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  1. Optogenetic Control of Mouse Outer Hair Cells.

    Science.gov (United States)

    Wu, Tao; Ramamoorthy, Sripriya; Wilson, Teresa; Chen, Fangyi; Porsov, Edward; Subhash, Hrebesh; Foster, Sarah; Zhang, Yuan; Omelchenko, Irina; Bateschell, Michael; Wang, Lingyan; Brigande, John V; Jiang, Zhi-Gen; Mao, Tianyi; Nuttall, Alfred L

    2016-01-19

    Normal hearing in mammals depends on sound amplification by outer hair cells (OHCs) presumably by their somatic motility and force production. However, the role of OHC force production in cochlear amplification and frequency tuning are not yet fully understood. Currently, available OHC manipulation techniques for physiological or clinical studies are limited by their invasive nature, lack of precision, and poor temporal-spatial resolution. To overcome these limitations, we explored an optogenetic approach based on channelrhodopsin 2 (ChR-2), a direct light-activated nonselective cation channel originally discovered in Chlamydomonas reinhardtii. Three approaches were compared: 1) adeno-associated virus-mediated in utero transfer of the ChR-2 gene into the developing murine otocyst, 2) expression of ChR-2(H134R) in an auditory cell line (HEI-OC1), and 3) expression of ChR-2 in the OHCs of a mouse line carrying a ChR-2 conditional allele. Whole cell recording showed that blue light (470 nm) elicited the typical nonselective cation current of ChR-2 with reversal potential around zero in both mouse OHCs and HEI-OC1 cells and generated depolarization in both cell types. In addition, pulsed light stimulation (10 Hz) elicited a 1:1 repetitive depolarization and ChR-2 currents in mouse OHCs and HEI-OC1 cells, respectively. The time constant of depolarization in OHCs, 1.45 ms, is 10 times faster than HEI-OC1 cells, which allowed light stimulation up to rates of 10/s to elicit corresponding membrane potential changes. Our study demonstrates that ChR-2 can successfully be expressed in mouse OHCs and HEI-OC1 cells and that these present a typical light-sensitive current and depolarization. However, the amount of ChR-2 current induced in our in vivo experiments was insufficient to result in measurable cochlear effects. PMID:26789771

  2. Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin.

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    Fangfang Wang

    Full Text Available Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR. Expression vectors that contained the Tet operator and amelogenin-coding (Amelx cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx. MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP, osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional

  3. Control of RFM strain endogenous retrovirus in RFM mouse cells

    International Nuclear Information System (INIS)

    RFM/Un mice express an endogenous type C retrovirus throughout their life span in many tissues; primary or established embryo fibroblast cell cultures do not express a virus but can be induced by exposure to 5-iodo-2'-deoxyuridine. All of our sources yielded a single ecotropic virus (RFV) which appeared to be related more closely to the endogenous N-tropic virus (WN1802N) of BALB/c mice than to Gross leukemia virus on the basis of two-dimensional gel electropherograms of virion proteins. No xenotropic or recombinant viruses were isolated by cocultivation techniques. RFV is N-tropic, and RFM/Un cells possess the Fv-1/sup n/ allele, as indicated by restriction of B-tropic virus and susceptibility to Gross strain N-tropic virus. However, RFM cells are highly resistant to RFV and other endogenous N-tropic viruses. This resistance is expressed by two-hit titration kinetics and by inhibition of viral linear duplex DNA formation. This is similar to the effects of the Fv-1 locus, but preliminary work has shown no apparent genetic linkage between the two restrictions. The relative strength of the restriction, the presence of a single class of ecotropic virus, and the absence of recombinant viruses suggest that in RFM mice virus is expressed only in cells in which it is induced and not by cell-to-cell transmission

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

    International Nuclear Information System (INIS)

    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% CO2 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 G2/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

  5. Mouse Leydig Tumor Cells

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    Bo-Syong Pan

    2011-01-01

    Full Text Available Cordycepin is a natural pure compound extracted from Cordyceps sinensis (CS. We have demonstrated that CS stimulates steroidogenesis in primary mouse Leydig cell and activates apoptosis in MA-10 mouse Leydig tumor cells. It is highly possible that cordycepin is the main component in CS modulating Leydig cell functions. Thus, our aim was to investigate the steroidogenic and apoptotic effects with potential mechanism of cordycepin on MA-10 mouse Leydig tumor cells. Results showed that cordycepin significantly stimulated progesterone production in dose- and time-dependent manners. Adenosine receptor (AR subtype agonists were further used to treat MA-10 cells, showing that A1, A 2A , A 2B , and A3, AR agonists could stimulate progesterone production. However, StAR promoter activity and protein expression remained of no difference among all cordycepin treatments, suggesting that cordycepin might activate AR, but not stimulated StAR protein to regulate MA-10 cell steroidogenesis. Meanwhile, cordycepin could also induce apoptotic cell death in MA-10 cells. Moreover, four AR subtype agonists induced cell death in a dose-dependent manner, and four AR subtype antagonists could all rescue cell death under cordycepin treatment in MA-10 cells. In conclusion, cordycepin could activate adenosine subtype receptors and simultaneously induce steroidogenesis and apoptosis in MA-10 mouse Leydig tumor cells.

  6. A gonogenic stimulated transition of mouse embryonic stem cells with enhanced control of diverse differentiation pathways.

    Science.gov (United States)

    Moshfegh, Cameron; Aires, Lina; Kisielow, Malgorzata; Vogel, Viola

    2016-01-01

    Embryonic stem (ES) cells share markers with undifferentiated primordial germ cells (PGCs). Here, we discovered that a cellular state with some molecular markers of male gonocyte induction, including a G1/S phase arrest and upregulation of specific genes such as Nanos2, Tdrd1, Ddx4, Zbtb16 and Plk1s1, can be chemically induced in male mouse ES cells in vitro, which we termed gonogenic stimulated transition (GoST). After longer culture of the resulting GoST cells without chemical stimulation, several molecular markers typical for early gonocytes were detected including the early gonocyte marker Tex101. Motivated by previous studies that found multipotency in cell lines derived from neonatal male germ cells in vitro, we then compared the differentiation potential of GoST cells to that of ES cells in vitro. Interestingly, GoST cells showed equal neurogenic, but enhanced cardiogenic and hepatogenic differentiation compared to ES cells in vitro. This work shows for the first time that some important molecular markers of the first developmental sexual differentiation program can be induced in male mouse ES cells in vitro and defines a novel concept to generate cells with enhanced multipotency. PMID:27157261

  7. Nitric Oxide Prevents Mouse Embryonic Stem Cell Differentiation Through Regulation of Gene Expression, Cell Signaling, and Control of Cell Proliferation.

    Science.gov (United States)

    Tapia-Limonchi, Rafael; Cahuana, Gladys M; Caballano-Infantes, Estefania; Salguero-Aranda, Carmen; Beltran-Povea, Amparo; Hitos, Ana B; Hmadcha, Abdelkrim; Martin, Franz; Soria, Bernat; Bedoya, Francisco J; Tejedo, Juan R

    2016-09-01

    Nitric oxide (NO) delays mouse embryonic stem cell (mESC) differentiation by regulating genes linked to pluripotency and differentiation. Nevertheless, no profound study has been conducted on cell differentiation regulation by this molecule through signaling on essential biological functions. We sought to demonstrate that NO positively regulates the pluripotency transcriptional core, enforcing changes in the chromatin structure, in addition to regulating cell proliferation, and signaling pathways with key roles in stemness. Culturing mESCs with 2 μM of the NO donor diethylenetriamine/NO (DETA/NO) in the absence of leukemia inhibitory factor (LIF) induced significant changes in the expression of 16 genes of the pluripotency transcriptional core. Furthermore, treatment with DETA/NO resulted in a high occupancy of activating H3K4me3 at the Oct4 and Nanog promoters and repressive H3K9me3 and H3k27me3 at the Brachyury promoter. Additionally, the activation of signaling pathways involved in pluripotency, such as Gsk3-β/β-catenin, was observed, in addition to activation of PI3 K/Akt, which is consistent with the protection of mESCs from cell death. Finally, a decrease in cell proliferation coincides with cell cycle arrest in G2/M. Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that NO is necessary but not sufficient for the maintenance of pluripotency and the prevention of cell differentiation. J. Cell. Biochem. 117: 2078-2088, 2016. © 2016 Wiley Periodicals, Inc. PMID:26853909

  8. The tumorigenicity of mouse embryonic stem cells and in vitro differentiated neuronal cells is controlled by the recipients' immune response.

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    Ralf Dressel

    Full Text Available Embryonic stem (ES cells have the potential to differentiate into all cell types and are considered as a valuable source of cells for transplantation therapies. A critical issue, however, is the risk of teratoma formation after transplantation. The effect of the immune response on the tumorigenicity of transplanted cells is poorly understood. We have systematically compared the tumorigenicity of mouse ES cells and in vitro differentiated neuronal cells in various recipients. Subcutaneous injection of 1x10(6 ES or differentiated cells into syngeneic or allogeneic immunodeficient mice resulted in teratomas in about 95% of the recipients. Both cell types did not give rise to tumors in immunocompetent allogeneic mice or xenogeneic rats. However, in 61% of cyclosporine A-treated rats teratomas developed after injection of differentiated cells. Undifferentiated ES cells did not give rise to tumors in these rats. ES cells turned out to be highly susceptible to killing by rat natural killer (NK cells due to the expression of ligands of the activating NK receptor NKG2D on ES cells. These ligands were down-regulated on differentiated cells. The activity of NK cells which is not suppressed by cyclosporine A might contribute to the prevention of teratomas after injection of ES cells but not after inoculation of differentiated cells. These findings clearly point to the importance of the immune response in this process. Interestingly, the differentiated cells must contain a tumorigenic cell population that is not present among ES cells and which might be resistant to NK cell-mediated killing.

  9. Hand gestures mouse cursor control

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    Marian-Avram Vincze

    2014-05-01

    Full Text Available The paper describes the implementation of a human-computer interface for controlling the mouse cursor. The test reveal the fact: a low-cost web camera some processing algorithms are quite enough to control the mouse cursor on computers. Even if the system is influenced by the illuminance level on the plane of the hand, the current study may represent a start point for some studies on the hand tracking and gesture recognition field.

  10. Huntingtin acts non cell-autonomously on hippocampal neurogenesis and controls anxiety-related behaviors in adult mouse.

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    Patrick Pla

    Full Text Available Huntington's disease (HD is a fatal neurodegenerative disease, characterized by motor defects and psychiatric symptoms, including mood disorders such as anxiety and depression. HD is caused by an abnormal polyglutamine (polyQ expansion in the huntingtin (HTT protein. The development and analysis of various mouse models that express pathogenic polyQ-HTT revealed a link between mutant HTT and the development of anxio-depressive behaviors and various hippocampal neurogenesis defects. However, it is unclear whether such phenotype is linked to alteration of HTT wild-type function in adults. Here, we report the analysis of a new mouse model in which HTT is inducibly deleted from adult mature cortical and hippocampal neurons using the CreER(T2/Lox system. These mice present defects in both the survival and the dendritic arborization of hippocampal newborn neurons. Our data suggest that these non-cell autonomous effects are linked to defects in both BDNF transport and release upon HTT silencing in hippocampal neurons, and in BDNF/TrkB signaling. The controlled deletion of HTT also had anxiogenic-like effects. Our results implicate endogenous wild-type HTT in adult hippocampal neurogenesis and in the control of mood disorders.

  11. Cancer cell expression of autotaxin controls bone metastasis formation in mouse through lysophosphatidic acid-dependent activation of osteoclasts.

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    Marion David

    Full Text Available BACKGROUND: Bone metastases are highly frequent complications of breast cancers. Current bone metastasis treatments using powerful anti-resorptive agents are only palliative indicating that factors independent of bone resorption control bone metastasis progression. Autotaxin (ATX/NPP2 is a secreted protein with both oncogenic and pro-metastatic properties. Through its lysosphospholipase D (lysoPLD activity, ATX controls the level of lysophosphatidic acid (LPA in the blood. Platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. We asked whether ATX was involved in the bone metastasis process. We characterized the role of ATX in osteolytic bone metastasis formation by using genetically modified breast cancer cells exploited on different osteolytic bone metastasis mouse models. METHODOLOGY/PRINCIPAL FINDINGS: Intravenous injection of human breast cancer MDA-B02 cells with forced expression of ATX (MDA-B02/ATX to immunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss, tumor burden, and a higher number of active osteoclasts at the metastatic site. Mouse breast cancer 4T1 cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. These cells expressed active ATX and silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at the bone metastatic site. In vitro, osteoclast differentiation was enhanced in presence of MDA-B02/ATX cell conditioned media or recombinant autotaxin that was blocked by the autotaxin inhibitor vpc8a202. In vitro, addition of LPA to active charcoal-treated serum restored the capacity of the serum to support RANK-L/MCSF-induced osteoclastogenesis. CONCLUSION/SIGNIFICANCE: Expression of autotaxin by cancer cells controls osteolytic bone metastasis formation. This work demonstrates a new role for LPA as a

  12. Manipulation of Mouse Embryonic Stem Cells for Knockout Mouse Production

    OpenAIRE

    Limaye, Advait; Hall, Bradford; Kulkarni, Ashok B.

    2009-01-01

    The establishment of mouse embryonic stem (ES) cell liness has allowed for the generation of the knockout mouse. ES cells that are genetically altered in culture can then be manipulated to derive a whole mouse containing the desired mutation. To successfully generate a knockout mouse, however, the ES cells must be carefully cultivated in a pluripotent state throughout the gene targeting experiment. This unit describes detailed step-by-step protocols, reagents, equipment, and strategies needed...

  13. A tight control of Rif1 by Oct4 and Smad3 is critical for mouse embryonic stem cell stability

    OpenAIRE

    Li, P; Ma, X; Adams, I.R.; Yuan, P.

    2015-01-01

    Prolonged culture of embryonic stem cells (ESCs) leads them to adopt embryonal carcinoma cell features, creating enormous dangers for their further application. The mechanism involved in ESC stability has not, however, been extensively studied. We previously reported that SMAD family member 3 (Smad3) has an important role in maintaining mouse ESC stability, as depletion of Smad3 results in cancer cell-like properties in ESCs and Smad3−/− ESCs are prone to grow large, malignant teratomas. To u...

  14. A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apoptosis

    Institute of Scientific and Technical Information of China (English)

    Yue-juan QIN; Zhen-lin ZHANG; Lu-yang YU; Jin-wei HE; Ya-nan HOU; Tian-jin LIU; Jia-cai WU; Song-hua WU; Li-he GUO

    2006-01-01

    Aim: To construct an A20 expression vector under the control of mouse osteocalcin promoter (OC-A20), and investigate osteoblastic MC3T3-E1 cell line, which stably overexpresses A20 protein prevented tumor necrosis factor (TNF)-alpha-induced apoptosis. Methods: OC-A20 vector was constructed by fusing a fragment of the mouse osteocalcin gene-2 promoter with human A20 complementary DNA. Then the mouse MC3T3-E1 cell line, stably transfected by A20, was established. The expression of A20 mRNA and A20 protein in the cells were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. To determine the specificity of A20 expression in osteoblast, the mouse osteoblastic MC3T3-E1 cell line and mouse embryo fibro-blast NIH3T3 cell line were transiently transfected with OC-A20. The anti-apoptotic role of A20 in MC3T3-E1 cells was determined by Flow cytometric analysis (FACS), terminal dUTP nick endo-labeling (TUNEL) and DNA gel electrophoresis analysis (DNA Ladder), respectively. Results: Weak A20 expression was found in MC3T3-El cells with the primers of mouse A20. A20 mRNA and A20 protein expression were identified in MC3T3-E1 cells transfected with OC-A20 using RT-PCR and Western blot analysis. Only A20 mRNA expression was found in MC3T3-E1 cell after MC3T3-E1 cells and NIH3T3 cells were transient transfected with OC-A20. A decrease obviously occurred in the rate of apoptosis in the OC-A20 group compared with the empty vector (pcDNA3) group by FACS (P<0.001). A significant increase in TUNEL positive staining was found in the pcDNA group compared with OC-A20 group (P<0.001). Simultaneously, similar effects were demonstrated in DNA gel electrophoresis analysis. Conclusion: We constructed an osteoblast-specific expression vector that expressed A20 protein in MC3T3-E1 cells and confirmed that A20 protects osteoblast against TNF-alpha-induced apoptosis.

  15. Inflammation Controls Sensitivity of Human and Mouse Intestinal Epithelial Cells to Galectin-1.

    Science.gov (United States)

    Muglia, Cecilia I; Papa Gobbi, Rodrigo; Smaldini, Paola; Orsini Delgado, María Lucía; Candia, Martín; Zanuzzi, Carolina; Sambuelli, Alicia; Rocca, Andrés; Toscano, Marta A; Rabinovich, Gabriel A; Docena, Guillermo H

    2016-07-01

    Galectins play key roles in the inflammatory cascade. In this study, we aimed to analyze the effect of galectin-1 (Gal-1) in the function of intestinal epithelial cells (IECs) isolated from healthy and inflamed mucosa. IECs isolated from mice or patients with inflammatory bowel diseases (IBD) were incubated with different pro-inflammatory cytokines, and Gal-1 binding, secretion of homeostatic factors and viability were assessed. Experimental models of food allergy and colitis were used to evaluate the in vivo influence of inflammation on Gal-1 binding and modulation of IECs. We found an enhanced binding of Gal-1 to: (a) murine IECs exposed to IL-1β, TNF, and IL-13; (b) IECs from inflamed areas in intestinal tissue from IBD patients; (c) small bowel of allergic mice; and (d) colon from mice with experimental colitis. Our results showed that low concentrations of Gal-1 favored a tolerogenic micro-environment, whereas high concentrations of this lectin modulated viability of IECs through mechanisms involving activation of caspase-9 and modulation of Bcl-2 protein family members. Our results showed that, when added in the presence of diverse pro-inflammatory cytokines such as tumor necrosis factor (TNF), IL-13 and IL-5, Gal-1 differentially promoted the secretion of growth factors including thymic stromal lymphopoietin (TSLP), epidermal growth factor (EGF), IL-10, IL-25, and transforming growth factor (TGF-β1 ). In conclusion, we found an augmented binding of Gal-1 to IECs when exposed in vitro or in vivo to inflammatory stimuli, showing different effects depending on Gal-1 concentration. These findings highlight the importance of the inflammatory micro-environment of mucosal tissues in modulating IECs susceptibility to the immunoregulatory lectin Gal-1 and its role in epithelial cell homeostasis. J. Cell. Physiol. 231: 1575-1585, 2016. © 2015 Wiley Periodicals, Inc. PMID:26566180

  16. Early Cell Fate Decisions of Human Embryonic Stem Cells and Mouse Epiblast Stem Cells Are Controlled by the Same Signalling Pathways

    OpenAIRE

    Ludovic Vallier; Thomas Touboul; Zhenzhi Chng; Minodora Brimpari; Nicholas Hannan; Enrique Millan; Smithers, Lucy E.; Matthew Trotter; Peter Rugg-Gunn; Anne Weber; Pedersen, Roger A.

    2009-01-01

    Human embryonic stem cells have unique value for regenerative medicine, as they are capable of differentiating into a broad variety of cell types. Therefore, defining the signalling pathways that control early cell fate decisions of pluripotent stem cells represents a major task. Moreover, modelling the early steps of embryonic development in vitro may provide the best approach to produce cell types with native properties. Here, we analysed the function of key developmental growth factors suc...

  17. Connexins and steroidogenesis in mouse Leydig cells

    OpenAIRE

    Li, Dan; Sekhon, Poonam; Barr, Kevin J.; Márquez-Rosado, Lucrecia; Lampe, Paul D; Kidder, Gerald M.

    2013-01-01

    Connexin43 has been recognized as forming gap junctions in Leydig cells. However, previous work had shown that mouse Leydig cells lacking this connexin do not suffer any limitation of their ability to produce testosterone when stimulated with luteinizing hormone. The objective of the present study was to identify additional connexins in mouse Leydig cells that could be required for steroidogenesis. An RT-PCR screen involving isolated adult Leydig cells identified connexin36 and connexin45 as ...

  18. COUP-TFII controls mouse pancreatic β-cell mass through GLP-1-β-catenin signaling pathways.

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    Marie Boutant

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

  19. pH-sensitive immunoliposomes mediate target-cell-specific delivery and controlled expression of a foreign gene in mouse.

    OpenAIRE

    Wang, C Y; Huang, L

    1987-01-01

    A plasmid containing the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under the control of a mammalian cAMP-regulated promoter was entrapped in H-2Kk antibody-coated liposomes composed of dioleoyl phosphatidylethanolamine, cholesterol, and oleic acid (pH-sensitive immunoliposomes). The entrapped or free DNA was injected intraperitoneally into immunodeficient (nude) BALB/c mice bearing ascites tumor generated by H-2Kk-positive RDM-4 lymphoma cells. About 20% of the injected im...

  20. NTPDase2 and Purinergic Signaling Control Progenitor Cell Proliferation in Neurogenic Niches of the Adult Mouse Brain

    OpenAIRE

    Gampe, Kristine; Stefani, Jennifer; Hammer, Klaus; Brendel, Peter; Pötzsch, Alexandra; Enikolopov, Grigori; Enjyoji, Keiichi; Acker-Palmer, Amparo; Robson, Simon C.; Zimmermann, Herbert

    2015-01-01

    Nerve cells are continuously generated from stem cells in the adult mammalian subventricular zone (SVZ) and hippocampal dentate gyrus. We have previously noted that stem/progenitor cells in the SVZ and the subgranular layer (SGL) of the dentate gyrus express high levels of plasma membrane-bound nucleoside triphosphate diphosphohydrolase 2 (NTPDase2), an ectoenzyme that hydrolyzes extracellular nucleoside di- and triphosphates. We inferred that deletion of NTPDase2 would increase local extrace...

  1. Development of neural precursor cells from mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    WU Xuan; LI Hai-di; Li Shu-nong; XU Hai-wei; XU Ling

    2001-01-01

    Objective: To explore the serum-free culture conditions for differentiating mouse embryonic stem cells (ES cells)into neural precursor cells (NPC) and compare the effects of human embryonic fibroblasts (HEF) as the feeder layer of ES with that of mouse embryonic fibroblasts (MEF)in vitro. Methods: Mouse ES cells were cultured in or not in feeder layer cells medium containing or not leukemia inhibitory factor to suppress their differentiation. Immunocytochemical method was used to identify NPC by detecting nestin antigen and alkaline phosphatase. Results: The ES cells cultured in HEF were positive to alkaline phosphatase. Serum-free medium allowed the differentiation of ES cells into NPC. Conclusion:HEF could replace MEF and keep the undifferentiated condition of ES cells with more benefits. NPC of high purity could be cultured from ES cells by serum-free culture method.

  2. Somatic Cell Nuclear Transfer in the Mouse

    Science.gov (United States)

    Kishigami, Satoshi; Wakayama, Teruhiko

    Somatic cell nuclear transfer (SCNT) has become a unique and powerful tool for epigenetic reprogramming research and gene manipulation in animals since “Dolly,” the first animal cloned from an adult cell was reported in 1997. Although the success rates of somatic cloning have been inefficient and the mechanism of reprogramming is still largely unknown, this technique has been proven to work in more than 10 mammalian species. Among them, the mouse provides the best model for both basic and applied research of somatic cloning because of its abounding genetic resources, rapid sexual maturity and propagation, minimal requirements for housing, etc. This chapter describes a basic protocol for mouse cloning using cumulus cells, the most popular cell type for NT, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. In particular, we focus on a new, more efficient mouse cloning protocol using trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, which increases both in vitro and in vivo developmental rates from twofold to fivefold. This new method including TSA will be helpful to establish mouse cloning in many laboratories.

  3. Germ cell transplantation in infertility mouse

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    This work investigated the spermatogenesis in an infertility BALB/c-nu mouse model by reinfusing germline stem cells into seminiferous tubules.Donor germ cells were isolated from male FVB/NJ-GFP transgenic mice.Seminiferous tubule microiniection was applied to achieve intratubular germ cell transfer.The germ cells were injected into exposed testes of the infertility mice.We used green fluorescence and DNA analysis of donor cells from GFP transgenic mice as genetic marker.The natural mating and Southern blot methods were applied to analyze the effect of sperm cell transplantation and the sperm function after seminiferous tubule microinjecUon.The spermatogenesis was morphologically observed from the seminiferous tubules in 41/60(68.33%)of the injected recipient mice using allogeneic donor cells.In the colonized testes,matured spermatozoa were seen in the lumen of the seminiferous tubules.In this research,BALB/c-nu infertility mouse model,the recipient animal,was used to avoid immunological rejection of donor cells,and germ cell transplantation was applied to overcome infertility caused by busulfan treatment.These results demonstrate that this technique of germ cell transplantation is of great use.Germ cell transplantation could be potentially valuable to oncological patients.

  4. Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens.

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    David S Gokhin

    Full Text Available The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α₂β₂-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1 and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.

  5. Mouse Hematopoietic Stem Cells, Unlike Human and Mouse Embryonic Stem Cells, Exhibit Checkpoint–Apoptosis Coupling

    OpenAIRE

    Rohrabaugh, Sara; Mantel, Charlie; Broxmeyer, Hal E.

    2008-01-01

    Previously, we reported that the spindle assembly checkpoint (SAC), which is coupled in somatic cells, is uncoupled from apoptosis-initiation in mouse and human embryonic stem cells (ESCs). This condition allows ESCs to tolerate and proliferate as polyploidy/aneuploid cells. Proper function of the SAC is vital to prevent polyploidy/aneuploidy during ex vivo hematopoietic stem cell (HSC) expansion. Here we address, for the first time, whether HSCs are more like ESCs or somatic cells with respe...

  6. Mouse cell culture - Methods and protocols

    Directory of Open Access Journals (Sweden)

    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........

  7. Intraspinal transplantation of mouse and human neural precursor cells

    OpenAIRE

    Weinger, Jason G.; Chen, Lu; Coleman, Ronald; Leang, Ronika; Plaisted, Warren C.; Loring, Jeanne F.; Lane, Thomas E

    2013-01-01

    This unit describes the preparation and transplantation of human neural precursor cells (hNPCs) and mouse neural precursor cells (mNPCs) into the thoracic region of the mouse spinal cord. The techniques in this unit also describe how to prepare the mouse for surgery by performing a laminectomy to expose the spinal cord for transplantation. Here we show NPCs genetically labeled with eGFP transplanted into the spinal cord of a mouse following viralmediated demyelination can efficiently be detec...

  8. mEst1A (mouse Ever Shorter Telomeres 1A) regulates telomere length and RNA quality control in murine stem cells

    OpenAIRE

    Lee, Hyemin

    2012-01-01

    Est1A/SMG6 controls telomere elongation by mediating telomerase recruitment. In addition, it is an essential component of the endonucleolytic branch of the Nonsense-mediated mRNA decay (NMD) pathway, which controls RNA quality by eliminating mRNAs that harbour premature termination codons (PTC). In vivo function of Est1A/SMG6 has not been investigated in genetic mouse models. Here, we show in conditional knockout mice that germ line deletion of Est1A/SMG6 leads to embryonic lethality. Cr...

  9. Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium

    OpenAIRE

    Lu, Xiaoyan; Chen, Dong; Liu, Zhiping; Li, Chaoyang; Liu, Ying; Zhou, Jin; Wan, Pengxia; Mou, Yong-Gao; Wang, Zhichong

    2010-01-01

    Purpose To determine whether mouse embryonic stem cell conditioned medium (ESC-CM) increases the proliferative capacity of human corneal endothelial cells (HCECs) in vitro. Methods Primary cultures of HCECs were established from explants of the endothelial cell layer, including the Descemet’s membrane. Cells were cultured in human corneal endothelium medium (CEM) containing 25% ESC-CM for the experimental group and CEM alone for the control group. Phase-contrast microscopy and reverse-transcr...

  10. Differentiations of transplanted mouse spermatogonial stem cells in the adult mouse renal parenchyma in vivo

    Institute of Scientific and Technical Information of China (English)

    Da-peng WU; Da-lin HE; Xiang LI; Zhao-hui LIU

    2008-01-01

    Aim:Spermatogonial stem cells can initiate the process of cellular differentia-tion to generate mature spermatozoa, but whether it possess the characteristic of pluripotency and plasticity, similar to embryonic stem cells, has not been elucidated. This study was designed to evaluate the differentiation potential of spermatogonial stem cells into renal cells in vivo. Methods: Neonatal mouse spermatogonial stem cells were transplanted into mature male mice lacking en-dogenous spermatogenesis. The restoration of fertility in recipient males was observed. Spermatogonial stem cells were then injected into renal parenchyma of mature female mice to make a new extracellular environment for differentia-tion. Fluorescence in situ hybridization technology (FISH) was used to detect the expression of chromosome Y in recipient renal tissues. To determine the type of cells differentiated from spermatogonial stem cells, the expression of ricinus communis agglutinin, vimentin, CD45, and F4/80 proteins were examined in the renal tissues by immunohistochemistry. Results: The proliferation of seminiferous epithelial cells was distinctly observed in seminiferous tubules of transplanted testes, whereas no regeneration of spermatogenesis was observed in non-transplanted control testes. In transplanted female renal tissues, FISH showed a much stronger immuno-fluorescence signal of chromosome Y in the nucleolus of epithelial cells of the renal tubule and podocytes of the glomerulus. Conclusion: The spermatogonial stem cells were successfully purified from mouse testicles. This finding demonstrated that spermatogonial stem cells could not only restore damaged spermatogenesis, but were also capable of differentiat-ing into mature renal parenchyma cells in vivo.

  11. Germinal vesicle material drives meiotic cell cycle of mouse oocyte through the 3'UTR-dependent control of cyclin B1 synthesis.

