baseline profiles of chemokines (CX3CL1, CXCL12, CCL2, CCL3, and CXCL10) and cytokines (IL-1α, IL-1β, IL-6, IL-10, and TNFα) showed significant differences between human and rodents. The response of chemokines/cytokines to oxygen-glucose deprivation was also significantly different between species. After 4 h oxygen-glucose deprivation and 4 h reoxygenation, human and rat neurons showed similar changes with a downregulation in many chemokines, whereas mouse neurons showed a mixed response with up- and down-regulated genes. For astrocytes, subnetwork response patterns were more similar in rats and mice compared to humans. For microglia, rat cells showed an upregulation in all chemokines/cytokines, mouse cells had many down-regulated genes, and human cells showed a mixed response with up- and down-regulated genes. This study provides proof-of-concept that species differences exist in chemokine/cytokine subnetworks in brain cells that may be relevant to stroke pathophysiology. Further investigation of differential gene pathways across species is warranted.

  19. Lipofection improves gene targeting efficiency in E14 TG2a mouse embryonic stem cells

    OpenAIRE

    Sandra M. López-Heydeck

    2009-01-01

    Electroporation has been the method of election for transfection of murine embryonic stem cells for over 15 years; however, it is a time consuming protocol because it requires large amounts of DNA and cells, as well as expensive and delicate equipment. Lipofection is a transfection method that requires lower amounts of cells and DNA than electroporation, and has proven to be effi cient in a large number of cell lines. It has been shown that after lipofection, mouse embryonic stem cells remain...

  20. Transformation of mouse embryo (C3H 10T1/2) cells by alpha particles

    International Nuclear Information System (INIS)

    Lloyd, E.L.; Gemmell, A.; Henning, C.B.; Gemmell, D.S.; Zabransky, B.J.

    1977-01-01

    Mammalian cells in culture (C3H mouse 10T1/2 cells) have been shown here for the first time to be transformed by alpha irradiation when cells were irradiated with 5.6 MeV alpha particles from a Tandem Van de Graaff machine. Malignant tumors were induced following inoculation of the transformed cells into syngeneic hosts. Unirradiated control cells injected at the same concentration have, so far, failed to produce tumors. The morphology of the transformed foci was remarkably similar to that obtained by x rays and chemicals but different from virally transformed cells. When the cells were seeded at low density in the exponential growth phase, the transformation frequency per surviving cell increased approximately as the cube of the dose and peaked at an alpha particle fluence between 1.5 and 2.5 x 10 7 alpha particles per cm 2 (205 to 342 rads). The frequency of the transformation was found to be greatly dependent on the number of cells per dish irradiated. Irradiation of larger numbers resulted in much lower frequencies of transformation. The maximum transformation frequency observed in nine separate experiments was 4 percent of the surviving cells. At doses greater than 200 rads the transformation frequency per surviving cell remained constant. The present results permit us to conclude that alpha irradiation may, indeed, be able to exert a direct effect on the genome of the cell to produce malignancy without any external immunological or hormonal influences

  1. Generation of single-copy transgenic mouse embryos directly from ES cells by tetraploid embryo complementation

    Directory of Open Access Journals (Sweden)

    Zhao Roong

    2001-12-01

    Full Text Available Abstract Background Transgenic mice have been used extensively to analyze gene function. Unfortunately, traditional transgenic procedures have only limited use in analyzing alleles that cause lethality because lines of founder mice cannot be established. This is frustrating given that such alleles often reveal crucial aspects of gene function. For this reason techniques that facilitate the generation of embryos expressing such alleles would be of enormous benefit. Although the transient generation of transgenic embryos has allowed limited analysis of lethal alleles, it is expensive, time consuming and technically challenging. Moreover a fundamental limitation with this approach is that each embryo generated is unique and transgene expression is highly variable due to the integration of different transgene copy numbers at random genomic sites. Results Here we describe an alternative method that allows the generation of clonal mouse embryos harboring a single-copy transgene at a defined genomic location. This was facilitated through the production of Hprt negative embryonic stem cells that allow the derivation of embryos by tetraploid embryo complementation. We show that targeting transgenes to the hprt locus in these ES cells by homologous recombination can be efficiently selected by growth in HAT medium. Moreover, embryos derived solely from targeted ES cells containing a single copy LacZ transgene under the control of the α-myosin heavy chain promoter exhibited the expected cardiac specific expression pattern. Conclusion Our results demonstrate that tetraploid embryo complementation by F3 hprt negative ES cells facilitates the generation of transgenic mouse embryos containing a single copy gene at a defined genomic locus. This approach is simple, extremely efficient and bypasses any requirement to generate chimeric mice. Moreover embryos generated by this procedure are clonal in that they are all derived from a single ES cell lines. This

  2. Radiosensitivity and cell kinetics of the human solid cancer transplanted to nude mouse

    International Nuclear Information System (INIS)

    Ikeuchi, Shunji

    1983-01-01

    This study was undertaken to analyse the relationship between radiosensitivity and cell kinetics of human solid cancer in experimental nude mouse system. Four strains of tumors used for the experiment were poorly differentiated squamous cell carcinoma of the lung (Lu-9), oat cell carcinoma of the lung (Lu-24), well differentiated squamous cell carcinoma of the tongue (To-1) and moderately differentiated squamous cell carcinoma of the esophagus (Es-4) which were serially transplantable to BALB/c nude mice. Radiosensitivity was evaluated by tumor growth in terms of inhibition rate, histological change and host reaction after irradiation. Cell kinetics were studied by autoradiography with pulse administration of 3 H-thymidine to mice. Although Lu-24 was most radiosensitive, followed by To-1, Es-4 and Lu-9 in the order of sensitivity, it was suggested that they might be more radioresistant in nude mice without T-cell function than in human. Regarding squamous cell carcinomas, well differentiated type was more radiosensitive than poorly differentiated one. All of these tumors in nude mouse revealed distinct percent labeled mitosis curves with two clear peaks which were quite different from those in human body. Lu-24 showed a characteristic pattern with a long time lag before visible growth, short G 1 , and low growth fraction, compared to other three tumors. Three strains of squamous cell carcinoma demonstrated similar cell kinetic factors which were almost the same as those in human body reported previously. The differences in volume doubling time of tumor, growth fraction and cell loss factor were partially related to those of radiosensitivities among tumors except for Lu-24. The theoretical volume doubling time was proved to be most reliable for estimation of effectiveness of irradiation, but the labeling index was not a valuable indicator for it. (author)

  3. Maternal retinoids control type 3 innate lymphoid cells and set the offspring immunity

    Science.gov (United States)

    van de Pavert, Serge A.; Ferreira, Manuela; Domingues, Rita G.; Ribeiro, Hélder; Molenaar, Rosalie; Moreira-Santos, Lara; Almeida, Francisca F.; Ibiza, Sales; Barbosa, Inês; Goverse, Gera; Labão-Almeida, Carlos; Godinho-Silva, Cristina; Konijn, Tanja; Schooneman, Dennis; O'Toole, Tom; Mizee, Mark R.; Habani, Yasmin; Haak, Esther; Santori, Fabio R.; Littman, Dan R.; Schulte-Merker, Stefan; Dzierzak, Elaine; Simas, J. Pedro; Mebius, Reina E.; Veiga-Fernandes, Henrique

    2014-04-01

    The impact of nutritional status during fetal life on the overall health of adults has been recognized; however, dietary effects on the developing immune system are largely unknown. Development of secondary lymphoid organs occurs during embryogenesis and is considered to be developmentally programmed. Secondary lymphoid organ formation depends on a subset of type 3 innate lymphoid cells (ILC3) named lymphoid tissue inducer (LTi) cells. Here we show that mouse fetal ILC3s are controlled by cell-autonomous retinoic acid (RA) signalling in utero, which pre-sets the immune fitness in adulthood. We found that embryonic lymphoid organs contain ILC progenitors that differentiate locally into mature LTi cells. Local LTi cell differentiation was controlled by maternal retinoid intake and fetal RA signalling acting in a haematopoietic cell-autonomous manner. RA controlled LTi cell maturation upstream of the transcription factor RORγt. Accordingly, enforced expression of Rorgt restored maturation of LTi cells with impaired RA signalling, whereas RA receptors directly regulated the Rorgt locus. Finally, we established that maternal levels of dietary retinoids control the size of secondary lymphoid organs and the efficiency of immune responses in the adult offspring. Our results reveal a molecular link between maternal nutrients and the formation of immune structures required for resistance to infection in the offspring.

  4. Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment.

    Science.gov (United States)

    Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M

    2016-03-01

    T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin (I) as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278.

  5. Differentiation of hematopoietic stem cells in irradiated mouse thymic lobes. Kinetics and phenotype of progeny

    International Nuclear Information System (INIS)

    Spangrude, G.J.; Scollay, R.

    1990-01-01

    To define cell populations which participate in the very early stages of T cell development in the mouse thymus, we enriched hematopoietic stem cells from mouse bone marrow and injected them into thymic lobes of irradiated Ly-5 congenic recipients. The progeny of the stem cells were identified and their phenotypes were determined by two-color flow cytometry for the expression of various cell surface differentiation Ag during the course of their subsequent intrathymic development. The majority of the differentiation which occurred in the first 10 days after intrathymic cell transfer was myeloid in nature; hence, this study demonstrates that the irradiated thymus is not strictly selective for T cell development. Further, the maximum rate of T cell development was observed after intrathymic injection of 200 stem cells. Donor-derived cells which did not express Ag characteristic of the myeloid lineage could be detected and their phenotypes could be determined by flow cytometry as early as 7 days after intrathymic injection. At this time, the cells were still very similar phenotypically to the bone marrow hematopoietic stem cells. Exceptions to this were the expression of stem cell Ag 2 and a decrease in the level of MHC class I Ag expression. After 9 days, the donor-derived cells expressed high levels of the Thy-1 Ag and proceeded to change in cell surface phenotype as differentiation continued. These cell phenotypes are described for the time frame ending 18 days after injection, when most donor-derived cells were phenotypically small CD4+ CD8+ (double-positive) thymocytes

  6. The phenotype of FancB-mutant mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu Lingchuan; Hasty, Paul

    2011-01-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslinking agent mitomycin C (MMC), increased spontaneous and MMC-induced chromosomal abnormalities, reduced spontaneous sister chromatid exchanges (SCEs), reduced gene targeting, reduced MMC-induced Rad51 foci and absent MMC-induced FancD2 foci. Since FancB is on the X chromosome and since ES cells are typically XY, FancB is an excellent target for an epistatic analysis to elucidate FA's role in ICL repair.

  7. 14-3-3σ regulates β-catenin-mediated mouse embryonic stem cell proliferation by sequestering GSK-3β.

    Directory of Open Access Journals (Sweden)

    Tzu-Ching Chang

    Full Text Available Pluripotent embryonic stem cells are considered to be an unlimited cell source for tissue regeneration and cell-based therapy. Investigating the molecular mechanism underlying the regulation of embryonic stem cell expansion is thus important. 14-3-3 proteins are implicated in controlling cell division, signaling transduction and survival by interacting with various regulatory proteins. However, the function of 14-3-3 in embryonic stem cell proliferation remains unclear.In this study, we show that all seven 14-3-3 isoforms were detected in mouse embryonic stem cells. Retinoid acid suppressed selectively the expression of 14-3-3σ isoform. Knockdown of 14-3-3σ with siRNA reduced embryonic stem cell proliferation, while only 14-3-3σ transfection increased cell growth and partially rescued retinoid acid-induced growth arrest. Since the growth-enhancing action of 14-3-3σ was abrogated by β-catenin knockdown, we investigated the influence of 14-3-3σ overexpression on β-catenin/GSK-3β. 14-3-3σ bound GSK-3β and increased GSK-3β phosphorylation in a PI-3K/Akt-dependent manner. It disrupted β-catenin binding by the multiprotein destruction complex. 14-3-3σ overexpression attenuated β-catenin phosphorylation and rescued the decline of β-catenin induced by retinoid acid. Furthermore, 14-3-3σ enhanced Wnt3a-induced β-catenin level and GSK-3β phosphorylation. DKK, an inhibitor of Wnt signaling, abolished Wnt3a-induced effect but did not interfere GSK-3β/14-3-3σ binding.Our findings show for the first time that 14-3-3σ plays an important role in regulating mouse embryonic stem cell proliferation by binding and sequestering phosphorylated GSK-3β and enhancing Wnt-signaled GSK-3β inactivation. 14-3-3σ is a novel target for embryonic stem cell expansion.

  8. Let-7b-mediated suppression of basigin expression and metastasis in mouse melanoma cells

    International Nuclear Information System (INIS)

    Fu, Tzu-Yen; Chang, Chia-Che; Lin, Chun-Ting; Lai, Cong-Hao; Peng, Shao-Yu; Ko, Yi-Ju; Tang, Pin-Chi

    2011-01-01

    Basigin (Bsg), also called extracellular matrix metalloproteinase inducer (EMMPRIN), is highly expressed on the surface of tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases (mmps). It has been shown that Bsg plays an important role in growth, development, cell differentiation, and tumor progression. MicroRNAs (miRNAs) are a class of short endogenous non-protein coding RNAs of 20-25 nucleotides (nt) that function as post-transcriptional regulators of gene expression by base-pairing to their target mRNAs and thereby mediate cleavage of target mRNAs or translational repression. In this study, let-7b, one of the let-7 family members, was investigated for its effect on the growth and invasiveness of the mouse melanoma cell line B16-F10. We have shown that let-7b can suppress the expression of Bsg in B16-F10 cells and also provided evidence that this suppression could result in the indirect suppression of mmp-9. The ability of B16-F10 cells transfected with let-7b to invade or migrate was significantly reduced. In addition, let-7b transfected B16-F10 cells displayed an inhibition of both cellular proliferation and colony formation. Furthermore, it was shown that the overexpression of let-7b in B16-F10 cells could reduce lung metastasis. Taken together, the present study identifies let-7b as a tumor suppressor that represses cancer cell proliferation and migration as well as tumor metastasis in mouse melanoma cells.

  9. Let-7b-mediated suppression of basigin expression and metastasis in mouse melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Tzu-Yen [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Chang, Chia-Che [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, 91 Hsueh Shih Road, Taichung 40402, Taiwan (China); Lin, Chun-Ting [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Lai, Cong-Hao [Institute of Biomedical Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Department of Life Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Peng, Shao-Yu; Ko, Yi-Ju [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China); Tang, Pin-Chi, E-mail: pctang@dragon.nchu.edu.tw [Department of Animal Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan (China)

    2011-02-15

    Basigin (Bsg), also called extracellular matrix metalloproteinase inducer (EMMPRIN), is highly expressed on the surface of tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases (mmps). It has been shown that Bsg plays an important role in growth, development, cell differentiation, and tumor progression. MicroRNAs (miRNAs) are a class of short endogenous non-protein coding RNAs of 20-25 nucleotides (nt) that function as post-transcriptional regulators of gene expression by base-pairing to their target mRNAs and thereby mediate cleavage of target mRNAs or translational repression. In this study, let-7b, one of the let-7 family members, was investigated for its effect on the growth and invasiveness of the mouse melanoma cell line B16-F10. We have shown that let-7b can suppress the expression of Bsg in B16-F10 cells and also provided evidence that this suppression could result in the indirect suppression of mmp-9. The ability of B16-F10 cells transfected with let-7b to invade or migrate was significantly reduced. In addition, let-7b transfected B16-F10 cells displayed an inhibition of both cellular proliferation and colony formation. Furthermore, it was shown that the overexpression of let-7b in B16-F10 cells could reduce lung metastasis. Taken together, the present study identifies let-7b as a tumor suppressor that represses cancer cell proliferation and migration as well as tumor metastasis in mouse melanoma cells.