    Science.gov (United States)

    Hoffmann, Steffen; Tsurumi, Chizuko; Kubiak, Jacek Z; Polanski, Zbigniew

    2006-04-01

    We compared the profile of histone H1 kinase activity, reflecting Maturation Promoting Factor (MPF) activity in oocytes bisected at the germinal vesicle (GV) stage and allowed to mature as separate oocyte halves in vitro. Whereas the oocyte halves containing the nucleus exhibited the same profile of increased kinase activity as that typical for intact oocytes, the anuclear halves revealed strong inhibition of the increase in this activity soon after germinal vesicle breakdown (GVBD). In contrast, the profile of MAP kinase activity did not differ significantly between anuclear and nucleus-containing oocyte halves throughout maturation. Of the two MPF components, CDK1 and cyclin B1, the amount of the latter was significantly reduced in anuclear halves, a reduction due to low-level synthesis and not to enhanced degradation. Expression of three reporter luciferase RNAs constructed, respectively, to contain cyclin B1-specific 3'UTR, the globin-specific 3'UTR, or no 3'UTR sequence was enhanced in nuclear halves, with significantly greater enhancement for the construct containing cyclin B1-specific 3'UTR as compared to the two other RNAs. We conclude that the profile of activity of MPF during mouse oocyte maturation is controlled by an unknown GV-associated factor(s) acting via 3'UTR-dependent control of cyclin B1 synthesis. These results require the revision of the hitherto prevailing view that the control of MPF activity during mouse oocyte maturation is independent of GV-derived material. PMID:16490186

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

    Directory of Open Access Journals (Sweden)

    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. Isolation of mouse cell proteoglycan mutants

    International Nuclear Information System (INIS)

    The sulfated proteoglycans on the surface of cultured mammalian cells have been implicated in a variety of phenomena. To obtain more direct evidence for the role of these molecules in specific cellular functions, they are isolating mutants that produce altered sulfated proteoglycans from a cloned line of Swiss mouse 3T3 cells. This cell type was selected because it exhibits contact inhibition of growth and there is extensive information on its' cell surface and extracellular proteoglycans and other glycoproteins. Cells were chemically mutagenized and subjected to one or more cycles of radiation suicide in the presence of 35S-sulfate. By replica plating, 150 clones, which appear to incorporate abnormal amounts of 35S-sulfate, have been selected. After recloning three times via the replica plating technique, the proteoglycans of 29 clones have thus far been analyzed. They have identified four clones which appear to make altered amounts of either cell surface heparan sulfate or chondroitin sulfate. The biochemical bases for the altered levels of the proteoglycans are under study. Of particular interest, however, is the fact that in this limited collection of mutants the chemical alterations correlate with specific altered cellular morphologies

  14. 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.

  15. Interleukin-1β-Targeted Vaccine Improves Glucose Control and β-Cell Function in a Diabetic KK-Ay Mouse Model

    Science.gov (United States)

    Yu, Xiao-lin; Liu, Xiang-meng; Liu, Dong-qun; Zhu, Jie; Ji, Hui; Liu, Rui-tian

    2016-01-01

    Interleukin-1β (IL-1β) has been implicated as a key proinflammatory cytokine involved in the pancreatic islet inflammation of type 2 diabetes mellitus (T2DM). Excess IL-1β impairs islet function by inducing insulin resistance and β-cell apoptosis. Therefore, specifically reducing IL-1β activity provides a therapeutic improvement for T2DM by sustaining the inhibition of IL-1β-mediated islet inflammation. In this study, we developed an IL-1β-targeted epitope peptide vaccine adjuvanted with polylactic acid microparticles (1βEPP) and applied it to a diabetic KK-Ay mouse model. Results showed that the 1βEPP elicited high antibody responses, which neutralized the biological activity of IL-1β, and induced barely detectable inflammatory activity. 1βEPP immunization reduced body weight gain, protected KK-Ay mice from hyperglycemia, improved glucose tolerance and insulin sensitivity, and decreased the serum levels of free fatty acids, total cholesterol and triglyceride. Moreover, 1βEPP restored β-cell mass; inhibited β-cell apoptosis; decreased the expression of IL-1β; and interrupted NF-κB activation by reducing IKKβ and pRelA levels. These studies indicated that the IL-1β-targeted vaccine may be a promising immunotherapeutic for T2DM treatment. PMID:27152706

  16. Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Jain, Neha; Lee, Eun Jung

    2016-01-01

    The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes. PMID:25751085

  17. The International Mouse Strain Resource (IMSR): cataloging worldwide mouse and ES cell line resources.

    Science.gov (United States)

    Eppig, Janan T; Motenko, Howie; Richardson, Joel E; Richards-Smith, Beverly; Smith, Cynthia L

    2015-10-01

    The availability of and access to quality genetically defined, health-status known mouse resources is critical for biomedical research. By ensuring that mice used in research experiments are biologically, genetically, and health-status equivalent, we enable knowledge transfer, hypothesis building based on multiple data streams, and experimental reproducibility based on common mouse resources (reagents). Major repositories for mouse resources have developed over time and each has significant unique resources to offer. Here we (a) describe The International Mouse Strain Resource that offers users a combined catalog of worldwide mouse resources (live, cryopreserved, embryonic stem cells), with direct access to repository sites holding resources of interest and (b) discuss the commitment to nomenclature standards among resources that remain a challenge in unifying mouse resource catalogs. PMID:26373861

  18. Effects of UVC-irradiation on cultured mouse embryonic cells

    International Nuclear Information System (INIS)

    Effects of UVC-irradiation on the cultured differentiating mouse embryonic cells were investigated. Embryonic mesenchymal cells, isolated from fore-and hind-limbs or mid brain of Day 11 mouse embryos, and 3T3 cells, a reference mouse fibroblast cell line, were irradiated with UVC at a dose range of 0∼30 J/m2. Dose-dependent inhibition was found for both cellular proliferation and differentiation, dose-dependent induction of DNA cyclobutane pyrimidine dimers and (6-4) photoproducts were found in the embryonic cells. Mesenchymal chondrogenesis was more sensitive to the UVC than proliferation, and the UVC-induced DNA damage and their repair kinetics in the cultured embryonic cells were similar to those in mouse 3T3 cells. No effects of treatments by the fluorescent light pre or post UVC-irradiation were found on the repair kinetics of DNA damage in all of the cells

  19. Generation and gene expression profiling of 48 transcription-factor-inducible mouse embryonic stem cell lines.

    Science.gov (United States)

    Yamamizu, Kohei; Sharov, Alexei A; Piao, Yulan; Amano, Misa; Yu, Hong; Nishiyama, Akira; Dudekula, Dawood B; Schlessinger, David; Ko, Minoru S H

    2016-01-01

    Mouse embryonic stem cells (ESCs) can differentiate into a wide range - and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously, we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this "NIA Mouse ESC Bank," we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs. PMID:27150017

  20. Ion channels modulating mouse dendritic cell functions.

    Science.gov (United States)

    Matzner, Nicole; Zemtsova, Irina M; Nguyen, Thi Xuan; Duszenko, Michael; Shumilina, Ekaterina; Lang, Florian

    2008-11-15

    Ca(2+)-mediated signal transduction pathways play a central regulatory role in dendritic cell (DC) responses to diverse Ags. However, the mechanisms leading to increased [Ca(2+)](i) upon DC activation remained ill-defined. In the present study, LPS treatment (100 ng/ml) of mouse DCs resulted in a rapid increase in [Ca(2+)](i), which was due to Ca(2+) release from intracellular stores and influx of extracellular Ca(2+) across the cell membrane. In whole-cell voltage-clamp experiments, LPS-induced currents exhibited properties similar to the currents through the Ca(2+) release-activated Ca(2+) channels (CRAC). These currents were highly selective for Ca(2+), exhibited a prominent inward rectification of the current-voltage relationship, and showed an anomalous mole fraction and a fast Ca(2+)-dependent inactivation. In addition, the LPS-induced increase of [Ca(2+)](i) was sensitive to margatoxin and ICAGEN-4, both inhibitors of voltage-gated K(+) (Kv) channels Kv1.3 and Kv1.5, respectively. MHC class II expression, CCL21-dependent migration, and TNF-alpha and IL-6 production decreased, whereas phagocytic capacity increased in LPS-stimulated DCs in the presence of both Kv channel inhibitors as well as the I(CRAC) inhibitor SKF-96365. Taken together, our results demonstrate that Ca(2+) influx in LPS-stimulated DCs occurs via Ca(2+) release-activated Ca(2+) channels, is sensitive to Kv channel activity, and is in turn critically important for DC maturation and functions. PMID:18981098

  1. DMSO exhibits similar cytotoxicity effects to thalidomide in mouse breast cancer cells

    OpenAIRE

    Öz, Ece Simsek; Aydemir, Esra; Fışkın, Kayahan

    2012-01-01

    The purpose of this study was to evaluate the cytotoxic effect of thalidomide on 4T1 and 4THMpc mouse breast cancer cell lines. Mouse breast cancer cells (4T1) and cells derived from metastatic lesions (4THMpc) were treated with various doses of thalidomide [10-2-100 µM dissolved in dimethyl sulfoxide (DMSO) as recommended] and 1.4 µM DMSO (maximum DMSO concentration in the highest thalidomide dose) as a DMSO control against the untreated control groups. MTT was used to evaluate the cytotoxic...

  2. CIRCADIAN CLOCK AND CELL CYCLE GENE EXPRESSION: IN MOUSE MAMMARY EPITHELIAL CELLS AND IN THE DEVELOPING MOUSE MAMMARY GLAND

    OpenAIRE

    Metz, Richard P.; Qu, Xiaoyu; Laffin, Brian; Earnest, David; Porter, Weston W.

    2006-01-01

    Mouse mammary epithelial cells (HC-11) and mammary tissues were analyzed for developmental changes in circadian clock, cellular proliferation and differentiation marker genes. Expression of the clock genes, Per1 and Bmal1, were elevated in differentiated HC-11 cells whereas Per2 mRNA levels were higher in undifferentiated cells. This differentiation-dependent profile of clock gene expression was consistent with that observed in mouse mammary glands as Per1 and Bmal1 mRNA levels were elevated ...

  3. Mouse Control using a Web Camera based on Colour Detection

    OpenAIRE

    2014-01-01

    In this paper we present an approach for Human computer Interaction (HCI), where we have tried to control the mouse cursor movement and click events of the mouse using hand gestures. Hand gestures were acquired using a camera based on colour detection technique. This method mainly focuses on the use of a Web Camera to develop a virtual human computer interaction device in a cost effective manner.

  4. Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency.

    Science.gov (United States)

    Fiorenzano, Alessandro; Pascale, Emilia; D'Aniello, Cristina; Acampora, Dario; Bassalert, Cecilia; Russo, Francesco; Andolfi, Gennaro; Biffoni, Mauro; Francescangeli, Federica; Zeuner, Ann; Angelini, Claudia; Chazaud, Claire; Patriarca, Eduardo J; Fico, Annalisa; Minchiotti, Gabriella

    2016-01-01

    Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo. PMID:27586544

  5. Apoptosis of matured T lymphocytes induced by mouse sertoli cells in cocultures in vitro

    Institute of Scientific and Technical Information of China (English)

    LIU Yang; LIN Zi-hao; ZHU Xiao-hai; LIU Shan-rong

    2001-01-01

    Objective: To study whether mouse sertoli cells can induce the apoptosis of matured T lymphocytes in cocultures in vitro. Methods: With TUNEL, DNA electrophoresis, eleetro-mierography and flow cytometry, we examined the apoptosis and its rates of mouse matured T lymphocytes in control group (T lymphocytes only), group A (T lymphocytes + culture medium of sertoli cells), group B (T lymphocytes + sertoli cells). Results: Under electro-micrography, chromatin condensation, karyopyknosis, karyorhexis and apoptotic body were observed in some T lymphocytes in 3 groups; some nucleuses were stained dark blue with TUNEL; a typical DNA ladder was found with DNA electrophoresis. The apoptotic rates of T lymphocytes in group A and B were significantly higher than that in control group (P<0.01). The apoptotic rate of T lymphocytes in group B was significantly higher than that in group A (P<0.01). Conclusion: In coculture condition in vitro,mouse sertoli cells can induce the apoptosis of matured T lymphocytes.

  6. 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....... 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...... 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....

  7. CML Mouse Model Generated from Leukemia Stem Cells.

    Science.gov (United States)

    Hu, Yiguo

    2016-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disorder with a high number of well-differentiated neutrophils in peripheral blood and myeloid cells in bone marrow (BM). CML is derived from the hematopoietic stem cells (HSCs) with the Philadelphia chromosome (Ph(+), t(9;22)-(q34;q11)), resulting in generating a fusion oncogene, BCR/ABL1. HSCs with Ph(+) are defined as leukemia stem cells (LSCs), a subpopulation cell at the apex of hierarchies in leukemia cells and responsible for the disease continuous propagation. Several kinds of CML models have been developed to reveal the mechanism of CML pathogenesis and evaluate therapeutic drugs in the past three decades. Here, we describe the procedures to generate a CML mouse model by introducing BCR/ABL1 into Lin(-)Sca1(+) cKit(+) population cells purified from mouse bone marrow. In CML retroviral transduction/transplantation mouse models, this modified model can mimic CML pathogenesis on high fidelity. PMID:27581136

  8. Hair cell damage recruited Lgr5-expressing cells are hair cell progenitors in neonatal mouse utricle

    Directory of Open Access Journals (Sweden)

    Jinchao Lin

    2015-04-01

    Full Text Available Damage-activated stem/progenitor cells play important roles in regenerating lost cells and in tissue repair. Previous studies reported that the mouse utricle has limited hair cell regeneration ability after hair cell ablation. However, the potential progenitor cell population regenerating new hair cells remains undiscovered. In this study, we first found that Lgr5, a Wnt target gene that is not usually expressed in the neonatal mouse utricle, can be activated by 24 hour neomycin treatment in a sub-population of supporting cells in the striolar region of the neonatal mouse utricle. Lineage tracing demonstrated that these Lgr5-positive supporting cells could regenerate new hair cells in explant culture. We isolated the damage-activated Lgr5-positive cells with flow cytometry and found that these Lgr5-positive supporting cells could regenerate hair cells in vitro, and self-renew to form spheres, which maintained the capacity to differentiate into hair cells over seven generations of passages. Our results suggest that damage-activated Lgr5-positive supporting cells act as hair cell progenitors in the neonatal mouse utricle, which may help to uncover a potential route to regenerate hair cell in mammals.

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

    International Nuclear Information System (INIS)

    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

  10. Brown adipogenesis of mouse embryonic stem cells in alginate microstrands

    Science.gov (United States)

    Unser, Andrea Mannarino

    The ability of brown adipocytes (fat cells) to dissipate energy as heat shows great promise for the treatment of obesity and other metabolic disorders. Employing pluripotent stem cells, with an emphasis on directed differentiation, may overcome many issues currently associated with primary fat cell cultures. However, brown adipocytes are difficult to transplant in vivo due to the instability of fat, in terms of necrosis and neovascularization, once injected. Thus, 3D cell culture systems that have the potential to mimic adipogenic microenvironments are needed, not only to advance brown fat implantation, but also to better understand the role of brown adipocytes in treating obesity. To address this need, we created 3D "Brown-Fat-in-Microstrands" by microfluidic synthesis of alginate hydrogel microstrands that encapsulated cells and directly induced cell differentiation into brown adipocytes, using mouse embryonic stem cells (ESCs) as a model of pluripotent stem cells and brown preadipocytes as a positive control. The effect of hydrogel formation parameters on brown adipogenesis was studied, leading to the establishment of "Brown-Fat-in-Microstrands". Brown adipocyte differentiation within microstrands was confirmed by lipid droplet accumulation, immunocytochemistry and qPCR analysis of gene expression of brown adipocyte marker uncoupling protein 1 (UCP1) in addition to adipocyte marker expression. Compared to a 2D approach, 3D differentiated "Brown-Fat-in-Microstrands" exhibited higher level of brown adipocyte marker expression. The functional analysis of "Brown-Fat-in-Microstrands" was attempted by measuring the mitochondrial activity of ESC-differentiated brown adipocytes in 3D using Seahorse XF24 3 Extracellular Flux Analyzer. The ability to create "Brown-Fat-in-Microstrands" from pluripotent stem cells opens up a new arena to understanding brown adipogenesis and its implications in obesity and metabolic disorders.

  11. GATA-1 directly regulates Nanog in mouse embryonic stem cells

    International Nuclear Information System (INIS)

    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

  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. GREG cells, a dysferlin-deficient myogenic mouse cell line

    International Nuclear Information System (INIS)

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large (∼ 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1–43 dye. Under the wounding conditions used, the majority (∼ 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  14. GREG cells, a dysferlin-deficient myogenic mouse cell line

    Energy Technology Data Exchange (ETDEWEB)

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S. [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Morree, Antoine de [Center for Human Genetics, Leiden University Medical Center, Leiden (Netherlands); Pekkurnaz, Gulcin [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Nagaraju, Kanneboyina [Research Center for Genetic Medicine, Children' s National Medical Center, Washington, DC 20010 (United States); Zimmerberg, Joshua, E-mail: zimmerbj@mail.nih.gov [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)

    2012-01-15

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large ({approx} 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority ({approx} 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  15. Stem cell transplantation in mouse models for Huntington's disease

    OpenAIRE

    Johann, Verena

    2005-01-01

    Cell replacement therapies for neurodegenerative diseases using stem cells require above all a good survival of the graft and therefore an understanding of the possible influence of the surrounding degenerating tissue on the grafted cells. In this thesis, I report on the experiments of stem cell transplantation in mouse models for Huntington´s disease. The most common rodent model for HD is the QA-lesion model, where quinolinic acid is injected unilaterally into the striatum of adult rats. We...

  16. A mouse model of intestinal stem cell function and regeneration

    OpenAIRE

    Slorach, E M; Campbell, F. C.; Dorin, J. R.

    1999-01-01

    We present here an in vivo mouse model for intestinal stem cell function and differentiation that uses postnatal intestinal epithelial cell aggregates to generate a differentiated murine small intestinal mucosa with full crypt-villus architecture. The process of neomucosal formation is highly similar to that of intestinal regeneration. Both in vivo grafting and primary culture of these cells reveal two different epithelial cell populations, which display properties consistent with intestinal ...

  17. Cell-SELEX-based selection and characterization of a G-quadruplex DNA aptamer against mouse dendritic cells.

    Science.gov (United States)

    Moghadam, M; Sankian, M; Abnous, K; Varasteh, A; Taghdisi, S M; Mahmoudi, M; Ramezani, M; Gholizadeh, Z; Ganji, A

    2016-07-01

    Targeting of dendritic cells (DCs) by aptamers increases antigen capture and presentation to the immune system. Our aim was to produce aptamers against DC molecules using the cell-SELEX procedure. For this purpose, 18 rounds of cell-SELEX were performed on mouse macrophage J774A.1 and CT26 as target and control cells, respectively. The selected aptamers were truncated and their binding to mouse macrophages, and immature and mature DCs analyzed. Two macrophage-specific aptamers, Seq6 and Seq7, were identified. A truncated form of Seq7, Seq7-4, 33 nucleotides in length and containing the G-quadruplex, bound macrophages and immature DCs with KD values in the nanomolar range. We anticipate that Seq7-4 has potential as a therapeutic tool in targeting of mouse macrophages and immature DCs to efficiently improve different immunotherapy approaches. PMID:27232653

  18. Mouse models for cancer stem cell research

    OpenAIRE

    Cheng, Le; Ramesh, Anirudh V.; Flesken-Nikitin, Andrea; Choi, Jinhyang; Nikitin, Alexander Yu.

    2009-01-01

    Cancer stem cell concept assumes that cancers are mainly sustained by a small pool of neoplastic cells, known as cancer stem cells or tumor initiating cells, which are able to reproduce themselves and produce phenotypically heterogeneous cells with lesser tumorigenic potential. Cancer stem cells represent an appealing target for development of more selective and efficient therapies. However, direct testing of the cancer stem cell concept and assessment of its therapeutic implications in human...

  19. Cell control report

    CERN Document Server

    2013-01-01

    Please note this is a Short Discount publication. This extensive report provides an essential overview of cells and their use as factory automation building blocks. The following issues are discussed in depth: Cell integration Cell software and standards Future technologies applied to cells Plus Cell control applications including: - rotary parts manufacturing - diesel engine component development - general cell control development at the General Electric Corporation - a vendor list.

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

    International Nuclear Information System (INIS)

    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

  1. Isolation, Culture, and Maintenance of Mouse Intestinal Stem Cells

    Science.gov (United States)

    O’Rourke, Kevin P.; Ackerman, Sarah; Dow, Lukas E; Lowe, Scott W

    2016-01-01

    In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro.

  2. Mouse cloning and somatic cell reprogramming using electrofused blastomeres

    OpenAIRE

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

    2010-01-01

    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 s...

  3. Replacement of Diseased Mouse Liver by Hepatic Cell Transplantation

    Science.gov (United States)

    Rhim, Jonathan A.; Sandgren, Eric P.; Degen, Jay L.; Palmiter, Richard D.; Brinster, Ralph L.

    1994-02-01

    Adult liver has the unusual ability to fully regenerate after injury. Although regeneration is accomplished by the division of mature hepatocytes, the replicative potential of these cells is unknown. Here, the replicative capacity of adult liver cells and their medical usefulness as donor cells for transplantation were investigated by transfer of adult mouse liver cells into transgenic mice that display an endogenous defect in hepatic growth potential and function. The transplanted liver cell populations replaced up to 80 percent of the diseased recipient liver. These findings demonstrate the enormous growth potential of adult hepatocytes, indicating the feasibility of liver cell transplantation as a method to replace lost or diseased hepatic parenchyma.

  4. Immunofluorescent Staining of Mouse Intestinal Stem Cells

    Science.gov (United States)

    O’Rourke, Kevin P.; Dow, Lukas E; Lowe, Scott W

    2016-01-01

    Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, permeabilize, stain and mount intestinal organoids for analysis by immunofluorescent confocal microscopy.

  5. Spontaneous generation of germline characteristics in mouse fibrosarcoma cells

    Science.gov (United States)

    Ma, Zhan; Hu, Yao; Jiang, Guoying; Hou, Jun; Liu, Ruilai; Lu, Yuan; Liu, Chunfang

    2012-10-01

    Germline/embryonic-specific genes have been found to be activated in somatic tumors. In this study, we further showed that cells functioning as germline could be present in mouse fibrosarcoma cells (L929 cell line). Early germline-like cells spontaneously appeared in L929 cells and further differentiated into oocyte-like cells. These germline-like cells can, in turn, develop into blastocyst-like structures in vitro and cause teratocarcinomas in vivo, which is consistent with natural germ cells in function. Generation of germline-like cells from somatic tumors might provide a novel way to understand why somatic cancer cells have strong features of embryonic/germline development. It is thought that the germline traits of tumors are associated with the central characteristics of malignancy, such as immortalization, invasion, migration and immune evasion. Therefore, germline-like cells in tumors might provide potential targets to tumor biology, diagnosis and therapy.

  6. miRNA regulation of cytotoxic effects in mouse Sertoli cells exposed to nonylphenol

    Directory of Open Access Journals (Sweden)

    Kim Seung Jun

    2011-09-01

    Full Text Available Abstract Background It is known that some environmental chemicals affect the human endocrine system. The harmful effects of endocrine disrupting chemical (EDC nonylphenol (NP have been studied since the 1980s. It is known that NP adversely affects physiological functions by mimicking the natural hormone 17 beta-estradiol. In the present study, we analyzed the expression of miRNAs and their target genes in mouse Sertoli TM4 cells to better understand the regulatory roles of miRNAs on Sertoli cells after NP exposure. Methods Mouse TM4 Sertoli cells were treated with NP for 3 or 24 h, and global gene and miRNA expression were analyzed using Agilent mouse whole genome and mouse miRNA v13 arrays. Results We identified genes that were > 2-fold differentially expressed in NP-treated cells and control cells (P Ppara may regulate the expression of certain miRNAs, including miR-378, miR-125a-3p miR-20a, miR-203, and miR-101a, after exposure to NP. Additionally, comprehensive analysis of predicted target genes for miRNAs showed that the expression of genes with roles in cell proliferation, the cell cycle, and cell death were regulated by miRNA in NP-treated TM4 cells. Levels of expression of the miRNAs miR-135a* and miR-199a-5p were validated by qRT-PCR. Finally, miR-135a* target gene analysis suggests that the generation of reactive oxygen species (ROS following exposure to NP exposure may be mediated by miR-135a* through regulation of the Wnt/beta-catenin signaling pathway. Conclusions Collectively, these data help to determine NP's actions on mouse TM4 Sertoli cells and increase our understanding of the molecular mechanisms underlying the adverse effects of xenoestrogens on the reproductive system.

  7. Single Targeted Exon Mutation Creates a True Congenic Mouse for Competitive Hematopoietic Stem Cell Transplantation: The C57BL/6-CD45.1STEM Mouse

    OpenAIRE

    Francois E. Mercier; David B. Sykes; David T. Scadden

    2016-01-01

    Defining the molecular regulators of hematopoietic stem and progenitor cells (HSPCs) requires in vivo functional analyses. Competitive bone marrow transplants (BMTs) compare control and test HSPCs to demonstrate the functional role of a genetic change or chemical perturbation. Competitive BMT is enabled by antibodies that specifically recognize hematopoietic cells from congenic mouse strains due to variants of the cell surface protein CD45, designated CD45.1 and CD45.2. The current congenic c...

  8. Effects of simulated microgravity on mouse Sertoli cells in culture

    Science.gov (United States)

    Angela, Masini Maria; Prato, Paola; Linda, Scarabelli; Lanza, Cristina; Palmero, Silvio; Pointis, Georges; Ricci, Franco; Strollo, Felice

    With the advent of space flights questions concerning the effects of microgravity (0xG) on hu-man reproduction physiology have got priority Spermatogenesis is a complex, highly ordered process of cell division and differentiation by which spermatogonial cells give rise to mature spermatozoa. Sertoli cells play a crucial role in the development of germ cells and the regulation of spermatogenesis. In this study the influence of 0xG on Sertoli cells was evaluated. A Sertoli cell line from mouse testis (42GPA9) was analyzed for cytoskeletal (using the 3D reconstruction generated from a stack of confocal images) and SHBG changes by immunohistochemistry, for antioxidant agents by RT-PCR and for culture medium lactate concentrations by wet chemistry. Cells were cultured for 6, 24 and 48 hrs on a three-dimensional Random Positioning Machine (3D-RPM); static controls (1xG) were positioned on the supporting frame. At the end of each experiment, cultured cells were either fixed in paraformaldehyde or RNA-extracted or used for culture medium lactate measurements as needed. At 0xG Sertoli cytoskeleton got disorganized, microtubules fragmented and SHBG undetectable already after 24 hrs, with alterations wors-ening further until 48 hrs; various antioxidant systems (SOD, GST, PARP, MTs) appreciably increased during the first 24 hrs but significantly decreased at 48 hrs. No changes occurred in 1xG samples. At least initially, 0xG seems to perturb antioxidant protection strategies allowing the testes to support sperm production, thus generating an aging-like state of oxidative stress. Lactate production at 0xG slightly decreased only after 24 hrs. Further experiments need to be carried out in space to investigate upon steroidogenesis and germ cell differentiation within the testis, to rule out eventually pending male infertility consequences, which would be a problem nowadays, when life expectancy increases and male fertility might become a social issue often extending into 60 years

  9. Ganglioside GM1 influences the proliferation rate of mouse induced pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Young-Kug Choo

    2012-12-01

    Full Text Available Gangliosides play important roles in the control of severalbiological processes, including proliferation and transmembranesignaling. In this study, we demonstrate the effect ofganglioside GM1 on the proliferation of mouse inducedpluripotent stem cells (miPSCs. The proliferation rate ofmiPSCs was lower than in mouse embryonic stem cells(mESCs. Fluorescence activated cell sorting analysis showedthat the percentage of cells in the G2/M phase in miPSCs waslower than that in mESCs. GM1 was expressed in mESCs, butnot miPSCs. To confirm the role of GM1 in miPSC proliferation,miPSCs were treated with GM1. GM1-treated miPSCsexhibited increased cell proliferation and a larger number ofcells in the G2/M phase. Furthermore, phosphorylation ofmitogen-activated protein kinases was increased in GM1-treated miPSCs.

  10. TSH stimulates adipogenesis in mouse embryonic stem cells

    OpenAIRE

    Lu, Min; Lin, Reigh-Yi

    2008-01-01

    Although TSH is the main regulator of thyroid growth and function, TSH binding activity in fat has long been reported. Since the TSH receptor (TSHR) has been detected in both preadipocytes and adipocytes, we hypothesized that it may play a role in adipose differentiation. Here, we use an in vitro model of adipogenesis from mouse embryonic stem (ES) cells to define TSH function. Directed differentiation of ES cells into the adipose lineage can be achieved over a 3-week period. Although adipocy...

  11. 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.

  12. Subretinal transplantation of mouse retinal progenitor cells

    Institute of Scientific and Technical Information of China (English)

    Caihui Jiang; Maonian Zhang; Henry Klassen; Michael Young

    2011-01-01

    The development of cell replacement techniques is promising as a potential treatment for photoreceptor loss. However, the limited integration ability of donor and recipient cells presents a challenge following transplantation. In the present study, retinal progenitor cells (RPCs) were harvested from the neural retinas of enhanced green fluorescent protein mice on postnatal day 1, and expanded in a neurobasal medium supplemented with fetal bovine serum without endothelial growth factor. Using a confocal microscope, immunohistochemistry demonstrated that expanded RPCs in vitro maintain retinal stem cell properties and can be differentiated into photoreceptor cells. Three weeks after transplantation, subretinal transplanted RPCs were found to have migrated and integrated into the outer nuclear layer of recipient retinas with laser injury, some of the integrated cells had differentiated into photoreceptors, and a subpopulation of these cells expressed photoreceptor specific synaptic protein, appearing to form synaptic connections with bipolar cells. These results suggest that subretinal transplantation of RPCs may provide a feasible therapeutic strategy for the loss of retinal photoreceptor cells.

  13. Cell death in the Purkinje cells of the cerebellum of senescence accelerated mouse (SAMP(8)).

    Science.gov (United States)

    Zhu, Yonghong; Lee, Cleo C L; Lam, W P; Wai, Maria S M; Rudd, John A; Yew, David T

    2007-10-01

    The cerebella of SAMP(8) (accelerated aging mouse) and SAMR(1) controls were analyzed by Western Blotting of tyrosine hydroxylase and choline acetyltransferase, as well as by TUNEL and histological silver staining. Both tyrosine hydroxylase and choline acetyltransferase levels were higher in SAMR(1) than in SAMP(8). There was also an age-related decrease in enzyme levels in SAMP(8), with the reduction of tyrosine hydroxylase being more apparent. Concomitantly, there was an age-related increase of apoptosis in the medial neocerebellum and the vermis as revealed by TUNEL, with changes being significant in the SAMP(8) strain. Histologically, some Purkinje cells appeared to disappear during aging. Taken together, the data suggests that the aging SAMP(8) strain displays differential Purkinje cell death in the medial cerebellum and that some of the dying cells are likely to be catecholaminergic. PMID:17415677

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

    International Nuclear Information System (INIS)

    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

  15. Radiation response of spermatogonial stem cells in the mouse

    International Nuclear Information System (INIS)

    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 (D0 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.)

  16. Intraperitoneal Injection of Multiplacentas Pooled Cells Treatment on a Mouse Model with Aplastic Anemia

    Science.gov (United States)

    Li, Jun; Chen, Hong; Lv, Yan-Bo; Wang, Qiang; Xie, Zheng-Jun; Ma, Li-Hua; He, Jie; Xue, Wei; Yu, Shan; Guo, Jun; Wang, Ting-Hua; Wu, Tian-Xi; Pan, Xing-Hua

    2016-01-01

    Coinfusion of hematopoietic and mesenchymal stem cells is more effective than hematopoietic stem cell transplantation alone. It is necessary to explore a safe and routine mixed stem cell intraperitoneal transplantation method. Multiplacentas pooled cells were intraperitoneally injected into a radiation- and immunity-induced mouse aplastic anemia model with single time. Then, mouse survival time, peripheral blood hemoglobin count, bone marrow architecture, and donor cell engraftment were assessed. The recipient mouse exhibited donor cell engraftment in both bone marrow and peripheral blood. Survival time and peripheral blood hemoglobin count increased in placenta pooled cells treated mice, compared with model-only controls (P = 0.048 and P = 0.000, resp.). However, placentas pooled cells failed to cause a significant decrease in bone marrow pimelosis area (P = 0.357). Intraperitoneally transplanted multiplacentas pooled cells can survive and engraft into a host body through blood circulation, which can increase the life span of an aplastic anemia model mice, and delay but not abrogate the development of aplastic anemia. Furthermore, they appear to play a role in increasing peripheral blood hemoglobin level response for increasing the life span of aplastic anemia model mice. PMID:26997957

  17. Connexins protect mouse pancreatic β cells against apoptosis

    OpenAIRE

    Klee, Philippe; Allagnat, Florent; Pontes, Helena; Cederroth, Manon; Charollais, Anne; Caille, Dorothée; Britan, Aurore; Haefliger, Jacques-Antoine; Meda, Paolo

    2011-01-01

    Type 1 diabetes develops when most insulin-producing β cells of the pancreas are killed by an autoimmune attack. The in vivo conditions modulating the sensitivity and resistance of β cells to this attack remain largely obscure. Here, we show that connexin 36 (Cx36), a trans-membrane protein that forms gap junctions between β cells in the pancreatic islets, protects mouse β cells against both cytotoxic drugs and cytokines that prevail in the islet environment at the onset of type 1 diabetes. W...

  18. Mouse Incisor Stem Cell Niche and Myb Transcription Factors.

    Science.gov (United States)

    Svandova, E; Vesela, B; Smarda, J; Hampl, A; Radlanski, R J; Matalova, E

    2015-10-01

    Dental hard tissues are formed particularly by odontoblasts (dentin) and ameloblasts (enamel). Whereas the reparation of dentin is often observed, enamel does not regenerate in most species. However, in mouse incisor, a population of somatic stem cells in the cervical loop is responsible for the incisor regeneration. Understanding of the specificities of these cells is therefore of an interest in basic research as well as regenerative therapies. The Myb transcription factors are involved in essential cellular processes. B-Myb is often linked to the stem cell phenotype, and c-Myb expression marks undifferentiated and proliferating cells such as the stem cells. In the presented study, temporo-spatial expression of B-Myb and c-Myb proteins was correlated with localisation of putative somatic stem cells in the mouse incisor cervical loop by immunohistochemistry. B-Myb expression was localised mostly in the zone of transit-amplifying cells, and c-Myb was found in the inner enamel epithelium, the surrounding mesenchyme and in differentiated cells. Taken together, neither B-Myb nor c-Myb was exclusively present or abundant in the area of the incisor stem cell niche. Their distribution, however, supports recently reported novel functions of c-Myb in differentiation of hard tissue cells. PMID:25182175

  19. Mouse cloning and somatic cell reprogramming using electrofused blastomeres

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

    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.

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

    OpenAIRE

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

    2012-01-01

    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...

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

    International Nuclear Information System (INIS)

    Highlights: • LPA5 inhibits the cell growth and motile activities of 3T3 cells. • LPA5 suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA5 on the cell motile activities inhibited by LPA1 in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA5 in 3T3 cells. • LPA signaling via LPA5 acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA1–LPA6) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA5 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 LPA1 and LPA5 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 LPA5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA1

  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. Voltage-gated Ca2+ channel in mouse myeloma cells.

    OpenAIRE

    Fukushima, Y; Hagiwara, S.

    1983-01-01

    Electrical properties of the cell membrane were studied in the neoplastic lymphocyte, mouse myeloma cell line S194, by using the whole-cell patch clamp technique. Inward Ca2+ currents due to voltage-gated Ca2+ channels were found. The current, which decayed exponentially after reaching a peak, was first activated at about -50 mV and attained its maximum peak amplitude at about -20 mV in a 10 mM Ca2+ solution. Outward current was negligible for the potential range more negative than +30 mV. Th...

  4. Transcriptome analysis of mouse stem cells and early embryos.

    Directory of Open Access Journals (Sweden)

    Alexei A Sharov

    2003-12-01

    Full Text Available Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

  5. Transcriptome Analysis of Mouse Stem Cells and Early Embryos

    Directory of Open Access Journals (Sweden)

    Sharov Alexei A

    2003-01-01

    Full Text Available Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

  6. Cell Control Engineering

    DEFF Research Database (Denmark)

    Lynggaard, Hans Jørgen Birk; Alting, Leo

    1996-01-01

    The engineering process of creating cell control systems is described, and a Cell Control Engineering (CCE) concept is defined. The purpose is to assist people, representing different disciplines in the organisation, to implement cell controllers by addressing the complexity of having many systems...... in physically and logically different and changing manufacturing environments. The defined CCE concept combines state-of-the-art of commercially available enabling technologies for automation system software development, generic cell control models and guidelines for the complete engineering process....... It facilitates the understanding of the task and structure of cell controllers and uses this knowledge directly in the implementation of the system. By applying generic models CCE facilitates reuse of software components and maintenance of applications. In many enterprises, software makes up an...

  7. Immunoregulatory properties of mouse limbal stem cells

    Czech Academy of Sciences Publication Activity Database

    Holáň, Vladimír; Pokorná, Kateřina; Procházková, Jana; Krulová, M.; Zajícová, Alena

    2010-01-01

    Roč. 184, č. 4 (2010), s. 2124-2129. ISSN 0022-1767 R&D Projects: GA AV ČR KAN200520804; GA MŠk 1M0506; GA ČR GD310/08/H077 Institutional research plan: CEZ:AV0Z50520514 Keywords : Limbal stem cells * immunoregulation * apoptosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.745, year: 2010

  8. Isolation, culture and characterization of primary mouse RPE cells.

    Science.gov (United States)

    Fernandez-Godino, Rosario; Garland, Donita L; Pierce, Eric A

    2016-07-01

    Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1-2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD). PMID:27281648

  9. Immunosuppressive and carcinogenic effect of UV light; Apoptosis and lysis of mouse spleen cells

    International Nuclear Information System (INIS)

    Harmful effects of UV light involves cell killing, mutagenesis and neoplastic transformation of exposed cells. The effect of UV light depends on the wavelength, dose irradiation, type of exposed cells, endogeny substance, experimental condition that can increase or decrease sensibility of the cells on the UV light. The loss of viability is the end point of cellular dysfunction, however, UV irradiation may affect cells function before it is perish. We examined the killing modes of cell death, necrosis or apoptosis, depending on the way of the cellular elements involved in process. Necrosis is a pothologic response involving a dramatic increase in cell volume that ultimately leads to cell lysis, usually caused by damaging of cell membrane. Apoptosis or genetically programmed cell death is result of complex cell mechanisms. Mouse spleen cells were diluted to 8.5·106/mL in Hanks' solution. Cells spread in thin layers (2mm) in glass Petri dishes were exposed to the different doses of UVC light. After irradiation and staining with fluorescein diacetate (FDA) and propidium iodide (PI) solution, the viable cells, as well as apoptosis were observed under fluorescent microscope. Immediately after UV irradiation the number of viable cell rapidly decreased. Lower doses of UVC light caused apoptosis as well as necrosis of exposed cells. Around 10% of spleen cells commit suicide after UVC exposure. There was significantly higher percentage of apoptotic cell than in control non irradiated sample. The highest fluence (1280J/m2) of UVC light, lysed considerable number of lymphocytes, around 80%. Small fraction (15%) of cells survived used UVC light. Noticed resistance of spleen cells is not completely clear, but results suggest that it due to the present of mature lymphocytes, which could not proliferate, and are less sensitive to UV light. In spite of this our results confirm that UVC light is capable to cause DNA damage, apoptosis and necrosis of mouse spleen cells. (author)

  10. Connexins protect mouse pancreatic β cells against apoptosis.

    Science.gov (United States)

    Klee, Philippe; Allagnat, Florent; Pontes, Helena; Cederroth, Manon; Charollais, Anne; Caille, Dorothée; Britan, Aurore; Haefliger, Jacques-Antoine; Meda, Paolo

    2011-12-01

    Type 1 diabetes develops when most insulin-producing β cells of the pancreas are killed by an autoimmune attack. The in vivo conditions modulating the sensitivity and resistance of β cells to this attack remain largely obscure. Here, we show that connexin 36 (Cx36), a trans-membrane protein that forms gap junctions between β cells in the pancreatic islets, protects mouse β cells against both cytotoxic drugs and cytokines that prevail in the islet environment at the onset of type 1 diabetes. We documented that this protection was at least partially dependent on intercellular communication, which Cx36 and other types of connexin channels establish within pancreatic islets. We further found that proinflammatory cytokines decreased expression of Cx36 and that experimental reduction or augmentation of Cx36 levels increased or decreased β cell apoptosis, respectively. Thus, we conclude that Cx36 is central to β cell protection from toxic insults. PMID:22056383

  11. Transplanting defrozen mouse bone marrow cells

    International Nuclear Information System (INIS)

    The regeneration was studied of blood formation in the spleen and the bone marrow of lethally irradiated mice 30 and 60 days after the transplantation of defrozen bone marrow. Also studied were the counts of leukocytes, thrombocytes and reticulocytes in the peripheral blood. Hematopoiesis changes were described and it was shown that after the transplantation of defrozen bone marrow, regeneration and progressive normalization of hematopoiesis took place in the lethally irradiated recipients. It was found that the freezing procedure used was tender and preserved the proliferation capacity of the stem hemopoietic cells. (author)

  12. Types of Parvalbumin-Containing Retinotectal Ganglion Cells in Mouse

    International Nuclear Information System (INIS)

    The calcium-binding protein parvalbumin (PV) occurs in the retinal ganglion cells (RGCs) of various vertebrate species. In the present study, we aimed to identify the types of PV-containing RGCs that project to the superior colliculus (SC) in the mouse. We injected retrograde tracer dextran into the mouse SC to label RGCs. PV-containing RGCs were first identified by immunocytochemistry and then neurons double-labeled with dextran and PV were iontophoretically injected with a lipophilic dye, DiI. Subsequently, confocal microscopy was used to characterize the morphologic classification of the PV-immunoreactive (IR) retinotectal ganglion cells on the basis of dendritic field size, branching pattern, and stratification within the inner plexiform layer. Among the 8 different types of PV-containing RGCs in the mouse retina, we found all 8 types of RGCs projecting to the SC. The RGCs were heterogeneous in morphology. The combined approach of using tracer injection and a single cell injection after immunocytochemistry on a particular protein will provide valuable data to further understand the functional features of the RGCs which constitute the retinotectal pathway

  13. The Inhibitory Effects of Mouse ICOS-Ig Gene-Modified Mouse Dendritic Cells on T Cells

    Institute of Scientific and Technical Information of China (English)

    GuohuaWang; LijuanZhu; PingHu; HuifenZhu; PingLei; WenjunLiao; BingYu; FeiliGong; GuanxinShen

    2004-01-01

    The main approach to reduce graft rejection has been focused on the development of immunosuppressive agents at present. Although these strategies have reportedly reduced graft rejection, there has been a reciprocal increase in more severe immunosuppression and lethal infections, as well as severe side effects. Blockade of costimulatory T cell response has been proved as one of useful strategies to reduce graft rejection. Furthermore, it has been shown that infusion of dendritic cells (DCs) with a potent negative regulatory ability for T cells could prolong allograft survival. In this study mouse DCs (mDCs) were transfected with the recombinant plasmid pcDNA3.0 containing mouse inducible costimulator-Ig (mICOS-Ig) cDNA by electroporation. The transient expression of mICOS-Ig in mDC could be detected by ELISA and SDS-PAGE. Mouse ICOS~Ig fusion protein expressed in mDC and mICOS-Ig gene-modified mDC could inhibit lymphocyte proliferation in mixed lymphocyte culture (MLC) in vitro. Furthermore, mICOS-Ig gene-modified mDC could inhibit lymphocyte proliferation in recipient mice. These results suggested that mICOS-Ig gene-modified mDC exerted inhibitory effects on T cells, and might be suitable for treatment or prevention of graft rejection and immunopathologic diseases. Cellular & Molecular Immunology. 2004;1(2):153-157.

  14. The Inhibitory Effects of Mouse ICOS-Ig Gene-Modified Mouse Dendritic Cells on T Cells

    Institute of Scientific and Technical Information of China (English)

    Guohua Wang; Lijuan Zhu; Ping Hu; Huifen Zhu; Ping Lei; Wenjun Liao; Bing Yu; Feili Gong; Guanxin Shen

    2004-01-01

    The main approach to reduce graft rejection has been focused on the development of immunosuppressive agents at present. Although these strategies have reportedly reduced graft rejection, there has been a reciprocal increase in more severe immunosuppression and lethal infections, as well as severe side effects. Blockade of costimulatory T cell response has been proved as one of useful strategies to reduce graft rejection. Furthermore,it has been shown that infusion of dendritic cells (DCs) with a potent negative regulatory ability for T cells could prolong allograft survival. In this study mouse DCs (mDCs) were transfected with the recombinant plasmid pcDNA3.0 containing mouse inducible costimulator-Ig (mICOS-Ig) cDNA by electroporation. The transient expression of mICOS-Ig in mDC could be detected by ELISA and SDS-PAGE. Mouse ICOS-Ig fusion protein expressed in mDC and mICOS-Ig gene-modified mDC could inhibit lymphocyte proliferation in mixed lymphocyte culture (MLC) in vitro. Furthermore, mICOS-Ig gene-modified mDC could inhibit lymphocyte proliferation in recipient mice. These results suggested that mICOS-Ig gene-modified mDC exerted inhibitory effects on T cells, and might be suitable for treatment or prevention of graft rejection and immunopathologicdiseases.

  15. Transfection of mouse ribosomal DNA into rat cells: faithful transcription and processing.

    OpenAIRE

    Vance, V B; Thompson, E A; Bowman, L H

    1985-01-01

    Truncated mouse ribosomal DNA (rDNA) genes were stably incorporated into rat HTC-5 cells by DNA-mediated cell transfection techniques. The mouse rDNA genes were accurately transcribed in these rat cells indicating that there is no absolute species specificity of rDNA transcription between mouse and rat. No more than 170 nucleotides of the 5' nontranscribed spacer was required for the accurate initiation of mouse rDNA transcription in rat cells. Further, the mouse transcripts were accurately c...

  16. Mouse embryonic retina delivers information controlling cortical neurogenesis.

    Directory of Open Access Journals (Sweden)

    Ciro Bonetti

    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.

  17. Automatic Stem Cell Detection in Microscopic Whole Mouse Cryo-Imaging.

    Science.gov (United States)

    Wuttisarnwattana, Patiwet; Gargesha, Madhusudhana; Van't Hof, Wouter; Cooke, Kenneth R; Wilson, David L

    2016-03-01

    With its single cell sensitivity over volumes as large as or larger than a mouse, cryo-imaging enables imaging of stem cell biodistribution, homing, engraftment, and molecular mechanisms. We developed and evaluated a highly automated software tool to detect fluorescently labeled stem cells within very large ( ∼ 200 GB) cryo-imaging datasets. Cell detection steps are: preprocess, remove immaterial regions, spatially filter to create features, identify candidate pixels, classify pixels using bagging decision trees, segment cell patches, and perform 3D labeling. There are options for analysis and visualization. To train the classifier, we created synthetic images by placing realistic digital cell models onto cryo-images of control mice devoid of cells. Very good cell detection results were (precision=98.49%, recall=99.97%) for synthetic cryo-images, (precision=97.81%, recall=97.71%) for manually evaluated, actual cryo-images, and false positives in control mice. An α-multiplier applied to features allows one to correct for experimental variations in cell brightness due to labeling. On dim cells (37% of standard brightness), with correction, we improved recall (49.26%→ 99.36%) without a significant drop in precision (99.99%→ 99.75%) . With tail vein injection, multipotent adult progenitor cells in a graft-versus-host-disease model in the first days post injection were predominantly found in lung, liver, spleen, and bone marrow. Distribution was not simply related to blood flow. The lung contained clusters of cells while other tissues contained single cells. Our methods provided stem cell distribution anywhere in mouse with single cell sensitivity. Methods should provide a rational means of evaluating dosing, delivery methods, cell enhancements, and mechanisms for therapeutic cells. PMID:26552080

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

    Energy Technology Data Exchange (ETDEWEB)

    L, Thi Thanh Hoa; Tae, Seong Ho; Min, Jung Joon [Chonnam National University Medical School, Gwangju (Korea, Republic of)

    2007-07-01

    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.

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

    International Nuclear Information System (INIS)

    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

  20. Measuring microRNA expression in mouse hematopoietic stem cells.

    Science.gov (United States)

    Hu, Wenhuo; Park, Christopher Y

    2014-01-01

    MicroRNAs (miRNAs) are important regulators of diverse biologic processes. In the hematopoietic system, miRNAs have been shown to regulate lineage fate decisions, mature immune effector cell function, apoptosis, and cell cycling, and a more limited number of miRNAs has been shown to regulate hematopoietic stem cell (HSC) self-renewal. Many of these miRNAs were initially identified as candidate regulators of HSC function by comparing miRNA expression in hematopoietic stem and progenitors cells (HSPCs) to their mature progeny. While the measurement of miRNA expression in rare cell populations such as HSCs poses practical challenges due to the low amount of RNA present, a number of techniques have been developed to measure miRNAs in small numbers of cells. Here, we describe our protocol for measuring miRNAs in purified mouse HSCs using a highly sensitive real-time quantitative PCR strategy that utilizes microfluidic array cards containing pre-spotted TaqMan probes that allows the detection of mature miRNAs in small reaction volumes. We also describe a simple data analysis method to evaluate miRNA expression profiling data using an open-source software package (HTqPCR) using mouse HSC miRNA profiling data generated in our lab. PMID:25062625

  1. caBIG® Spotlight - Solving Research Problems: Analyze Mouse Embryonic Stem Cell Transcriptional Profiles —

    Science.gov (United States)

    Read a case study to learn more about how Dr. Bradley Merrill of the University of Illinois at Chicago and his lab were able to perform their first gene expression array experiment comparing a mutant mouse embryonic stem cell line to a non-mutant control line using GenePattern, an application supported by the Molecular Analysis Tools Knowledge Center which provides bioinformatics tools for gene expression, proteomic and SNP analysis.

  2. Endoderm lineage labelling using BAC reporter constructs in ES cells and mouse embryos

    OpenAIRE

    Imhof, Sascha

    2009-01-01

    The endoderm is one of the three embryonic germ layers that gives rise to inner organs like lung, liver and pancreas. BAC recombineering was used to generate transgenic embryonic stem cells and mouse lines containing fluorescent reporters under the control of important endodermal genes like Foxa2, Sox17, Hhex, Nkx2.1 and Pdx1. This allowed the analysis of the specific expression patterns of those genes during endoderm and organ formation. In particular, the analysis of the Hhex reporter expre...

  3. Chlorambucil effectively induces deletion mutations in mouse germ cells.

    OpenAIRE

    Russell, L B; Hunsicker, P R; Cacheiro, N L; Bangham, J W; Russell, W. L.; Shelby, M D

    1989-01-01

    The chemotherapeutic agent chlorambucil was found to be more effective than x-rays or any chemical investigated to date in inducing high yields of mouse germ-line mutations that appear to be deletions or other structural changes. Induction of mutations involving seven specific loci was studied after exposures of various male germ-cell stages to chlorambucil at 10-25 mg/kg. A total of 60,750 offspring was scored. Mutation rates in spermatogonial stem cells were not significantly increased over...

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

    International Nuclear Information System (INIS)

    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 G1 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

  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. Assisting People with Multiple Disabilities and Minimal Motor Behavior to Control Environmental Stimulation through a Mouse Wheel

    Science.gov (United States)

    Shih, Ching-Hsiang; Shih, Ching-Tien; Lin, Kun-Tsan; Chiang, Ming-Shan

    2009-01-01

    This study assessed whether two people with profound multiple disabilities and minimal motor behavior would be able to control environmental stimulation using thumb poke ability with a mouse wheel and a newly developed mouse driver (i.e., a new mouse driver replacing standard mouse driver, and turning a mouse into a precise thumb poke detector).…

  7. The Effect of Cordycepin on Steroidogenesis and Apoptosis in MA-10 Mouse Leydig Tumor Cells

    OpenAIRE

    Bo-Syong Pan; Chun-Yu Lin; Bu-Miin Huang

    2011-01-01

    Cordycepin is a natural pure compound extracted from Cordyceps sinensis (CS). We have demonstrated that CS stimulates steroidogenesis in primary mouse Leydig cell and activates apoptosis in MA-10 mouse Leydig tumor cells. It is highly possible that cordycepin is the main component in CS modulating Leydig cell functions. Thus, our aim was to investigate the steroidogenic and apoptotic effects with potential mechanism of cordycepin on MA-10 mouse Leydig tumor cells. Results showed that cordycep...

  8. Control of Fuel Cells

    OpenAIRE

    ZENITH, Federico

    2007-01-01

    This thesis deals with control of fuel cells, focusing on high-temperature proton-exchange-membrane fuel cells. Fuel cells are devices that convert the chemical energy of hydrogen, methanol or other chemical compounds directly into electricity, without combustion or thermal cycles. They are efficient, scalable and silent devices that can provide power to a wide variety of utilities, from portable electronics to vehicles, to nation-wide electric grids. Whereas studies about the design of fuel ...

  9. Control of Fuel Cells

    OpenAIRE

    ZENITH, Federico

    2007-01-01

    This thesis deals with control of fuel cells, focusing on high-temperature proton-exchange-membrane fuel cells.Fuel cells are devices that convert the chemical energy of hydrogen, methanol or other chemical compounds directly into electricity, without combustion or thermal cycles. They are efficient, scalable and silent devices that can provide power to a wide variety of utilities, from portable electronics to vehicles, to nation-wide electric grids.Whereas studies about the design of fuel ce...

  10. Radioprotection of mouse CNS endothelial cells in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lyubimova, N.; Coultas, P.; Martin, R. [Peter McCallum Cancer Institute, Melbourne, VIC (Australia)

    1996-12-31

    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 {gamma}-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

  11. Mast cells and gastrointestinal dysmotility in the cystic fibrosis mouse.

    Directory of Open Access Journals (Sweden)

    Robert C De Lisle

    Full Text Available BACKGROUND: Cystic fibrosis (CF has many effects on the gastrointestinal tract and a common problem in this disease is poor nutrition. In the CF mouse there is an innate immune response with a large influx of mast cells into the muscularis externa of the small intestine and gastrointestinal dysmotility. The aim of this study was to evaluate the potential role of mast cells in gastrointestinal dysmotility using the CF mouse (Cftr(tm1UNC, Cftr knockout. METHODOLOGY: Wild type (WT and CF mice were treated for 3 weeks with mast cell stabilizing drugs (ketotifen, cromolyn, doxantrazole or were treated acutely with a mast cell activator (compound 48/80. Gastrointestinal transit was measured using gavage of a fluorescent tracer. RESULTS: In CF mice gastric emptying at 20 min post-gavage did not differ from WT, but was significantly less than in WT at 90 min post-gavage. Gastric emptying was significantly increased in WT mice by doxantrazole, but none of the mast cell stabilizers had any significant effect on gastric emptying in CF mice. Mast cell activation significantly enhanced gastric emptying in WT mice but not in CF mice. Small intestinal transit was significantly less in CF mice as compared to WT. Of the mast cell stabilizers, only doxantrazole significantly affected small intestinal transit in WT mice and none had any effect in CF mice. Mast cell activation resulted in a small but significant increase in small intestinal transit in CF mice but not WT mice. CONCLUSIONS: The results indicate that mast cells are not involved in gastrointestinal dysmotility but their activation can stimulate small intestinal transit in cystic fibrosis.

  12. Serotonin competence of mouse beta cells during pregnancy.

    Science.gov (United States)

    Goyvaerts, Lotte; Schraenen, Anica; Schuit, Frans

    2016-07-01

    Pregnancy is a key mammalian reproductive event in which growth and differentiation of the fetus imposes extra metabolic and hormonal demands on the mother. Its successful outcome depends on major changes in maternal blood circulation, metabolism and endocrine function. One example is the endocrine pancreas, where beta cells undergo a number of changes in pregnancy that result in enhanced functional beta cell mass in order to compensate for the rising metabolic needs for maternal insulin. During the last 5 years, a series of studies have increased our understanding of the molecular events involved in this functional adaptation. In the mouse, a prominent functional change during pregnancy is the capacity of some beta cells to produce serotonin. In this review we will discuss the mechanism and potential effects of pregnancy-related serotonin production in beta cells, considering functional consequences at the local intra-islet and systemic level. PMID:27056372

  13. Regulation of apoptosis and cell cycle in irradiated mouse brain

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Won Yong; Song, Mi Hee; Hung, Eun Ji; Seong, Jin Sil; Suh, Chang Ok [College of Medicine, Yonsei Univ., Seoul (Korea, Republic of)

    2001-06-01

    To investigate the regulation of apoptosis and cell cycle in mouse brain irradiation. 8-week old male mice, C57B 1/6J were given whole body {gamma} -radiation with a single dose of 25 Gy using Cobalt 60 irradiator. At different times 1, 2, 4, 8 and 24hr after irradiation, mice were killed and brain tissues were collected. Apoptotic cells were scored by TUNEL assay. Expression of p53, Bcl-2, and Bax and cell cycle regulating molecules; cyclins BI, D1, E and cdk2, cdk4, p34{sup cdc2} were analysed by Western blotting. Cell cycle was analysed by flow cytometry. The peak of radiation induced apoptosis is shown at 8 hour after radiation. With a single 25 Gy irradiation, the peak of apoptotic index in C57B1/6J is 24.0{+-}0.25 (p<0.05) at 8 hour after radiation. Radiation upregulated the expression of p53/tubulin, Bax/tubulin, and Bcl-2/tubulin with 1.3, 1.1 and 1.45 fold increase, respectively were shown at the peak level at 8 hour after radiation. The levels of cell cycle regulating molecules after radiation are not changed significantly except cyclin D1 with 1.3 fold increase. Fractions of Go-G 1, G2-M and S phase in the cell cycle does not specific changes by time. In mouse brain tissue, radiation induced apoptosis is particularly shown in a specific area, subependyma. These results and lack of radiation induced changes in cell cycle offer better understanding of radiation response of normal brain tissue.

  14. Isolation and Enrichment of Mouse Female Germ Line Stem Cells

    Directory of Open Access Journals (Sweden)

    Somayeh Khosravi-Farsani

    2015-01-01

    Full Text Available Objective: The existence of female germ-line stem cells (FGSCs has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in enrichment of FGSCs and in establishment of the best procedure. Materials and Methods: In this experimental study, after digesting neonate ovary from C57Bl/6 mice, we performed 2 different isolation experiments: magnetic activated cell sorting (MACS and pre-plating. MACS was applied using two different antibodies against mouse vasa homolog (MVH and stage-specific embryonic antigen-1 (SSEA1 markers. After the cells were passaged and proliferated in vitro, colony-forming cells were characterized using reverse transcription-polymerase chain reaction (RT-PCR (for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 and Zp3, alkaline phosphatase (AP activity test and immunocytochemistry. Results: Data showed that colonies can be seen more frequently in pre-plating technique than that in MACS. Using the SSEA1 antibody with MACS, 1.98 ± 0.49% (Mean ± SDV positive cells were yield as compared to the total cells sorted. The colonies formed after pre-plating expressed pluripotency and germ stem cell markers (Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl whereas did not express Zp3 and Scp3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins, and AP activity measured by AP-kit showed positive reaction. Conclusion: We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries.