  10. Introduction of N-cadherin-binding motif to alginate hydrogels for controlled stem cell differentiation.

    Science.gov (United States)

    Lee, Jae Won; An, Hyoseok; Lee, Kuen Yong

    2017-07-01

    Control of stem cell fate and phenotype using biomimetic synthetic extracellular matrices (ECMs) is an important tissue engineering approach. Many studies have focused on improving cell-matrix interactions. However, proper control of cell-cell interactions using synthetic ECMs could be critical for tissue engineering, especially with undifferentiated stem cells. In this study, alginate hydrogels were modified with a peptide derived from the low-density lipoprotein receptor-related protein 5 (LRP5), which is known to bind to N-cadherin, as a cell-cell interaction motif. In vitro changes in the morphology and differentiation of mouse bone marrow stromal cells (D1 stem cells) cultured in LRP5-alginate hydrogels were investigated. LRP5-alginate gels successfully induced stem cell aggregation and enhanced chondrogenic differentiation of D1 stem cells, compared to RGD-alginate gels, at low cell density. This approach to tailoring synthetic biomimetic ECMs using cell-cell interaction motifs may be critical in tissue engineering approaches using stem cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Lentiviral vectors containing mouse Csf1r control elements direct macrophage-restricted expression in multiple species of birds and mammals

    Directory of Open Access Journals (Sweden)

    Clare Pridans

    2014-01-01

    Full Text Available The development of macrophages requires signaling through the lineage-restricted receptor Csf1r. Macrophage-restricted expression of transgenic reporters based upon Csf1r requires the highly conserved Fms-intronic regulatory element (FIRE. We have created a lentiviral construct containing mouse FIRE and promoter. The lentivirus is capable of directing macrophage-restricted reporter gene expression in mouse, rat, human, pig, cow, sheep, and even chicken. Rat bone marrow cells transduced with the lentivirus were capable of differentiating into macrophages expressing the reporter gene in vitro. Macrophage-restricted expression may be desirable for immunization or immune response modulation, and for gene therapy for lysosomal storage diseases and some immunodeficiencies. The small size of the Csf1r transcription control elements will allow the insertion of large “cargo” for applications in gene therapy and vaccine delivery.

  12. A Rapid Embryonic Stem Cell-Based Mouse Model for B-cell Lymphomas Driven by Epstein-Barr Virus Protein LMP1.

    Science.gov (United States)

    Ba, Zhaoqing; Meng, Fei-Long; Gostissa, Monica; Huang, Pei-Yi; Ke, Qiang; Wang, Zhe; Dao, Mai N; Fujiwara, Yuko; Rajewsky, Klaus; Zhang, Baochun; Alt, Frederick W

    2015-06-01

    The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/β and γ/δ T cells ("CLT" mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, although introduction of additional activating or knockout mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive, and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT embryonic stem (ES) cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which, like germline CLT mice, harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation ("RDBC") approach die rapidly in association with B-cell lymphoproliferation and lymphoma. Because CLT lymphomas routinely express the activation-induced cytidine deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models. ©2015 American Association for Cancer Research.

  13. Enhanced adherence of mouse fibroblast and vascular cells to plasma modified polyethylene

    Energy Technology Data Exchange (ETDEWEB)

    Reznickova, Alena, E-mail: alena.reznickova@vscht.cz [Department of Solid State Engineering, Institute of Chemical Technology Prague, 166 28 Prague 6 (Czech Republic); Novotna, Zdenka, E-mail: zdenka1.novotna@vscht.cz [Department of Solid State Engineering, Institute of Chemical Technology Prague, 166 28 Prague 6 (Czech Republic); Kolska, Zdenka [Faculty of Science, J.E. Purkyně University, 400 96 Usti nad Labem (Czech Republic); Kasalkova, Nikola Slepickova [Department of Solid State Engineering, Institute of Chemical Technology Prague, 166 28 Prague 6 (Czech Republic); Rimpelova, Silvie [Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, 166 28 Prague 6 (Czech Republic); Svorcik, Vaclav [Department of Solid State Engineering, Institute of Chemical Technology Prague, 166 28 Prague 6 (Czech Republic)

    2015-07-01

    Since the last decade, tissue engineering has shown a sensational promise in providing more viable alternatives to surgical procedures for harvested tissues, implants and prostheses. Biomedical polymers, such as low-density polyethylene (LDPE), high-density polyethylene (HDPE) and ultra-high molecular weight polyethylene (UHMWPE), were activated by Ar plasma discharge. Degradation of polymer chains was examined by determination of the thickness of ablated layer. The amount of an ablated polymer layer was measured by gravimetry. Contact angle, measured by goniometry, was studied as a function of plasma exposure and post-exposure aging times. Chemical structure of modified polymers was characterized by angle resolved X-ray photoelectron spectroscopy. Surface chemistry and polarity of the samples were investigated by electrokinetic analysis. Changes in surface morphology were followed using atomic force microscopy. Cytocompatibility of plasma activated polyethylene foils was studied using two distinct model cell lines; VSMCs (vascular smooth muscle cells) as a model for vascular graft testing and connective tissue cells L929 (mouse fibroblasts) approved for standardized material cytotoxicity testing. Specifically, the cell number, morphology, and metabolic activity of the adhered and proliferated cells on the polyethylene matrices were studied in vitro. It was found that the plasma treatment caused ablation of the polymers, resulting in dramatic changes in their surface morphology and roughness. ARXPS and electrokinetic measurements revealed oxidation of the polymer surface. It was found that plasma activation has a positive effect on the adhesion and proliferation of VSMCs and L929 cells. - Highlights: • Plasma activation of LDPE, HDPE and UHMWPE • Study of surface properties by several techniques: ARXPS, AFM, zeta-potential, and goniometry • Investigation of adhesion and spreading of vascular smooth muscle cells (VSMCs) and mouse fibroblasts (L929)

  14. Radiation effects on cultured mouse embryos in relation to cell division cycle

    International Nuclear Information System (INIS)

    Domon, M.

    1982-01-01

    The authors have worked with mouse embryos in vitro asking first, what are the suitable parameters to define the radiation sensitivity of embryos, and second what is a major factor determining it. The LD 50 was adopted as a parameter of the radiation sensitivity of a population in a mouse embryo system in culture. The fertilized ova were collected into Whitten's medium at various times during the pronuclear and 2-cell stages of development. They were irradiated in chambers with X-rays at doses of 0 to 800 rads. After the embryos were cultured, a set of the lethal fractions for various X-ray doses were obtained. Regarding the radiation sensitivity variation of the embryos, the LD 50 varied from 100 to 200 rads during the pronuclear stage and from 100 to 600 rads during the 2-cell stage. The embryos during the pronuclear stage were most radioresistant at early G 2 phase, followed by an increase in the sensitivity. The embryos during the 2-cell stage were also most radioresistant at early G 2 phase and were more sensitive when they got close to either the first or the second cleavage division. Furthermore, it seems that the factor 6 of the large variation was due to the extremely long G 2 period, 14 hrs for the 2-cell embryos. That is, the pooled 2-cell embryos were in a relative sense well synchronized with G 2 phase. In contrast, the synchrony was poor during the pronuclear stage, which led to less variation of the LD 50 for the pronuclear embryos. It is concluded that during the early cleavage stages of mice, radiosensitivity is mainly governed by the content of cells of various cell cycle ages in the embryo. (Namekawa, K.)

  15. Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Shirai, Hidenori; Fujimori, Hiroaki [Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Gunji, Akemi [Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Maeda, Daisuke [Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); ADP-Ribosylation in Oncology Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Hirai, Takahisa [Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Department of Radiation Oncology, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Poetsch, Anna R. [ADP-Ribosylation in Oncology Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Harada, Hiromi [Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Yoshida, Tomoko [Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Kyoritsu College of Pharmacy, 1-5-30 Shibakoen, Minatoku, Tokyo 105-8512 (Japan); Sasai, Keisuke [Department of Radiation Oncology, Juntendo University Faculty of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan); Okayasu, Ryuichi [International Open Laboratory, National Institute of Radiological Science, 4-9-1 Anagawa, Inage, Chiba 263-8555 (Japan); Masutani, Mitsuko, E-mail: mmasutan@ncc.go.jp [Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); ADP-Ribosylation in Oncology Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2013-05-24

    Highlights: •Parg{sup −/−} ES cells were more sensitive to γ-irradiation than Parp-1{sup −/−} ES cells. •Parg{sup −/−} cells were more sensitive to carbon-ion irradiation than Parp-1{sup −/−} cells. •Parg{sup −/−} cells showed defects in DSB repair after carbon-ion irradiation. •PAR accumulation was enhanced after carbon-ion irradiation compared to γ-irradiation. -- Abstract: Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg{sup −/−} and poly(ADP-ribose) polymerase-1 deficient (Parp-1{sup −/−}) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg{sup −/−} cells were more sensitive to γ-irradiation than Parp-1{sup −/−} cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg{sup −/−} cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg{sup −/−} ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1{sup −/−} cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg{sup −/−} ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/μm) and Fe-ion irradiation (200 keV/μm) were also examined. Parg{sup −/−} cells were more sensitive to LET 70 keV/μm carbon-ion irradiation than Parp-1{sup −/−} cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24 h. The induction level of p53 phophorylation at ser18 was

  16. Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation

    International Nuclear Information System (INIS)

    Shirai, Hidenori; Fujimori, Hiroaki; Gunji, Akemi; Maeda, Daisuke; Hirai, Takahisa; Poetsch, Anna R.; Harada, Hiromi; Yoshida, Tomoko; Sasai, Keisuke; Okayasu, Ryuichi; Masutani, Mitsuko

    2013-01-01

    Highlights: •Parg −/− ES cells were more sensitive to γ-irradiation than Parp-1 −/− ES cells. •Parg −/− cells were more sensitive to carbon-ion irradiation than Parp-1 −/− cells. •Parg −/− cells showed defects in DSB repair after carbon-ion irradiation. •PAR accumulation was enhanced after carbon-ion irradiation compared to γ-irradiation. -- Abstract: Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg −/− and poly(ADP-ribose) polymerase-1 deficient (Parp-1 −/− ) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg −/− cells were more sensitive to γ-irradiation than Parp-1 −/− cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg −/− cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg −/− ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1 −/− cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg −/− ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/μm) and Fe-ion irradiation (200 keV/μm) were also examined. Parg −/− cells were more sensitive to LET 70 keV/μm carbon-ion irradiation than Parp-1 −/− cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24 h. The induction level of p53 phophorylation at ser18 was not different between wild-type and Parg −/− cells. The augmented

  17. Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

    Science.gov (United States)

    Shuda, Masahiro; Guastafierro, Anna; Geng, Xuehui; Shuda, Yoko; Ostrowski, Stephen M; Lukianov, Stefan; Jenkins, Frank J; Honda, Kord; Maricich, Stephen M; Moore, Patrick S; Chang, Yuan

    2015-01-01

    Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

  18. Hedgehog Signaling Promotes the Proliferation and Subsequent Hair Cell Formation of Progenitor Cells in the Neonatal Mouse Cochlea

    Science.gov (United States)

    Chen, Yan; Lu, Xiaoling; Guo, Luo; Ni, Wenli; Zhang, Yanping; Zhao, Liping; Wu, Lingjie; Sun, Shan; Zhang, Shasha; Tang, Mingliang; Li, Wenyan; Chai, Renjie; Li, Huawei

    2017-01-01

    Hair cell (HC) loss is the major cause of permanent sensorineural hearing loss in mammals. Unlike lower vertebrates, mammalian cochlear HCs cannot regenerate spontaneously after damage, although the vestibular system does maintain limited HC regeneration capacity. Thus HC regeneration from the damaged sensory epithelium has been one of the main areas of research in the field of hearing restoration. Hedgehog signaling plays important roles during the embryonic development of the inner ear, and it is involved in progenitor cell proliferation and differentiation as well as the cell fate decision. In this study, we show that recombinant Sonic Hedgehog (Shh) protein effectively promotes sphere formation, proliferation, and differentiation of Lgr5+ progenitor cells isolated from the neonatal mouse cochlea. To further explore this, we determined the effect of Hedgehog signaling on cell proliferation and HC regeneration in cultured cochlear explant from transgenic R26-SmoM2 mice that constitutively activate Hedgehog signaling in the supporting cells of the cochlea. Without neomycin treatment, up-regulation of Hedgehog signaling did not significantly promote cell proliferation or new HC formation. However, after injury to the sensory epithelium by neomycin treatment, the over-activation of Hedgehog signaling led to significant supporting cell proliferation and HC regeneration in the cochlear epithelium explants. RNA sequencing and real-time PCR were used to compare the transcripts of the cochleae from control mice and R26-SmoM2 mice, and multiple genes involved in the proliferation and differentiation processes were identified. This study has important implications for the treatment of sensorineural hearing loss by manipulating the Hedgehog signaling pathway. PMID:29311816

  19. Hedgehog Signaling Promotes the Proliferation and Subsequent Hair Cell Formation of Progenitor Cells in the Neonatal Mouse Cochlea

    Directory of Open Access Journals (Sweden)

    Yan Chen

    2017-12-01

    Full Text Available Hair cell (HC loss is the major cause of permanent sensorineural hearing loss in mammals. Unlike lower vertebrates, mammalian cochlear HCs cannot regenerate spontaneously after damage, although the vestibular system does maintain limited HC regeneration capacity. Thus HC regeneration from the damaged sensory epithelium has been one of the main areas of research in the field of hearing restoration. Hedgehog signaling plays important roles during the embryonic development of the inner ear, and it is involved in progenitor cell proliferation and differentiation as well as the cell fate decision. In this study, we show that recombinant Sonic Hedgehog (Shh protein effectively promotes sphere formation, proliferation, and differentiation of Lgr5+ progenitor cells isolated from the neonatal mouse cochlea. To further explore this, we determined the effect of Hedgehog signaling on cell proliferation and HC regeneration in cultured cochlear explant from transgenic R26-SmoM2 mice that constitutively activate Hedgehog signaling in the supporting cells of the cochlea. Without neomycin treatment, up-regulation of Hedgehog signaling did not significantly promote cell proliferation or new HC formation. However, after injury to the sensory epithelium by neomycin treatment, the over-activation of Hedgehog signaling led to significant supporting cell proliferation and HC regeneration in the cochlear epithelium explants. RNA sequencing and real-time PCR were used to compare the transcripts of the cochleae from control mice and R26-SmoM2 mice, and multiple genes involved in the proliferation and differentiation processes were identified. This study has important implications for the treatment of sensorineural hearing loss by manipulating the Hedgehog signaling pathway.

  20. Effect of low dose radiation on cytochrome c and caspase-3 protein expressions in spermatogenic cells of mouse testis

    International Nuclear Information System (INIS)

    Wang Zhicheng; Zhao Hongguang; Piao Chunnan; Liu Guangwei; Liu Shuchun; Lv Zhe; Gong Shouliang

    2006-01-01

    Objective: To investigate the effect of low dose radiation on the expressions of cytochrome e (Cyt c) and caspase-3 proteins in spermatogenic cells of mouse testis. Methods: The relationships of dose- and time-effect of Cyt c and caspase-3 protein expressions with different dose of X-rays were observed in the spermatogenic cells of mouse testis with immunohistochemical technique (SABC). Results: After irradiation with 0, 0.025, 0.05, 0.075, 0.1 and 0.2 Gy, Cyt c and caspase-3 proteins expressed differently in all kinds of spermatogenic cells, and principally in spermatogonia and spermatocytes, and less in spermatids and spermatozoa. And the expressions increased with the increasing of irradiation dose. The expressions of both proteins after irradiation with 0.075 Gy increased with the lapse of time and reached to the peak at 12 h, and then decreased. Conclusion: Dose-and time-effect exists on the low-dose irradiation induced expressions of Cyt e and caspase-3 proteins in spermatogenic cells of mouse testis. (authors)

  1. Effects of lithium chloride as a potential radioprotective agent on radiation response of DNA synthesis in mouse germinal cells.