  15. Regulation of apoptosis and cell cycle in irradiated mouse brain

    International Nuclear Information System (INIS)

    To investigate the regulation of apoptosis and cell cycle in mouse brain irradiation. 8-week old male mice, C57B 1/6J were given whole body γ -radiation with a single dose of 25 Gy using Cobalt 60 irradiator. At different times 1, 2, 4, 8 and 24hr after irradiation, mice were killed and brain tissues were collected. Apoptotic cells were scored by TUNEL assay. Expression of p53, Bcl-2, and Bax and cell cycle regulating molecules; cyclins BI, D1, E and cdk2, cdk4, p34cdc2 were analysed by Western blotting. Cell cycle was analysed by flow cytometry. The peak of radiation induced apoptosis is shown at 8 hour after radiation. With a single 25 Gy irradiation, the peak of apoptotic index in C57B1/6J is 24.0±0.25 (p<0.05) at 8 hour after radiation. Radiation upregulated the expression of p53/tubulin, Bax/tubulin, and Bcl-2/tubulin with 1.3, 1.1 and 1.45 fold increase, respectively were shown at the peak level at 8 hour after radiation. The levels of cell cycle regulating molecules after radiation are not changed significantly except cyclin D1 with 1.3 fold increase. Fractions of Go-G 1, G2-M and S phase in the cell cycle does not specific changes by time. In mouse brain tissue, radiation induced apoptosis is particularly shown in a specific area, subependyma. These results and lack of radiation induced changes in cell cycle offer better understanding of radiation response of normal brain tissue

  16. 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. PMID:26428408

  17. 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

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

    International Nuclear Information System (INIS)

    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.

  19. Germline Competent Pluripotent Mouse Stem Cells Generated by Plasmid Vectors.

    Science.gov (United States)

    Chen, Chien-Hong; Su, Yu-Hsiu; Lee, Kun-Hsiung; Chuang, Chin-Kai

    2016-07-01

    We developed nonintegrated methods to reprogram mouse embryonic fibroblast (MEF) cells into induced pluripotent stem cells (iPSCs) using pig pOct4, pSox2, and pc-Myc as well as human hKLF4, hAID, and hTDG that were carried by plasmid vectors. The 4F method employed pOct4, pSox2, pc-Myc, and hKLF4 to derive iPSC clones with naive embryonic stem cell (ESC)-like morphology. These 4F clones expressed endogenous mouse Nanog protein and could generate chimeras. In addition to the four conventional reprogramming factors used in the 4F method, hAID and hTDG were utilized in a 6F method to increase the conversion efficiency of reprogramming by approximately five-fold. One of the 6F plasmid derived iPSC (piPSC) clones was shown to be germline transmission competent. PMID:26980563

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Naloxone acts as a potent analgesic in transgenic mouse models of sickle cell anemia

    OpenAIRE

    Lunzer, Mary M.; Yekkirala, Ajay; Hebbel, Robert P.; Portoghese, Philip S.

    2007-01-01

    Sickle cell anemia is a common genetic disorder in African Americans. Opioid analgesics are traditionally the treatment for the severe pain associated with this disease. Here we reveal that the opioid antagonist naloxone possesses potent analgesic activity in two transgenic mouse models of sickle cell anemia (NY1DD and hBERK1) and not in their respective controls (ICR-CD1 and C57BL/6J) when administered by three parenteral routes [intracerebroventricular (i.c.v.), intrathecal, and subcutaneou...

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

    OpenAIRE

    Ching-Gong Lin; Mon-Han Wu; Te-Sheng Chang; Sorgan Shou-Ku Tai

    2009-01-01

    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 ...

  3. Identification of cells in primate bone marrow resembling the hemopoietic stem cell in the mouse

    NARCIS (Netherlands)

    Dicke, K.A.; Noord, M.J. van; Maat, B.

    1973-01-01

    The colony forming unit culture (CFU C) in the thin layer agar colony technique is considered to be representative for hemopoietic stem cells (HSC), according to studies in mouse and monkey bone marrow. Using this in vitro assay as a guide, stem cell concentrates were prepared from monkey and human

  4. Isolation and Characterization of Node/Notochord-like Cells from Mouse Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Winzi, Maria Karin; Maddox-Hyttel, Poul; Dale, J 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....../notochord-like cells in vitro will aid the biochemical characterization of these developmentally important structures....

  5. Generation and characterisation of Friedreich ataxia YG8R mouse fibroblast and neural stem cell models.

    Directory of Open Access Journals (Sweden)

    Chiranjeevi Sandi

    Full Text Available BACKGROUND: Friedreich ataxia (FRDA is an autosomal recessive neurodegenerative disease caused by GAA repeat expansion in the first intron of the FXN gene, which encodes frataxin, an essential mitochondrial protein. To further characterise the molecular abnormalities associated with FRDA pathogenesis and to hasten drug screening, the development and use of animal and cellular models is considered essential. Studies of lower organisms have already contributed to understanding FRDA disease pathology, but mammalian cells are more related to FRDA patient cells in physiological terms. METHODOLOGY/PRINCIPAL FINDINGS: We have generated fibroblast cells and neural stem cells (NSCs from control Y47R mice (9 GAA repeats and GAA repeat expansion YG8R mice (190+120 GAA repeats. We then differentiated the NSCs in to neurons, oligodendrocytes and astrocytes as confirmed by immunocytochemical analysis of cell specific markers. The three YG8R mouse cell types (fibroblasts, NSCs and differentiated NSCs exhibit GAA repeat stability, together with reduced expression of frataxin and reduced aconitase activity compared to control Y47R cells. Furthermore, YG8R cells also show increased sensitivity to oxidative stress and downregulation of Pgc-1α and antioxidant gene expression levels, especially Sod2. We also analysed various DNA mismatch repair (MMR gene expression levels and found that YG8R cells displayed significant reduction in expression of several MMR genes, which may contribute to the GAA repeat stability. CONCLUSIONS/SIGNIFICANCE: We describe the first fibroblast and NSC models from YG8R FRDA mice and we confirm that the NSCs can be differentiated into neurons and glia. These novel FRDA mouse cell models, which exhibit a FRDA-like cellular and molecular phenotype, will be valuable resources to further study FRDA molecular pathogenesis. They will also provide very useful tools for preclinical testing of frataxin-increasing compounds for FRDA drug therapy

  6. Genome-wide profiling to analyze the effects of FXR activation on mouse renal proximal tubular cells

    OpenAIRE

    Gui, Ting; Gai, Zhibo

    2015-01-01

    To assess the effect of farnesoid X receptor (FXR), a bile acid nuclear receptor, on renal proximal tubular cells, primary cultured mouse kidney proximal tubular cells were treated with GW4064 (a FXR agonist) or DMSO (as controls) overnight. Analysis of gene expression in the proximal tubular cells by whole genome microarrays indicated that FXR activation induced genes involved in fatty acid degradation and oxidation reduction. Among them, genes involved in glutathione metabolism were mostly ...

  7. Emotional change-associated T cell mobilization at the early stage of a mouse model of multiple sclerosis

    OpenAIRE

    Giuseppa ePiras; Lorenza eRattazzi; Adam eMcDermott; Robert eDeacon; Fulvio eD'acquisto

    2013-01-01

    Autoimmune diseases like multiple sclerosis are known to be associated with debilitating emotional disorders that manifest long before the flaring of motor dysfunctions. Given the emerging role of T cells in controlling both emotions and autoimmunity, in this study we explored possible correlation between T cell activation and changes in emotional behavior in a mouse model of multiple sclerosis. Our results showed a significant increase in blood circulating T cells as soon as at day 4 post-im...

  8. Emotional Change-Associated T Cell Mobilization at the Early Stage of a Mouse Model of Multiple Sclerosis

    OpenAIRE

    Piras, Giuseppa; Rattazzi, Lorenza; McDermott, Adam; Deacon, Robert; D’Acquisto, Fulvio

    2013-01-01

    Autoimmune diseases like multiple sclerosis (MS) are known to be associated with debilitating emotional disorders that manifest long before the flaring of motor dysfunctions. Given the emerging role of T cells in controlling both emotions and autoimmunity, in this study we explored possible correlation between T cell activation and changes in emotional behavior in a mouse model of MS. Our results showed a significant increase in blood circulating T cells as soon as at day 4 post immunization....

  9. Enhanced survival in vitro of human corneal endothelial cells using mouse embryonic stem cell conditioned medium

    Science.gov (United States)

    Lu, Xiaoyan; Chen, Dong; Liu, Zhiping; Li, Chaoyang; Liu, Ying; Zhou, Jin; Wan, Pengxia; Mou, Yong-gao

    2010-01-01

    Purpose To determine whether mouse embryonic stem cell conditioned medium (ESC-CM) increases the proliferative capacity of human corneal endothelial cells (HCECs) in vitro. Methods Primary cultures of HCECs were established from explants of the endothelial cell layer, including the Descemet’s membrane. Cells were cultured in human corneal endothelium medium (CEM) containing 25% ESC-CM for the experimental group and CEM alone for the control group. Phase-contrast microscopy and reverse-transcription polymerase chain reaction (RT–PCR) were used to identify HCECs. The eruption time and HCEC morphology were observed under phase-contrast microscopy. We detected the protein expression of zona occludens protein-1 (ZO-1; a tight junction protein) and the Na+-K+-ATPase by western blot analysis and immunocytochemistry. The mRNA expression of the Na+-K+-ATPase, voltage-dependent anion channel 3 (VDAC3), solute carrier family 4, sodium bicarbonate cotransporter member 4 (SLC4A4), and chloride channel protein 3 (CLCN3) were detected by RT–PCR. To explore the proliferation capacity of HCECs, the colony forming efficiency (CFE) was determined by Giemsa staining and the cellular proliferation marker of Ki-67 protein (Ki-67) positive cells were detected by immunocytochemistry and flow cytometry. Progression of the cell cycle and apoptosis were analyzed by flow cytometry. Negative regulation of the cell cycle, as measured by cyclin-dependent kinase inhibitor p21 (p21) levels, was detected by western blot analysis and immunocytochemistry. Results In primary culture, HCECs in the 25%ESC-CM group erupted with polygonal appearance on day 2, while those in the CEM group erupted with slightly larger cells on day 3–4. HCECs in the 25%ESC-CM group could be subcultured until passage 6 without enlargement of cell volume, while those in the CEM group were enlarged and lost their polygonal appearance by passage 2. HCECs in both the 25%ESC-CM and CEM groups expressed ZO-1, Na

  10. Autonomous replicating sequences from mouse cells which can replicate in mouse cells in vivo and in vitro.

    OpenAIRE

    Ariga, H; Itani, T; Iguchi-Ariga, S M

    1987-01-01

    We have already reported that the cloned mouse DNA fragment (pMU65) could replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro (H. Ariga, Z. Tsuchihashi, M. Naruto, and M. Yamada, Mol. Cell. Biol. 5:563-568, 1985). The plasmid p65-tk, containing the thymidine kinase (tk) gene of herpes simplex virus and the BglII-EcoRI region of pMU65 homologous to the simian virus 40 origin of DNA replication, was constructed. The p65-tk persisted episomally in tk+ transformants aft...

  11. 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.

  12. Differentiation of mouse erythroleukemia cells is blocked by late up-regulation of a c-myb transgene.

    OpenAIRE

    McClinton, D; Stafford, J; Brents, L; Bender, T. P.; Kuehl, W M

    1990-01-01

    During chemically induced differentiation of Friend virus-infected mouse erythroleukemia (MEL) cell lines, there is a biphasic down-regulation of the c-myb proto-oncogene. A plasmid containing a murine c-myb cDNA controlled by a mouse metallothionein I promoter was transfected into the C19 MEL cell line. For six transfected clones, it was found that expression of the exogenous c-myb mRNA could be up-regulated by the addition of 120 microM ZnCl2 and that the N,N'-hexamethylenebisacetamide-indu...

  13. Comparison of the rat and mouse cell lines commercially available for CALUX bioassays

    Energy Technology Data Exchange (ETDEWEB)

    Goeyens, L.; Windal, I.; Wouwe, N. van [Institute of Public Health, Brussels (Belgium); Scippo, M.L.; Eppe, G.; Pauw, E. de; Maghin-Rogister, G. [Liege Univ. (Belgium)

    2004-09-15

    CALUX (Chemical-activated luciferase gene expression) is nowadays more and more widely used, both for the control of the norms applied to the food chain and to environmental contamination evaluation. In that purpose, two cell lines are commercially available: one rat cell line, commercialized by Bio Detection System (BDS, The Netherlands) and one mouse cell line, commercialized by Xenobiotic Detection System (XDS, USA). Both suppliers propose different clean-up methods and a slightly different method in the preparation, dosage and reading of the plates. Until now, almost no comparison of the cell lines has been performed, or the comparison includes many variables (extraction, purification, method of preparation, dosage and reading of the plate) so that it is difficult to evaluate which variables are mainly responsible of the observed differences. The objective of the research presented here is to perform a direct comparison of the 2 cell lines, and evaluate which variables can be responsible of the discrepancy observed between the results.

  14. Response of maternal immune cells of irradiation of mouse embryos

    International Nuclear Information System (INIS)

    This work began as an attempt to explain the paradox of pregnancy - the survival and growth of the semi-allogenic embryo in an immunologically hostile environment. In 1982 and 1983 we reported the tracing of quinacrine labelled maternal leukocytes (WBC) in maternal, placental and embryonic mouse tissues by fluorescence microscopy. We found that cells in the placenta phagocytose labelled WBC, so that after 1-2 hours the labelled nuclear DNA is found as brightly fluorescing particles in the cytoplasm of the phagocytes with no evidence of it in the nuclei. Identical cells were observed in slide preparations of embryos which had been carefully separated from their placentas. We also found a small population of intact labelled lymphocytes, clearly maternal in origin, in the embryos. This seems to be another paradox - placental phagocytes are observed to be phagocytosing maternal WBC in the placenta and embryo, but there are also free maternal cells in the placenta and embryo. A theoretical explanation is that maternal lymphocytes alloreactive against the embryo will attempt to react with placental cells and in the process be phagocytosed, while other maternal cells will be able to enter the embryo where they could have a surveillance function, removing dead or mutant embryonic cells. To test this theory a series of experiments were carried out and are reported

  15. 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.

  16. Effects of vitamin A on in vitro maturation of pre-pubertal mouse spermatogonial stem cells.

    Directory of Open Access Journals (Sweden)

    Albanne Travers

    Full Text Available Testicular tissue cryopreservation is the only potential option for fertility preservation in pre-pubertal boys exposed to gonadotoxic treatment. Completion of spermatogenesis after in vitro maturation is one of the future uses of harvested testicular tissue. The purpose of the current study was to evaluate the effects of vitamin A on in vitro maturation of fresh and frozen-thawed mouse pre-pubertal spermatogonial stem cells in an organ culture system. Pre-pubertal CD1 mouse fresh testes were cultured for 7 (D7, 9 (D9 and 11 (D11 days using an organ culture system. Basal medium was supplemented with different concentrations of retinol (Re or retinoic acid (RA alone or in combination. Seminiferous tubule morphology (tubule diameter, intra-tubular cell type, intra-tubular cell death and proliferation (PCNA antibody and testosterone level were assessed at D7, D9 and D11. Pre-pubertal mouse testicular tissue were frozen after a soaking temperature performed at -7 °C, -8 °C or -9 °C and after thawing, were cultured for 9 days, using the culture medium preserving the best fresh tissue functionality. Retinoic acid at 10(-6M and retinol at 3.3.10(-7M, as well as retinol 10(-6M are favourable for seminiferous tubule growth, maintenance of intra-tubular cell proliferation and germ cell differentiation of fresh pre-pubertal mouse spermatogonia. Structural and functional integrity of frozen-thawed testicular tissue appeared to be well-preserved after soaking temperature at -8 °C, after 9 days of organotypic culture using 10(-6M retinol. RA and Re can control in vitro germ cell proliferation and differentiation. Re at a concentration of 10(-6M maintains intra-tubular cell proliferation and the ability of spermatogonia to initiate spermatogenesis in fresh and frozen pre-pubertal mouse testicular tissue using a soaking temperature at -8 °C. Our data suggested a possible human application for in vitro maturation of cryopreserved pre-pubertal testicular

  17. Chondrogenic differentiation of mouse embryonic stem cells promoted by mature chondrocytes

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    In order to direct embryonic stem (ES) cells to differentiate into chondrocytes, a chondrogenic envi-ronment provided by mature chondrocytes was investigated. Flk-1 positive cells sorted from pre-differentiated mouse ES cells were mixed with adult porcine articular chondrocytes, seeded on biodegradable scaffolds, and then implanted subcutaneously into nude mice. The cell-scaffold com-plexes formed cartilage tissues after 4 weeks, which was demonstrated by histology and anti-type II collagen antibody staining. Positive staining of mouse Major Histocompatibility Complex class I molecules confirmed that part of the chondrocytes were derived from mouse ES cells. The current study established a new approach for directing ES cell differentiation.

  18. 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.

  19. Ginseng Berry Extract Promotes Maturation of Mouse Dendritic Cells.

    Directory of Open Access Journals (Sweden)

    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.

  20. Intraperitoneal Injection of Multiplacentas Pooled Cells Treatment on a Mouse Model with Aplastic Anemia

    OpenAIRE

    Jun Li; Hong Chen; Yan-Bo Lv; Qiang Wang; Zheng-Jun Xie; Li-Hua Ma; Jie He; Wei Xue; Shan Yu; Jun Guo; Ting-Hua Wang; Tian-Xi Wu; Xing-Hua Pan

    2016-01-01

    Coinfusion of hematopoietic and mesenchymal stem cells is more effective than hematopoietic stem cell transplantation alone. It is necessary to explore a safe and routine mixed stem cell intraperitoneal transplantation method. Multiplacentas pooled cells were intraperitoneally injected into a radiation- and immunity-induced mouse aplastic anemia model with single time. Then, mouse survival time, peripheral blood hemoglobin count, bone marrow architecture, and donor cell engraftment were asses...

  1. PANIC-ATTAC: A Mouse Model for Inducible and Reversible β-Cell Ablation

    OpenAIRE

    Wang, Zhao V.; Mu, James; Schraw, Todd D.; Gautron, Laurent; Elmquist, Joel K.; Zhang, Bei B.; Brownlee, Michael; Scherer, Philipp E

    2008-01-01

    OBJECTIVE—Islet transplantations have been performed clinically, but their practical applications are limited. An extensive effort has been made toward the identification of pancreatic β-cell stem cells that has yielded many insights to date, yet targeted reconstitution of β-cell mass remains elusive. Here, we present a mouse model for inducible and reversible ablation of pancreatic β-cells named the PANIC-ATTAC (pancreatic islet β-cell apoptosis through targeted activation of caspase 8) mous...

  2. Differential identification of ID sequence of DNA in cells of rats, mouse, and rat-mouse radiation chimeras

    International Nuclear Information System (INIS)

    It is shown that ID-sequence can be used for identification of donor cells of rat-mouse radiation chimeras. The fact of intensive binding of labelled probe with cytoplasmic cell components of neutrophilic series of differentiation is noted. Trace were observed at that both for rats and radiation chimeras, and some times-for mice. The reasons of observed phenomenon are interesting from the viewpoint of working out techniques and characterizing functional peculiarities of cells of different lines and differentiation stages

  3. ATRX dysfunction induces replication defects in primary mouse cells.

    Directory of Open Access Journals (Sweden)

    David Clynes

    Full Text Available The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells.

  4. The action of ultraviolet radiation on the cell proliferation of two-cell mouse embryos

    International Nuclear Information System (INIS)

    The two-cell mouse embryo has a unique cell cycle of a short DNA synthesis (S) phase and an extremely long post-DNA synthesis (G2) phase. An attempt was made to investigate the radiation biology of the long G2 phase using UV radiation as a probe. Two cell mouse embryos, at various positions in the cell cycle, were UV-irradiated in phosphate-buffered saline. The embryos were cultured for a few hours to 3 days to assay for their cell proliferative characteristics. The embryos were most sensitive to the killing action of UV radiation in the late G2 phase. The embryos divided more than two times after low UV fluences before dying and experienced G2 phase delays. These results can be contrasted to the situation in somatic cells, in which the action of UV radiation is S phase selective. One possibility is that the target for the action of UV radiation is different in two-cell mouse embryos from that in somatic cells and that the target is similar to that for X-ray effects. (author)

  5. Specialized mouse embryonic stem cells for studying vascular development

    Directory of Open Access Journals (Sweden)

    Glaser DE

    2014-10-01

    Full Text Available Drew E Glaser,1 Andrew B Burns,2 Rachel Hatano,2 Magdalena Medrzycki,3 Yuhong Fan,3 Kara E McCloskey1 1School of Engineering, University of California, Merced, CA, USA; 2School of Natural Sciences, University of California, Merced, CA, USA; 3School of Biology and the Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, USAAbstract: 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

  6. Regeneration of tracheal epithelium using mouse induced pluripotent stem cells.

    Science.gov (United States)

    Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Yoshie, Susumu; Otsuki, Koshi; Miyake, Masao; Hazama, Akihiro; Wada, Ikuo; Omori, Koichi

    2016-04-01

    Conclusion The findings demonstrated the potential use of induced pluripotent stem cells for regeneration of tracheal epithelium. Objective Autologous tissue implantation techniques using skin or cartilage are often applied in cases of tracheal defects with laryngeal inflammatory lesions and malignant tumor invasion. However, these techniques are invasive with an unstable clinical outcome. The purpose of this study was to investigate regeneration in a tracheal defect site of nude rats after implantation of ciliated epithelium that was differentiated from induced pluripotent stem cells. Method Embryoid bodies were formed from mouse induced pluripotent stem cells. They were cultured with growth factors for 5 days, and then cultured at the air-liquid interface. The degree of differentiation achieved prior to implantation was determined by histological findings and the results of real-time polymerase chain reaction. Embryoid bodies including ciliated epithelium were embedded into collagen gel that served as an artificial scaffold, and then implanted into nude rats, creating an 'air-liquid interface model'. Histological evaluation was performed 7 days after implantation. Results The ciliated epithelial structure survived on the lumen side of regenerated tissue. It was demonstrated histologically that the structure was composed of ciliated epithelial cells. PMID:26755348

  7. Radioprotection of mouse CNS endothelial cells in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Lyubimova, N.; Coultas, P.; Martin, R. [Peter MacCallum Cancer institute, Melbourne, Victoria (Australia)

    1997-03-01

    After treatments with monoamine oxidase inhibitors and L-DOPA, the blood brain barrier causes a build-up of dopamine in brain capillary endothelial cells. Conversion of the dopamine to a fluorophore provides a marker which can be used to measure endothelial cell density by fluorescence microscopy. Earlier studies used this technique to monitor post-irradiation changes in endothelial cell density in rodent brain and showed loss within 24 hours of a sub-population of about 15 % of the endothelial cells. As a first step, rather than use the later endpoints of radionecrosis it was decided to examine directly whether Hoechst 33342 could protect against this rapid initial endothelial cell loss. Ten minutes after intravenous injection of Joechst 33342, in mouse brain the ligand was confined to endothelial cells and, for irradiation at this time, there was protection against endothelial cell loss over the first 24 hours after after exposure. Ablation of the sensitive subpopulation in unprotected mice took place over a dose range of 1 to 3 Gy {gamma}-rays but doses between 12 to 20 Gy were required in the presence of the ligand. This protection equated to a high dose modification factor of approximately 7 and may reflect suppression of apoptosis in this 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 these results suggest a potential use of DNA-binding radioprotectors with limited penetration in investigations of the relative significance of endothelial and parenchymal damage in normal tissue responses to ionising radiation. (authors)

  8. Radioprotection of mouse CNS endothelial cells in vivo

    International Nuclear Information System (INIS)

    After treatments with monoamine oxidase inhibitors and L-DOPA, the blood brain barrier causes a build-up of dopamine in brain capillary endothelial cells. Conversion of the dopamine to a fluorophore provides a marker which can be used to measure endothelial cell density by fluorescence microscopy. Earlier studies used this technique to monitor post-irradiation changes in endothelial cell density in rodent brain and showed loss within 24 hours of a sub-population of about 15 % of the endothelial cells. As a first step, rather than use the later endpoints of radionecrosis it was decided to examine directly whether Hoechst 33342 could protect against this rapid initial endothelial cell loss. Ten minutes after intravenous injection of Joechst 33342, in mouse brain the ligand was confined to endothelial cells and, for irradiation at this time, there was protection against endothelial cell loss over the first 24 hours after after exposure. Ablation of the sensitive subpopulation in unprotected mice took place over a dose range of 1 to 3 Gy γ-rays but doses between 12 to 20 Gy were required in the presence of the ligand. This protection equated to a high dose modification factor of approximately 7 and may reflect suppression of apoptosis in this 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 these results suggest a potential use of DNA-binding radioprotectors with limited penetration in investigations of the relative significance of endothelial and parenchymal damage in normal tissue responses to ionising radiation. (authors)

  9. Similarity on neural stem cells and brain tumor stem cells in transgenic brain tumor mouse models

    OpenAIRE

    Qiao, Guanqun; Li, Qingquan; Peng, Gang; Ma, Jun; Fan, Hongwei; Li, Yingbin

    2013-01-01

    Although it is believed that glioma is derived from brain tumor stem cells, the source and molecular signal pathways of these cells are still unclear. In this study, we used stable doxycycline-inducible transgenic mouse brain tumor models (c-myc+/SV40Tag+/Tet-on+) to explore the malignant trans-formation potential of neural stem cells by observing the differences of neural stem cells and brain tumor stem cells in the tumor models. Results showed that chromosome instability occurred in brain t...

  10. Sel Kumulus Sebagai Feeder Layer pada Kultur Stem Cells Embrionic Mencit (CUMULUS CELLS AS FEEDER LAYER IN CULTURE OF MOUSE EMBRYONIC STEM CELLS)

    OpenAIRE

    Thomas Mata Hine; Arief Boediono; Iman Supriatna; Dondin Sajuthi

    2014-01-01

    The purpose of this study was to verify the effectiveness of cumulus cells as a feeder layer in supportingthe growth of mouse embryonic stem cells. Embryos at blastocyst stage were exposed in pronase solution,and then incubated in rabbit anti-mouse antibody and guinea pig complement to lyse and separate thetrophoblast cells from the inner cell mass. Inner cell mass subsequently cultured in a petri dish containinga cumulus feeder layer, mouse embryonic fibroblasts, or without a feeder layer, i...

  11. Impact of let-7g on Proliferation and Lactation of Mouse Mammary Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Feng Li; Li Qing-zhang; Cui Wei; Ding Wei

    2012-01-01

    let-7g, a member of the let-7 family, regulates gene expression at the post-transcriptional level. The study explored a series of biological effects of mouse mammary epithelial cells that let-7g was produced. The differential expression of let-7g was detected by qRT-PCR in different developmental stages of the mouse mammary gland, let-7g expression and impact of let-7g on mouse mammary epithelial cells were analyzed by CASY-technology, qRT-PCR, Western blotting and HPLC inhibited let-7g expression of mouse mammary epithelial ceils through gene silencing. The results showed that qRT-PCR identified let-7g as being down-regulated in mouse mammary epithelial cells after it was inhibited. Mouse mammary epithelial cells with low expression of let-7g displayed higher expression of TGFβR I protein than those with high expression of let-7g, suggesting that low let-7g expression contributed to TGFβR I over-expression. Finally, the expression of let-7g was down-regulated, which significantly enhanced the proliferation of mouse mammary epithelial cells, and increased expression of β-Casein. The data indicated that let-7g could negatively regulate the expression of target Tgfbrl by complementary combination in mouse mammary epithelial cells, and then regulate the cell proliferation and expression of β-Casein by suppressing the TGFβR I expression.

  12. Generation of airway epithelial cells with native characteristics from mouse induced pluripotent stem cells.

    Science.gov (United States)

    Yoshie, Susumu; Imaizumi, Mitsuyoshi; Nakamura, Ryosuke; Otsuki, Koshi; Ikeda, Masakazu; Nomoto, Yukio; Wada, Ikuo; Omori, Koichi

    2016-05-01

    Airway epithelial cells derived from induced pluripotent stem (iPS) cells are expected to be a useful source for the regeneration of airway epithelium. Our preliminary study of embryoid body (EB) formation and the air-liquid interface (ALI) method suggested that mouse iPS cells can differentiate into airway epithelial cells. However, whether the cells generated from mouse iPS cells had the character and phenotype of native airway epithelial cells remained uninvestigated. In this study, we generated airway epithelial cells from EBs by culturing them under serum-free conditions supplemented with Activin and bFGF and by the ALI method and characterized the iPS cell-derived airway epithelial cells in terms of their gene expression, immunoreactivity, morphology, and function. Analysis by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) revealed that the expression of the undifferentiated cell marker Nanog decreased time-dependently after the induction of differentiation, whereas definitive endoderm markers Foxa2 and Cxcr4 were transiently up-regulated. Thereafter, the expression of airway epithelium markers such as Tubb4a, Muc5ac, and Krt5 was detected by RT-PCR and immunostaining. The formation of tight junctions was also confirmed by immunostaining and permeability assay. Analysis by hematoxylin and eosin staining and scanning electron microscopy indicated that the cells generated from mouse iPS cells formed airway-epithelium-like tissue and had cilia, the movement of which was visualized and observed to be synchronized. These results demonstrate that the airway epithelial cells generated by our method have native characteristics and open new perspectives for the regeneration of injured airway epithelium. PMID:26590823

  13. Single cell studies of mouse embryonic stem cell (mESC) differentiation by electrical impedance measurements in a microfluidic device.