    Science.gov (United States)

    Bhattacharjee, D; Rajan, R; Krishnamoorthy, L; Singh, B B

    1997-06-01

    Mouse spermatogonial germ cells are highly sensitive to ionizing radiation. Lithium salts are reported to stimulate the postirradiation recovery of hematopoietic marrow cells. We have, therefore, examined whether administered lithium chloride (LiCl) would also be able to protect the mouse germinal cells against radiation injury. Taking DNA synthesis as an endpoint, our results show that the testicular DNA-specific activity in irradiated mice was higher by 61% on average when they had been pretreated with LiCl both 24 h and 1 h prior to gamma-irradiation (2.0 Gy). It was also observed that the DNA synthetic activity in the germinal cells fully recovered after LiCl pretreatment at doses of 40 mg per kg body weight prior to total body irradiation of 0.05-0.25 Gy, whereas at doses of 0.5-6.0 Gy, following the same procedure of LiCl pretreatment, only an incomplete recovery was observed. The dose reduction factor for LiCl is 1.84. The current findings indicate that pretreatment with LiCl provides considerable protection against radiation damage in mouse spermatogonia.

  2. Regenerative therapy for vestibular disorders using human induced pluripotent stem cells (iPSCs): neural differentiation of human iPSC-derived neural stem cells after in vitro transplantation into mouse vestibular epithelia.

    Science.gov (United States)

    Taura, Akiko; Nakashima, Noriyuki; Ohnishi, Hiroe; Nakagawa, Takayuki; Funabiki, Kazuo; Ito, Juichi; Omori, Koichi

    2016-10-01

    Vestibular ganglion cells, which convey sense of motion from vestibular hair cells to the brainstem, are known to degenerate with aging and after vestibular neuritis. Thus, regeneration of vestibular ganglion cells is important to aid in the recovery of balance for associated disorders. The present study derived hNSCs from induced pluripotent stem cells (iPSCs) and transplanted these cells into mouse utricle tissues. After a 7-day co-culture period, histological and electrophysiological examinations of transplanted hNSCs were performed. Injected hNSC-derived cells produced elongated axon-like structures within the utricle tissue that made contact with vestibular hair cells. A proportion of hNSC-derived cells showed spontaneous firing activities, similar to those observed in cultured mouse vestibular ganglion cells. However, hNSC-derived cells around the mouse utricle persisted as immature neurons or occasionally differentiated into putative astrocytes. Moreover, electrophysiological examination showed hNSC-derived cells around utricles did not exhibit any obvious spontaneous firing activities. Injected human neural stem cells (hNSCs) showed signs of morphological maturation including reconnection to denervated hair cells and partial physiological maturation, suggesting hNSC-derived cells possibly differentiated into neurons.

  3. The loss-of-allele assay for ES cell screening and mouse genotyping.

    Science.gov (United States)

    Frendewey, David; Chernomorsky, Rostislav; Esau, Lakeisha; Om, Jinsop; Xue, Yingzi; Murphy, Andrew J; Yancopoulos, George D; Valenzuela, David M

    2010-01-01

    Targeting vectors used to create directed mutations in mouse embryonic stem (ES) cells consist, in their simplest form, of a gene for drug selection flanked by mouse genomic sequences, the so-called homology arms that promote site-directed homologous recombination between the vector and the target gene. The VelociGene method for the creation of targeted mutations in ES cells employs targeting vectors, called BACVecs, that are based on bacterial artificial chromosomes. Compared with conventional short targeting vectors, BacVecs provide two major advantages: (1) their much larger homology arms promote high targeting efficiencies without the need for isogenicity or negative selection strategies; and (2) they enable deletions and insertions of up to 100kb in a single targeting event, making possible gene-ablating definitive null alleles and other large-scale genomic modifications. Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus. To exploit the advantages of BACVecs for gene targeting, we inverted the conventional screening logic in developing the loss-of-allele (LOA) assay, which quantifies the number of copies of the native locus to which the mutation was directed. In a correctly targeted ES cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector. The LOA assay reveals a correctly targeted clone as having lost one copy of the native target gene and gained one copy of the drug resistance gene or other inserted marker. The combination of these quantitative assays makes LOA genotyping unequivocal and amenable to automated scoring. We use the quantitative polymerase chain reaction

  4. Acid stimulation (sour taste elicits GABA and serotonin release from mouse taste cells.

    Directory of Open Access Journals (Sweden)

    Yijen A Huang

    Full Text Available Several transmitter candidates including serotonin (5-HT, ATP, and norepinephrine (NE have been identified in taste buds. Recently, γ-aminobutyric acid (GABA as well as the associated synthetic enzymes and receptors have also been identified in taste cells. GABA reduces taste-evoked ATP secretion from Receptor cells and is considered to be an inhibitory transmitter in taste buds. However, to date, the identity of GABAergic taste cells and the specific stimulus for GABA release are not well understood. In the present study, we used genetically-engineered Chinese hamster ovary (CHO cells stably co-expressing GABA(B receptors and Gαqo5 proteins to measure GABA release from isolated taste buds. We recorded robust responses from GABA biosensors when they were positioned against taste buds isolated from mouse circumvallate papillae and the buds were depolarized with KCl or a stimulated with an acid (sour taste. In contrast, a mixture of sweet and bitter taste stimuli did not trigger GABA release. KCl- or acid-evoked GABA secretion from taste buds was Ca(2+-dependent; removing Ca(2+ from the bathing medium eliminated GABA secretion. Finally, we isolated individual taste cells to identify the origin of GABA secretion. GABA was released only from Presynaptic (Type III cells and not from Receptor (Type II cells. Previously, we reported that 5-HT released from Presynaptic cells inhibits taste-evoked ATP secretion. Combined with the recent findings that GABA depresses taste-evoked ATP secretion, the present results indicate that GABA and 5-HT are inhibitory transmitters in mouse taste buds and both likely play an important role in modulating taste responses.

  5. Wallerian degeneration slow mouse neurons are protected against cell death caused by mechanisms involving mitochondrial electron transport dysfunction.

    Science.gov (United States)

    Tokunaga, Shinji; Araki, Toshiyuki

    2012-03-01

    Ischemia elicits a variety of stress responses in neuronal cells, which result in cell death. wld(S) Mice bear a mutation that significantly delays Wallerian degeneration. This mutation also protects all neuronal cells against other types of stresses resulting in cell death, including ischemia. To clarify the types of stresses that neuronal cell bodies derived from wld(S) mice are protected from, we exposed primary cultured neurons derived from wld(S) mice to various components of hypoxic stress. We found that wld(S) mouse neurons are protected against cellular injury induced by reoxygenation following hypoxic stress. Furthermore, we found that wld(S) mouse neurons are protected against functional impairment of the mitochondrial electron transport chain. These data suggest that Wld(S) protein expression may provide protection against neuronal cell death caused by mechanisms involving mitochondrial electron transport dysfunction. Copyright © 2011 Wiley Periodicals, Inc.

  6. S1 nuclease analysis of α-globin gene expression in preleukemic patients with acquired hemoglobin H disease after transfer to mouse erythroleukemia cells

    International Nuclear Information System (INIS)

    Helder, J.; Deisseroth, A.

    1987-01-01

    The loss of α-globin gene transcriptional activity rarely occurs as an acquired abnormality during the evolution of myeloproliferative disease or preleukemia. To test whether the mutation responsible for the loss of α-globin gene expression (hemoglobin H disease) in these patients is linked with the α-globin genes on chromosome 16, the authors transferred chromosome 16 from preleukemic patients with acquired hemoglobin H disease to mouse erythroleukemia cells and measured the transcriptional activity of the human α-globin genes. After transfer to mouse erythroleukemia cells, the expression of human α-globin genes from the peripheral blood or marrow cells of preleukemic patients with acquired hemoglobin H disease was similar to that of human α-globin genes transferred to mouse erythroleukemia cells from normal donors. These data showed that factor(s) in the mouse erythroleukemia cell can genetically complement the α-globin gene defect in these preleukemia patients with acquired hemoglobin H disease and suggest that altered expression of a gene in trans to the α-globin gene may be responsible for the acquisition of hemoglobin H disease in these patients

  7. Overexpression of Cardiac-Specific Kinase TNNI3K Promotes Mouse Embryonic Stem Cells Differentiation into Cardiomyocytes.

    Science.gov (United States)

    Wang, Yin; Wang, Shi-Qiang; Wang, Li-Peng; Yao, Yu-Hong; Ma, Chun-Yan; Ding, Jin-Feng; Ye, Jue; Meng, Xian-Min; Li, Jian-Jun; Xu, Rui-Xia

    2017-01-01

    Backgroud/Aims: The biological function of cardiac troponin I-interacting kinase (TNNI3K), a cardiac-specific functional kinase, is largely unknown. We investigated the effect of human TNNI3K (hTNNI3K) on the differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. First, the time-space expression of endogenous Tnni3k was detected by real-time polymerase chain reaction (PCR) and western blotting at 16 different time-points over a period of 28 days. Further, action potentials and calcium current with/without 5 µM nifedipine were measured by patch clamp for mESC-derived cardiomyocytes. HTNNI3K and mouse-derived siRNA were transfected into mESC using lentivirus vector to induce hTNNI3K overexpression and knock-down, respectively. The number of troponin-T (cTnT) positive cells was greater in the group with TNNI3K overexpression as compared to that in control group, while less such cells were detected in the mTnni3k knock-down group as evaluated on flow cytometry (FCM) and ImageXpress Micro system. After upregulation of connexin43, cardiac troponin-I (Ctni), Ctni, Gata4 were detected in mESCs with TNNI3K overexpression; however, overexpression of α-Actinin and Mlc2v was not detected. Interestingly, Ctnt, connexin40 and connexin45, the markers of ventricular, atrial, and pacemaker cells, respectively, were detected in by real-time PCR in TNNI3K overexpression group. our study indicated that TNNI3K overexpression promoted mESC differentiating into beating cardiomyocytes and induced up-regulating expression of cTnT by PKCε signal pathway, which suggested a modulation of TNNI3K activity as a potential therapeutic approach for ischemic cardiac disease. © 2017 The Author(s) Published by S. Karger AG, Basel.

  8. Effect of 935-MHz phone-simulating electromagnetic radiation on endometrial glandular cells during mouse embryo implantation.

    Science.gov (United States)

    Liu, Wenhui; Zheng, Xinmin; Qu, Zaiqing; Zhang, Ming; Zhou, Chun; Ma, Ling; Zhang, Yuanzhen

    2012-10-01

    This study examined the impact of 935MHz phone-simulating electromagnetic radiation on embryo implantation of pregnant mice. Each 7-week-old Kunming (KM) female white mouse was set up with a KM male mouse in a single cage for mating overnight after induction of ovulation. In the first three days of pregnancy, the pregnant mice was exposed to electromagnetic radiation at low-intensity (150 μW/cm(2), ranging from 130 to 200 μW/cm(2), for 2- or 4-h exposure every day), mid-intensity (570 μW/cm(2), ranging from 400 to 700 μW/cm(2), for 2- or 4-h exposure every day) or high-intensity (1400 μW/cm(2), ranging from 1200 to 1500 μW/cm(2), for 2- or 4-h exposure every day), respectively. On the day 4 after gestation (known as the window of murine embryo implantation), the endometrium was collected and the suspension of endometrial glandular cells was made. Laser scanning microscopy was employed to detect the mitochondrial membrane potential and intracellular calcium ion concentration. In high-intensity, 2- and 4-h groups, mitochondrial membrane potential of endometrial glandular cells was significantly lower than that in the normal control group (Pelectromagnetic radiation and longer length of the radiation are required to inflict a remarkable functional and structural damage to mitochondrial membrane. Our data demonstrated that electromagnetic radiation with a 935-MHz phone for 4 h conspicuously decreased mitochondrial membrane potential and lowered the calcium ion concentration of endometrial glandular cells. It is suggested that high-intensity electromagnetic radiation is very likely to induce the death of embryonic cells and decrease the chance of their implantation, thereby posing a high risk to pregnancy.

  9. PD-1/CTLA-4 Blockade Inhibits Epstein-Barr Virus-Induced Lymphoma Growth in a Cord Blood Humanized-Mouse Model.

    Directory of Open Access Journals (Sweden)

    Shi-Dong Ma

    2016-05-01

    Full Text Available Epstein-Barr virus (EBV infection causes B cell lymphomas in humanized mouse models and contributes to a variety of different types of human lymphomas. T cells directed against viral antigens play a critical role in controlling EBV infection, and EBV-positive lymphomas are particularly common in immunocompromised hosts. We previously showed that EBV induces B cell lymphomas with high frequency in a cord blood-humanized mouse model in which EBV-infected human cord blood is injected intraperitoneally into NOD/LtSz-scid/IL2Rγnull (NSG mice. Since our former studies showed that it is possible for T cells to control the tumors in another NSG mouse model engrafted with both human fetal CD34+ cells and human thymus and liver, here we investigated whether monoclonal antibodies that block the T cell inhibitory receptors, PD-1 and CTLA-4, enhance the ability of cord blood T cells to control the outgrowth of EBV-induced lymphomas in the cord-blood humanized mouse model. We demonstrate that EBV-infected lymphoma cells in this model express both the PD-L1 and PD-L2 inhibitory ligands for the PD-1 receptor, and that T cells express the PD-1 and CTLA-4 receptors. Furthermore, we show that the combination of CTLA-4 and PD-1 blockade strikingly reduces the size of lymphomas induced by a lytic EBV strain (M81 in this model, and that this anti-tumor effect requires T cells. PD-1/CTLA-4 blockade markedly increases EBV-specific T cell responses, and is associated with enhanced tumor infiltration by CD4+ and CD8+ T cells. In addition, PD-1/CTLA-4 blockade decreases the number of both latently, and lytically, EBV-infected B cells. These results indicate that PD-1/CTLA-4 blockade enhances the ability of cord blood T cells to control outgrowth of EBV-induced lymphomas, and suggest that PD-1/CTLA-4 blockade might be useful for treating certain EBV-induced diseases in humans.

  10. Cell flux through S phase in the mouse duodenal epithelium determined by cell sorting and radioautography

    International Nuclear Information System (INIS)

    Bjerknes, M.; Cheng, H.

    1982-01-01

    An accumulation of cells in early S phase was observed in normal mouse duodenal epithelium studied with flow cytometry. To determine if this accumulation of cells was the result of a lower rate of DNA synthesis, animals were given a single injection of 3 H-thymidine and the epithelium collected one hour later. The epithelium was processed for flow cytometry. Seven sort windows were established in different portions of the DNA histogram. Cells from each window were sorted onto glass slides that were then processed for radioautography. The number of silver grains over the nuclei of each sorted population was counted. It was found that cells in early S phase had significantly fewer grains over their nuclei than did mid- or late-S phase cells. We conclude that the accumulation of cells in early S phase is due, at least in part, to a lower rate of DNA synthesis in early than in mid or late S phase

  11. Endogenous retinoic acid activity in principal cells and intercalated cells of mouse collecting duct system.

    Directory of Open Access Journals (Sweden)

    Yuen Fei Wong

    2011-02-01

    Full Text Available Retinoic acid is the bioactive derivative of vitamin A, which plays an indispensible role in kidney development by activating retinoic acid receptors. Although the location, concentration and roles of endogenous retinoic acid in post-natal kidneys are poorly defined, there is accumulating evidence linking post-natal vitamin A deficiency to impaired renal concentrating and acidifying capacity associated with increased susceptibility to urolithiasis, renal inflammation and scarring. The aim of this study is to examine the presence and the detailed localization of endogenous retinoic acid activity in neonatal, young and adult mouse kidneys, to establish a fundamental ground for further research into potential target genes, as well as physiological and pathophysiological roles of endogenous retinoic acid in the post-natal kidneys.RARE-hsp68-lacZ transgenic mice were employed as a reporter for endogenous retinoic acid activity that was determined by X-gal assay and immunostaining of the reporter gene product, β-galactosidase. Double immunostaining was performed for β-galactosidase and markers of kidney tubules to localize retinoic acid activity. Distinct pattern of retinoic acid activity was observed in kidneys, which is higher in neonatal and 1- to 3-week-old mice than that in 5- and 8-week-old mice. The activity was present specifically in the principal cells and the intercalated cells of the collecting duct system in all age groups, but was absent from the glomeruli, proximal tubules, thin limbs of Henle's loop and distal tubules.Endogenous retinoic acid activity exists in principal cells and intercalated cells of the mouse collecting duct system after birth and persists into adulthood. This observation provides novel insights into potential roles for endogenous retinoic acid beyond nephrogenesis and warrants further studies to investigate target genes and functions of endogenous retinoic acid in the kidney after birth, particularly in the

  12. Striking a balance: regulation of transposable elements by Zfp281 and Mll2 in mouse embryonic stem cells.

    Science.gov (United States)

    Dai, Qian; Shen, Yang; Wang, Yan; Wang, Xin; Francisco, Joel Celio; Luo, Zhuojuan; Lin, Chengqi

    2017-12-01

    Transposable elements (TEs) compose about 40% of the murine genome. Retrotransposition of active TEs such as LINE-1 (L1) tremendously impacts genetic diversification and genome stability. Therefore, transcription and transposition activities of retrotransposons are tightly controlled. Here, we show that the Krüppel-like zinc finger protein Zfp281 directly binds and suppresses a subset of retrotransposons, including the active young L1 repeat elements, in mouse embryonic stem (ES) cells. In addition, we find that Zfp281-regulated L1s are highly enriched for 5-hydroxymethylcytosine (5hmC) and H3K4me3. The COMPASS-like H3K4 methyltransferase Mll2 is the major H3K4me3 methylase at the Zfp281-regulated L1s and required for their proper expression. Our studies also reveal that Zfp281 functions partially through recruiting the L1 regulators DNA hydroxymethylase Tet1 and Sin3A, and restricting Mll2 at these active L1s, leading to their balanced expression. In summary, our data indicate an instrumental role of Zfp281 in suppressing the young active L1s in mouse ES cells. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Cell context-specific expression of primary cilia in the human testis and ciliary coordination of Hedgehog signalling in mouse Leydig cells

    DEFF Research Database (Denmark)

    Berg Nygaard, Marie; Almstrup, Kristian; Lindbæk, Louise

    2015-01-01

    Primary cilia are sensory organelles that coordinate numerous cellular signalling pathways during development and adulthood. Defects in ciliary assembly or function lead to a series of developmental disorders and diseases commonly referred to as ciliopathies. Still, little is known about...... cells of mature seminiferous epithelium, but present in Sertoli cell-only tubules in Klinefelter syndrome testis. Peritubular cells in atrophic testis produce overly long cilia. Furthermore cultures of growth-arrested immature mouse Leydig cells express primary cilia that are enriched in components...

  14. Molecular mechanisms involved in casein gene expression and secretion in mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    Lee, E.Y.H.P.; Lee, W.H.; Parry, G.; Bissell, M.J.

    1985-01-01

    Mouse mammary epithelial cells (MMEC) secrete a group of milk-specific proteins including various caseins and whey proteins. Dissociated mammary epithelial cells maintain expression of most of their differentiated functions only if cells are plated on a suitable substratum. Casein production and section, cell morphology, and production of α-lactalbumin have been used as markers to assess the degree of differentiation of mammary cells in culture. The general consensus is that cells express their differentiated properties at high levels and for longer periods of time on such substrata. In this paper, the authors demonstrate that modulation of the expression of caseins by floating collagen gels is manifested at several regulatory points

  15. Cell cycle of spermatogonial colony forming stem cells in the CBA mouse after neutron irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Bootsma, A.L. (Rijksuniversiteit Utrecht (Netherlands). Academisch Ziekenhuis); Davids, J.A.G. (Netherlands Energy Research Foundation, Petten (Netherlands))

    1988-03-01

    In the CBA mouse testis, about 10% of the stem cell population is highly resistant to neutron irradiation (D/sub 0/, 0.75 Gy). Following a dose of 1.50 Gy these cells rapidly increase their sensitivity towards a second neutron dose and progress fairly synchronously through their first post-irradiation cell cycle. From experiments in which neutron irradiation was combined with hydroxyurea, it appeared that in this cycle the S-phase is less radiosensitive (D/sub 0/, 0.43 Gy) than the other phases of the cell cycle (D/sub 0/, 0.25 Gy). From experiments in which hydroxyurea was injected twice after irradiation, the speed of inflow of cells in S and the duration of S and the cell cycle could be calculated. Between 32 and 36 hr after irradiation cells start to enter the S-phase at a speed of 30% of the population every 12 hr. At 60 hr 50% of the population has already passed the S-phase while 30% is still in S. The data point to a cell cycle time of about 36 hr, while the S-phase lasts 12 hr at the most. (author).

  16. An investigation of the shedding of macromolecules from the Ehrlich mouse ascites tumor cell

    International Nuclear Information System (INIS)

    Edwards, E.H.

    1984-01-01

    The spontaneous release, or shedding, of cell surface components into the extracellular medium may be important in the determination of several features of the cancer cell phenotype. The release of macromolecules from the Erhlich mouse ascites tumor cell was studied under a variety of experimental conditions to elucidate the origin and the underlying mechanisms of release. The extrinsic macromolecules are a diverse group with apparent molecular weights ranging from 13,500 to 400,000 daltons. External labeling of the cell surface with tritiated 4,4'-diisothiocyano-1,2-diphenylethane-2,2-disulfonic acid ([ 3 H]H 2 DIDS) reveals a slow loss of labeled components at 4 degrees C, while at 21 degrees C and 37 degrees C an initial rapid loss is followed by a slower release. In vitro metabolic labeling with [1- 14 C]-D-glucosamine hydrochloride, D-[2- 3 H]-mannose and various [ 3 H]-L-amino acids results in the appearance of labeled macromolecules in the medium suggesting tumor, not mouse, origin. These data suggest that the extrinsic macromolecules originate from the cell surface. Macromolecules are shed by a temperature and pH sensitive process. These results suggest that a limited proteolytic digestion, or sublethal autolysis, of the cell surface may occur in this system. The macromolecules shed by the Ehrlich cell originate from the surface and are probably released by sublethal autolysis, direct secretion and a passive process

  17. Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment

    Directory of Open Access Journals (Sweden)

    Pek Siew Lim

    2016-03-01

    Full Text Available T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA and calcium ionomycin (I as stimuli. We were also interested to examine the importance of new protein synthesis in regulating the expression of genes involved in T cell activation. Thus we have pre-treated mouse EL4 T cells with cycloheximide, a protein synthesis inhibitor, and left the cells unstimulated or stimulated with PMA/I for 4 h. We performed microarray expression profiling of these cells to correlate the gene expression with chromatin state of T cells upon T cell activation [1]. Here, we detail further information and analysis of the microarray data, which shows that T cell activation leads to differential expression of genes and inducible genes can be further classified as primary and secondary response genes based on their protein synthesis dependency. The data is available in the Gene Expression Omnibus under accession number GSE13278. Keywords: EL4 T cell, Microarray, T cell activation, Inducible genes

  18. Immunohistochemical Examination for the Distribution of Podoplanin-Expressing Cells in Developing Mouse Molar Tooth Germs

    Science.gov (United States)

    Imaizumi, Yuri; Amano, Ikuko; Tsuruga, Eichi; Kojima, Hiroshi; Sawa, Yoshihiko

    2010-01-01

    We recently reported the expression of podoplanin in the apical bud of adult mouse incisal tooth. This study was aimed to investigate the distribution of podoplanin-expressing cells in mouse tooth germs at several developing stages. At the bud stage podoplanin was expressed in oral mucous epithelia and in a tooth bud. At the cap stage podoplanin was expressed on inner and outer enamel epithelia but not in mesenchymal cells expressing the neural crest stem cell marker nestin. At the early bell stage nestin and podoplanin were expressed in cervical loop and odontoblasts. At the root formation stage both nestin and podoplanin were weakly expressed in odontoblasts generating radicular dentin. Podoplanin expression was also found in the Hertwig epithelial sheath. These results suggest that epithelial cells of developing tooth germ acquire the ability to express nestin, and that tooth germ epithelial cells maintain the ability to express podoplanin in oral mucous epithelia. The expression of podoplanin in odontoblasts was induced as tooth germ development advanced, but was suppressed with the completion of the primary dentin, suggesting that podoplanin may be involved in the cell growth of odontoblasts. Nestin may function as an intermediate filament that binds podoplanin in odontoblasts. PMID:21060740

  19. Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice.

    Science.gov (United States)

    Shao, Jiawei; Xue, Shuai; Yu, Guiling; Yu, Yuanhuan; Yang, Xueping; Bai, Yu; Zhu, Sucheng; Yang, Linfeng; Yin, Jianli; Wang, Yidan; Liao, Shuyong; Guo, Sanwei; Xie, Mingqi; Fussenegger, Martin; Ye, Haifeng

    2017-04-26

    With the increasingly dominant role of smartphones in our lives, mobile health care systems integrating advanced point-of-care technologies to manage chronic diseases are gaining attention. Using a multidisciplinary design principle coupling electrical engineering, software development, and synthetic biology, we have engineered a technological infrastructure enabling the smartphone-assisted semiautomatic treatment of diabetes in mice. A custom-designed home server SmartController was programmed to process wireless signals, enabling a smartphone to regulate hormone production by optically engineered cells implanted in diabetic mice via a far-red light (FRL)-responsive optogenetic interface. To develop this wireless controller network, we designed and implanted hydrogel capsules carrying both engineered cells and wirelessly powered FRL LEDs (light-emitting diodes). In vivo production of a short variant of human glucagon-like peptide 1 (shGLP-1) or mouse insulin by the engineered cells in the hydrogel could be remotely controlled by smartphone programs or a custom-engineered Bluetooth-active glucometer in a semiautomatic, glucose-dependent manner. By combining electronic device-generated digital signals with optogenetically engineered cells, this study provides a step toward translating cell-based therapies into the clinic. Copyright © 2017, American Association for the Advancement of Science.

  20. REDOX DISRUPTING POTENTIAL OF TOXCAST CHEMICALS RANKED BY ACTIVITY IN MOUSE EMBRYONIC STEM CELLS

    Science.gov (United States)

    To gain insight regarding the adverse outcome pathways leading to developmental toxicity following exposure to chemicals, we evaluated ToxCast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay and identified a redox sensitive pathway that correlated with al...

  1. Fingerprinting of neurotoxic compounds using a mouse embryonic stem cell dual luminescence reporter assay

    NARCIS (Netherlands)

    Colaianna, M.; Ilmjärv, S.; Peterson, H.; Ilse Kern, I.; Julien, S.; Baquié, M.; allocca, G.; Bosgra, S.; Sachinidis, A.; Hengstler, J.G.; Leist, M.; Krause, K.H.

    2017-01-01

    Identification of neurotoxic drugs and environmental chemicals is an important challenge. However, only few tools to address this topic are available. The aim of this study was to develop a neurotoxicity/developmental neurotoxicity (DNT) test system, using the pluripotent mouse embryonic stem cell

  2. Resistance of 3T3 mouse cells to UV light in relation to excision and transfer of dimers to daughter strands

    Energy Technology Data Exchange (ETDEWEB)

    Menck, C F.M.; Meneghini, R [Sao Paulo Univ. (Brazil). Inst. de Quimica

    1982-04-01

    Mouse cells (3T3 line) and human fibroblasts are equally sensitive to UV light. At fluences of 2.0-2.5 J/m/sup 2/ mouse cells excise only 20% of the pyrimidine dimers as compared to 80% excised by human fibroblasts. This fluence allows 37% survival in both cases. Hence, mouse cells are more resistant to the same burden of unexcised dimers. The reason for this increased tolerance to dimers does not seem to be due to a recombinational mechanism, as judged by the fact that only ca. 5% of the dimers are transferred from parental to daughter strands. The transfer of dimers was measured by the Micrococcus luteus UV endonuclease assay, irradiating cells at G/sub 1/ to avoid artifacts arising from introduction of dimers in nascent strands. The possibility of other mechanisms being involved in the process of tolerance to DNA lesions is discussed.

  3. Stimulation of cytolytic T lymphocytes by azaguanine-resistant mouse tumor cells in selective hat medium

    International Nuclear Information System (INIS)

    Snick, J. van; Uyttenhove, C.; Pel, A. van; Boon, T.

    1981-01-01

    Primed syngeneic or umprimed allogeneic mouse spleen cells were stimulated with azaguanine-resistant P815 tumor cells that were killed by the addition of aminopterin to the stimulation medium. The recovery of lymphocytes and their cytolytic activity and specificity were similar to those obtained after stimulation with irradiated cells. This method conveniently replaces the inactivation of stimulatory cells by irradiation or mitomycin treatment. Moreover, it has the advantage of inactivating not only the stimulatory cells but also the tumor cells that often contaminate the spleens of tumor-bearing animals, provided these animals have been inoculated with azaguanine-resistant tumor cell mutants. (Auth.)

  4. Localization of tritiated vitamin A in lymph nodes of the mouse: an autoradiographic study of vitamin A-storing cells

    International Nuclear Information System (INIS)

    Hirosawa, K.; Yamada, E.

    1981-01-01

    Localization of tritiated vitamin A in lymph nodes of the mouse was investigated by the use of light- and electron-microscopic autoradiography. Young male mice were fed a diet containing 3H-vitamin A acetate for a week. Lymph nodes were removed and prepared for autoradiography. Lipid droplets in fibroblast-like cells showed high concentrations of radioactivity. These cells were distributed around lymphatic sinuses and blood vessels. The cells can, therefore, be classified as ''vitamin A-storing cells'' according to criteria proposed earlier by Yamada and Hirosawa (1976). Control animals from the same litter were maintained on ordinary laboratory food for the same period and examined by electron microscopy. Lipid-droplet-containing cells were found in the same areas as in the experimental animals but in fewer numbers. This suggests that the increased number of vitamin A-containing lipid droplets is due to hypervitaminosis A in the experimental animals. The presence of some cells containing these droplets in the control animals would imply that even under normal feeding conditions the animals ingested excess amounts of vitamin A, which was retained in lipid droplets. The stored vitamin A probably participates in metabolic processes such as the formation of glycoproteins in ground substance

  5. Saccharomyces cerevisiae-derived virus-like particle parvovirus B19 vaccine elicits binding and neutralizing antibodies in a mouse model for sickle cell disease.

    Science.gov (United States)

    Penkert, Rhiannon R; Young, Neal S; Surman, Sherri L; Sealy, Robert E; Rosch, Jason; Dormitzer, Philip R; Settembre, Ethan C; Chandramouli, Sumana; Wong, Susan; Hankins, Jane S; Hurwitz, Julia L

    2017-06-22

    Parvovirus B19 infections are typically mild in healthy individuals, but can be life threatening in individuals with sickle cell disease (SCD). A Saccharomyces cerevisiae-derived B19 VLP vaccine, now in pre-clinical development, is immunogenic in wild type mice when administered with the adjuvant MF59. Because SCD alters the immune response, we evaluated the efficacy of this vaccine in a mouse model for SCD. Vaccinated mice with SCD demonstrated similar binding and neutralizing antibody responses to those of heterozygous littermate controls following a prime-boost-boost regimen. Due to the lack of a mouse parvovirus B19 challenge model, we employed a natural mouse pathogen, Sendai virus, to evaluate SCD respiratory tract responses to infection. Normal mucosal and systemic antibody responses were observed in these mice. Results demonstrate that mice with SCD can respond to a VLP vaccine and to a respiratory virus challenge, encouraging rapid development of the B19 vaccine for patients with SCD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells.