    Science.gov (United States)

    Zhou, Ying; Basu, Srinjan; Laue, Ernest; Seshia, Ashwin A

    2016-07-15

    Biological populations of cells show considerable cell-to-cell variability. Study of single cells and analysis of cell heterogeneity are considered to be critical in understanding biological processes such as stem cell differentiation and cancer development. Recent advances in lab-on-a-chip techniques have allowed single-cell capture in microfluidic channels with the possibility of precise environmental control and high throughput of experiments with minimal usage of samples and reagents. In recent years, label-free techniques such as electrical impedance spectroscopy have emerged as a non-invasive approach to studying cell properties. In this study, we have designed and fabricated a microfluidic device that combines hydrodynamic trapping of single cells in pre-defined locations with the capability of running electrical impedance measurements within the same device. We have measured mouse embryonic stem cells (mESCs) at different states during differentiation (t=0h, 24h and 48h) and quantitatively analysed the changes in electrical parameters of cells during differentiation. A marked increase in the magnitude of the cell impedance is found during cell differentiation, which can be attributed to an increase in cell size. The analysis of the measurements shows that the nucleus-to-cytoplasm ratio decreases during this process. The degree of cell heterogeneity is observed to be the highest when the cells are at the transition state (24h), compare with cells at undifferentiated (0h) and fully differentiated (48h) states. The device enables highly efficient single cell trapping and provides sensitive, label-free electrical impedance measurements of individual cells, enabling the possibility of quantitatively analysing their physical state as well as studying the associated heterogeneity of a cell population. PMID:26963790

  14. Single cell studies of mouse embryonic stem cell (mESC) differentiation by electrical impedance measurements in a microfluidic device

    Science.gov (United States)

    Zhou, Ying; Basu, Srinjan; Laue, Ernest; Seshia, Ashwin A.

    2016-01-01

    Biological populations of cells show considerable cell-to-cell variability. Study of single cells and analysis of cell heterogeneity are considered to be critical in understanding biological processes such as stem cell differentiation and cancer development. Recent advances in lab-on-a-chip techniques have allowed single-cell capture in microfluidic channels with the possibility of precise environmental control and high throughput of experiments with minimal usage of samples and reagents. In recent years, label-free techniques such as electrical impedance spectroscopy have emerged as a non-invasive approach to studying cell properties. In this study, we have designed and fabricated a microfluidic device that combines hydrodynamic trapping of single cells in pre-defined locations with the capability of running electrical impedance measurements within the same device. We have measured mouse embryonic stem cells (mESCs) at different states during differentiation (t=0 h, 24 h and 48 h) and quantitatively analysed the changes in electrical parameters of cells during differentiation. A marked increase in the magnitude of the cell impedance is found during cell differentiation, which can be attributed to an increase in cell size. The analysis of the measurements shows that the nucleus-to-cytoplasm ratio decreases during this process. The degree of cell heterogeneity is observed to be the highest when the cells are at the transition state (24 h), compare with cells at undifferentiated (0 h) and fully differentiated (48 h) states. The device enables highly efficient single cell trapping and provides sensitive, label-free electrical impedance measurements of individual cells, enabling the possibility of quantitatively analysing their physical state as well as studying the associated heterogeneity of a cell population. PMID:26963790

  15. Genotoxic effects of acrylamide and glycidamide in mouse lymphoma cells.

    Science.gov (United States)

    Mei, Nan; Hu, Jiaxiang; Churchwell, Mona I; Guo, Lei; Moore, Martha M; Doerge, Daniel R; Chen, Tao

    2008-02-01

    In addition to occupational exposures to acrylamide (AA), concerns about AA health risks for the general population have been recently raised due to the finding of AA in food. In this study, we evaluated the genotoxicity of AA and its metabolite glycidamide (GA) in L5178Y/Tk(+/-) mouse lymphoma cells. The cells were treated with 2-18 mM of AA or 0.125-4 mM of GA for 4 h without metabolic activation. The DNA adducts, mutant frequencies and the types of mutations for the treated cells were examined. Within the dose range tested, GA induced DNA adducts of adenine and guanine [N3-(2-carbamoyl-2-hydroxyethyl)-adenine and N7-(2-carbamoyl-2-hydroxyethyl)-guanine] in a linear dose-dependent manner. The levels of guanine adducts were consistently about 60-fold higher across the dose range than those of adenine. In contrast, no GA-derived DNA adducts were found in the cells treated with any concentrations of AA, consistent with a lack of metabolic conversion of AA to GA. However, the mutant frequency was significantly increased by AA at concentrations of 12 mM and higher. GA was mutagenic starting with the 2mM dose, suggesting that GA is much more mutagenic than AA. The mutant frequencies were increased with increasing concentrations of AA and GA, mainly due to an increase of proportion of small colony mutants. To elucidate the underlying mutagenic mechanism, we examined the loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11 for mutants induced by AA or GA. Compared to GA induced mutations, AA induced more mutants whose LOH extended to D11Mit22 and D11Mit74, an alteration of DNA larger than half of the chromosome. Statistical analysis of the mutational spectra revealed a significant difference between the types of mutations induced by AA and GA treatments (P=0.018). These results suggest that although both AA and GA generate mutations through a clastogenic mode of action in mouse lymphoma cells, GA induces mutations via a DNA adduct

  16. Preparation of Single Cell Suspensions from Mouse Aorta

    Science.gov (United States)

    Hu, Desheng; Yin, Changjun; Mohanta, Sarajo K.; Weber, Christian; Habenicht, Andreas J. R.

    2016-01-01

    fluorescence activated cell sorter (FACS) analyses from single cell suspensions to quantify the cells of interest. This protocol describes isolation of single cells from mouse aorta for FACS and other analysis. PMID:27335895

  17. Proliferation of cultured mouse choroid plexus epithelial cells.

    Directory of Open Access Journals (Sweden)

    Basam Z Barkho

    Full Text Available The choroid plexus (ChP epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF that bathes and nourishes the central nervous system (CNS. In addition to the CSF, ChP epithelial cells (CPECs produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and intensify multiple disease phenotypes, and CPEC regeneration would represent a potential therapeutic approach for these diseases. However, previous reports suggest that CPECs rarely divide, although this has not been extensively studied in response to extrinsic factors. Utilizing a cell-cycle reporter mouse line and live cell imaging, we identified scratch injury and the growth factors insulin-like growth factor 1 (IGF-1 and epidermal growth factor (EGF as extrinsic cues that promote increased CPEC expansion in vitro. Furthermore, we found that IGF-1 and EGF treatment enhances scratch injury-induced proliferation. Finally, we established whole tissue explant cultures and observed that IGF-1 and EGF promote CPEC division within the intact ChP epithelium. We conclude that although CPECs normally have a slow turnover rate, they expand in response to external stimuli such as injury and/or growth factors, which provides a potential avenue for enhancing ChP function after brain injury or neurodegeneration.

  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. Mouse fibroblast L929 cells are less permissive to infection by Nelson Bay orthoreovirus compared to other mammalian cell lines.

    Science.gov (United States)

    Mok, Lawrence; Wynne, James W; Grimley, Samantha; Shiell, Brian; Green, Diane; Monaghan, Paul; Pallister, Jackie; Bacic, Antony; Michalski, Wojtek P

    2015-07-01

    In recent years, bats have been identified as a natural reservoir for a diverse range of viruses. Nelson Bay orthoreovirus (NBV) was first isolated from the heart blood of a fruit bat (Pteropus poliocephalus) in 1968. While the pathogenesis of NBV remains unknown, other related members of this group have caused acute respiratory disease in humans. Thus the potential for NBV to impact human health appears plausible. Here, to increase our knowledge of NBV, we examined the replication and infectivity of NBV using different mammalian cell lines derived from bat, human, mouse and monkey. All cell lines supported the replication of NBV; however, L929 cells showed a greater than 2 log reduction in virus titre compared with the other cell lines. Furthermore, NBV did not induce major cytopathic effects in the L929 cells, as was observed in other cell lines. Interestingly, the related Pteropine orthoreoviruses, Pulau virus (PulV) and Melaka virus (MelV) were able to replicate to high titres in L929 cells but infection resulted in reduced cytopathic effect. Our study demonstrates a unique virus-host interaction between NBV and L929 cells, where cells effectively control viral infection/replication and limit the formation of syncytia. By elucidating the molecular mechanisms that control this unique relationship, important insights will be made into the biology of this fusogenic virus. PMID:25748429

  20. Isolation and characterization of alveolar epithelial type II cells derived from mouse embryonic stem cells.

    Science.gov (United States)

    Sun, Huanhuan; Quan, Yuan; Yan, Qing; Peng, Xinmiao; Mao, Zhengmei; Wetsel, Rick A; Wang, Dachun

    2014-06-01

    The use of embryonic stem cells (ESCs) to regenerate distal lung epithelia damaged by injuries or diseases requires development of safe and efficient methodologies that direct ESC differentiation into transplantable distal lung epithelial progenitors. Time-consuming culture procedure and low differentiation efficiency are major problems that are associated with conventional differentiation approaches via embryoid body formation. The use of a growth factor cocktail or a lung-specific cell-conditioned medium to enrich definitive endoderm for efficient differentiation of mouse ESCs (mESC) into alveolar epithelial progenitor type II cells (ATIICs) has been reported, but not yet successful for generating a homogenous population of ATIICs for tissue regeneration purpose, and it remains unclear whether or not those mESC-derived ATIICs possess normal biological functions. Here, we report a novel method using a genetically modified mESC line harboring an ATIIC-specific neomycin(R) transgene in Rosa 26 locus. We showed that ATIICs can be efficiently differentiated from mESCs as early as day 7 by culturing them directly on Matrigel-coated plates in DMEM containing 15% knockout serum replacement. With this culture condition, the genetically modified mESCs can be selectively differentiated into a homogenous population (>99%) of ATIICs. Importantly, the mESC-derived ATIICs (mESC-ATIICs) exhibited typical lamellar bodies and expressed surfactant protein A, B, and C as normal control ATIICs. When cultured with an air-liquid-interface culture system in Small Airway Epithelial Cell Growth Medium, the mESC-ATIICs can be induced to secrete surfactant proteins after being treated with dibutyryl cAMP+dexamethasone. These mESC-ATIICs can synthesize and secrete surfactant lipid in response to secretagogue, demonstrating active surfactant metabolism in mESC-ATIICs as that seen in normal control ATIICs. In addition, we demonstrated that the selected mESC-ATIICs can be maintained on Matrigel

  1. A transgenic mouse model of metastatic prostate cancer originating from neuroendocrine cells

    OpenAIRE

    Garabedian, Emily M.; Humphrey, Peter A.; Jeffrey I Gordon

    1998-01-01

    A transgenic mouse model of metastatic prostate cancer has been developed that is 100% penetrant in multiple pedigrees. Nucleotides −6500 to +34 of the mouse cryptdin-2 gene were used to direct expression of simian virus 40 T antigen to a subset of neuroendocrine cells in all lobes of the FVB/N mouse prostate. Transgene expression is initiated between 7 and 8 weeks of age and leads to development of prostatic intraepithelial neoplasia within a week. Prostatic intraepithelial neoplasia progres...

  2. Redirection of Human Cancer Cells upon the Interaction with the Regenerating Mouse Mammary Gland Microenvironment

    Directory of Open Access Journals (Sweden)

    Sonia M. Rosenfield

    2013-01-01

    Full Text Available Tumorigenesis is often described as a result of accumulated mutations that lead to growth advantage and clonal expansion of mutated cells. There is evidence in the literature that cancer cells are influenced by the microenvironment. Our previous studies demonstrated that the mouse mammary gland is capable of redirecting mouse cells of non-mammary origins as well as Mouse Mammary Tumor Virus (MMTV-neu transformed cells toward normal mammary epithelial cell fate during gland regeneration. Interestingly, the malignant phenotype of MMTV-neu transformed cells was suppressed during serial transplantation experiments. Here, we discuss our studies that demonstrated the potential of the regenerating mouse mammary gland to redirect cancer cells of different species into a functional tumor-free mammary epithelial cell progeny. Immunochemistry for human specific CD133, mitochondria, cytokeratins as well as milk proteins and FISH for human specific probe identified human epithelial cell progeny in ducts, lobules, and secretory acini. Fluorescent In Situ Hybridization (FISH for human centromeric DNA and FACS analysis of propidium iodine staining excluded the possibility of mouse-human cell fusion. To our knowledge this is the first evidence that human cancer cells of embryonic or somatic origins respond to developmental signals generated by the mouse mammary gland microenvironment during gland regeneration in vivo.

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

    International Nuclear Information System (INIS)

    The effects of diamide (diazenedicarboxylic acid bis (N,N'-dimethylamide)) on the transport of potassium in mouse L-cells have been investigated using 86Rb+ 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

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

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    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.

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

    Directory of Open Access Journals (Sweden)

    V.B. CÂRSTEA

    2013-12-01

    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.

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

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    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.

  7. Genome-wide profiling to analyze the effects of FXR activation on mouse renal proximal tubular cells.

    Science.gov (United States)

    Gui, Ting; Gai, Zhibo

    2015-12-01

    To assess the effect of farnesoid X receptor (FXR), a bile acid nuclear receptor, on renal proximal tubular cells, primary cultured mouse kidney proximal tubular cells were treated with GW4064 (a FXR agonist) or DMSO (as controls) overnight. Analysis of gene expression in the proximal tubular cells by whole genome microarrays indicated that FXR activation induced genes involved in fatty acid degradation and oxidation reduction. Among them, genes involved in glutathione metabolism were mostly induced. Here we describe in details the contents and quality controls for the gene expression and related results associated with the data uploaded to Gene Expression Omnibus (accession number GSE70296). PMID:26697325

  8. Genome-wide profiling to analyze the effects of FXR activation on mouse renal proximal tubular cells

    Directory of Open Access Journals (Sweden)

    Ting Gui

    2015-12-01

    Full Text Available To assess the effect of farnesoid X receptor (FXR, a bile acid nuclear receptor, on renal proximal tubular cells, primary cultured mouse kidney proximal tubular cells were treated with GW4064 (a FXR agonist or DMSO (as controls overnight. Analysis of gene expression in the proximal tubular cells by whole genome microarrays indicated that FXR activation induced genes involved in fatty acid degradation and oxidation reduction. Among them, genes involved in glutathione metabolism were mostly induced. Here we describe in details the contents and quality controls for the gene expression and related results associated with the data uploaded to Gene Expression Omnibus (accession number GSE70296.

  9. Interspecific complementation between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation

    International Nuclear Information System (INIS)

    Interspecific and intraspecific hybrids were formed between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation and their radiosensitivities were examined. Chinese hamster cell mutants irs1, irs2 and irs3 and mouse mammary carcinoma cell mutants SX9 and SX10 have been found to belong to five different complementation groups. A radiosensitive mouse lymphoma cell line L5178Y-S has been demonstrated to be different from the X-ray sensitive mouse cell mutants M10 and LX830, both of which are derived from L5178Y cells, in their complementation groups. L5178Y-S is also distinct from SX9 and SX10. (author)

  10. Impact of methoxyacetic acid on mouse Leydig cell gene expression

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    Waxman David J

    2010-06-01

    Full Text Available Abstract Background Methoxyacetic acid (MAA is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, which is associated with various developmental and reproductive toxicities, including neural toxicity, blood and immune disorders, limb degeneration and testicular toxicity. Testicular toxicity is caused by degeneration of germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Methods Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and changes in gene expression were monitored by genome-wide transcriptional profiling. Results A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes. 60 DNA-binding proteins responded to MAA rapidly but transiently, and may contribute to the downstream effects of MAA seen for many mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. In contrast, many of the genes responding to MAA at later time points encode membrane proteins that contribute to cell adhesion and membrane signaling. Conclusions These findings

  11. Nuclear RNA sequencing of the mouse erythroid cell transcriptome.

    Directory of Open Access Journals (Sweden)

    Jennifer A Mitchell

    Full Text Available In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.

  12. Cell-penetrable mouse forkhead box protein 3 alleviates experimental arthritis in mice by up-regulating regulatory T cells.

    Science.gov (United States)

    Liu, Xia; Ji, Baoju; Sun, Mengyi; Wu, Weijiang; Huang, Lili; Sun, Aihua; Zong, Yangyong; Xia, Sheng; Shi, Liyun; Qian, Hui; Xu, Wenrong; Shao, Qixiang

    2015-07-01

    Regulatory T cells (T(regs)) have potential applications in clinical disease therapy, such as autoimmune diseases and transplant rejection. However, their numbers are limited. Forkhead box protein 3 (FoxP3) is a key transcription factor that controls T(reg) development and function. Here, we generated a cell-permeable fusion protein, protein transduction domain (PTD)-conjugated mouse FoxP3 protein (PTD-mFoxP3), and evaluated whether PTD-mFoxp3 can alleviate rheumatoid arthritis (RA) in the collagen-induced arthritis (CIA) mouse model. As expected, PTD-mFoxP3 was transduced into cells effectively, and inhibited T cell activation and attenuated the cell proliferation. It decreased interleukin (IL) 2 and interferon (IFN)-γ expression, and increased IL-10 expression in activated CD4(+)CD25(-) T cells. PTD-mFoxP3-transduced CD4(+)CD25(-) T cells attenuated proliferation of activated CD4(+)CD25(-) T cells. In addition, PTD-mFoxP3 blocked the Th17 differentiation programme in vitro and down-regulated IL-17 production from T cells by modulating induction and levels of retinoid-related orphan receptor gamma t (RORγt). Intra-articular delivery of PTD-mFoxP3 delayed disease incidence remarkably and alleviated autoimmune symptoms of CIA mice. Moreover, protective effects of PTD-mFoxP3 were associated with regulating the balance of T helper type 17 (Th17) and T(regs). These results suggest that PTD-mFoxP3 may be a candidate for RA therapy. PMID:25809415

  13. Multiple intravenous administrations of human umbilical cord blood cells benefit in a mouse model of ALS.

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    Svitlana Garbuzova-Davis

    Full Text Available BACKGROUND: A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS is the use of cell-based therapies that can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (25 × 10⁶ cells of mononuclear cells from human umbilical cord blood (MNC hUCB administered intravenously to pre-symptomatic G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages. METHODOLOGY/PRINCIPAL FINDINGS: Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice received 10⁶ or 2.5 × 10⁶ cells from 13 weeks of age. A third, pre-symptomatic, group received 10⁶ cells from 9 weeks of age. Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in mouse spinal cords. Results showed that mice receiving 10⁶ cells pre-symptomatically or 2.5 × 10⁶ cells symptomatically significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice. Astrocytes and microglia were significantly reduced in all cell-treated groups. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This multiple cell infusion approach may promote future clinical studies.

  14. Functional effect of mouse embryonic stem cell implantation after spinal cord injury

    OpenAIRE

    Lee, Tae-Hoon

    2013-01-01

    We transplanted mouse embryonic stem cells (mESCs) to improve functional loss in a rat model of clip-compression spinal cord injury (SCI). The mouse embryonic stem cells were transplanted to injured cord 7 days after injury. We include minimizing the progression of secondary injury, manipulating the neuroinhibitory environment of the spinal cord, replacing lost tissue with transplanted cells and substantial improvement of motor. A number of potential approaches optimize functional recovery af...

  15. Immunohistochemical Examination for the Distribution of Podoplanin-Expressing Cells in Developing Mouse Molar Tooth Germs

    OpenAIRE

    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...

  16. The roles of ERAS during cell lineage specification of mouse early embryonic development

    OpenAIRE

    Zhao, Zhen-Ao; Yu, Yang; Ma, Huai-Xiao; Wang, Xiao-Xiao; Lu, Xukun; Zhai, Yanhua; Zhang, Xiaoxin; Wang, Haibin; Li, Lei

    2015-01-01

    Eras encodes a Ras-like GTPase protein that was originally identified as an embryonic stem cell-specific Ras. ERAS has been known to be required for the growth of embryonic stem cells and stimulates somatic cell reprogramming, suggesting its roles on mouse early embryonic development. We now report a dynamic expression pattern of Eras during mouse peri-implantation development: its expression increases at the blastocyst stage, and specifically decreases in E7.5 mesoderm. In accordance with it...

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

    International Nuclear Information System (INIS)

    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 FANXA7-control group was 77%, and the lymph node metastasis rate of the FANXA7-down group was 100% (p < 0.05). In contrast, the lymph node metastasis rate of the PANXA7-up group was 0% and that of the PANXA7-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

  18. A combination of upstream and proximal elements is required for efficient expression of the mouse renin promoter in cultured cells.

    OpenAIRE

    Tamura, K.; Tanimoto, K; Murakami, K.; Fukamizu, A

    1992-01-01

    Renin, a key enzyme controlling blood pressure, is produced mainly in the kidney. To identify the transcriptional regulatory elements of the mouse Ren-1c gene, the promoter regions were fused to the CAT reporter gene and transfected into embryonic kidney-derived 293 cells and four extrarenal cell lines, HeLa, HepG2, HT1080 and NIH3T3 cells. Transient transfection assay showed that sequences from -365 to +16 of the renin gene could direct transcription of the CAT hybrid gene only in 293 cells....

  19. Differentiation patterns of mouse embryonic stem cells and induced pluripotent stem cells into neurons.

    Science.gov (United States)

    Nakamura, Mai; Kamishibahara, Yu; Kitazawa, Ayako; Kawaguchi, Hideo; Shimizu, Norio

    2016-05-01

    Mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have the ability to differentiate in vitro into various cell lineages including neurons. The differentiation of these cells into neurons has potential applications in regenerative medicine. Previously, we reported that a chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse ES and iPS cells into neurons. Here, we used real-time PCR to investigate the differentiation patterns of ES and iPS cells into neurons when DRG-CM was added. DRG-CM promoted the expression levels of βIII-tubulin gene (a marker of postmitotic neurons) in ES and iPS cells. ES cells differentiated into neurons faster than iPS cells, and the maximum peaks of gene expression involved in motor, sensory, and dopaminergic neurons were different. Rho kinase (ROCK) inhibitors could be very valuable at numerous stages in the production and use of stem cells in basic research and eventual cell-based therapies. Thus, we investigated whether the addition of a ROCK inhibitor Y-27632 and DRG-CM on the basis of the differentiation patterns promotes the neuronal differentiation of ES cells. When the ROCK inhibitor was added to the culture medium at the initial stages of cultivation, it stimulated the neuronal differentiation of ES cells more strongly than that stimulated by DRG-CM. Moreover, the combination of the ROCK inhibitor and DRG-CM promoted the neuronal differentiation of ES cells when the ROCK inhibitor was added to the culture medium at day 3. The ROCK inhibitor may be useful for promoting neuronal differentiation of ES cells. PMID:25354731

  20. Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation

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    Barbara Arbeithuber

    2015-01-01

    Full Text Available Several studies have revealed that aquaporins play a role in tumor progression and invasion. In breast carcinomas, high levels of aquaporin 5 (AQP5, a membrane protein involved in water transport, have been linked to increased cell proliferation and migration, thus facilitating tumor progression. Despite the potential role of AQP5 in mammary oncogenesis, the mechanisms controlling mammary AQP5 expression are poorly understood. In other tissues, AQP5 expression has been correlated with its promoter methylation, yet, very little is known about AQP5 promoter methylation in the mammary gland. In this work, we used the mouse mammary gland cell line EpH4, in which we controlled AQP5 expression via the steroid hormone dexamethasone (Dex to further investigate mechanisms regulating AQP5 expression. In this system, we observed a rapid drop of AQP5 mRNA levels with a delay of several hours in AQP5 protein, suggesting transcriptional control of AQP5 levels. Yet, AQP5 expression was independent of its promoter methylation, or to the presence of negative glucocorticoid receptor elements (nGREs in its imminent promoter region, but was rather influenced by the cell proliferative state or cell density. We conclude that AQP5 promoter methylation is not a universal mechanism for AQP5 regulation and varies on cell and tissue type.

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

    International Nuclear Information System (INIS)

    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 Li2CO3 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

    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.

  3. Identification of NR0B1 as a novel androgen receptor co-repressor in mouse Sertoli cells.

    Science.gov (United States)

    Li, Yu-Chi; Luo, Man-Ling; Guo, Huan; Wang, Tian-Tian; Lin, Shou-Ren; Chen, Jian-Bo; Ma, Qian; Gu, Yan-Li; Jiang, Zhi-Mao; Gui, Yao-Ting

    2016-09-01

    Nuclear receptor subfamily 0 group B member 1 (Nr0b1) is an atypical member of the nuclear receptor family that is predominantly expressed in mouse Sertoli cells (SCs). Mutations of NR0B1 in humans cause adrenal failure and hypogonadotropic hypogonadism. The targeted mutagenesis of Nr0b1 in mice has revealed a primary gonadal defect characterized by the overexpression of aromatase and cellular obstruction of the seminiferous tubules and efferent ductules, leading to germ cell death and infertility. The transgenic expression of Nr0b1 under the control of the Müllerian-inhibiting substance promoter (MIS-Nr0b1), which is selectively expressed in SCs, improves fertility. Testicular androgen receptor (AR) was also expressed in SCs. Many genes are directly regulated by androgen and its AR, which are involved in spermatogenesis and male infertility. As the association between NR0B1 and AR remains unclear in mouse SCs, we decided to further explore the relationship between them. In the present study, we have identified NR0B1 as a novel AR co-repressor in mouse SCs. Using RT‑qPCR and immunofluorescence, we determined that NR0B1 was mainly expressed in mouse SCs in an age-dependent manner from 2-8 weeks of age postnatally. The inhibition of the effects of AR on AR target genes by NR0B1, in an androgen‑dependent manner, was further demonstrated by western blot analysis and RT-qPCR in TM4 cells, a mouse Sertoli cell line. Finally, in vitro luciferase and co-immunoprecipitation assays validated that NR0B1, as an AR co-repressor, significantly inhibited the transcriptional activation of its target genes. These results suggest that novel inhibitory mechanisms underlie the effects of NR0B1 in modulating androgen-dependent gene transcription in mouse SCs. PMID:27431683

  4. Gradual regeneration of mouse testicular stem cells after exposure to ionizing radiation

    International Nuclear Information System (INIS)

    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

  5. Serum amine oxidase activity contributes to crisis in mouse embryo cell lines.

    Science.gov (United States)

    Parchment, R E; Lewellyn, A; Swartzendruber, D; Pierce, G B

    1990-06-01

    This paper reports the results of experiments to test the hypothesis that crisis of spontaneous transformation is caused by the hydrogen peroxide and/or aldehydes generated from endogenous polyamines by serum amine oxidase [amine: oxygen oxidoreductase (deaminating), EC 1.4.3.6]. After 4-5 weeks of culture, crisis occurred in 16 of 29 cell lines derived from limb buds of embryos from SJL/J, C3H, and CD-1 mice. In contrast, after the same time in culture but in medium supplemented with aminoguanidine, which inhibits serum amine oxidase, crisis occurred in only 1 of 41 cell lines. Protection against crisis was maximal in cell lines of SJL/J embryos, in which the incidence of crisis fell from 7 of 9 in untreated controls of 0 to 12 in the presence of 2 mM aminoguanidine. 2-Mercaptoethanol at 150-300 microM, which protects cells from serum amine oxidase-dependent polyamine toxicity, also protected the cell lines against crisis. These protected cell lines retained proliferative potential, diploid DNA content, and the mixture of cell types found in the primary cultures. These results indicate that cytotoxic catabolites generated by serum amine oxidase caused at least a large portion, but perhaps not all, of the cellular damage that leads to crisis in mouse embryo cell lines. PMID:2349241

  6. Tcf7l2 localization of putative stem/progenitor cells in mouse conjunctiva.

    Science.gov (United States)

    Quan, Yadan; Zhang, Xinchun; Xu, Siying; Li, Kang; Zhu, Feng; Li, Qian; Cai, Xianxian; Lu, Rong

    2016-08-01

    Conjunctival integrity and preservation is indispensable for vision. The self-renewing capacity of conjunctival cells controls conjunctival homeostasis and regeneration; however, the source of conjunctival self-renewal and the underlying mechanism is currently unclear. Here, we characterize the biochemical phenotype and proliferative potential of conjunctival epithelial cells in adult mouse by detecting proliferation-related signatures and conducting clonal analysis. Further, we show that transcription factor 7-like 2 (T-cell-specific transcription factor 4), a DNA binding protein expressed in multiple types of adult stem cells, is highly correlated with proliferative signatures in basal conjunctival epithelia. Clonal studies demonstrated that Transcription factor 7-like 2 (Tcf7l2) was coexpressed with p63α and proliferating cell nuclear antigen (PCNA) in propagative colonies. Furthermore, Tcf7l2 was actively transcribed concurrently with conjunctival epithelial proliferation in vitro. Collectively, we suggest that Tcf7l2 may be involved in maintenance of stem/progenitor cells properties of conjunctival epithelial stem/progenitor cells, and with the fornix as the optimal site to isolate highly proliferative conjunctival epithelial cells in adult mice. PMID:27281479

  7. Oxaliplatin enhances gap junction-mediated coupling in cell cultures of mouse trigeminal ganglia.

    Science.gov (United States)

    Poulsen, Jeppe Nørgaard; Warwick, Rebekah; Duroux, Meg; Hanani, Menachem; Gazerani, Parisa

    2015-08-01

    Communications between satellite glial cells and neighboring neurons within sensory ganglia may contribute to neuropathic and inflammatory pain. To elucidate the role of satellite glial cells in chemotherapy-induced pain, we examined the effects of oxaliplatin on the gap junction-mediated coupling between these cells. We also examined whether the gap junction blocker, carbenoxolone, can reverse the coupling. Primary cultures of mice trigeminal ganglia, 24-48h after cell isolation, were used. Satellite glial cells were injected with Lucifer yellow in the presence or absence of oxaliplatin (60 μM). In addition, the effect of carbenoxolone (100 μM) on coupling, and the expression of connexin 43 proteins were evaluated. Dye coupling between adjacent satellite glial cells was significantly increased (2.3-fold, P<0.05) following a 2h incubation with oxaliplatin. Adding carbenoxolone to the oxaliplatin-treated cultures reversed oxaliplatin-evoked coupling to baseline (P<0.05). Immunostaining showed no difference between expression of connexin 43 in control and oxaliplatin-treated cultures. Our findings indicated that oxaliplatin-increased gap junction-mediated coupling between satellite glial cells in primary cultures of mouse trigeminal ganglia, and carbenoxolone reversed this effect. Hence, it is proposed that increased gap junction-mediated coupling was seen between satellite glial cells in TG. This observation together with our previous data obtained from a behavioral study suggests that this phenomenon might contribute to chemotherapy-induced nociception following oxaliplatin treatment. PMID:25999145

  8. CD34 defines an osteoprogenitor cell population in mouse bone marrow stromal cells

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Al-Shammary, Asma; Skagen, Peter;

    2015-01-01

    Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) and their progenitors have been identified based on retrospective functional criteria. CD markers are employed to define cell populations with distinct functional characteristics. However, defining and...... prospective isolation of mouse bone marrow osteoprogenitors....... prospective isolation of BMSCs and committed progenitors are lacking. Here, we compared the transcriptome profile of CD markers expressed at baseline and during the course of osteoblast and adipocyte differentiation of two well-characterized osteogenic-committed murine BMSCs (mBMSC(Bone)) and adipogenic...

  9. 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.

  10. Photomutagenicity of retinyl palmitate by ultraviolet a irradiation in mouse lymphoma cells.

    Science.gov (United States)

    Mei, Nan; Xia, Qingsu; Chen, Ling; Moore, Martha M; Fu, Peter P; Chen, Tao

    2005-11-01

    Retinyl palmitate (RP), a storage form of vitamin A, is frequently used as a cosmetic ingredient, with more than 700 RP-containing cosmetic products on the U.S. market in 2004. There are concerns for the possible genotoxicity and carcinogenicity of RP when it is exposed to sunlight. To evaluate the photomutagenicity of RP in cells when exposed to ultraviolet A (UVA) light, L5178Y/Tk+/- mouse lymphoma cells were treated with different doses of RP alone/or in the presence of UVA light. Treatment of the cells with RP alone at the dose range of 25-100 microg/ml did not increase mutant frequencies (MFs) over the negative control, whereas treatment of cells with 1-25 microg/ml RP under UVA light (82.8 mJ/cm2/min for 30 min) produced a dose-dependent mutation induction. The mean induced MF (392 x 10(-6)) for treatment with 25 microg/ml RP under UVA exposure was about threefold higher than that for UVA alone (122 x 10(-6)), a synergistic effect. To elucidate the underlying mechanism of action, we examined the mutants for loss of heterozygosity (LOH) at four microsatellite loci spanning the entire chromosome 11, on which the Tk gene is located. The mutational spectrum for the RP + UVA treatment was significantly different from the negative control, but not significantly different from UVA exposure alone. Ninety four percent of the mutants from RP + UVA treatment lost the Tk+ allele, and 91% of the deleted sequences extended more than 6 cM in chromosome length, indicating clastogenic events affecting a large segment of the chromosome. These results suggest that RP is photomutagenic in combination with UVA exposure in mouse lymphoma cells, with a clastogenic mode-of-action. PMID:16107546

  11. Equivalence of human and mouse CD4 in enhancing antigen responses by a mouse class II-restricted T cell hybridoma

    OpenAIRE

    1989-01-01

    We have examined the ability of hCD4 to interact functionally with mouse class II MHC molecules using the mouse T cell hybridoma BI-141, specific for beef insulin. We have previously shown that expression of mouse CD4 results in a marked enhancement of IL-2 release by BI-141 cells in response to beef insulin or, in a cross-reactive response, to pork insulin, on the appropriate mouse APCs. We now demonstrate that expression of hCD4 results in an equivalent stimulation of antigen responses by t...

  12. Immunologic analyses of mouse cystathionase in normal and leukemic cells

    International Nuclear Information System (INIS)

    Rabbit antisera have been raised against mouse liver cystathionase and shown to possess enzyme neutralizing activity. Agar gel double immunodiffusion analyses demonstrated that both mouse liver cystathionase and rat liver cystathionase react with the antisera, the latter enzyme being completely cross-reactive with the former. Following radioiodination of the purified rat liver enzyme, a double antibody radioimmunoassay was developed in which greater than 90% of the labeled protein could be specifically precipitated with the anti-mouse cystathionase antibodies. In this test the purified rat liver and mouse liver enzymes were virtually indistinguishable, generating superimposable competition displacement curves on a protein mass basis. These results indicate that both enzymes are immunologically identical, thus validating the use of the rat in lieu of the murine liver enzyme as radiolabeled tracer in an assay for mouse cystathionase. In addition, competition radioimmunoassays demonstrated that the immunological reactivities of both the purified rat liver and mouse liver enzymes were equally heat sensitive. The sensitivity of the assay was determined to be 1 ng of enzyme protein/0.22 mL of assay mixture, and the assay could be used to detect the presence of enzyme protein in tissue homogenates of single mouse organs. Mouse or rat cross-reactivity with human liver cystathionase was incomplete; but, with the exception of heart and spleen, parallel radioimmunoassay competition displacement curves were obtained for cystathionase from different mouse organs including thymus. Extracts of 7-, 9-, and 10-month-old spontaneous AKR mouse thymomas were tested in the radioimmunoassay along with extracts of age-matched thymuses which were grossly tumor free. A reaction of nonidentity was observed for all of the tumor extracts while a reaction identical with that of the pure liver enzyme was found with all of the normal thymus extracts

  13. Mouse Clone Model” for evaluating the immunogenicity and tumorigenicity of pluripotent stem cells

    OpenAIRE

    Zhang, Gang; Zhang, Yi

    2015-01-01

    To investigate the immune-rejection and tumor-formation potentials of induced pluripotent stem cells and other stem cells, we devised a model—designated the “Mouse Clone Model”—which combined the theory of somatic animal cloning, tetraploid complementation, and induced pluripotent stem cells to demonstrate the applicability of stem cells for transplantation therapy.

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

    International Nuclear Information System (INIS)

    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

  15. Chemotherapy of WAP-T mouse mammary carcinomas aggravates tumor phenotype and enhances tumor cell dissemination.

    Science.gov (United States)

    Jannasch, Katharina; Wegwitz, Florian; Lenfert, Eva; Maenz, Claudia; Deppert, Wolfgang; Alves, Frauke

    2015-07-01

    In this study, the effects of the standard chemotherapy, cyclophosphamide/adriamycin/5-fluorouracil (CAF) on tumor growth, dissemination and recurrence after orthotopic implantation of murine G-2 cells were analyzed in the syngeneic immunocompetent whey acidic protein-T mouse model (Wegwitz et al., PLoS One 2010; 5:e12103; Schulze-Garg et al., Oncogene 2000; 19:1028-37). Single-dose CAF treatment reduced tumor size significantly, but was not able to eradicate all tumor cells, as recurrent tumor growth was observed 4 weeks after CAF treatment. Nine days after CAF treatment, residual tumors showed features of regressive alterations and were composed of mesenchymal-like tumor cells, infiltrating immune cells and some tumor-associated fibroblasts with an intense deposition of collagen. Recurrent tumors were characterized by coagulative necrosis and less tumor cell differentiation compared with untreated tumors, suggesting a more aggressive tumor phenotype. In support, tumor cell dissemination was strongly enhanced in mice that had developed recurrent tumors in comparison with untreated controls, although only few disseminated tumor cells could be detected in various organs 9 days after CAF application. In vitro experiments revealed that CAF treatment of G-2 cells eliminates the vast majority of epithelial tumor cells, whereas tumor cells with a mesenchymal phenotype survive. These results together with the in vivo findings suggest that tumor cells that underwent epithelial-mesenchymal transition and/or exhibit stem-cell-like properties are difficult to eliminate using one round of CAF chemotherapy. The model system described here provides a valuable tool for the characterization of the effects of chemotherapeutic regimens on recurrent tumor growth and on tumor cell dissemination, thereby enabling the development and preclinical evaluation of novel therapeutic strategies to target mammary carcinomas. PMID:25449528

  16. Gene function in early mouse embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Campbell Pearl A

    2007-03-01

    Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

  17. Similarity on neural stem cells and brain tumor stem cells in transgenic brain tumor mouse models

    Institute of Scientific and Technical Information of China (English)

    Guanqun Qiao; Qingquan Li; Gang Peng; Jun Ma; Hongwei Fan; Yingbin Li

    2013-01-01

    Although it is believed that glioma is derived from brain tumor stem cells, the source and molecular signal pathways of these cells are stil unclear. In this study, we used stable doxycycline-inducible transgenic mouse brain tumor models (c-myc+/SV40Tag+/Tet-on+) to explore the malignant trans-formation potential of neural stem cells by observing the differences of neural stem cel s and brain tumor stem cells in the tumor models. Results showed that chromosome instability occurred in brain tumor stem cells. The numbers of cytolysosomes and autophagosomes in brain tumor stem cells and induced neural stem cel s were lower and the proliferative activity was obviously stronger than that in normal neural stem cells. Normal neural stem cells could differentiate into glial fibril ary acidic protein-positive and microtubule associated protein-2-positive cells, which were also negative for nestin. However, glial fibril ary acidic protein/nestin, microtubule associated protein-2/nestin, and glial fibril ary acidic protein/microtubule associated protein-2 double-positive cells were found in induced neural stem cells and brain tumor stem cel s. Results indicate that induced neural stem cells are similar to brain tumor stem cells, and are possibly the source of brain tumor stem cells.

  18. Multiple cell and population-level interactions with mouse embryonic stem cell heterogeneity.

    Science.gov (United States)

    Cannon, Danielle; Corrigan, Adam M; Miermont, Agnes; McDonel, Patrick; Chubb, Jonathan R

    2015-08-15

    Much of development and disease concerns the generation of gene expression differences between related cells sharing similar niches. However, most analyses of gene expression only assess population and time-averaged levels of steady-state transcription. The mechanisms driving differentiation are buried within snapshots of the average cell, lacking dynamic information and the diverse regulatory history experienced by individual cells. Here, we use a quantitative imaging platform with large time series data sets to determine the regulation of developmental gene expression by cell cycle, lineage, motility and environment. We apply this technology to the regulation of the pluripotency gene Nanog in mouse embryonic stem cells. Our data reveal the diversity of cell and population-level interactions with Nanog dynamics and heterogeneity, and how this regulation responds to triggers of pluripotency. Cell cycles are highly heterogeneous and cycle time increases with Nanog reporter expression, with longer, more variable cycle times as cells approach ground-state pluripotency. Nanog reporter expression is highly stable over multiple cell generations, with fluctuations within cycles confined by an attractor state. Modelling reveals an environmental component to expression stability, in addition to any cell-autonomous behaviour, and we identify interactions of cell density with both cycle behaviour and Nanog. Rex1 expression dynamics showed shared and distinct regulatory effects. Overall, our observations of multiple partially overlapping dynamic heterogeneities imply complex cell and environmental regulation of pluripotent cell behaviour, and suggest simple deterministic views of stem cell states are inappropriate. PMID:26209649

  19. Preliminary evaluation of treatment efficacy of umbilical cord blood-derived mesenchymal stem cell-differentiated cardiac pro-genitor cells in a myocardial injury mouse model

    Directory of Open Access Journals (Sweden)

    Truc Le-Buu Pham

    2015-12-01

    Full Text Available Recently, stem cell therapy has been investigated as a strategy to prevent or reverse damage to heart tissue. Although the results of cell transplantation in animal models and patients with myocardial ischemia are promising, the selection of the appropriate cell type remains an issue that requires consideration. In this study, we aimed to evaluate the effect of cardiac progenitor cell transplantation in a mouse model of myocardial ischemia. The cardiac progenitor cells used for transplantation were differentiated from umbilical cord blood mesenchymal stem cells. Animal models injected with phosphate-buffered saline (PBS and healthy mice were used as controls. Cell grafting was assessed by changes in blood pressure and histological evaluation. After 14 days of transplantation, the results demonstrated that the blood pressure of transplanted mice was stable, similar to healthy mice, whereas it fluctuated in PBS-injected mice. Histological analysis showed that heart tissue had regenerated in transplanted mice, but remained damaged in PBS-injected mice. Furthermore, trichrome staining revealed that the transplanted mice did not generate significant amount of scar tissue compared with PBS-injected control mice. In addition, the cardiac progenitor cells managed to survive and integrate with local cells in cell-injected heart tissue 14 days after transplantation. Most importantly, the transplanted cells did not exhibit tumorigenesis. In conclusion, cardiac progenitor cell transplantation produced a positive effect in a mouse model of myocardial ischemia. [Biomed Res Ther 2015; 2(12.000: 435-445

  20. 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.

  1. Evaluation of depigmenting activity by 8-hydroxydaidzein in mouse B16 melanoma cells and human volunteers.

    Science.gov (United States)

    Tai, Sorgan Shou-Ku; Lin, Ching-Gong; Wu, Mon-Han; Chang, Te-Sheng

    2009-10-01

    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 microM 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 microM. 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. PMID:20057943

  2. Global microRNA characterization reveals that miR-103 is involved in IGF-1 stimulated mouse intestinal cell proliferation.

    Directory of Open Access Journals (Sweden)

    Yalin Liao

    Full Text Available MicroRNAs play extensive roles in cellular development. Analysis of the microRNA expression pattern during intestinal cell proliferation in early life is likely to unravel molecular mechanisms behind intestinal development and have implications for therapeutic intervention. In this study, we isolated mouse intestinal crypt cells, examined the differences in microRNA expression upon IGF-1 stimulated proliferation and identified miR-103 as a one of the key regulators. Mouse intestinal crypt cells were cultured and treated with IGF-1 for 24 h. MicroRNA microarray showed that multiple microRNAs are regulated by IGF-1, and miR-103 was the most sharply down-regulated. Expression of miR-103 in mouse intestinal crypt cells was confirmed by real-time Q-PCR. Sequence analyses showed that, among the 1040 predicted miR-103 target genes, CCNE1, CDK2, and CREB1 contain complementary sequences to the miR-103 seed region that are conserved between human and mouse. We further demonstrated that miR-103 controls the expression level of these three genes in mouse crypt cells by luciferase assay and immunoblotting assay. Taken together, our data suggest that in mouse intestinal crypt cells, miR-103 is part of the G1/S transition regulatory network, which targets CCNE1, CDK2, and CREB1 during IGF-1 stimulated proliferation.

  3. 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

  4. Generation of Stable Pluripotent Stem Cells From NOD Mouse Tail-Tip Fibroblasts

    OpenAIRE

    Liu, Jun; Ashton, Michelle P.; Sumer, Huseyin; O’Bryan, Moira K.; Brodnicki, Thomas C.; Verma, Paul J.

    2011-01-01

    OBJECTIVE The NOD mouse strain has been widely used to investigate the pathology and genetic susceptibility for type 1 diabetes. Induced pluripotent stem cells (iPSCs) derived from this unique mouse strain would enable new strategies for investigating type 1 diabetes pathogenesis and potential therapeutic targets. The objective of this study was to determine whether somatic fibroblasts from NOD mice could be reprogrammed to become iPSCs, providing an alternative source of stem cells for the p...

  5. Functional Characterization of Stem Cell Activity in the Mouse Mammary Gland

    OpenAIRE

    Bruno, Robert D.; Smith, Gilbert H.

    2011-01-01

    Any portion of the mouse mammary gland is capable of recapitulating a clonally derived complete and functional mammary tree upon transplantation into an epithelial divested mammary fat-pad of a recipient host. As such, it is an ideal model tissue for the study somatic stem cell function. This review will outline what is known regarding the function of stem/progenitor cells in the mouse mammary gland, including how progenitor populations can be functionally defined, the evidence for and potent...

  6. Cardiomyocyte Marker Expression in Mouse Embryonic Fibroblasts by Cell-Free Cardiomyocyte Extract and Epigenetic Manipulation

    OpenAIRE

    Tahereh Talaei-Khozani; Fatemeh Heidari; Tahereh Esmaeilpour; Zahra Vojdani; Zohrah Mostafavi-Pour; Leili Rohani

    2014-01-01

    Background: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium. Methods: Mouse embryonic fibroblasts were treated with Trichostatin A (TSA) and 5-Aza-2-Deoxycytidine (5-aza-dC). The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21...

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

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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.

  10. Effects of beta radiation from organically bound tritium on cultured mouse embryonic mid brain cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Bing [Univ. of Tokyo (Japan)]|[Toho Univ. School of Medicine, Tokyo (Japan); Watanabe, Keiko; Yamada, Takeshi [Toho Univ. School of Medicine, Tokyo (Japan); Shima, Akihiro [Univ. of Tokyo (Japan)

    1996-12-01

    Effects of beta radiation from four kinds of organically bound tritium compounds were investigated on cultured mouse embryonic mid brain cells, isolated form 11-d-old mouse embryos. The MBC showed a critical time when they were more radiosensitive. Although dose-dependent inhibition was found for both cellular proliferation and differentiation. the differential was more sensitive to radiation than proliferation when compared at ID50, the inhibitory dose that reduced assessment value by 50% of the control. Dose-dependent decrease of DNA and protein contents were also observed. The relative biological effectiveness values, ranging from 4.6 to 8.7, of beta ray from organically bound tritium compounds were obtained when compared with x irradiation at their ID50s on the inhibition of cell proliferation and differentiation, and on the decrease of DNA and protein contents of the cultures. Th mixed exposure to x ray and one kind of organically bound tritium compound or to any two kinds of organically bound tritium compound resulted in a more efficiently inhibitory effect on differentiation than the exposure to x ray or to any one kind of organically bound tritium compound d alone, especially when methyl-{sup 3}H-thymidine was used. Doses as low as 0.05 Gy (5 cGy) from a mixed exposure resulted in detectable inhibitory effects. 21 refs., 4 figs., 1 tab.

  11. Effects of beta radiation from organically bound tritium on cultured mouse embryonic mid brain cells

    International Nuclear Information System (INIS)

    Effects of beta radiation from four kinds of organically bound tritium compounds were investigated on cultured mouse embryonic mid brain cells, isolated form 11-d-old mouse embryos. The MBC showed a critical time when they were more radiosensitive. Although dose-dependent inhibition was found for both cellular proliferation and differentiation. the differential was more sensitive to radiation than proliferation when compared at ID50, the inhibitory dose that reduced assessment value by 50% of the control. Dose-dependent decrease of DNA and protein contents were also observed. The relative biological effectiveness values, ranging from 4.6 to 8.7, of beta ray from organically bound tritium compounds were obtained when compared with x irradiation at their ID50s on the inhibition of cell proliferation and differentiation, and on the decrease of DNA and protein contents of the cultures. Th mixed exposure to x ray and one kind of organically bound tritium compound or to any two kinds of organically bound tritium compound resulted in a more efficiently inhibitory effect on differentiation than the exposure to x ray or to any one kind of organically bound tritium compound d alone, especially when methyl-3H-thymidine was used. Doses as low as 0.05 Gy (5 cGy) from a mixed exposure resulted in detectable inhibitory effects. 21 refs., 4 figs., 1 tab

  12. Hypoxia Enhances Differentiation of Mouse Embryonic Stem Cells into Definitive Endoderm and Distal Lung Cells

    OpenAIRE

    Pimton, Pimchanok; Lecht, Shimon; Stabler, Collin T.; Johannes, Gregg; Schulman, Edward S.; Lelkes, Peter I.

    2014-01-01

    We investigated the effects of hypoxia on spontaneous (SP)- and activin A (AA)-induced definitive endoderm (DE) differentiation of mouse embryonic stem cells (mESCs) and their subsequent differentiation into distal pulmonary epithelial cells. SP differentiation for 6 days of mESCs toward endoderm at hypoxia of 1% O2, but not at 3% or 21% (normoxia), increased the expression of Sox17 and Foxa2 by 31- and 63-fold above maintenance culture, respectively. Treatment of mESCs with 20 ng/mL AA for 6...

  13. Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

    International Nuclear Information System (INIS)

    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.

  14. Co-culture of Mouse Embryonic Stem Cells with Sertoli Cells Promote in vitro Generation of Germ Cells

    OpenAIRE

    Mohammad Miryounesi; Karim Nayernia; Mahdi Dianatpour; Fatemeh Mansouri; Mohammad Hossein Modarressi

    2013-01-01

      Objective(s): Sertoli cells support in vivo germ cell production; but, its exact mechanism has not been well understood. The present study was designed to analyze the effect of Sertoli cells in differentiation of mouse embryonic stem cells (mESCs) to germ cells.   Materials and Methods: A fusion construct composed of a Stra8 gene promoter and the coding region of enhanced green fluorescence protein was produced to select differentiated mESCs. To analyze sertoli cells’ effect in differentiat...

  15. Blood-borne donor mast cell precursors migrate to mast cell-rich brain regions in the adult mouse

    OpenAIRE

    Nautiyal, Katherine M.; Liu, Charles; Dong, Xin; Silver, Rae

    2011-01-01

    Mast cells are hematopoietic immune cells located throughout the body, including within the brain. Reconstitution of mast cell deficient KitW-sh/W-sh mice has proven valuable in determining peripheral mast cell function. Here we study the brain mast cell population using a novel method of blood transfusion for reconstitution. We show that blood transfusion results in mast cells of donor origin in the WT mouse, including in the brain and are restricted to regions bearing host mast cells. In co...

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

    International Nuclear Information System (INIS)

    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

  17. 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.

  18. Mouse T-cells restrict replication of human immunodeficiency virus at the level of integration

    Directory of Open Access Journals (Sweden)

    Goffinet Christine

    2008-07-01

    Full Text Available Abstract Background The development of an immunocompetent, genetically modified mouse model to study HIV-1 pathogenesis and to test antiviral strategies has been hampered by the fact that cells from native mice do not or only inefficiently support several steps of the HIV-1 replication cycle. Upon HIV-1 infection, mouse T-cell lines fail to express viral proteins, but the underlying replication barrier has thus far not been unambiguously identified. Here, we performed a kinetic and quantitative assessment of consecutive steps in the early phase of the HIV-1 replication cycle in T-cells from mice and humans. Results Both T-cell lines and primary T-cells from mice harbor a severe post-entry defect that is independent of potential species-specTR transactivation. Reverse transcription occurred efficiently following VSV-G-mediated entry of virions into mouse T-cells, and abundant levels of 2-LTR circles indicated successful nuclear import of the pre-integration complex. To probe the next step in the retroviral replication cycle, i.e. the integration of HIV-1 into the host cell genome, we established and validated a nested real-time PCR to specifically quantify HIV-1 integrants exploiting highly repetitive mouse B1 elements. Importantly, we demonstrate that the frequency of integrant formation is diminished 18- to > 305-fold in mouse T-cell lines compared to a human counterpart, resulting in a largely abortive infection. Moreover, differences in transgene expression from residual vector integrants, the transcription off which is cyclin T1-independent, provided evidence for an additional, peri-integrational deficit in certain mouse T-cell lines. Conclusion In contrast to earlier reports, we find that mouse T-cells efficiently support early replication steps up to and including nuclear import, but restrict HIV-1 at the level of chromosomal integration.

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

    Energy Technology Data Exchange (ETDEWEB)

    Su, Xin; Guan, Wuqiang; Yu, Yong-Chun; Fu, Yinghui, E-mail: fuyh@fudan.edu.cn

    2014-07-18

    Highlights: • No new neurons and astrocytes are generated in adult mouse cerebellum. • Very few mash1{sup +} or nestin{sup +} 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{sup +} 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{sup +} cells, very few are mash1{sup +} or nestin{sup +} 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{sup +} 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.

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

    International Nuclear Information System (INIS)

    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

  1. Astaxanthin Protects Steroidogenesis from Hydrogen Peroxide-Induced Oxidative Stress in Mouse Leydig Cells

    Directory of Open Access Journals (Sweden)

    Jyun-Yuan Wang

    2015-03-01

    Full Text Available Androgens, especially testosterone produced in Leydig cells, play an essential role in development of the male reproductive phenotype and fertility. However, testicular oxidative stress may cause a decline in testosterone production. Many antioxidants have been used as reactive oxygen species (ROS scavengers to eliminate oxidative stress to protect steroidogenesis. Astaxanthin (AST, a natural extract from algae and plants ubiquitous in the marine environment, has been shown to have antioxidant activity in many previous studies. In this study, we treated primary mouse Leydig cells or MA-10 cells with hydrogen peroxide (H2O2 to cause oxidative stress. Testosterone and progesterone production was suppressed and the expression of the mature (30 kDa form of StAR protein was down-regulated in MA-10 cells by H2O2 and cAMP co-treatment. However, progesterone production and expression of mature StAR protein were restored in MA-10 cells by a one-hour pretreatment with AST. AST also reduced ROS levels in cells so that they were lower than the levels in untreated controls. These results provide additional evidence of the potential health benefits of AST as a potential food additive to ease oxidative stress.

  2. Mesenchymal cells engulf and clear apoptotic footplate cells in macrophageless PU.1 null mouse embryos.

    Science.gov (United States)

    Wood, W; Turmaine, M; Weber, R; Camp, V; Maki, R A; McKercher, S R; Martin, P

    2000-12-01

    Apoptosis is one of the key tools used by an embryo to regulate cell numbers and sculpt body shape. Although massive numbers of cells die during development, they are so rapidly phagocytosed that very few corpses are ever seen in most embryonic tissues. In this paper, we focus on the catastrophic cell death that occurs as the developing footplate is remodelled to transform webbed regions into free interdigital spaces. In the wild-type embryo, these dead cells are rapidly engulfed and cleared by macrophages. We show that in a macrophageless mouse embryo, null for the haemopoetic-lineage-specific transcription factor, PU.1, the task of phagocytosis is taken over by 'stand-in' mesenchymal neighbours in a clear example of cell redundancy. However, it takes three times as many of these mesenchymal phagocytes to complete the task and, at each stage of the clearance process - in the recognition of apoptotic debris, its engulfment and finally its digestion - they appear to be less efficient than macrophages. A molecular explanation for this may be that several of the engulfment genes expressed by macrophages, including the ABC1 transporter (believed to be part of the phagocytic machinery conserved from Caenorhabditis elegans to mouse), are not upregulated by these 'stand-in' phagocytes. PMID:11076747

  3. Holographic photolysis for multiple cell stimulation in mouse hippocampal slices.

    Directory of Open Access Journals (Sweden)

    Morad Zahid

    Full Text Available BACKGROUND: Advanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable molecules and, recently, light-gated channels. These approaches require precise and yet flexible light excitation patterns. For synchronous stimulation of subsets of cells, they also require large excitation areas with millisecond and micrometric resolution. We have recently developed a new method for such optical control using a phase holographic modulation of optical wave-fronts, which minimizes power loss, enables rapid switching between excitation patterns, and allows a true 3D sculpting of the excitation volumes. In previous studies we have used holographic photololysis to control glutamate uncaging on single neuronal cells. Here, we extend the use of holographic photolysis for the excitation of multiple neurons and of glial cells. METHODS/PRINCIPAL FINDINGS: The system combines a liquid crystal device for holographic patterned photostimulation, high-resolution optical imaging, the HiLo microscopy, to define the stimulated regions and a conventional Ca(2+ imaging system to detect neural activity. By means of electrophysiological recordings and calcium imaging in acute hippocampal slices, we show that the use of excitation patterns precisely tailored to the shape of multiple neuronal somata represents a very efficient way for the simultaneous excitation of a group of neurons. In addition, we demonstrate that fast shaped illumination patterns also induce reliable responses in single glial cells. CONCLUSIONS/SIGNIFICANCE: We show that the main advantage of holographic illumination is that it allows for an efficient excitation of multiple cells with a spatiotemporal resolution unachievable with other existing approaches. Although this paper focuses on the photoactivation of caged molecules, our approach will surely prove very efficient for other probes, such as light-gated channels, genetically

  4. An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow

    Directory of Open Access Journals (Sweden)

    Shuo Huang

    2015-01-01

    Full Text Available Mesenchymal stem cells (MSCs from bone marrow are main cell source for tissue repair and engineering, and vehicles of cell-based gene therapy. Unlike other species, mouse bone marrow derived MSCs (BM-MSCs are difficult to harvest and grow due to the low MSCs yield. We report here a standardised, reliable, and easy-to-perform protocol for isolation and culture of mouse BM-MSCs. There are five main features of this protocol. (1 After flushing bone marrow out of the marrow cavity, we cultured the cells with fat mass without filtering and washing them. Our method is simply keeping the MSCs in their initial niche with minimal disturbance. (2 Our culture medium is not supplemented with any additional growth factor. (3 Our method does not need to separate cells using flow cytometry or immunomagnetic sorting techniques. (4 Our method has been carefully tested in several mouse strains and the results are reproducible. (5 We have optimised this protocol, and list detailed potential problems and trouble-shooting tricks. Using our protocol, the isolated mouse BM-MSCs were strongly positive for CD44 and CD90, negative CD45 and CD31, and exhibited tri-lineage differentiation potentials. Compared with the commonly used protocol, our protocol had higher success rate of establishing the mouse BM-MSCs in culture. Our protocol may be a simple, reliable, and alternative method for culturing MSCs from mouse bone marrow tissues.

  5. Inhibitory effect of cordycepin on experimental hepatic metastasis of B16-F0 mouse melanoma cells.

    Science.gov (United States)

    Sato, Ayuko; Yoshikawa, Noriko; Kubo, Erika; Kakuda, Mami; Nishiuchi, Arisa; Kimoto, Yoko; Takahashi, Yuta; Kagota, Satomi; Shinozuka, Kazumasa; Nakamura, Kazuki

    2013-01-01

    In a previous study performed by our group, we demonstrated that the water extract of Cordyceps sinensis (WECS) significantly prevented tumor metastasis from the spleen to the liver, using B16-F0 mouse melanoma cells as a model. In this study, we investigated the anti-metastatic activity of cordycepin (3'-deoxyadenosine), one of the components of WECS, using an identical model of mice injected with B16-F0 cells into the spleen. All mice inoculated with B16-F0 cells died due to liver metastases via the portal vein from the spleen. Control mice not administered cordycepin exhibited higher serum levels of alanine aminotransferase (ALT) due to damage to the liver by metastasized B16-F0 cells from the spleen, and survival times ranged from 17 to 22 days after tumor inoculation. Cordycepin was intraperitoneally administered to mice, and resulted in significantly lower serum ALT levels and longer survival times than those observed in control mice. Taken together, these results indicate that cordycepin may be the active ingredient in C. sinensis exerting an anti-metastatic effect, and may be a potential candidate anti-metastatic agent. PMID:24292575

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

    International Nuclear Information System (INIS)

    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)

  7. Interaction of Mouse Pem Protein and Cell Division Cycle 37 Homolog

    Institute of Scientific and Technical Information of China (English)

    Fen GUO; Yue-Qin LI; Shi-Qian LI; Zhi-Wen LUO; Xin ZHANG; Dong-Sheng TANG; Tian-Hong ZHOU

    2005-01-01

    Mouse Pem, a homeobox gene, encodes a protein consisting of 210 amino acid residues. To study the function of mouse Pem protein, we used the yeast two-hybrid system to screen the library of 7-day mouse embryo with full-length mouse Pem eDNA. Fifty-two colonies were obtained after 1.57×108 colonies were screened by nutrition limitation and β-galactosidase assay. Seven individual insert fragments were obtained from the library, and three of them were identified, one of which was confirmed to be the cell division cycle 37 (Cdc37) homolog gene by sequencing. The interaction between mouse Pem and Cdc37homolog was then confirmed by glutathione S-transferase pull-down assay, and the possible interaction model was suggested.