    Science.gov (United States)

    Foxman, Ellen F; Storer, James A; Fitzgerald, Megan E; Wasik, Bethany R; Hou, Lin; Zhao, Hongyu; Turner, Paul E; Pyle, Anna Marie; Iwasaki, Akiko

    2015-01-20

    Most isolates of human rhinovirus, the common cold virus, replicate more robustly at the cool temperatures found in the nasal cavity (33-35 °C) than at core body temperature (37 °C). To gain insight into the mechanism of temperature-dependent growth, we compared the transcriptional response of primary mouse airway epithelial cells infected with rhinovirus at 33 °C vs. 37 °C. Mouse airway cells infected with mouse-adapted rhinovirus 1B exhibited a striking enrichment in expression of antiviral defense response genes at 37 °C relative to 33 °C, which correlated with significantly higher expression levels of type I and type III IFN genes and IFN-stimulated genes (ISGs) at 37 °C. Temperature-dependent IFN induction in response to rhinovirus was dependent on the MAVS protein, a key signaling adaptor of the RIG-I-like receptors (RLRs). Stimulation of primary airway cells with the synthetic RLR ligand poly I:C led to greater IFN induction at 37 °C relative to 33 °C at early time points poststimulation and to a sustained increase in the induction of ISGs at 37 °C relative to 33 °C. Recombinant type I IFN also stimulated more robust induction of ISGs at 37 °C than at 33 °C. Genetic deficiency of MAVS or the type I IFN receptor in infected airway cells permitted higher levels of viral replication, particularly at 37 °C, and partially rescued the temperature-dependent growth phenotype. These findings demonstrate that in mouse airway cells, rhinovirus replicates preferentially at nasal cavity temperature due, in part, to a less efficient antiviral defense response of infected cells at cool temperature.

  7. Transcriptomic analysis of mouse EL4 T cells upon T cell activation and in response to protein synthesis inhibition via cycloheximide treatment

    OpenAIRE

    Lim, Pek Siew; Hardy, Kristine; Peng, Kaiman; Shannon, Frances M.

    2015-01-01

    T cell activation involves the recognition of a foreign antigen complexed to the major histocompatibility complex on the antigen presenting T cell to the T cell receptor. This leads to activation of signaling pathways, which ultimately leads to induction of key cytokine genes responsible for eradication of foreign antigens. We used the mouse EL4 T cell as a model system to study genes that are induced as a result of T cell activation using phorbol myristate acetate (PMA) and calcium ionomycin...

  8. Defined Conditions for the Isolation and Expansion of Basal Prostate Progenitor Cells of Mouse and Human Origin

    Directory of Open Access Journals (Sweden)

    Thomas Höfner

    2015-03-01

    Full Text Available Methods to isolate and culture primary prostate epithelial stem/progenitor cells (PESCs have proven difficult and ineffective. Here, we present a method to grow and expand both murine and human basal PESCs long term in serum- and feeder-free conditions. The method enriches for adherent mouse basal PESCs with a Lin−SCA-1+CD49f+TROP2high phenotype. Progesterone and sodium selenite are additionally required for the growth of human Lin−CD49f+TROP2high PESCs. The gene-expression profiles of expanded basal PESCs show similarities to ESCs, and NF-kB function is critical for epithelial differentiation of sphere-cultured PESCs. When transplanted in combination with urogenital sinus mesenchyme, expanded mouse and human PESCs generate ectopic prostatic tubules, demonstrating their stem cell activity in vivo. This novel method will facilitate the molecular, genomic, and functional characterization of normal and pathologic prostate glands of mouse and human origin.

  9. Proteomic analysis of mouse thymoma EL4 cells treated with bis (tri-n-butyltin)oxide (TBTO)

    NARCIS (Netherlands)

    Osman, A.M.; Kol, S.; Peijnenburg, A.A.C.M.; Blokland, M.H.; Pennings, J.L.A.; Kleinjans, J.C.S.; Loveren, van H.

    2009-01-01

    Here, we report the results of proteomic analysis of the mouse thymoma EL4 cell line exposed to bis(tri-n-butylin)oxide (TBTO), an immunotoxic organotin compound. The objective of the work was to examine whether TBTO affects the expression of proteins in this cell line and to compare the

  10. [Regulatory T cells inhibit proliferation of mouse lymphoma cell line EL4 in vitro].

    Science.gov (United States)

    Zhang, Chen; Kong, Yan; Guo, Jun; Ying, Zhi-Tao; Yuan, Zhi-Hong; Zhang, Yun-Tao; Zheng, Wen; Song, Yu-Qin; Li, Ping-Ping; Zhu, Jun

    2010-10-01

    This study was aimed to investigate the effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cells and its mechanism in vitro. C57BL/6 mouse Treg cells were isolated by magnetic cell sorting (MACS). The purity of Treg cells and their expression of Foxp3 were identified by flow cytometry (FCM) and PT-PCR respectively. The suppression of Treg cells on EL4 cells was detected by 3H-TdR method. At the same time, enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of cytokine TGF-β1 and IL-10. The results showed that CD4+CD25+ T cells could be successfully isolated by MACS with the purity reaching 94.52% and the expression of Foxp3 reaching 84.72%. After sorting, the expression of Foxp3 mRNA could be detected by RT-PCR. 3H-TdR assay confirmed that regulatory T cells could suppress the proliferation of EL4 cells with or without antigen presenting cells (APC) or dendritic cells (DC), APC or DC might effectively enhance the suppression. In addition, DC alone also suppressed the proliferation. TGF-β1 and IL-10 could be detected in the supernatant by ELISA. It is concluded that the Treg cells can obviously suppress the proliferation of T cell lymphoma cells in vitro, APC or DC can enhance this suppressive effect, while the DC alone also can suppress the proliferation of EL4 cells, the TGF-β1 and IL-10 cytokine pathway may be one of the mechanisms of suppression.

  11. Cell and tissue kinetics of the subependymal layer in mouse brain following heavy charged particle irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Manley, N.B.; Fabrikant, J.I.; Alpen, E.L.

    1988-12-01

    The following studies investigate the cellular response and cell population kinetics of the subependymal layer in the mouse brain exposed to heavy charged particle irradiation. Partial brain irradiation with helium and neon ions was confined to one cortex of the brain. Both the irradiated and the unirradiated contralateral cortex showed similar disturbances of the cell and tissue kinetics in the subependymal layers. The irradiated hemisphere exhibited histological damage, whereas the unirradiated side appeared normal histologically. This study concerns the cell population and cell cycle kinetics of the subependymal layer in the mouse brain, and the effects of charged particle irradiations on this cell population. Quantitative high resolution autoradiography was used to study the kinetic parameters in this cell layer. This study should help in understanding the effects of these high-energy heavy ions on normal mammalian brain tissue. The response of the mammalian brain exposure to charged particle ionizing radiation may be extremely variable. It varies from minimal physiological changes to overt tissue necrosis depending on a number of factors such as: the administered dose, dose-rate, the volume of the irradiated tissue, and the biological end-point being examined.

  12. The phenotype of FancB-mutant mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Tae Moon; Ko, Jun Ho; Choi, Yong Jun; Hu Lingchuan [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245 (United States); Hasty, Paul, E-mail: hastye@uthscsa.edu [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245 (United States)

    2011-07-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by bone marrow failure, developmental defects and cancer. There are multiple FA genes that enable the repair of interstrand crosslinks (ICLs) in coordination with a variety of other DNA repair pathways in a way that is poorly understood. Here we present the phenotype of mouse embryonic stem (ES) cells mutated for FancB. We found FancB-mutant cells exhibited reduced cellular proliferation, hypersensitivity to the crosslinking agent mitomycin C (MMC), increased spontaneous and MMC-induced chromosomal abnormalities, reduced spontaneous sister chromatid exchanges (SCEs), reduced gene targeting, reduced MMC-induced Rad51 foci and absent MMC-induced FancD2 foci. Since FancB is on the X chromosome and since ES cells are typically XY, FancB is an excellent target for an epistatic analysis to elucidate FA's role in ICL repair.

  13. Differential radiosensitivity of mouse embryonic neurons and glia in cell culture

    International Nuclear Information System (INIS)

    Dambergs, R.; Kidson, C.

    1977-01-01

    The responses of neurons and glial cells to ultraviolet and γ-radiation were studied in cell cultures of embryonic mouse brains. A decrease in the ratio of glia to neurons occurred after both forms of irradiation. [ 3 H]thymidine labelling followed by autoradiography revealed that all glia were capable of replication, whereas 70 percent of neurons were non-replicating under the conditions of the study. Ultraviolet radiation caused a decrease in the proportion of replicating neurons but did not affect the proportion of replicating glia, whereas γ-radiation caused a decrease in DNA replication in both cell types. Levels of ultraviolet radiation-induced unscheduled DNA synthesis were lower in neurons than in glia. It is concluded that sensitivity to both ionizing and ultraviolet radiation of neurons and glial cells in embryonic brain cultures is determined primarily by the capacity for and state of DNA replication. Neurons which have already reached the stage of terminal differentiation are more resistant than replicating neurons of glial cells

  14. Tachykinins stimulate a subset of mouse taste cells.

    Directory of Open Access Journals (Sweden)

    Jeff Grant

    Full Text Available The tachykinins substance P (SP and neurokinin A (NKA are present in nociceptive sensory fibers expressing transient receptor potential cation channel, subfamily V, member 1 (TRPV1. These fibers are found extensively in and around the taste buds of several species. Tachykinins are released from nociceptive fibers by irritants such as capsaicin, the active compound found in chili peppers commonly associated with the sensation of spiciness. Using real-time Ca(2+-imaging on isolated taste cells, it was observed that SP induces Ca(2+ -responses in a subset of taste cells at concentrations in the low nanomolar range. These responses were reversibly inhibited by blocking the SP receptor NK-1R. NKA also induced Ca(2+-responses in a subset of taste cells, but only at concentrations in the high nanomolar range. These responses were only partially inhibited by blocking the NKA receptor NK-2R, and were also inhibited by blocking NK-1R indicating that NKA is only active in taste cells at concentrations that activate both receptors. In addition, it was determined that tachykinin signaling in taste cells requires Ca(2+-release from endoplasmic reticulum stores. RT-PCR analysis further confirmed that mouse taste buds express NK-1R and NK-2R. Using Ca(2+-imaging and single cell RT-PCR, it was determined that the majority of tachykinin-responsive taste cells were Type I (Glial-like and umami-responsive Type II (Receptor cells. Importantly, stimulating NK-1R had an additive effect on Ca(2+ responses evoked by umami stimuli in Type II (Receptor cells. This data indicates that tachykinin release from nociceptive sensory fibers in and around taste buds may enhance umami and other taste modalities, providing a possible mechanism for the increased palatability of spicy foods.

  15. Generation of glucose-responsive functional islets with a three-dimensional structure from mouse fetal pancreatic cells and iPS cells in vitro.

    Directory of Open Access Journals (Sweden)

    Hiroki Saito

    Full Text Available Islets of Langerhans are a pancreatic endocrine compartment consisting of insulin-producing β cells together with several other hormone-producing cells. While some insulin-producing cells or immature pancreatic cells have been generated in vitro from ES and iPS cells, islets with proper functions and a three-dimensional (3D structure have never been successfully produced. To test whether islets can be formed in vitro, we first examined the potential of mouse fetal pancreatic cells. We found that E16.5 pancreatic cells, just before forming islets, were able to develop cell aggregates consisting of β cells surrounded by glucagon-producing α cells, a structure similar to murine adult islets. Moreover, the transplantation of these cells improved blood glucose levels in hyperglycemic mice. These results indicate that functional islets are formed in vitro from fetal pancreatic cells at a specific developmental stage. By adopting these culture conditions to the differentiation of mouse iPS cells, we developed a two-step system to generate islets, i.e. immature pancreatic cells were first produced from iPS cells, and then transferred to culture conditions that allowed the formation of islets from fetal pancreatic cells. The islets exhibited distinct 3D structural features similar to adult pancreatic islets and secreted insulin in response to glucose concentrations. Transplantation of the islets improved blood glucose levels in hyperglycemic mice. In conclusion, the two-step culture system allows the generation of functional islets with a 3D structure from iPS cells.

  16. Specialized mouse embryonic stem cells for studying vascular development.

    Science.gov (United States)

    Glaser, Drew E; Burns, Andrew B; Hatano, Rachel; Medrzycki, Magdalena; Fan, Yuhong; McCloskey, Kara E

    2014-01-01

    Vascular progenitor cells are desirable in a variety of therapeutic strategies; however, the lineage commitment of endothelial and smooth muscle cell from a common progenitor is not well-understood. Here, we report the generation of the first dual reporter mouse embryonic stem cell (mESC) lines designed to facilitate the study of vascular endothelial and smooth muscle development in vitro. These mESC lines express green fluorescent protein (GFP) under the endothelial promoter, Tie-2, and Discomsoma sp. red fluorescent protein (RFP) under the promoter for alpha-smooth muscle actin (α-SMA). The lines were then characterized for morphology, marker expression, and pluripotency. The mESC colonies were found to exhibit dome-shaped morphology, alkaline phosphotase activity, as well as expression of Oct 3/4 and stage-specific embryonic antigen-1. The mESC colonies were also found to display normal karyotypes and are able to generate cells from all three germ layers, verifying pluripotency. Tissue staining confirmed the coexpression of VE (vascular endothelial)-cadherin with the Tie-2 GFP+ expression on endothelial structures and smooth muscle myosin heavy chain with the α-SMA RFP+ smooth muscle cells. Lastly, it was verified that the developing mESC do express Tie-2 GFP+ and α-SMA RFP+ cells during differentiation and that the GFP+ cells colocalize with the vascular-like structures surrounded by α-SMA-RFP cells. These dual reporter vascular-specific mESC permit visualization and cell tracking of individual endothelial and smooth muscle cells over time and in multiple dimensions, a powerful new tool for studying vascular development in real time.

  17. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

    International Nuclear Information System (INIS)

    Lee, Jingu; Park, Sangkyu; Roh, Sangho

    2015-01-01

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy

  18. Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jingu; Park, Sangkyu; Roh, Sangho, E-mail: sangho@snu.ac.kr

    2015-05-15

    A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. The cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy.

  19. Supraphysiologic control over HIV-1 replication mediated by CD8 T cells expressing a re-engineered CD4-based chimeric antigen receptor.

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    Rachel S Leibman

    2017-10-01

    Full Text Available HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent in vitro at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy.

  20. An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

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    David A. Turner

    2014-06-01

    Full Text Available Embryonic Stem cells derived from the epiblast tissue of the mammalian blastocyst retain the capability to differentiate into any adult cell type and are able to self-renew indefinitely under appropriate culture conditions. Despite the large amount of knowledge that we have accumulated to date about the regulation and control of self-renewal, efficient directed differentiation into specific tissues remains elusive. In this work, we have analysed in a systematic manner the interaction between the dynamics of loss of pluripotency and Activin/Nodal, BMP4 and Wnt signalling in fate assignment during the early stages of differentiation of mouse ES cells in culture. During the initial period of differentiation, cells exit from pluripotency and enter an Epi-like state. Following this transient stage, and under the influence of Activin/Nodal and BMP signalling, cells face a fate choice between differentiating into neuroectoderm and contributing to Primitive Streak fates. We find that Wnt signalling does not suppress neural development as previously thought and that it aids both fates in a context dependent manner. Our results suggest that as cells exit pluripotency they are endowed with a primary neuroectodermal fate and that the potency to become endomesodermal rises with time. We suggest that this situation translates into a “race for fates” in which the neuroectodermal fate has an advantage.

  1. Resveratrol Enhances Self-Renewal of Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Li, Na; Du, Zhaoyu; Shen, Qiaoyan; Lei, Qijing; Zhang, Ying; Zhang, Mengfei; Hua, Jinlian

    2017-07-01

    Resveratrol (RSV) has been shown to affect the differentiation of several types of stem cells, while the detailed mechanism is elusive. Here, we aim to investigate the function of RSV in self-renewal of mouse embryonic stem cells (ESCs) and the related mechanisms. In contrast with its reported roles, we found unexpectedly that differentiated ESCs or iPSCs treated by RSV would not show further differentiation, but regained a naïve pluripotency state with higher expressions of core transcriptional factors and with the ability to differentiate into all three germ layers when transplanted in vivo. In accordance with these findings, RSV also enhanced cell cycle progression of ESCs via regulating cell cycle-related proteins. Finally, enhanced activation of JAK/STAT3 signaling pathway and suppressed activation of mTOR were found essential in enhancing the self-renewal of ESCs by RSV. Our finding discovered a novel function of RSV in enhancing the self-renewal of ESCs, and suggested that the timing of treatment and concentration of RSV determined the final effect of it. Our work may contribute to understanding of RSV in the self-renewal maintenance of pluripotent stem cells, and may also provide help to the generation and maintenance of iPSCs in vitro. J. Cell. Biochem. 118: 1928-1935, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  2. Inducible and reversible suppression of Npm1 gene expression using stably integrated small interfering RNA vector in mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Wang Beibei; Lu Rui; Wang Weicheng; Jin Ying

    2006-01-01

    The tetracycline (Tc)-inducible small interference RNA (siRNA) is a powerful tool for studying gene function in mammalian cells. However, the system is infrequently utilized in embryonic stem (ES) cells. Here, we present First application of the Tc-inducible, stably integrated plasmid-based siRNA system in mouse ES cells to down-regulate expression of Npm1, an essential gene for embryonic development. The physiological role of Npm1 in ES cells has not been defined. Our data show that the knock-down of Npm1 expression by this siRNA system was not only highly efficient, but also Tc- dose- and induction time-dependent. Particularly, the down-regulation of Npm1 expression was reversible. Importantly, suppression of Npm1 expression in ES cells resulted in reduced cell proliferation. Taken together, this system allows for studying gene function in a highly controlled manner, otherwise difficult to achieve in ES cells. Moreover, our results demonstrate that Npm1 is essential for ES cell proliferation

  3. Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy

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    Haiying Guo

    2018-01-01

    Full Text Available Dermal papilla (DP plays important roles in hair follicle regeneration. Long-term culture of mouse DP cells can provide enough cells for research and application of DP cells. We optimized the culture strategy for DP cells from three dimensions: stepwise dissection, collagen I coating, and optimized culture medium. Based on the optimized culture strategy, we immortalized primary DP cells with SV40 large T antigen, and established several immortalized DP cell strains. By comparing molecular expression and morphologic characteristics with primary DP cells, we found one cell strain named iDP6 was similar with primary DP cells. Further identifications illustrate that iDP6 expresses FGF7 and α-SMA, and has activity of alkaline phosphatase. During the process of characterization of immortalized DP cell strains, we also found that cells in DP were heterogeneous. We successfully optimized culture strategy for DP cells, and established an immortalized DP cell strain suitable for research and application of DP cells.

  4. Tissue- and Cell Type-Specific Expression of the Long Noncoding RNA Klhl14-AS in Mouse

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    Sara Carmela Credendino

    2017-01-01

    Full Text Available lncRNAs are acquiring increasing relevance as regulators in a wide spectrum of biological processes. The extreme heterogeneity in the mechanisms of action of these molecules, however, makes them very difficult to study, especially regarding their molecular function. A novel lncRNA has been recently identified as the most enriched transcript in mouse developing thyroid. Due to its genomic localization antisense to the protein-encoding Klhl14 gene, we named it Klhl14-AS. In this paper, we highlight that mouse Klhl14-AS produces at least five splicing variants, some of which have not been previously described. Klhl14-AS is expressed with a peculiar pattern, characterized by diverse relative abundance of its isoforms in different mouse tissues. We examine the whole expression level of Klhl14-AS in a panel of adult mouse tissues, showing that it is expressed in the thyroid, lung, kidney, testis, ovary, brain, and spleen, although at different levels. In situ hybridization analysis reveals that, in the context of each organ, Klhl14-AS shows a cell type-specific expression. Interestingly, databases report a similar expression profile for human Klhl14-AS. Our observations suggest that this lncRNA could play cell type-specific roles in several organs and pave the way for functional characterization of this gene in appropriate biological contexts.

  5. Angelica Sinensis Polysaccharide Prevents Hematopoietic Stem Cells Senescence in D-Galactose-Induced Aging Mouse Model

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    Xinyi Mu

    2017-01-01

    Full Text Available Age-related regression in hematopoietic stem/progenitor cells (HSC/HPCs limits replenishment of the blood and immune system and hence contributes to hematopoietic diseases and declined immunity. In this study, we employed D-gal-induced aging mouse model and observed the antiaging effects of Angelica Sinensis Polysaccharide (ASP, a major active ingredient in dong quai (Chinese Angelica Sinensis, on the Sca-1+ HSC/HPCs in vivo. ASP treatment prevents HSC/HPCs senescence with decreased AGEs levels in the serum, reduced SA-β-Gal positive cells, and promoted CFU-Mix formation in the D-gal administrated mouse. We further found that multiple mechanisms were involved: (1 ASP treatment prevented oxidative damage as total antioxidant capacity was increased and levels of reactive oxygen species (ROS, 8-OHdG, and 4-HNE were declined, (2 ASP reduced the expression of γ-H2A.X which is a DNA double strand breaks (DSBs marker and decreased the subsequent ectopic expressions of effectors in p16Ink4a-RB and p19Arf-p21Cip1/Waf senescent pathways, and (3 ASP inhibited the excessive activation of Wnt/β-catenin signaling in aged HSC/HPCs, as the expressions of β-catenin, phospho-GSK-3β, and TCF-4 were decreased, and the cyto-nuclear translocation of β-catenin was inhibited. Moreover, compared with the positive control of Vitamin E, ASP exhibited a better antiaging effect and a weaker antioxidation ability, suggesting a novel protective role of ASP in the hematopoietic system.

  6. α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

    International Nuclear Information System (INIS)

    Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-01-01

    We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blot and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated

  7. α2 Integrin, extracellular matrix metalloproteinase inducer, and matrix metalloproteinase-3 act sequentially to induce differentiation of mouse embryonic stem cells into odontoblast-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Kawai, Rie; Hase, Naoko; Hiyama, Taiki; Yamaguchi, Hideyuki [Department of Endodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650 (Japan); Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, Nagoya, Aichi 464-8651 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, Nagoya 464-8650 (Japan)

    2015-02-01

    We previously reported that interleukin 1β acts via matrix metalloproteinase (MMP)-3 to regulate cell proliferation and suppress apoptosis in α2 integrin-positive odontoblast-like cells differentiated from mouse embryonic stem (ES) cells. Here we characterize the signal cascade underpinning odontoblastic differentiation in mouse ES cells. The expression of α2 integrin, extracellular matrix metalloproteinase inducer (Emmprin), and MMP-3 mRNA and protein were all potently increased during odontoblastic differentiation. Small interfering RNA (siRNA) disruption of the expression of these effectors potently suppressed the expression of the odontoblastic biomarkers dentin sialophosphoprotein, dentin matrix protein-1 and alkaline phosphatase, and blocked odontoblast calcification. Our siRNA, western blot and blocking antibody analyses revealed a unique sequential cascade involving α2 integrin, Emmprin and MMP-3 that drives ES cell differentiation into odontoblasts. This cascade requires the interaction between α2 integrin and Emmprin and is potentiated by exogenous MMP-3. Finally, although odontoblast-like cells potently express α2, α6, αV, β1, and β3, integrins, we confirmed that β1 integrin acts as the trigger for ES cell differentiation, apparently in complex with α2 integrin. These results demonstrate a unique and unanticipated role for an α2 integrin-, Emmprin-, and MMP-3-mediated signaling cascade in driving mouse ES cell differentiation into odontoblast-like cells. - Highlights: • Odontoblast differentiation requires activation of α2 integrin, Emmprin and MMP-3. • α2 integrin, Emmprin and MMP-3 form a sequential signaling cascade. • β1 integrin acts a specific trigger for odontoblast differentiation. • The role of these effectors is highly novel and unanticipated.

  8. Transient developmental Purkinje cell axonal torpedoes in healthy and ataxic mouse cerebellum

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    Lovisa Ljungberg

    2016-11-01

    Full Text Available Information is carried out of the cerebellar cortical microcircuit via action potentials propagated along Purkinje cell axons. In several human neurodegenerative diseases, focal axonal swellings on Purkinje cells – known as torpedoes – have been associated with Purkinje cell loss. Interestingly, torpedoes are also reported to appear transiently during development in rat cerebellum. The function of Purkinje cell axonal torpedoes in health as well as in disease is poorly understood. We investigated the properties of developmental torpedoes in the postnatal mouse cerebellum of wildtype and transgenic mice. We found that Purkinje cell axonal torpedoes transiently appeared on axons of Purkinje neurons, with the largest number of torpedoes observed at postnatal day 11 (P11. This was after peak developmental apoptosis had occurred, when Purkinje cell counts in a lobule were static, suggesting that most developmental torpedoes appear on axons of neurons that persist into adulthood. We found that developmental torpedoes were not associated with a presynaptic GABAergic marker, indicating that they are not synapses. They were seldom found at axonal collateral branch points, and lacked microglia enrichment, suggesting that they are unlikely to be involved in axonal refinement. Interestingly, we found several differences between developmental torpedoes and disease-related torpedoes: developmental torpedoes occured largely on myelinated axons, and were not associated with changes in basket cell innervation on their parent soma. Disease-related torpedoes are typically reported to contain neurofilament; while the majority of developmental torpedoes did as well, a fraction of smaller developmental torpedoes did not. These differences indicate that developmental torpedoes may not be functionally identical to disease-related torpedoes. To study this further, we used a mouse model of spinocerebellar ataxia type 6 (SCA6, and found elevated disease

  9. Pharmacologic inhibition of MLK3 kinase activity blocks the in vitro migratory capacity of breast cancer cells but has no effect on breast cancer brain metastasis in a mouse xenograft model.

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    Kun Hyoe Rhoo

    Full Text Available Brain metastasis of breast cancer is an important clinical problem, with few therapeutic options and a poor prognosis. Recent data have implicated mixed lineage kinase 3 (MLK3 in controlling the in vitro migratory capacity of breast cancer cells, as well as the metastasis of MDA-MB-231 breast cancer cells from the mammary fat pad to distant lymph nodes in a mouse xenograft model. We therefore set out to test whether MLK3 plays a role in brain metastasis of breast cancer cells. To address this question, we used a novel, brain penetrant, MLK3 inhibitor, URMC099. URMC099 efficiently inhibited the migration of breast cancer cells in an in vitro cell monolayer wounding assay, and an in vitro transwell migration assay, but had no effect on in vitro cell growth. We also tested the effect of URMC099 on tumor formation in a mouse xenograft model of breast cancer brain metastasis. This analysis showed that URMC099 had no effect on the either the frequency or size of breast cancer brain metastases. We conclude that pharmacologic inhibition of MLK3 by URMC099 can reduce the in vitro migratory capacity of breast cancer cells, but that it has no effect on either the frequency or size of breast cancer brain metastases, in a mouse xenograft model.

  10. The effect of melatonin on mouse jejunal crypt cell survival and apoptosis

    International Nuclear Information System (INIS)

    Kang, Jin Oh; Ha, Eun Young; Baik, Hyung Hwan; Cho, Yong Ho; Hong, Seong Eon

    2000-01-01

    To evaluate protective mechanism of melatonin against radiation damage and its relationship with apoptosis in mouse jejunum. 168 mice were divided into 28 groups according to radiation dose and melatonin treatment. To analysis crypt survival, microcolony survival assay was done according to Withers and Elkind's method. To analysis apoptosis, TUNEL assay was done according to Labet-Moleur's method. Radiation protection effect of melatonin was demonstrated by crypt survival assay and its effect was stronger in high radiation dose area. Apoptosis index with 8 Gy irradiation was 18.4% in control group and 16.5% in melatonin treated group. After 18 Gy, apoptosis index was 17.2%in control group and 15.4% in melatonin treated group. Apoptosis index did not show statistically significant difference between melatonin shows clear protective effect in mouse jejunum against radiation damage but its protective effect seems not to be related with apoptosis protection effect

  11. Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency.

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    Sander Barnhoorn

    2014-10-01

    Full Text Available As part of the Nucleotide Excision Repair (NER process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS, or the infantile lethal cerebro-oculo-facio-skeletal (COFS syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional Xpg-/- mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4-5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.

  12. In vivo transplantation of enteric neural crest cells into mouse gut; Engraftment, functional integration and long-term safety

    NARCIS (Netherlands)

    J.E. Cooper (Julie E.); C. Mccann; D. Natarajan (Dipa); S. Choudhury; W. Boesmans (Werend); J.-M. Delalande (Jean-Marie); P.V. Berghe (Pieter Vanden); A.J. Burns (Alan); N. Thapar (Nikhil)

    2016-01-01

    textabstractObjectives: Enteric neuropathies are severe gastrointestinal disorders with unsatisfactory outcomes. We aimed to investigate the potential of enteric neural stem cell therapy approaches for such disorders by transplanting mouse enteric neural crest cells (ENCCs) into ganglionic and

  13. Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4-/- mouse and bipolar cells in the rd1 mouse and human retina ex vivo.

    Science.gov (United States)

    De Silva, Samantha R; Charbel Issa, Peter; Singh, Mandeep S; Lipinski, Daniel M; Barnea-Cramer, Alona O; Walker, Nathan J; Barnard, Alun R; Hankins, Mark W; MacLaren, Robert E

    2016-11-01

    Gene therapy using adeno-associated viral (AAV) vectors for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4 -/- and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4 -/- mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treat retinal degenerations in which high levels of transgene expression are required.

  14. CD45lowc-Kithigh cells have hematopoietic properties in the mouse aorta-gonad-mesonephros region

    International Nuclear Information System (INIS)

    Nobuhisa, Ikuo; Yamasaki, Shoutarou; Ramadan, Ahmed; Taga, Tetsuya

    2012-01-01

    Long-term reconstituting hematopoietic stem cells first arise from the aorta of the aorta-gonad-mesonephros (AGM) region in a mouse embryo. We have previously reported that in cultures of the dispersed AGM region, CD45 low c-Kit + cells possess the ability to reconstitute multilineage hematopoietic cells, but investigations are needed to show that this is not a cultured artifact and to clarify when and how this population is present. Based on the expression profile of CD45 and c-Kit in freshly dissociated AGM cells from embryonic day 9.5 (E9.5) to E12.5 and aorta cells in the AGM from E13.5 to E15.5, we defined six cell populations (CD45 − c-Kit − , CD45 − c-Kit low , CD45 − c-Kit high , CD45 low c-Kit high , CD45 high c-Kit high , and CD45 high c-Kit very low ). Among these six populations, CD45 low c-Kit high cells were most able to form hematopoietic cell colonies, but their ability decreased after E11.5 and was undetectable at E13.5 and later. The CD45 low c-Kit high cells showed multipotency in vitro. We demonstrated further enrichment of hematopoietic activity in the Hoechst dye-effluxing side population among the CD45 low c-Kit high cells. Here, we determined that CD45 low c-Kit high cells arise from the lateral plate mesoderm using embryonic stem cell-derived differentiation system. In conclusion, CD45 low c-Kit high cells are the major hematopoietic cells of mouse AGM.