  8. The Defensive Role of Cumulus Cells Against Reactive Oxygen Species Insult in Metaphase II Mouse Oocytes.

    Science.gov (United States)

    Shaeib, Faten; Khan, Sana N; Ali, Iyad; Thakur, Mili; Saed, Mohammed G; Dai, Jing; Awonuga, Awoniyi O; Banerjee, Jashoman; Abu-Soud, Husam M

    2016-04-01

    We investigated the ability of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), hydroxyl radical ((·)OH), and hypochlorous acid (HOCl), to overcome the defensive capacity of cumulus cells and elucidate the mechanism through which ROS differentially deteriorate oocyte quality. Metaphase II mouse oocytes with (n = 1634) and without cumulus cells (n = 1633) were treated with increasing concentration of ROS, and the deterioration in oocyte quality was assessed by the changes in the microtubule morphology and chromosomal alignment. Oocyte and cumulus cell viability and cumulus cell number were assessed by indirect immunofluorescence, staining of gap junction protein, and trypan blue staining. The treated oocytes showed decreased quality as a function of increasing concentrations of ROS when compared to controls. Cumulus cells show protection against H2O2 and (·)OH insult at lower concentrations, but this protection was lost at higher concentrations (>50 μmol/L). At higher H2O2 concentrations, treatment dramatically influenced the cumulus cell number and viability with resulting reduction in the antioxidant capacity making the oocyte more susceptible to oxidative damage. However, cumulus cells offered no significant protection against HOCl at any concentration used. In all circumstances in which cumulus cells did not offer protection to the oocyte, both cumulus cell number and viability were decreased. Therefore, the deterioration in oocyte quality may be caused by one or more of the following: a decrease in the antioxidant machinery by the loss of cumulus cells, the lack of scavengers for specific ROS, and/or the ability of the ROS to overcome these defenses. PMID:26468254

  9. A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland

    International Nuclear Information System (INIS)

    This paper describes the derivation and characterization of a novel, conditionally immortal mammary epithelial cell line named KIM-2. These cells were derived from mid-pregnant mammary glands of a mouse harbouring one to two copies of a transgene comprised of the ovine β-lactoglobulin milk protein gene promoter, driving expression of a temperature-sensitive variant of simian virus-40 (SV40) large T antigen (T-Ag). KIM-2 cells have a characteristic luminal epithelial cell morphology and a stable, nontransformed phenotype at the semipermissive temperature of 37°C. In contrast, at the permissive temperature of 33°C the cells have an elongated spindle-like morphology and become transformed after prolonged culture. Differentiation of KIM-2 cells at 37°C, in response to lactogenic hormones, results in the formation of polarized dome-like structures with tight junctions. This is accompanied by expression of the milk protein genes that encode β-casein and whey acidic protein (WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37°C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon hormone withdrawal, as indicated by nuclear morphology and flow cytometric analysis. KIM-2 cells can be genetically modified by stable transfection and clonal lines isolated that retain the characteristics of untransfected cells. KIM-2 cells are a valuable addition, therefore, to currently available lines of mammary epithelial cells. Their capacity for extensive differentiation in the absence of exogenously added basement membrane, and ability to undergo apoptosis in response to physiological signals will provide an invaluable model system for the study of signal transduction pathways and transcriptional regulatory mechanisms that control differentiation and involution in the mammary gland

  10. The effects of poly L-lactic acid nanofiber scaffold on mouse spermatogonial stem cell culture

    Directory of Open Access Journals (Sweden)

    Eslahi N

    2013-11-01

    , as well as the ultrastructural study of cell clusters and SSCs transplantation to a recipient azoospermic mouse. The significance of the data was analyzed using the repeated measures and analysis of variance.Results: The findings indicated that the spermatogonial cells seeded on PLLA significantly increased in vitro spermatogonial cell cluster formations in comparison with the control groups (culture of SSCs not seeded on PLLA (P≤0.001. The viability rate for the frozen cells after thawing was 63.00% ± 3.56%. This number decreased significantly (40.00% ± 0.82% in spermatogonial cells obtained from the frozen-thawed testis tissue. Both groups, however, showed in vitro cluster formation. Although the expression of spermatogonial markers was maintained after 3 weeks of culture, there was a significant downregulation for some spermatogonial genes in the experimental groups compared with those of the control groups. Furthermore, transplantation assay and transmission electron microscopy studies suggested the presence of SSCs among the cultured cells.Conclusion: Although PLLA can increase the in vitro cluster formation of neonate fresh and frozen-thawed spermatogonial cells, it may also cause them to differentiate during cultivation. The study therefore has implications for SSCs proliferation and germ cell differentiation in vitro.Keywords: PLLA nanofibers, tissue cryopreservation, testis

  11. A New Movement Detector to Enable People with Multiple Disabilities to Control Environmental Stimulation with Hand Swing through a Commercial Mouse

    Science.gov (United States)

    Shih, Ching-Hsiang; Shih, Ching-Tien

    2009-01-01

    This study assessed whether two persons with profound multiple disabilities would be able to control environmental stimulation using hand swing and a standard mouse with a newly developed mouse driver (i.e. a new mouse driver replaces standard mouse driver, and turns a mouse into a precise two-dimensional motion detector). The study was performed…

  12. MSH regulation of tyrosinase in Cloudman S-91 mouse melanoma cell cultures

    International Nuclear Information System (INIS)

    Melanocyte Stimulating Hormone (MSH) causes an increase in tyrosinase activity (O-diphenol: O2 oxidoreductase) in Cloudman S-91 mouse melanoma cell cultures following a lag period of approximately 9 hours. Treatment of cells with 2 x 10-7M α- MSH for 6 days results in a 90 fold increase in the specific activity of the enzyme. The hormone mediated increase in tyrosinase activity is dependent upon continued transcription since the enzyme induction is suppressed by either cordycepin (1μg/ml) or α-amanitin (10μg/ml). To determine if MSH is increasing the synthesis rate of tyrosinase, cell cultures, either exposed to MSH for various times or left untreated, were pulsed with [3H]-leucine for 4 hours and tyrosinase immunoprecipitated with an anti-tyrosinase polyclonal antiserum raised in rabbits. The immunoprecipitates were solubilized and electrophoresed on SDS polyacrylamide gels. The proteins were electroblotted to nitrocellulose and the radioactivity in the tyrosinase bands determined. These studies have shown that while tyrosinase activity in hormone-treated cells may increase 90 fold, the rate of synthesis of the enzyme increases only 3 fold at most. Immunoprecipitation analysis of equivalence points of tyrosinase from control and MSH-treated cultures suggests the presence of inactive forms of the enzyme in melanoma cell cultures. These results suggest that, in addition to stimulating tyrosinase synthesis, MSH may also promote the activation of pre-existing enzyme molecules

  13. Binding of Candida albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages.

    OpenAIRE

    Han, Y.; Van Rooijen, N.; Cutler, J E

    1993-01-01

    We previously reported that Candida albicans yeast cells adhere to the macrophage-rich medullary and subcapsular sinus areas of mouse lymph node tissue. To determine whether the yeast cell-lymph node interaction is mediated by macrophages, the effect of specific elimination of macrophages on yeast cell binding was studied, and yeast cell adherence was correlated with the ingestion of India ink by lymph node cells. Macrophage elimination was done by use of liposome-containing dichloromethylene...

  14. Stem Cells Antigen-1 Enriches for a Cancer Stem Cell-Like Subpopulation in Mouse Gastric Cancer.

    Science.gov (United States)

    Park, Jun Won; Park, Jung Min; Park, Dong Min; Kim, Dae-Yong; Kim, Hark Kyun

    2016-05-01

    There is a strong need to identify markers to enrich gastric cancer stem cells (CSCs). However, CSC enrichment markers for mouse gastric cancers have not yet been determined. In our previous study, we generated primary mouse gastric cancer cell line NCC-S1 (S1) established from a Villin-cre;Smad4(F/F) ;Trp53(F/F) ;Cdh1(F/wt) mouse and its metastatic variant cell line NCC-S1M (S1M). Interestingly, S1M cells exhibited CSC-like features, such as increased tumorigenic potential and chemoresistance. By comparing gene expression profiles between S1 and S1M cells, we identified Stem Cells Antigen-1 (Sca-1) as a cell surface marker, which was mostly upregulated in S1M. Sca-1 was upregulated in tumorspheres from S1 cells or after cisplatin treatment in S1 cells. Immunofluorescence (IF) analysis showed that approximately 7% of cancer cells exhibited positivity for Sca-1 in primary mouse gastric cancer tissues. An in vivo-limiting dilution assay showed that Sca-1(high) mouse gastric cancer cells demonstrated increased tumorigenicity compared with Sca-1(negative) cells. The Sca-1 expression was downregulated by TGF-β pathway activation and Wnt pathway inhibition in mouse gastric cancer cells. Sca-1(high) cells showed relatively low TGF-β reporter activity and high TCF/LEF1 reporter activity compared with Sca-1(negative) cells. A chromatin immunoprecipitation analysis demonstrated that Sca-1 was a β-catenin/LEF1 target gene. Sca-1(high) allografts were more resistant to cisplatin/fluorouracil chemotherapy than Sca-1(negative) allografts, and overexpressed Bcl-xL. Eighty-five mouse genes overexpressed in Sca-1(high) S1 cells compared with Sca-1(negative) cells clustered 123 pretreatment gastric cancer patient samples according to survival following chemotherapy. Taken together, Sca-1 is a novel CSC enrichment marker that mediates TGF-β and Wnt/β-catenin signaling in mouse gastric cancer. Stem Cells 2016;34:1177-1187. PMID:26869189

  15. Transforming growth factor beta-regulated gene expression in a mouse mammary gland epithelial cell line

    International Nuclear Information System (INIS)

    Transforming growth factor beta (TGF-β) plays an essential role in a wide array of cellular processes. The most well studied TGF-β response in normal epithelial cells is growth inhibition. In some cell types, TGF-β induces an epithelial to mesenchymal transition (EMT). NMuMG is a nontransformed mouse mammary gland epithelial cell line that exhibits both a growth inhibitory response and an EMT response to TGF-β, rendering NMuMG cells a good model system for studying these TGF-β effects. A National Institutes of Aging mouse 15,000 cDNA microarray was used to profile the gene expression of NMuMG cells treated with TGF-β1 for 1, 6, or 24 hours. Data analyses were performed using GenePixPro and GeneSpring software. Selected microarray results were verified by northern analyses. Of the 15,000 genes examined by microarray, 939 were upregulated or downregulated by TGF-β. This represents approximately 10% of the genes examined, minus redundancy. Seven genes previously not known to be regulated by TGF-β at the transcriptional level (Akt and RhoB) or not at all (IQGAP1, mCalpain, actinin α3, Ikki, PP2A-PR53), were identified and their regulation by TGF-β verified by northern blotting. Cell cycle pathway examination demonstrated downregulation of cyclin D2, c-myc, Id2, p107, E2F5, cyclin A, cyclin B, and cyclin H. Examination of cell adhesion-related genes revealed upregulation of c-Jun, α-actinin, actin, myosin light chain, p120cas catenin (Catns), α-integrin, integrin β5, fibronectin, IQGAP1, and mCalpain. Using a cDNA microarray to examine TGF-β-regulated gene expression in NMuMG cells, we have shown regulation of multiple genes that play important roles in cell cycle control and EMT. In addition, we have identified several novel TGF-β-regulated genes that may mediate previously unknown TGF-β functions

  16. 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...

  17. Chromosome painting analysis of radiation-induced aberrant cell clones in the mouse

    International Nuclear Information System (INIS)

    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 137Cs 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

  18. Visualizing early splenic memory CD8+ T cells reactivation against intracellular bacteria in the mouse.

    Directory of Open Access Journals (Sweden)

    Marc Bajénoff

    Full Text Available Memory CD8(+ T cells represent an important effector arm of the immune response in maintaining long-lived protective immunity against viruses and some intracellular bacteria such as Listeria monocytogenes (L.m. Memory CD8(+ T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens. It is largely accepted that these functions are sufficient to explain how memory CD8(+ T cells can mediate rapid protection. However, it is important to point out that such improved functional features would be useless if memory cells were unable to rapidly find the pathogen loaded/infected cells within the infected organ. Growing evidences suggest that the anatomy of secondary lymphoid organs (SLOs fosters the cellular interactions required to initiate naive adaptive immune responses. However, very little is known on how the SLOs structures regulate memory immune responses. Using Listeria monocytogenes (L.m as a murine infection model and imaging techniques, we have investigated if and how the architecture of the spleen plays a role in the reactivation of memory CD8(+ T cells and the subsequent control of L.m growth. We observed that in the mouse, memory CD8(+ T cells start to control L.m burden 6 hours after the challenge infection. At this very early time point, L.m-specific and non-specific memory CD8(+ T cells localize in the splenic red pulp and form clusters around L.m infected cells while naïve CD8(+ T cells remain in the white pulp. Within these clusters that only last few hours, memory CD8(+ T produce inflammatory cytokines such as IFN-gamma and CCL3 nearby infected myeloid cells known to be crucial for L.m killing. Altogether, we describe how memory CD8(+ T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8(+ T cells provide a local response by secreting effector molecules around infected cells.

  19. Screening ToxCast™ Phase I Chemicals in a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) Assay

    Science.gov (United States)

    An Adherent Cell Differentiation and Cytotoxicity (ACDC) in vitro assay with mouse embryonic stem cells was used to screen the ToxCast Phase I chemical library for effects on cellular differentiation and cell number. The U.S. Environmental Protection Agency (EPA) established the ...

  20. Overexpression of Hepatitis B Virus-binding Protein, Squamous Cell Carcinoma Antigen 1, Extends Retention of Hepatitis B Virus in Mouse Liver

    Institute of Scientific and Technical Information of China (English)

    Hong-Bin XIA; Xi-Gu CHEN

    2006-01-01

    How receptors mediate the entry of hepatitis B virus (HBV) into the target liver cells is poorly understood. Recently, human squamous cell carcinoma antigen 1 (SCCA1) has been found to mediate binding and internalization of HBV to liver-derived cell lines in vitro. In this report, we investigate if SCCA1 is able to function as an HBV receptor and mediate HBV entry into mouse liver. SCCA1 transgene under the control of Rous sarcoma virus promoter was constructed in a minicircle DNA vector that was delivered to NOD/SCID mouse liver using the hydrodynamic technique. Subsequently, HBV-positive human serum was injected intravenously. We demonstrated that approximately 30% of the mouse liver cells expressed a high level of recombined SCCA1 protein for at least 37 d. The HBV surface antigen was found to persist in mouse liver for up to 17 d. Furthermore, HBV genome also persisted in mouse liver, as determined by polymerase chain reaction, for up to 17 d, and in mouse circulation for 7 d. These results suggest that SCAA1 might serve as an HBV receptor or co-receptor and play an important role in mediating HBV entry into hepatocytes, although its role in human HBV infection remains to be determined.

  1. Deep coverage mouse red blood cell proteome: a first comparison with the human red blood cell

    DEFF Research Database (Denmark)

    Pasini, Erica M; Kirkegaard, Morten; Salerno, Doris;

    2008-01-01

    Mice have close genetic/physiological relationships to humans, breed rapidly, and can be genetically modified, making them the most used mammal in biomedical research. Because the red blood cell (RBC) is the sole gas transporter in vertebrates, diseases of the RBC are frequently severe; much...... identified, and polypeptides migrating with anomalous apparent molecular weights were grouped into putatively ubiquitinated or partially degraded complexes. Overall there was close concordance between mouse and human proteomes, confirming the unexpected RBC complexity. Several novel findings in the human...

  2. Early remodeling of Müller cells in the rd/rd mouse model of retinal dystrophy.

    Science.gov (United States)

    Chua, Jacqueline; Nivison-Smith, Lisa; Fletcher, Erica L; Trenholm, Stuart; Awatramani, Gautam B; Kalloniatis, Michael

    2013-08-01

    We studied the anatomical remodeling and gliosis of retinal Müller cells in the rd/rd mouse model of photoreceptor degeneration. A computational calculation of glutamine synthetase immunoreactivity was developed so we could specifically quantify changes in Müller cell anatomy between control mice (C57Bl/6) and the dystrophic strain. We found no change in the number of Müller cell somata between mice strains, indicating no cell proliferation as a function of development and degeneration. The retinal area occupied by the total Müller cell body (soma and processes) was significantly less in the rd/rd mouse retina compared with control mice. When only the outer retina was considered, we found rd/rd Müller cell processes were dramatically reduced during the cone phase of photoreceptor degeneration. However, at older ages an increase in Müller cell processes was seen. Conversely, glial fibrillary acidic protein (GFAP) expression showed a significant increase during cone degeneration followed by a reduction in older ages. Müller cell electrophysiology, particularly K(+) currents and membrane potential, was similar between rd/rd and control Müller cells during cone degeneration. Together, these results show that glial remodeling in the rd/rd retina follows separate phases-an initial conservative glial response involving the loss of Müller cells processes, hyperexpression of GFAP, and preservation of normal electrophysiology followed by an active growth of Müller cell processes, glial seal formation, and attenuation of GFAP expression after complete photoreceptor loss. PMID:23348616

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

    International Nuclear Information System (INIS)

    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

  4. 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

  5. Cell-specific oxidative stress and cytotoxicity after wildfire coarse particulate matter instillation into mouse lung

    International Nuclear Information System (INIS)

    Our previous work has shown that coarse particulate matter (PM10-2.5) from wildfire smoke is more toxic to lung macrophages on an equal dose (by mass) basis than coarse PM isolated from normal ambient air, as evidenced by decreased numbers of macrophages in lung lavage fluid 6 and 24 hours after PM instillation into mouse lungs in vivo and by cytotoxicity to a macrophage cell line observed directly in vitro. We hypothesized that pulmonary macrophages from mice instilled with wildfire coarse PM would undergo more cytotoxicity than macrophages from controls, and that there would be an increase in oxidative stress in their lungs. Cytotoxicity was quantified as decreased viable macrophages and increased percentages of dead macrophages in the bronchoalveolar lavage fluid (BALF) of mice instilled with wildfire coarse PM. At 1 hour after PM instillation, we observed both decreased numbers of viable macrophages and increased dead macrophage percentages as compared to controls. An increase in free isoprostanes, an indicator of oxidative stress, from control values of 28.1 ± 3.2 pg/mL to 83.9 ± 12.2 pg/mL was observed a half-hour after PM instillation. By 1 hour after PM instillation, isoprostane values had returned to 30.4 ± 7.6 pg/mL, not significantly different from control concentrations. Lung sections from mice instilled with wildfire coarse PM showed rapid Clara cell responses, with decreased intracellular staining for the Clara cell secretory protein CCSP 1 hour after wildfire PM instillation. In conclusion, very rapid cytotoxicity occurs in pulmonary macrophages and oxidative stress responses are seen 0.5–1 hour after wildfire coarse PM instillation. These results define early cellular and biochemical events occurring in vivo and support the hypothesis that oxidative stress-mediated macrophage toxicity plays a key role in the initial response of the mouse lung to wildfire PM exposure. -- Highlights: ► We studied very early events (0.5–1 hour) after giving

  6. Vitamin E Modulates Cigarette Smoke Extract-induced Cell Apoptosis in Mouse Embryonic Cells

    Directory of Open Access Journals (Sweden)

    Zhao-Li Chen, Jian Tao, Jie Yang, Zhen-Li Yuan, Xing-Hua Liu, Min Jin, Zhi-Qiang Shen, Lu Wang, Hai-Feng Li, Zhi-Gang Qiu, Jing-Feng Wang, Xin-Wei Wang, Jun-Wen Li

    2011-01-01

    Full Text Available Vitamin E (VE can effectively prevent occurrence of lung cancer caused by passive smoking in mice. However, whether VE prevents smoking-induced cytotoxicity remains unclear. In this study, a primary culture of embryonic lung cells (ELCs was used to observe the cytotoxic effects of cigarette smoke extract (CSE, including its influence on cell survival, cell cycle, apoptosis, and DNA damage, and also to examine the effects of VE intervention on CSE-induced cytotoxicity. Our results showed that CSE could significantly inhibit the survival of ELCs with dose- and time-dependent effects. Furthermore, CSE clearly disturbed the cell cycle of ELCs by decreasing the proportion of cells at the S and G2/M phases and increasing the proportion of cells at the G0/G1 phase. CSE promoted cell apoptosis, with the highest apoptosis rate reaching more than 40%. CSE also significantly caused DNA damage of ELCs. VE supplementation could evidently inhibit or reverse the cytotoxic effects of CSE in a dose- and time-dependent manner. The mechanism of CSE effects on ELCs and that of VE intervention might involve the mitochondrial pathway of cytochrome c-mediated caspase activation. Our study validate that VE plays a clearly protective effect against CSE-induced cytotoxicity in mouse embryonic lung cells.

  7. Establishment of leptin-Responsive cell lines from adult mouse hypothalamus

    OpenAIRE

    Iwakura, Hiroshi; Dote, Katsuko; Bando, Mika; Koyama, Hiroyuki; Hosoda, Kiminori; Kangawa, Kenji; Nakao, Kazuwa

    2016-01-01

    Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus-derived cell lines, termed adult mouse hypothalamu...

  8. Establishment of Leptin-Responsive Cell Lines from Adult Mouse Hypothalamus

    OpenAIRE

    Hiroshi Iwakura; Katsuko Dote; Mika Bando; Hiroyuki Koyama; Kiminori Hosoda; Kenji Kangawa; Kazuwa Nakao

    2016-01-01

    Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus-derived cell lines, termed adult mouse hypothalamu...

  9. Establishment of Leptin-Responsive Cell Lines from Adult Mouse Hypothalamus

    OpenAIRE

    Iwakura, Hiroshi; Dote, Katsuko; Bando, Mika; Koyama, Hiroyuki; Hosoda, Kiminori; Kangawa, Kenji; Nakao, Kazuwa

    2016-01-01

    Leptin resistance is considered to be the primary cause of obesity. However, the cause of leptin resistance remains incompletely understood, and there is currently no cure for the leptin-resistant state. In order to identify novel drug-target molecules that could overcome leptin resistance, it would be useful to develop in vitro assay systems for evaluating leptin resistance. In this study, we established immortalized adult mouse hypothalamus—derived cell lines, termed adult mouse hypothalamu...

  10. Sel Kumulus Sebagai Feeder Layer pada Kultur Stem Cells Embrionic Mencit (CUMULUS CELLS AS FEEDER LAYER IN CULTURE OF MOUSE EMBRYONIC STEM CELLS

    Directory of Open Access Journals (Sweden)

    Thomas Mata Hine

    2014-08-01

    Full Text Available The purpose of this study was to verify the effectiveness of cumulus cells as a feeder layer in supportingthe growth of mouse embryonic stem cells. Embryos at blastocyst stage were exposed in pronase solution,and then incubated in rabbit anti-mouse antibody and guinea pig complement to lyse and separate thetrophoblast cells from the inner cell mass. Inner cell mass subsequently cultured in a petri dish containinga cumulus feeder layer, mouse embryonic fibroblasts, or without a feeder layer, in stem cells medium. Theresulting stem cells colony passaged in trypsin solution, pipetted repeatedly to produce subcolonies orsingle cells, and cultured as before in some new petri dishes. Characterization of stem cells was identifiedby using alkaline phosphatase staining. The results showed the effectiveness of cumulus cells as feederlayer for culture of mouse embryonic stem cells was comparable with mouse embryonic fibroblasts, andboth of them were better than without a feeder layer method. The number of primary colony, cell lines, andcolony growth rate increased 41.30, 8.70, and 54.20%, respectively, while doubling time was shorter 10:21hours compared to the growth without feeder layer method. These results prove that the cumulus feederlayer effectively supports the growth of mouse embryonic stem cells.

  11. Iron uptake mediated by binding of H-ferritin to the TIM-2 receptor in mouse cells.

    Science.gov (United States)

    Han, Jian; Seaman, William E; Di, Xiumin; Wang, Wei; Willingham, Mark; Torti, Frank M; Torti, Suzy V

    2011-01-01

    Ferritin binds specifically and saturably to a variety of cell types, and recently several ferritin receptors have been cloned. TIM-2 is a specific receptor for H ferritin (HFt) in the mouse. TIM-2 is a member of the T cell immunoglobulin and mucin domain containing (TIM) protein family and plays an important role in immunity. The expression of TIM-2 outside of the immune system indicates that this receptor may have broader roles. We tested whether ferritin binding to TIM-2 can serve as an iron delivery mechanism. TIM-2 was transfected into normal (TCMK-1) mouse kidney cells, where it was appropriately expressed on the cell surface. HFt was labeled with (55)Fe and (55)Fe-HFt was incubated with TIM-2 positive cells or controls. (55)Fe-HFt uptake was observed only in TIM-2 positive cells. HFt uptake was also seen in A20 B cells, which express endogenous TIM-2. TIM-2 levels were not increased by iron chelation. Uptake of (55)Fe-HFt was specific and temperature-dependent. HFt taken up by TIM-2 positive cells transited through the endosome and eventually entered a lysosomal compartment, distinguishing the HFt pathway from that of transferrin, the classical vehicle for cellular iron delivery. Iron delivered following binding of HFt to TIM-2 entered the cytosol and became metabolically available, resulting in increased levels of endogenous intracellular ferritin. We conclude that TIM-2 can function as an iron uptake pathway. PMID:21886823

  12. Expression of human epidermal growth factor pressures cDNA in transfected mouse NIH 3T3 cells

    International Nuclear Information System (INIS)

    Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 μM ZnCl2. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is ≅ 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with 125I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself

  13. 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-spec...... cell mass and curing diabetes in animals that have been chemically depleted of beta cells....

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

    International Nuclear Information System (INIS)

    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)

  15. Electrical determination of viability in saline-treated mouse myeloma cells.

    OpenAIRE

    Matsushita, T.; Brendzel, A M; Shotola, M A; Groh, K R

    1982-01-01

    Suspension of mouse myeloma cells in phosphate buffered saline (PBS) induced a significant amount of cell death. The lethal effects of PBS include an increase in cell lysis, a decreased ability of cells to exclude trypan blue, and a decrease in the colony-forming ability of these cells. Dead cells were also detected on a Coulter counter by the increase in the fraction of cells with a smaller electrical size distribution (ESD). Comparing mixtures of live and dead cells by ESD and trypan-blue e...

  16. The characteristics of NK cells in Schistosoma japonicum-infected mouse spleens.

    Science.gov (United States)

    Li, Lu; Cha, Hefei; Yu, Xiuxue; Xie, Hongyan; Wu, Changyou; Dong, Nuo; Huang, Jun

    2015-12-01

    Natural killer (NK) cells are classic innate immune cells that play roles in many types of infectious disease. Recently, some new characteristics of NK cells were discovered. In this study, C57BL/6 mice were infected with Schistosoma japonicum for 5-6 weeks and lymphocytes were isolated from the spleen to detect some of the NK cell characteristics by multiparametric flow cytometry. The results revealed that the S. japonicum infection induced a large amount of NK cells, although the percentage of NK cells was not increased significantly. At the same time, the results showed that infected mouse splenic NK cells expressed increased levels of CD25 and CD69 and produced more IL-2, IL-4, and IL-17 and less IFN-γ after stimulation with PMA and ionomycin. This meant that NK cells played a role in S. japonicum infection. Moreover, decreased NKG2A/C/E (CD94) expression levels were detected on the surface of NK cells from infected mouse spleens, which might serve as a NK cell activation mechanism. Additionally, high levels of IL-10, but not PD-1, were expressed on the infected mouse NK cells, which implied that functional exhaustion might exist in the splenic NK cells from S. japonicum-infected mice. Collectively, our results suggest that NK cells play important roles in the course of S. japonicum infection. PMID:26319521

  17. Folate receptor α regulates cell proliferation in mouse gonadotroph αT3-1 cells

    International Nuclear Information System (INIS)

    We have previously found that the mRNA and protein levels of the folate receptor alpha (FRα) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FRα has not fully been determined. We investigated the effect of FRα over-expression in the mouse gonadotroph αT3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FRα were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FRα promotes cell proliferation. These effects were abrogated in the same αT3-1 cells when transfected with a mutant FRα cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FRα over-expressing cells. In summary, our data suggests that FRα regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.

  18. MPG protection and goblet cell kinetics in mouse jejunum

    International Nuclear Information System (INIS)

    Experiments were undertaken to study the goblet cell changes in the jejunum of mice irradiated in the presence and absence of the protective drug 2-mercaptopropionylglycine (MPG). One group of adult Swiss albino mice of 6 to 8 weeks was injected with the drug (MPG) intraperitoneally and a second group was injected with distilled water in the same manner and served as a control. 15-30 minutes after injection animals from both the groups were exposed to a Co-60 source to give a total dose of 1000 R at the rate of 25 R/min. Three animals from each group were sacrificed at different successive intervals. 5μ paraffin sections of the jejunum were prepared and goblet cells were counted in the crypt and villus region. The results from the two groups were compared. The results from MPG treated mice indicated that there was a protection from the radiation-induced changes. The drug accelerated the regeneration process leading to the restoration of normal number of goblet cells by the last interval studied. (orig.)