  15. Discrimination of taste qualities among mouse fungiform taste bud cells.

    Science.gov (United States)

    Yoshida, Ryusuke; Miyauchi, Aya; Yasuo, Toshiaki; Jyotaki, Masafumi; Murata, Yoshihiro; Yasumatsu, Keiko; Shigemura, Noriatsu; Yanagawa, Yuchio; Obata, Kunihiko; Ueno, Hiroshi; Margolskee, Robert F; Ninomiya, Yuzo

    2009-09-15

    Multiple lines of evidence from molecular studies indicate that individual taste qualities are encoded by distinct taste receptor cells. In contrast, many physiological studies have found that a significant proportion of taste cells respond to multiple taste qualities. To reconcile this apparent discrepancy and to identify taste cells that underlie each taste quality, we investigated taste responses of individual mouse fungiform taste cells that express gustducin or GAD67, markers for specific types of taste cells. Type II taste cells respond to sweet, bitter or umami tastants, express taste receptors, gustducin and other transduction components. Type III cells possess putative sour taste receptors, and have well elaborated conventional synapses. Consistent with these findings we found that gustducin-expressing Type II taste cells responded best to sweet (25/49), bitter (20/49) or umami (4/49) stimuli, while all GAD67 (Type III) taste cells examined (44/44) responded to sour stimuli and a portion of them showed multiple taste sensitivities, suggesting discrimination of each taste quality among taste bud cells. These results were largely consistent with those previously reported with circumvallate papillae taste cells. Bitter-best taste cells responded to multiple bitter compounds such as quinine, denatonium and cyclohexamide. Three sour compounds, HCl, acetic acid and citric acid, elicited responses in sour-best taste cells. These results suggest that taste cells may be capable of recognizing multiple taste compounds that elicit similar taste sensation. We did not find any NaCl-best cells among the gustducin and GAD67 taste cells, raising the possibility that salt sensitive taste cells comprise a different population.

  16. Effects of lithium chloride as a potential radioprotective agent on radiation response of DNA synthesis in mouse germinal cells

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, D. [Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Rajan, R. [Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Krishnamoorthy, L. [Kidwai Memorial Institute of Oncology, Bangalore 560 029 (India); Singh, B.B. [Radiation Biology and Biochemistry Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India)

    1997-06-01

    Mouse spermatogonial germ cells are highly sensitive to ionizing radiation. Lithium salts are reported to stimulate the postirradiation recovery of hematopoietic marrow cells. We have, therefore, examined whether administered lithium chloride (LiCl) would also be able to protect the mouse germinal cells against radiation injury. Taking DNA synthesis as an endpoint, our results show that the testicular DNA-specific activity in irradiated mice was higher by 61% on average when they had been pretreated with LiCl both 24 h and 1 h prior to {gamma}-irradiation (2.0 Gy). It was also observed that the DNA synthetic activity in the germinal cells fully recovered after LiCl pretreatment at doses of 40 mg per kg body weight prior to total body irradiation of 0.05-0.25 Gy, whereas at doses of 0.5-6.0 Gy, following the same procedure of LiCl pretreatment, only an incomplete recovery was observed. The dose reduction factor for LiCl is 1.84. The current findings indicate that pretreatment with LiCl provides considerable protection against radiation damage in mouse spermatogonia. (orig.). With 3 tabs.

  17. Survival of mouse testicular stem cells after γ or neutron irradiation

    International Nuclear Information System (INIS)

    Lu, C.C.; Meistrich, M.L.; Thames, H.D. Jr.

    1980-01-01

    The survival of mouse testicular stem cells after γ or neutron irradiation was measured by counts of repopulated tubular cross sections and by the numbers of differentiated spermatogenic cells produced. The numbers of such cells were determined either by sperm head counts of the X-isozyme of lactate dehydrogenase enzyme levels. Qualitatively similar results were obtained with all three assays. The results have confirmed that, with C3H mice, stem-cell survival is higher when the γ-radiation dose is fractionated by a 24-h interval. Single-dose γ-radiaton survival curves for the stem cell had large shoulders and also showed the presence of a radioresistant subpopulation which predominated after doses greater than 600 rad. Part of the shoulder must have resulted from repair of sublethal damage since neutron irradiation produced survival curves with smaller shoulders. The relative biological effectiveness for stem-cell killing for these neutrons (mean energy, 22 MeV) varied from about 2.9 at 10 rad of γ radiation to 2.2 at 600 rad

  18. Single cell lineage analysis of mouse embryonic stem cells at the exit from pluripotency

    Directory of Open Access Journals (Sweden)

    Jamie Trott

    2013-08-01

    Understanding how interactions between extracellular signalling pathways and transcription factor networks influence cellular decision making will be crucial for understanding mammalian embryogenesis and for generating specialised cell types in vitro. To this end, pluripotent mouse Embryonic Stem (mES cells have proven to be a useful model system. However, understanding how transcription factors and signalling pathways affect decisions made by individual cells is confounded by the fact that measurements are generally made on groups of cells, whilst individual mES cells differentiate at different rates and towards different lineages, even in conditions that favour a particular lineage. Here we have used single-cell measurements of transcription factor expression and Wnt/β-catenin signalling activity to investigate their effects on lineage commitment decisions made by individual cells. We find that pluripotent mES cells exhibit differing degrees of heterogeneity in their expression of important regulators from pluripotency, depending on the signalling environment to which they are exposed. As mES cells differentiate, downregulation of Nanog and Oct4 primes cells for neural commitment, whilst loss of Sox2 expression primes cells for primitive streak commitment. Furthermore, we find that Wnt signalling acts through Nanog to direct cells towards a primitive streak fate, but that transcriptionally active β-catenin is associated with both neural and primitive streak commitment. These observations confirm and extend previous suggestions that pluripotency genes influence lineage commitment and demonstrate how their dynamic expression affects the direction of lineage commitment, whilst illustrating two ways in which the Wnt signalling pathway acts on this network during cell fate assignment.

  19. MiRNA-mediated regulation of cell signaling and homeostasis in the early mouse embryo.

    Science.gov (United States)

    Pernaute, Barbara; Spruce, Thomas; Rodriguez, Tristan A; Manzanares, Miguel

    2011-02-15

    At the time of implantation the mouse embryo is composed of three tissues the epiblast, trophectoderm and primitive endoderm. As development progresses the epiblast goes on to form the foetus whilst the trophectoderm and primitive endoderm give rise to extra-embryonic structures with important roles in embryo patterning and nutrition. Dramatic changes in gene expression occur during early embryo development and these require regulation at different levels. miRNAs are small non coding RNAs that have emerged over the last decade as important post-transcriptional repressors of gene expression. The roles played by miRNAs during early mammalian development are only starting to be elucidated. In order to gain insight into the function of miRNAs in the different lineages of the early mouse embryo we have analysed in depth the phenotype of embryos and extra-embryonic stem cells mutant for the miRNA maturation protein Dicer. This study revealed that miRNAs are involved in regulating cell signaling and homeostasis in the early embryo. Specifically, we identified a role for miRNAs in regulating the Erk signaling pathway in the extra-embryonic endoderm, cell cycle progression in extra-embryonic tissues and apoptosis in the epiblast.

  20. A Humanized Mouse Model Generated Using Surplus Neonatal Tissue

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    Matthew E. Brown

    2018-04-01

    Full Text Available Summary: Here, we describe the NeoThy humanized mouse model created using non-fetal human tissue sources, cryopreserved neonatal thymus and umbilical cord blood hematopoietic stem cells (HSCs. Conventional humanized mouse models are made by engrafting human fetal thymus and HSCs into immunocompromised mice. These mice harbor functional human T cells that have matured in the presence of human self-peptides and human leukocyte antigen molecules. Neonatal thymus tissue is more abundant and developmentally mature and allows for creation of up to ∼50-fold more mice per donor compared with fetal tissue models. The NeoThy has equivalent frequencies of engrafted human immune cells compared with fetal tissue humanized mice and exhibits T cell function in assays of ex vivo cell proliferation, interferon γ secretion, and in vivo graft infiltration. The NeoThy model may provide significant advantages for induced pluripotent stem cell immunogenicity studies, while bypassing the requirement for fetal tissue. : Corresponding author William Burlingham and colleagues created a humanized mouse model called the NeoThy. The NeoThy uses human neonatal, rather than fetal, tissue sources for generating a human immune system within immunocompromised mouse hosts. NeoThy mice are an attractive alternative to conventional humanized mouse models, as they enable robust and reproducible iPSC immunogenicity experiments in vivo. Keywords: NeoThy, humanized mouse, iPSC, PSC, immunogenicity, transplantation, immunology, hematopoietic stem cells, induced pluripotent stem cells, thymus

  1. Post-Transcriptional Control of Gene Expression in Mouse Early Embryo Development: A View from the Tip of the Iceberg

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    Claudio Sette

    2011-04-01

    Full Text Available Fertilization is a very complex biological process that requires the perfect cooperation between two highly specialized cells: the male and female gametes. The oocyte provides the physical space where this process takes place, most of the energetic need, and half of the genetic contribution. The spermatozoon mostly contributes the other half of the chromosomes and it is specialized to reach and to penetrate the oocyte. Notably, the mouse oocyte and early embryo are transcriptionally inactive. Hence, they fully depend on the maternal mRNAs and proteins stored during oocyte maturation to drive the onset of development. The new embryo develops autonomously around the four-cell stage, when maternal supplies are exhausted and the zygotic genome is activated in mice. This oocyte-to-embryo transition needs an efficient and tightly regulated translation of the maternally-inherited mRNAs, which likely contributes to embryonic genome activation. Full understanding of post-transcriptional regulation of gene expression in early embryos is crucial to understand the reprogramming of the embryonic genome, it might help driving reprogramming of stem cells in vitro and will likely improve in vitro culturing of mammalian embryos for assisted reproduction. Nevertheless, the knowledge of the mechanism(s underlying this fundamental step in embryogenesis is still scarce, especially if compared to other model organisms. We will review here the current knowledge on the post-transcriptional control of gene expression in mouse early embryos and discuss some of the unanswered questions concerning this fascinating field of biology.

  2. Rho-associated kinase activity is required for proper morphogenesis of the inner cell mass in the mouse blastocyst.

    Science.gov (United States)

    Laeno, Arlene May A; Tamashiro, Dana Ann A; Alarcon, Vernadeth B

    2013-11-01

    The blastocyst consists of the outer layer of trophectoderm and pluripotent inner cell mass (ICM), the precursor of the placenta and fetus, respectively. During blastocyst expansion, the ICM adopts a compact, ovoidal shape, whose proper morphology is crucial for normal embryogenesis. Rho-associated kinase (ROCK), an effector of small GTPase RHO signaling, mediates the diverse cellular processes of morphogenesis, but its role in ICM morphogenesis is unclear. Here, we demonstrate that ROCK is required for cohesion of ICM cells and formation of segregated tissues called primitive endoderm (PrE) and epiblast (Epi) in the ICM of the mouse blastocyst. Blastocyst treatment with ROCK inhibitors Y-27632 and Fasudil caused widening or spreading of the ICM, and intermingling of PrE and Epi. Widening of ICM was independent of trophectoderm because isolated ICMs as well as colonies of mouse embryonic stem cells (mESC) also spread upon Y-27632 treatment. PrE, Epi, and trophectoderm cell numbers were similar between control and treated blastocysts, suggesting that ROCK inhibition affected ICM morphology but not lineage differentiation. Rock1 and Rock2 knockdown via RNA interference in mESC also induced spreading, supporting the conclusion that morphological defects caused by the pharmacological inhibitors were due to ROCK inactivation. When blastocysts were transferred into surrogates, implantation efficiencies were unaffected by ROCK inhibition, but treated blastocysts yielded greater fetal loss. These results show that proper ICM morphology is dependent on ROCK activity and is crucial for fetal development. Our studies have wider implication for improving efficiencies of human assisted reproductive technologies that diminish pregnancy loss and promote successful births.

  3. The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells

    DEFF Research Database (Denmark)

    Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe

    2009-01-01

    We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell......-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However......, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell...

  4. Proliferating Cell Nuclear Antigen (PCNA) Regulates Primordial Follicle Assembly by Promoting Apoptosis of Oocytes in Fetal and Neonatal Mouse Ovaries

    Science.gov (United States)

    Zhang, Yuanwei; Jiang, Xiaohua; Zhang, Huan; Ma, Tieliang; Zheng, Wei; Sun, Rui; Shen, Wei; Sha, Jiahao; Cooke, Howard J.; Shi, Qinghua

    2011-01-01

    Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA), a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer guanulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries. PMID:21253613

  5. Proliferating cell nuclear antigen (PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries.

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    Bo Xu

    Full Text Available Primordial follicles, providing all the oocytes available to a female throughout her reproductive life, assemble in perinatal ovaries with individual oocytes surrounded by granulosa cells. In mammals including the mouse, most oocytes die by apoptosis during primordial follicle assembly, but factors that regulate oocyte death remain largely unknown. Proliferating cell nuclear antigen (PCNA, a key regulator in many essential cellular processes, was shown to be differentially expressed during these processes in mouse ovaries using 2D-PAGE and MALDI-TOF/TOF methodology. A V-shaped expression pattern of PCNA in both oocytes and somatic cells was observed during the development of fetal and neonatal mouse ovaries, decreasing from 13.5 to 18.5 dpc and increasing from 18.5 dpc to 5 dpp. This was closely correlated with the meiotic prophase I progression from pre-leptotene to pachytene and from pachytene to diplotene when primordial follicles started to assemble. Inhibition of the increase of PCNA expression by RNA interference in cultured 18.5 dpc mouse ovaries strikingly reduced the apoptosis of oocytes, accompanied by down-regulation of known pro-apoptotic genes, e.g. Bax, caspase-3, and TNFα and TNFR2, and up-regulation of Bcl-2, a known anti-apoptotic gene. Moreover, reduced expression of PCNA was observed to significantly increase primordial follicle assembly, but these primordial follicles contained fewer granulosa cells. Similar results were obtained after down-regulation by RNA interference of Ing1b, a PCNA-binding protein in the UV-induced apoptosis regulation. Thus, our results demonstrate that PCNA regulates primordial follicle assembly by promoting apoptosis of oocytes in fetal and neonatal mouse ovaries.

  6. The Hsc/Hsp70 co-chaperone network controls antigen aggregation and presentation during maturation of professional antigen presenting cells.

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    Nadja Kettern

    Full Text Available The maturation of mouse macrophages and dendritic cells involves the transient deposition of ubiquitylated proteins in the form of dendritic cell aggresome-like induced structures (DALIS. Transient DALIS formation was used here as a paradigm to study how mammalian cells influence the formation and disassembly of protein aggregates through alterations of their proteostasis machinery. Co-chaperones that modulate the interplay of Hsc70 and Hsp70 with the ubiquitin-proteasome system (UPS and the autophagosome-lysosome pathway emerged as key regulators of this process. The chaperone-associated ubiquitin ligase CHIP and the ubiquitin-domain protein BAG-1 are essential for DALIS formation in mouse macrophages and bone-marrow derived dendritic cells (BMDCs. CHIP also cooperates with BAG-3 and the autophagic ubiquitin adaptor p62 in the clearance of DALIS through chaperone-assisted selective autophagy (CASA. On the other hand, the co-chaperone HspBP1 inhibits the activity of CHIP and thereby attenuates antigen sequestration. Through a modulation of DALIS formation CHIP, BAG-1 and HspBP1 alter MHC class I mediated antigen presentation in mouse BMDCs. Our data show that the Hsc/Hsp70 co-chaperone network controls transient protein aggregation during maturation of professional antigen presenting cells and in this way regulates the immune response. Similar mechanisms may modulate the formation of aggresomes and aggresome-like induced structures (ALIS in other mammalian cell types.

  7. Redox Disrupting Potential of ToxCast™Chemicals Ranked by Activity in Mouse Embryonic Stem Cells

    Science.gov (United States)

    Little is known regarding the adverse outcome pathways responsible for developmental toxicity following exposure to chemicals. An evaluation of Toxoast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay revealed a redox sensitive pathway that correlated with...

  8. Characterization of calcium responses and electrical activity in differentiating mouse neural progenitor cells in vitro

    NARCIS (Netherlands)

    de Groot, Martje W G D M; Dingemans, Milou M L; Rus, Katinka H; de Groot, Aart; Westerink, Remco H S

    In vitro methods for developmental neurotoxicity (DNT) testing have the potential to reduce animal use and increase insight into cellular and molecular mechanisms underlying chemical-induced alterations in the development of functional neuronal networks. Mouse neural progenitor cells (mNPCs)

  9. High-fidelity Glucagon-CreER mouse line generated by CRISPR-Cas9 assisted gene targeting

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    Amanda M. Ackermann

    2017-03-01

    Full Text Available Objective: α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for β-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER “knock-in” mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreERT2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing. Methods: We utilized CRISPR-Cas9 technology to insert an IRES-CreERT2 sequence into the 3′ UTR of the Glucagon (Gcg locus in mouse embryonic stem cells (ESCs. Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreERT2 mice. Recombination efficiency in GCG+ pancreatic α-cells and glucagon-like peptide 1 positive (GLP1+ enteroendocrine L-cells was measured in Gcg-CreERT2;Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood. Results: Tamoxifen injection of Gcg-CreERT2;Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively, as well as in first-wave fetal α-cells (36% and adult enteroendocrine L-cells (33%. Mice homozygous for the Gcg-CreERT2 allele were phenotypically normal. Conclusions: We successfully derived a Gcg-CreERT2 mouse line that expresses CreERT2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice

  10. Chromosome painting analysis of radiation-induced aberrant cell clones in the mouse

    International Nuclear Information System (INIS)

    Spruill, M.D.; Nath, J.; Tucker, J.D.

    1997-01-01

    In a study of the persistence of radiation-induced translocations over the life span of the mouse, we observed a number of clonal cells in peripheral blood lymphocytes. The presence of clones caused the mean frequency of aberrations at various time points to be elevated which interfered with biodosimetry. For this reason, we have corrected our data for the presence of clones. Mice were given an acute dose of 0, 1, 2, 3 or 4 Gy 137 Cs at 8 weeks of age. Aberrations were measured by painting chromosomes 2 and 8 and cells were examined for clones at 3 months and every 3 months thereafter until 21 months. Clones were identified by comparing the color photographic slides of all abnormal cells from each animal. Determination of clonality was made on the basis of similar breakpoint locations or the presence of other identifying characteristics such as unusual aberrations. To correct the frequency of translocations for the presence of clones, each clone, regardless of how many cells it contained, was counted only once. This reflects the original aberration frequency since each clone originated as only one cell. Among mice exposed to 4 Gy, the mean frequencies of aberrant cell clones ranged from 3-29% of the total number of metaphase cells scored with the highest frequency being 1 year post exposure. 32-70% of reciprocal and 19-92% of non-reciprocal translocations were clonal. A dose response relationship for clones was evident until 21 months when the unexposed animals exhibited a mean frequency of aberrant cell clones >10% of the total number of cells scored. Almost 75% of reciprocal and 95% of non-reciprocal translocations in these unexposed control animals were of clonal origin. Correction for clonal expansion greatly reduced the means and their standard errors at most time points where clonal expansion was prevalent. The biodosimetry was much improved suggesting that correction is beneficial in long-term studies

  11. Mouse-induced pluripotent stem cells differentiate into odontoblast-like cells with induction of altered adhesive and migratory phenotype of integrin.

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    Nobuaki Ozeki

    Full Text Available Methods for differentiating induced pluripotent stem (iPS cells into odontoblasts generally require epithelial-mesenchymal interactions. Here, we sought to characterize the cells produced by a 'hanging drop' technique for differentiating mouse iPS cells into odontoblast-like cells that requires no such interaction. Cells were cultured by the hanging drop method on a collagen type-I (Col-I scaffold (CS combined with bone morphogenetic protein (BMP-4 (CS/BMP-4 without an epithelial-mesenchymal interaction. We evaluated the expression of odontoblast-related mRNA and protein, and the proliferation rate of these cells using reverse-transcription polymerase chain reaction, immunofluorescence staining, and BrdU cell proliferation enzyme-linked immunosorbent assay, respectively. The differentiated cells strongly expressed the mRNA for dentin sialophosphoprotein (DSPP and dentin matrix protein-1 (Dmp-1, which are markers of mature odontoblasts. Osteopontin and osteocalcin were not expressed in the differentiated cells, demonstrating that the differentiated iPS cells bore little resemblance to osteoblasts. Instead, they acquired odontoblast-specific properties, including the adoption of an odontoblastic phenotype, typified by high alkaline phosphatase (ALP activity and calcification capacity. The cell-surface expression of proteins such as integrins α2, α6, αV and αVβ3 was rapidly up-regulated. Interestingly, antibodies and siRNAs against integrin α2 suppressed the expression of DSPP and Dmp-1, reduced the activity of ALP and blocked calcification, suggesting that integrin α2 in iPS cells mediates their differentiation into odontoblast-like cells. The adhesion of these cells to fibronectin and Col-I, and their migration on these substrata, was significantly increased following differentiation into odontoblast-like cells. Thus, we have demonstrated that integrin α2 is involved in the differentiation of mouse iPS cells into odontoblast-like cells

  12. Chaotic electrical activity of living β-cells in the mouse pancreatic islet

    Science.gov (United States)

    Kanno, Takahiro; Miyano, Takaya; Tokuda, Isao; Galvanovskis, Juris; Wakui, Makoto

    2007-02-01

    To test for chaotic dynamics of the insulin producing β-cell and explore its biological role, we observed the action potentials with the perforated patch clamp technique, for isolated cells as well as for intact cells of the mouse pancreatic islet. The time series obtained were analyzed using nonlinear diagnostic algorithms associated with the surrogate method. The isolated cells exhibited short-term predictability and visible determinism, in the steady state response to 10 mM glucose, while the intact cells did not. In the latter case, determinism became visible after the application of a gap junction inhibitor. This tendency was enhanced by the stimulation with tolbutamide. Our observations suggest that, thanks to the integration of individual chaotic dynamics via gap junction coupling, the β-cells will lose memory of fluctuations occurring at any instant in their electrical activity more rapidly with time. This is likely to contribute to the functional stability of the islet against uncertain perturbations.

  13. Spallanzani's mouse: a model of restoration and regeneration.

    Science.gov (United States)

    Heber-Katz, E; Leferovich, J M; Bedelbaeva, K; Gourevitch, D

    2004-01-01

    The ability to regenerate is thought to be a lost phenotype in mammals, though there are certainly sporadic examples of mammalian regeneration. Our laboratory has identified a strain of mouse, the MRL mouse, which has a unique capacity to heal complex tissue in an epimorphic fashion, i.e., to restore a damaged limb or organ to its normal structure and function. Initial studies using through-and-through ear punches showed rapid full closure of the ear holes with cartilage growth, new hair follicles, and normal tissue architecture reminiscent of regeneration seen in amphibians as opposed to the scarring usually seen in mammals. Since the ear hole closure phenotype is a quantitative trait, this has been used to show-through extensive breeding and backcrossing--that the trait is heritable. Such analysis reveals that there is a complex genetic basis for this trait with multiple loci. One of the major phenotypes of the MRL mouse is a potent remodeling response with the absence or a reduced level of scarring. MRL healing is associated with the upregulation of the metalloproteinases MMP-2 and MMP-9 and the downregulation of their inhibitors TIMP-2 and TIMP-3, both present in inflammatory cells such as neutrophils and macrophages. This model has more recently been extended to the heart. In this case, a cryoinjury to the right ventricle leads to near complete scarless healing in the MRL mouse whereas scarring is seen in the control mouse. In the MRL heart, bromodeoxyuridine uptake by cardiomyocytes filling the wound site can be seen 60 days after injury. This does not occur in the control mouse. Function in the MRL heart, as measured by echocardiography, returns to normal.

  14. Age-related variability of some characters of karyotype instability in the mouse line CC57W/Mv

    International Nuclear Information System (INIS)

    Glazko, T.T.; Safonova, N.A.; Kovaleva, O.A.; Stolina, M.P.; Solomko, A.P.; Malyuta, S.S.; Glazko, V.I.; AN Ukrainskoj SSR, Kiev

    1995-01-01

    The investigations of relations between cytogenetical variability in cells of bone marrow of the mouse line CC57W/Mv and factors of age and radioactivity pollution (the specific vivarium in the 30-km Chernobyl zone) were carried out. The karyotype instability on some characters were similarly between young mice in the Chernobyl zone and old mice under control conditions. The old Chernobyl mice differentiated from old control ones by a low frequency of some cytogenetic anomalies and higher values of the mitotic index. The contribution of the intensity of cell division into observed variabilities of cytogenetic character between different mouse groups was discussed

  15. Ginseng Berry Extract Promotes Maturation of Mouse Dendritic Cells.

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    Wei Zhang

    Full Text Available Ginseng extract has been shown to possess certain anti-virus, anti-tumor and immune-activating effects. However, the immunostimulatory effect of ginseng berry extract (GB has been less well characterized. In this study, we investigated the effect of GB on the activation of mouse dendritic cells (DCs in vitro and in vivo. GB treatment induced up-regulation of co-stimulatory molecules in bone marrow-derived DCs (BMDCs. Interestingly, GB induced a higher degree of co-stimulatory molecule up-regulation than ginseng root extract (GR at the same concentrations. Moreover, in vivo administration of GB promoted up-regulation of CD86, MHC class I and MHC class II and production of IL-6, IL-12 and TNF-α in spleen DCs. GB also promoted the generation of Th1 and Tc1 cells. Furthermore, Toll like receptor 4 (TLR4 and myeloid differentiation primary response 88 (MyD88 signaling pathway were essential for DC activation induced by GB. In addition, GB strongly prompted the proliferation of ovalbumin (OVA-specific CD4 and CD8 T cells. Finally, GB induced DC activation in tumor-bearing mice and the combination of OVA and GB treatment inhibited B16-OVA tumor cell growth in C57BL/6 mice. These results demonstrate that GB is a novel tumor therapeutic vaccine adjuvant by promoting DC and T cell activation.

  16. Identifying Tmem59 related gene regulatory network of mouse neural stem cell from a compendium of expression profiles

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    Guo Xiuyun

    2011-09-01

    Full Text Available Abstract Background Neural stem cells offer potential treatment for neurodegenerative disorders, such like Alzheimer's disease (AD. While much progress has been made in understanding neural stem cell function, a precise description of the molecular mechanisms regulating neural stem cells is not yet established. This lack of knowledge is a major barrier holding back the discovery of therapeutic uses of neural stem cells. In this paper, the regulatory mechanism of mouse neural stem cell (NSC differentiation by tmem59 is explored on the genome-level. Results We identified regulators of tmem59 during the differentiation of mouse NSCs from a compendium of expression profiles. Based on the microarray experiment, we developed the parallelized SWNI algorithm to reconstruct gene regulatory networks of mouse neural stem cells. From the inferred tmem59 related gene network including 36 genes, pou6f1 was identified to regulate tmem59 significantly and might play an important role in the differentiation of NSCs in mouse brain. There are four pathways shown in the gene network, indicating that tmem59 locates in the downstream of the signalling pathway. The real-time RT-PCR results shown that the over-expression of pou6f1 could significantly up-regulate tmem59 expression in C17.2 NSC line. 16 out of 36 predicted genes in our constructed network have been reported to be AD-related, including Ace, aqp1, arrdc3, cd14, cd59a, cds1, cldn1, cox8b, defb11, folr1, gdi2, mmp3, mgp, myrip, Ripk4, rnd3, and sncg. The localization of tmem59 related genes and functional-related gene groups based on the Gene Ontology (GO annotation was also identified. Conclusions Our findings suggest that the expression of tmem59 is an important factor contributing to AD. The parallelized SWNI algorithm increased the efficiency of network reconstruction significantly. This study enables us to highlight novel genes that may be involved in NSC differentiation and provides a shortcut to

  17. TRANSGENIC GDNF POSITIVELY INFLUENCES PROLIFERATION, DIFFERENTIATION, MATURATION AND SURVIVAL OF MOTOR NEURONS PRODUCED FROM MOUSE EMBRYONIC STEM CELLS.

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    Daniel Édgar Cortés

    2016-09-01

    Full Text Available Embryonic stem cells (ESC are pluripotent and thus can differentiate into every cell type present in the body. Directed differentiation into motor neurons has been described for pluripotent cells. Although neurotrophic factors promote neuronal survival, their role in neuronal commitment is elusive. Here, we developed double-transgenic lines of mouse ESC that constitutively produce Glial cell-derived neurotrophic factor (GDNF and also contain a GFP reporter, driven by HB9, which is expressed only by postmitotic motor neurons. After lentiviral transduction, ESC lines integrated and expressed the human GDNF gene without altering pluripotency markers before differentiation. Further, GDNF-ESC showed significantly higher spontaneous release of this neurotrophin to the medium, when compared to controls. To study motor neuron induction, control and GDNF cell lines were grown as embryoid bodies and stimulated with retinoic acid and Sonic Hedgehog. In GDNF-overexpressing cells, a significant increase of proliferative Olig2+ precursors, which are specified as spinal motor neurons, was found. Accordingly, GDNF increases the yield of cells with the pan motor neuronal markers HB9, monitored by GFP expression, and Isl1. At terminal differentiation, almost all differentiated neurons express phenotypic markers of motor neurons in GDNF cultures, with lower proportions in control cells. To test if the effects of GDNF were present at early differentiation stages, exogenous recombinant human GDNF was added to control ESC, also resulting in enhanced motor neuron differentiation. This effect was abolished by the co-addition of neutralizing anti-GDNF antibodies, strongly suggesting that differentiating ESC are responsive to GDNF. Using the HB9::GFP reporter, motor neurons were selected for electrophysiological recordings. Motor neurons differentiated from GDNF-ESC, compared to control motor neurons, showed greater electrophysiological maturation, characterized by

  18. Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

    Science.gov (United States)

    Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

    1984-09-01

    In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the

  19. The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

    Science.gov (United States)

    Collombat, Patrick; Xu, Xiaobo; Ravassard, Philippe; Sosa-Pineda, Beatriz; Dussaud, Sébastien; Billestrup, Nils; Madsen, Ole D; Serup, Palle; Heimberg, Harry; Mansouri, Ahmed

    2009-08-07

    We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

  20. Osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 is promoted by mammalian transcriptional repressor Rbpj

    International Nuclear Information System (INIS)

    Wang, Shengchao; Kawashima, Nobuyuki; Sakamoto, Kei; Katsube, Ken-ichi; Umezawa, Akihiro; Suda, Hideaki

    2010-01-01

    Research highlights: → High Rbpj mRNA expression was observed in mesenchymal cells surrounding the bone of mouse embryos. → Overexpression of Rbpj depressed Notch-Hes1/Hey1 signaling. → Rbpj upregulated promoter activities of Runx2 and Ose2. → Rbpj promoted osteoblastic differentiation/maturation in Kusa-A1 cells. -- Abstract: Pluripotent mesenchymal stem cells possess the ability to differentiate into many cell types, but the precise mechanisms of differentiation are still unclear. Here, we provide evidence that Rbpj (recombination signal-binding protein for immunoglobulin kappa j region) protein, the primary nuclear mediator of Notch, is involved in osteogenesis. Overexpression of Rbpj promoted osteogenic differentiation of mouse Kusa-A1 cells in vitro and in vivo. Transient transfection of an Rbpj expression vector into Kusa-A1 cells upregulated promoter activities of Runx2 and Ose2. Enhanced osteogenic potentials including high alkaline phosphatase activity, rapid calcium deposition, and increased calcified nodule formation, were observed in established stable Rbpj-overexpressing Kusa-A1 (Kusa-A1/Rbpj) cell line. In vivo mineralization by Kusa-A1/Rbpj was promoted compared to that by Kusa-A1 host cells. Histological findings revealed that expression of Rbpj was primarily observed in osteoblasts. These results suggest that Rbpj may play essential roles in osteoblast differentiation.