  19. Lymphotactin enhances the in-vitro immune efficacy of dendritoma formed by dendritic cells and mouse hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    张浩; 蒋国平; 郑树森; 吴丽花; 朱峰; 杨振林

    2004-01-01

    Objective: To investigate the in-vitro antitumor immune responses of dendritoma formed by mouse hepatocellular carcinoma (HCC) cells and lymphotactin (Lptn) gene modified dendritic cells (DCs). Method: DCs prepared from mouse bone marrow were genetically modified by lymphotactin adenovirus, and fused with H22 cells by polyethylene glycol (PEG). RT-PCR and ELISA were employed to identify lymphotactin expression at mRNA and protein level. Cell phenotypes and fusion efficiency was detected by FACS. The stimulatory effect of DC on T cells was detected by mixed lymphocyte reaction. The cytotoxicity activity against H22 cells was assayed by LDH method. Results: Lymphotactin could be efficiently expressed by DCLptn/H22 hybridoma. DCLptn/H22 cells could induce potent T cell proliferation effect and generate strong cytotoxic T lymphocyte (CTL) reaction against allogenic H22 cells. Conclusion: Lymphotactin genetic modification could enhance the in vitro immune activity of the dendritoma.

  20. Single-molecule transcript counting of stem-cell markers in the mouse intestine

    NARCIS (Netherlands)

    Itzkovitz, S.; Lyubimova, A.; Blat, I.C.; Maynard, M.; van Es, J.H.; Lees, J.; Jacks, T.; Clevers, H.; van Oudenaarden, A.

    2012-01-01

    Determining the molecular identities of adult stem cells requires technologies for sensitive transcript detection in tissues. In mouse intestinal crypts, lineage-tracing studies indicated that different genes uniquely mark spatially distinct stem-cell populations, residing either at crypt bases or a

  1. Interstitial cells of Cajal in the striated musculature of the mouse esophagus

    DEFF Research Database (Denmark)

    Rumessen, J J; de Kerchove d'Exaerde, A; Mignon, S; Bernex, F; Timmermans, J P; Schiffmann, S N; Panthier, J J; Vanderwinden, J M

    2001-01-01

    Interstitial cells of Cajal (ICC) are important regulatory cells in the smooth muscle coats of the digestive tract. Expression of the Kit receptor tyrosine kinase was used in this study as a marker to study their distribution and development in the striated musculature of the mouse esophagus. Sec...

  2. Modified genetic response to X-irradiation of mouse spermatogonial stem cells surviving treatment with TEM

    International Nuclear Information System (INIS)

    Earlier studies have shown that the genetic response to X-irradiation of mouse spermatogonial stem-cell populations that are recovering from a previous radiation exposure may differ from that of a normal, unirradiated stem-cell population. Similar modified responses to X-irradiation have now been observed in stem spermatogonia that are recovering from treatment with the chemical mutagen, TEM. (orig.)

  3. 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...

  4. Development of functional human embryonic stem cell-derived neurons in mouse brain

    OpenAIRE

    Muotri, Alysson R.; Nakashima, Kinichi; Toni, Nicolas; Sandler, Vladislav M.; Gage, Fred H

    2005-01-01

    Human embryonic stem cells are pluripotent entities, theoretically capable of generating a whole-body spectrum of distinct cell types. However, differentiation of these cells has been observed only in culture or during teratoma formation. Our results show that human embryonic stem cells implanted in the brain ventricles of embryonic mice can differentiate into functional neural lineages and generate mature, active human neurons that successfully integrate into the adult mouse forebrain. Moreo...

  5. Establishment of Conditionally Immortalized Mouse Glomerular Parietal Epithelial Cells in Culture

    OpenAIRE

    Ohse, Takamoto; Pippin, Jeffrey W.; Vaughan, Michael R.; Brinkkoetter, Paul T.; Krofft, Ronald D.; Shankland, Stuart J.

    2008-01-01

    Parietal epithelial cells (PEC) are major constituents of crescents in crescentic glomerulonephritis. The purpose of these studies was to establish an immortalized PEC cell line with similar characteristics to PEC in vivo for use in future mechanistic studies. Glomeruli were isolated from H-2Kb tsA58 transgenic mice (ImmortoMouse) by standard differential sieving, and several candidate PEC cell lines were obtained by subcloning outgrowths of cells from capsulated glomeruli. One clone, designa...

  6. Radioresistance of cells responsible for delayed hypersensitivity reactions in the mouse

    International Nuclear Information System (INIS)

    The cells responsible for delayed hypersensitivity reactivity in the mouse act in a radioresistant fashion only if such irradiated cells are injected directly into the site of antigen challenge. Intravenous transfer of irradiated, primed spleen cells does not achieve a transfer of sensitivity to show delayed type hypersensitivity reactions. Transfer of sensitivity by the intravenous route can be effected by injection of non-irradiated spleen cells from primed mice. (U.S.)

  7. Binding and uptake of diphtheria toxin by toxin-resistant Chinese hamster ovary and mouse cells.

    OpenAIRE

    Didsbury, J R; Moehring, J M; Moehring, T. J.

    1983-01-01

    We investigated two phenotypically distinct types of diphtheria toxin-resistant mutants of Chinese hamster cells and compared their resistance with that of naturally resistant mouse cells. All are resistant due to a defect in the process of internalization and delivery of toxin to its target in the cytosol, elongation factor 2. By cell hybridization studies, analysis of cross-resistance, and determination of specific binding sites for 125I-labeled diphtheria toxin, we showed that these cell s...

  8. Epithelial basal cells are distinct from dendritic cells and macrophages in the mouse epididymis.

    Science.gov (United States)

    Shum, Winnie W; Smith, Tegan B; Cortez-Retamozo, Virna; Grigoryeva, Lubov S; Roy, Jeremy W; Hill, Eric; Pittet, Mikael J; Breton, Sylvie; Da Silva, Nicolas

    2014-05-01

    The epithelium that lines the epididymal duct establishes the optimal milieu in which spermatozoa mature, acquire motility, and are stored. This finely tuned environment also protects antigenic sperm against pathogens and autoimmunity, which are potential causes of transient or permanent infertility. The epididymal epithelium is pseudostratified and contains basal cells (BCs) that are located beneath other epithelial cells. Previous studies showed that in the mouse epididymis, BCs possess macrophage-like characteristics. However, we previously identified a dense population of cells belonging to the mononuclear phagocyte (MP) system (comprised of macrophages and dendritic cells) in the basal compartment of the mouse epididymis and showed that a subset of MPs express the macrophage marker F4/80. In the present study, we evaluate the distribution of BCs and MPs in the epididymis of transgenic CD11c-EYFP mice, in which EYFP is expressed exclusively in MPs, using antibodies against the BC marker keratin 5 (KRT5) and the macrophage marker F4/80. Immunofluorescence labeling for laminin, a basement membrane marker, showed that BCs and most MPs are located in the basal region of the epithelium. Confocal microscopy showed that in the initial segment, both BCs and MPs project intraepithelial extensions and establish a very intricate network. Flow cytometry experiments demonstrated that epididymal MPs and BCs are phenotypically distinct. BCs do not express F4/80, and MPs do not express KRT5. Therefore, despite their proximity and some morphological similarities with peritubular macrophages and dendritic cells, BCs do not belong to the MP system. PMID:24648397

  9. Epigenetic modifications and self-renewal regulation of mouse germline stem cells

    Institute of Scientific and Technical Information of China (English)

    Jiyoung Lee; Takashi Shinohara

    2011-01-01

    Germline stem (GS) cells were established from gonocytes and spermatogonia of postnatal mouse testes. GS cells proliferate in the presence of several kinds of cytokines, and a small percentage of GS cells also show spermatogonial stem cell (SSC) activity, i.e., they differentiate into sperm after being transplanted into infertile mouse testes without endogenous spermatogenesis. Interestingly, in GS cell culture, we also found that pluripotent stem cells (multipotent germline stem cells (mGS cells)) could be derived and these mGS cells do not have normal androgenetic genomic imprinting marks that are shown in GS cells, e.g., H19 hypermethylation. A new culture system for fetal male germ cells (embryonic GS (eGS) cells) has also been recently developed. Although these cells exhibited SSC potential, the offspring from cultured cells showed heritable imprinting defects in their DNA methylation patterns. In an attempt to understand the self-renewal machinery in SSCs, we transfected H-Ras and cylin D2 into GS cells, and successfully reconstructed the SSC self-renewal ability without using exogenous cytokines. Although these cells showed SSC activity in germ cell transplantation assays, we also found development of seminomatous tumors, possibly induced by excessive self-renewing signal. These stem cell culture systems are useful tools not only for understanding the mechanisms of self-renewal or epigenetic reprogramming but also for clarifying the mechanism of germ cell tumor development.

  10. Stepwise Development of MAIT Cells in Mouse and Human

    OpenAIRE

    Laurent Gapin

    2009-01-01

    Mucosal-associated invariant T cells are newly identified subpopulation of T cells. A new study highlights their developmental pathway and functional features that allow these cells to assume a unique position in the family of unconventional T cells.

  11. Control of T cell infiltration and tumor rejection by regulatory T cells, basophils and macrophages

    OpenAIRE

    Sektioglu, Ibrahim Murathan

    2015-01-01

    Most solid tumors are intrinsically resistant to immune rejection due to immunosuppressive mechanisms operative within the tumor microenvironment. Cancer patients frequently harbor elevated numbers of regulatory T cells (Tregs), which inhibit efficient anti-tumor T cell responses. We employed different mouse models for Treg depletion in order to study the mechanisms that control tumor rejection. Depletion of about 99% Tregs in Foxp3DTR knock-in mice resulted in complete rejection of transplan...

  12. F-actin mechanics control spindle centring in the mouse zygote

    Science.gov (United States)

    Chaigne, Agathe; Campillo, Clément; Voituriez, Raphaël; Gov, Nir S.; Sykes, Cécile; Verlhac, Marie-Hélène; Terret, Marie-Emilie

    2016-01-01

    Mitotic spindle position relies on interactions between astral microtubules nucleated by centrosomes and a rigid cortex. Some cells, such as mouse oocytes, do not possess centrosomes and astral microtubules. These cells rely only on actin and on a soft cortex to position their spindle off-centre and undergo asymmetric divisions. While the first mouse embryonic division also occurs in the absence of centrosomes, it is symmetric and not much is known on how the spindle is positioned at the exact cell centre. Using interdisciplinary approaches, we demonstrate that zygotic spindle positioning follows a three-step process: (1) coarse centring of pronuclei relying on the dynamics of an F-actin/Myosin-Vb meshwork; (2) fine centring of the metaphase plate depending on a high cortical tension; (3) passive maintenance at the cell centre. Altogether, we show that F-actin-dependent mechanics operate the switch between asymmetric to symmetric division required at the oocyte to embryo transition.

  13. GABA transporters control GABAergic neurotransmission in the mouse subplate.

    Science.gov (United States)

    Unichenko, P; Kirischuk, S; Luhmann, H J

    2015-09-24

    The subplate is a transient layer between the cortical plate and intermediate zone in the developing cortex. Thalamo-cortical axons form temporary synapses on subplate neurons (SPns) before invading the cortical plate. Neuronal activity within the subplate is of critical importance for the development of neocortical circuits and architecture. Although both glutamatergic and GABAergic inputs on SPns were reported, short-term plasticity of GABAergic transmission has not been investigated yet. GABAergic postsynaptic currents (GPSCs) were recorded from SPns in coronal neocortical slices prepared from postnatal day 3-4 mice using whole-cell patch-clamp technique. Evoked GPSCs (eGPSCs) elicited by electrical paired-pulse stimulation demonstrated paired-pulse depression at all interstimulus intervals tested. Baclofen, a specific GABAB receptor (GABABR) agonist, reduced eGPSC amplitudes and increased paired-pulse ratio (PPR), suggesting presynaptic location of functional GABABRs. Baclofen-induced effects were alleviated by (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid (CGP55845), a selective GABABR blocker. Moreover, CGP55845 increased eGPSC amplitudes and decreased PPR even under control conditions, indicating that GABABRs are tonically activated by ambient GABA. Because extracellular GABA concentration is mainly regulated by GABA transporters (GATs), we asked whether GATs release GABA. 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid (NNC-711) (10μM), a selective GAT-1 blocker, increased eGPSC decay time, decreased eGPSC amplitudes and PPR. The two last effects but not the first one were blocked by CGP55845, indicating that GAT-1 blockade causes an elevation of extracellular GABA concentration and in turn activation of extrasynaptic GABAARs and presynaptic GABABRs. 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3-piperidinecarboxylic acid (SNAP-5114), a specific GAT-2/3 blocker, failed

  14. Activity of neutral endopeptidase and aminopeptidase N in mouse thymic stromal cells which bind double-positive thymocytes.

    Science.gov (United States)

    Small, M; Kaiser, M; Tse, W; Heimfeld, S; Blumberg, S

    1996-04-01

    The activity of two peptidases was determined in immortalized lines of thymic stromal cells. A line of total stromal cells (T-TG-St) was grown from transgenic mouse expressing temperature-sensitive SV40 T antigen under the control of the regulatory elements of the mouse major histocompatibility complex class I gene. From these cells we isolated a subset (DP-TG-St) that binds thymocytes which are mainly CD4+8+. We also assayed a clone of fetal thymic epithelial cells (BA/10) that binds CD4+8+ thymocytes. Both lines of double -positive cell-binding stroma exhibited strong activity of two peptidases, neutral endopeptidase (NEP; EC 3.4.24.11) and aminopeptidase N (APN; EC 3.4.11.2). In contrast, the activity of both enzymes was very low in the total thymic stromal line. Use of the specific inhibitors confirmed that these two enzymes were responsible for the activity observed but also suggested the presence of additional unidentified aminopeptidase(s) in the same stromal cells. The high activity of the two peptidases on stromal cells that bind thymocytes at the double-positive stage raises the possibility that they might contribute to the microenvironment of the developing thymocytes. PMID:8625997

  15. Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures.

    Directory of Open Access Journals (Sweden)

    Cara L Sherwood

    Full Text Available Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE cell model we found that both micromolar (3.9 μM and submicromolar (0.8 μM arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-. We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.

  16. Involvement of insulin in early development of mouse one-cell stage embryos

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Recent studies have suggested that growth factors and hormones play important roles in cell prolif-eration and differentiation during early embryonic development. In the present study, we examined the expression and localization of insulin in the mouse oocytes and one-cell stage embryos by quantitative ELISA, RT-PCR, Western blot and immunofluorescence. In the mouse oocytes and one-cell stage em-bryos, expression of insulin was uniformly distributed in the cytoplasm. We also examined the expres-sion, activity and localization of mTOR (mammalian target of rapamycin) and p70S6K. The expression of mTOR and p70S6K was not significantly different at the cell cycle of mouse one-cell stage embryos. mTOR and S6K were distributed evenly in the cytoplasm at G1, G2 and M phase phase, but at S phase, the distribution of mTOR and S6K was around the pronucleus. At different phases, the activity of mTOR fluctuated. We also used the PI3K specific inhibitor-Wortmannin to investigate the cleavage rate of eggs. The result showed that the rate obviously decreased. When the mTOR specific inhibitor Rapa-mycin was used, the first mitotic division of the mouse one-cell stage embryo was delayed. These re-sults suggested that insulin was expressed both in mouse oocytes and one-cell stage embryos, and may play functional roles in regulation of mouse early embryogenesis by activating the signal pathway of PI3K/PKB/mTOR/S6K.

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

    International Nuclear Information System (INIS)

    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 106 receptors per cell. The cell line with the highest 125I-insulin binding (NIH 3T3 HIR3.5) had 6 x 106 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 107 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-08-01

    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/sup 6/ receptors per cell. The cell line with the highest /sup 125/I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10/sup 6/ receptors with a K/sub d/ of 10/sup -9/ M. This level was not dependent on exposure to metals but could be increased further to 2 x 10/sup 7/ receptors per cell by addition of sodium butyrate to the culture medium. The ..cap alpha.. and ..beta.. 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 ..cap alpha.. and ..beta.. 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

  19. 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...... gland of mice. Actively phagocytosing cells with a prominent lysosomal system were found in the pericapillary spaces of the mouse pineal gland following intravenous injection of horseradish peroxidase. The cells also exhibited strong acid phosphatase activity. Perivascular cells were immunopositive 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....

  20. 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.

    2014-01-01

    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 are unable to support development of human innate immune cells, including myeloid cells and NK cells. Here we describe a mouse strain, called MI(S)TRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked in to their respective mouse loci. The human cytokines support the development and function of monocytes/macrophages and natural killer cells derived from human fetal liver or adult CD34+ progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MI(S)TRG 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. PMID:24633240

  1. Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells

    International Nuclear Information System (INIS)

    A chimeric mouse-human antibody has been created that recognizes an antigen found on the surface of cells from many carcinomas. Immunoglobulin constant (C) domains of the mouse monoclonal antibody L6, C/sub γ2a/ and C/sub kappa/, were substituted by the human C/sub γ1/ and C/sub kappa/ by recombining cDNA modules encoding variable or C domains. The cDNA constructs were transfected into lymphoid cells for antibody production. The chimeric antibody and mouse L6 antibody bound to carcinoma cells with equal affinity and mediated complement-dependent cytolysis. In the presence of human effector cells, the chimeric antibody gave antibody-dependent cellular cytotoxicity at 100 times lower concentration than that needed for the mouse L6 antibody. The assay for lysis was carried out with 51Cr-labeled target calls. The chimeric antibody, but not the mouse L6 antibody, is effective against a melanoma line expressing small amounts of the L6 antigen. The findings point to the usefulness of the chimeric antibody approach for obtaining agents with strong antitumor activity for possible therapeutic use in man

  2. Effect of Different High CO2 Concentrations on the Development of 2-cell Mouse Embryos in vitro

    Institute of Scientific and Technical Information of China (English)

    Li-hua LU; Wei-jie ZHU

    2003-01-01

    Objective To investigate effects of different high CO2 concentrations on the development of 2-cell mouse embryos in vitroMethods At levels of 5% CO2 (control group), 5.7% CO2, 6.0% CO2 and 15% CO2, embryos were incubated in drops with CZB medium, respectively, and the drops were covered by paraffin oil which was treated with three-distilled water. In addition, at the level of 15% CO2, there were another two groups, in which paraffin oil was treated with phosphate-buffered saline (PBS) solution or the drops were uncovered. The development of embryos in all stages was noted.Results The developmental rates of blastocysts in five experimental groups were significantly lower than that of the control group (P0.05). At the level of 15% CO2, 15% embryos developed in the 4-cell stage with irregular blastomere and degenerated quickly in the group which paraffin oil was treated with distilled water; 2.2% embryos developed in the 4-cell stage in the group which paraffin oil was treated with PBS and the rest stagnated in the 2-cell stage. Conclusions High CO2 concentrations had toxic effect on the in vitro development of 2-cell mouse embryos, and was responsible for the inhibition of the embryos. It is important for the development of embryos in vitro to detect strictly CO2 concentration.

  3. Effect of bone marrow derived mesenchymal stem cells on lung pathology and inflammation in ovalbumin-induced asthma in mouse

    Directory of Open Access Journals (Sweden)

    Maryam Mohammadian

    2016-01-01

    Full Text Available Objective(s:Bone marrow-derived mesenchymal stem cells (BMSCs have attracted significant interest to treat asthma and its complication. In this study, the effects of BMSCs on lung pathology and inflammation in an ovalbumin-induced asthma model in mouse were examined. Materials and Methods:BALB/c mice were divided into three groups: control group (animals were not sensitized, asthma group (animals were sensitized by ovalbumin, asthma+BMSC group (animals were sensitized by ovalbumin and treated with BMSCs. BMSCs were isolated and characterized and then labeled with Bromodeoxyuridine (BrdU. After that the cells transferred into asthmatic mice. Histopathological changes of the airways, BMSCs migration and total and differential white blood cell (WBC count in bronchoalveolar lavage (BAL fluid were evaluated. Results:A large number of BrdU-BMSCs were found in the lungs of mice treated with BMSCs. The histopathological changes, BAL total WBC counts and the percentage of neutrophils and eosinophils were increased in asthma group compared to the control group. Treatment with BMSCs significantly decreased airway pathological indices, inflammatory cell infiltration, and also goblet cell hyperplasia. Conclusion:The results of this study revealed that BMSCs therapy significantly suppressed the lung pathology and inflammation in the ovalbumin induced asthma model in mouse.

  4. Ultrasound fails to induce proliferation of human brain and mouse endothelial cell lines

    Science.gov (United States)

    Rodemer, Claus; Jenne, Jürgen; Fatar, Marc; Hennerici, Michael G.; Meairs, Stephen

    2012-11-01

    Both in vitro and in vivo studies suggest that ultrasound (US) is capable of inducing angiogenesis. There is no information, however, on whether ultrasound can induce proliferation of brain endothelial cells. We therefore explored the angiogenic potential of ultrasound on a novel immortalised human brain endothelial cell line (hCMEC/D3) and on mouse brain microvascular endothelial cells (bEND3). Ultrasound failed to enhance cell proliferation in both cell lines at all acoustic pressures studied. Endothelial cell damage occurred at 0.24 MPa with significantly slower proliferation. Cells growing in Opticell{trade mark, serif} dishes did not show damage or reduced proliferation at these pressures.

  5. Endogenous retinoic acid activity in principal cells and intercalated cells of mouse collecting duct system.

    Directory of Open Access Journals (Sweden)

    Yuen Fei Wong

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

  6. Repopulation dynamics of single haematopoietic stem cells in mouse transplantation experiments: Importance of stem cell composition in competitor cells.

    Science.gov (United States)

    Ema, Hideo; Uchinomiya, Kouki; Morita, Yohei; Suda, Toshio; Iwasa, Yoh

    2016-04-01

    The transplantation of blood tissues from bone marrow into a lethally irradiated animal is an experimental procedure that is used to study how the blood system is reconstituted by haematopoietic stem cells (HSC). In a competitive repopulation experiment, a lethally irradiated mouse was transplanted with a single HSC as a test cell together with a number of bone marrow cells as competitor cells, and the fraction of the test cell progeny (percentage of chimerism) was traced over time. In this paper, we studied the stem cell kinetics in this experimental procedure. The balance between symmetric self-renewal and differentiation divisions in HSC determined the number of cells which HSC produce and the length of time for which HSC live after transplantation. The percentage of chimerism depended on the type of test cell (long-, intermediate-, or short-term HSC), as well as the type and number of HSC included in competitor cells. We next examined two alternative HSC differentiation models, one-step and multi-step differentiation models. Although these models differed in blood cell production, the percentage of chimerism appeared very similar. We also estimated the numbers of different types of HSC in competitor cells. Based on these results, we concluded that the experimental results inevitably include stochasticity with regard to the number and the type of HSC in competitor cells, and that, in order to detect different types of HSC, an appropriate number of competitor cells needs to be used in transplantation experiments. PMID:26802482

  7. Radioprotective effects of hawthorn fruit extract against gamma irradiation in mouse bone marrow cells

    International Nuclear Information System (INIS)

    The radioprotective effect of hawthorn (Crataegus microphylla) fruit extract against genotoxicity induced by gamma irradiation has been investigated in mouse bone marrow cells. A single intraperitoneal (ip) administration of hawthorn extract at doses of 25, 50, 100 and 200 mg/kg 1 h prior to gamma irradiation (2 Gy) reduced the frequencies of micronucleated polychromatic erythrocytes (MnPCEs). All four doses of hawthorn extract significantly reduced the frequencies of MnPCEs and increased the PCE/PCE+NCE ratio (polychromatic erythrocyte/polychromatic erythrocyte+normochromatic erythrocyte) in mice bone marrow compared with the non drug-treated irradiated control (p<0.02-0.00001). The maximum reduction in MnPCEs was observed in mice treated with extract at a dose of 200 mg/kg. Administration of amifostine at dose 100 mg/kg and hawthorn at dose 200 mg/kg reduced the frequency of MnPCE almost 4.8 and 5.7 fold; respectively, after being exposed to 2 Gy of gamma rays, compare with the irradiated control group. Crataegus extract exhibited concentration-dependent activity on 1, 1-diphenyl 2-picrylhydrazyl free radical showing that Crataegus contained high amounts of phenolic compounds and the high performance liquid chromatography (HPLC) analysis determined that it contained chlorogenic acid, epicatechin and hyperoside. It appeared that hawthorn extract with antioxidant activity reduced the genotoxicity induced by gamma irradiation in bone marrow cells. (author)

  8. 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.

  9. Cell swelling activates ATP-dependent voltage-gated chloride channels in M-1 mouse cortical collecting duct cells

    OpenAIRE

    1996-01-01

    In the present study we used whole-cell patch clamp recordings to investigate swelling-activated Cl-currents (ICl-swell) in M-1 mouse cortical collecting duct (CCD) cells. Hypotonic cell swelling reversibly increased the whole-cell Cl- conductance by about 30-fold. The I-V relationship was outwardly-rectifying and ICl-swell displayed a characteristic voltage-dependence with relatively fast inactivation upon large depolarizing and slow activation upon hyperpolarizing voltage steps. Reversal po...

  10. RYBP and Cbx7 Define Specific Biological Functions of Polycomb Complexes in Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Lluis Morey

    2013-01-01

    Full Text Available The Polycomb repressive complex 1 (PRC1 is required for decisions of stem cell fate. In mouse embryonic stem cells (ESCs, two major variations of PRC1 complex, defined by the mutually exclusive presence of Cbx7 or RYBP, have been identified. Here, we show that although the genomic localization of the Cbx7- and RYBP-containing PRC1 complexes overlaps in certain genes, it can also be mutually exclusive. At the molecular level, Cbx7 is necessary for recruitment of Ring1B to chromatin, whereas RYBP enhances the PRC1 enzymatic activity. Genes occupied by RYBP show lower levels of Ring1B and H2AK119ub and are consequently more highly transcribed than those bound by Cbx7. At the functional level, we show that genes occupied by RYBP are primarily involved in the regulation of metabolism and cell-cycle progression, whereas those bound by Cbx7 predominantly control early-lineage commitment of ESCs. Altogether, our results indicate that different PRC1 subtypes establish a complex pattern of gene regulation that regulates common and nonoverlapping aspects of ESC pluripotency and differentiation.

  11. Ex vivo expansions and transplantations of mouse bone marrow-derived hematopoietic stem/progenitor cells

    Institute of Scientific and Technical Information of China (English)

    WANG Jin-fu(王金福); WU Yi-fan(吴亦凡); HARRINTONG Jenny; McNIECE Ian K.

    2004-01-01

    To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic stem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded cells, we expanded mononuclear cells (MNCs) and CD34+/c-kit+ cells from mouse bone marrow and transplanted the expanded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34+/c-kit+ cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34+/c-kit+ cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells,CD34+ cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34+/c-kit+ cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of expanded cells by the co-culture with MSCs may result in more rapid engraftment ofneutrophils following infusion to transplant recipients.

  12. Effects of GDNF and LIF on mouse spermatogonial stem cells proliferation in vitro.

    Science.gov (United States)

    Wang, Peng; Suo, Li-Juan; Wang, Yan-Feng; Shang, Hua; Li, Guang-Xuan; Hu, Jian-Hong; Li, Qing-Wang

    2014-03-01

    Spermatogonial stem cells (SSCs) are the only type of cells that transmit genes to the subsequent generations. The proliferation, cultivation and identification of SSCs in vitro are critical to understanding of male infertility, genetic resources and conservation of endangered species. To investigate the effects of glial cell-derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) on the proliferation of mouse SSCs in vitro, supplement of GDNF and/or LIF were designed to culture SSCs. The testes of 6-8 d mouse were harvested and digested by two-step enzyme digestion method. The SSCs and Sertoli cells were separated by differential plating. Then the SSCs were identified by alkaline phosphatase staining, RT-PCR and indirect immunofluorescence cell analysis. The cellular proliferation capacity was measured by methyl thiazolyl tetrazolium assay. The results showed that addition of 20 and 40 ng/ml of GDNF could strongly promote growth of mouse SSCs (p  0.05). However, the combination of 20 ng/ml GDNF and 1,000 U/ml LIF could significantly enhance the invitro proliferation of mouse SSCs (p culture when the density of SSCs was 5-10 × 10(4) cells/ml. PMID:23896701

  13. Tough and Cell-Compatible Chitosan Physical Hydrogels for Mouse Bone Mesenchymal Stem Cells in Vitro.

    Science.gov (United States)

    Ding, Beibei; Gao, Huichang; Song, Jianhui; Li, Yaya; Zhang, Lina; Cao, Xiaodong; Xu, Min; Cai, Jie

    2016-08-01

    Most hydrogels involve synthetic polymers and organic cross-linkers that cannot simultaneously fulfill the mechanical and cell-compatibility requirements of biomedical applications. We prepared a new type of chitosan physical hydrogel with various degrees of deacetylation (DDs) via the heterogeneous deacetylation of nanoporous chitin hydrogels under mild conditions. The DD of the chitosan physical hydrogels ranged from 56 to 99%, and the hydrogels were transparent and mechanically strong because of the extra intra- and intermolecular hydrogen bonding interactions between the amino and hydroxyl groups on the nearby chitosan nanofibrils. The tensile strength and Young's modulus of the chitosan physical hydrogels were 3.6 and 7.9 MPa, respectively, for a DD of 56% and increased to 12.1 and 92.0 MPa for a DD of 99% in a swelling equilibrium state. In vitro studies demonstrated that mouse bone mesenchymal stem cells (mBMSCs) cultured on chitosan physical hydrogels had better adhesion and proliferation than those cultured on chitin hydrogels. In particular, the chitosan physical hydrogels promoted the differentiation of the mBMSCs into epidermal cells in vitro. These materials are promising candidates for applications such as stem cell research, cell therapy, and tissue engineering. PMID:27410199

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

    International Nuclear Information System (INIS)

    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

  15. Altered retinal cell differentiation in the AP-3 delta mutant (Mocha) mouse.

    Science.gov (United States)

    Baguma-Nibasheka, Mark; Kablar, Boris

    2009-11-01

    Adaptor-related protein complex 3 delta 1 (Ap3d1) encodes the delta 1 subunit of an adaptor protein regulating intracellular vesicle-mediated transport, and the Ap3d-deletion mutant (Mocha) mouse undergoes rapid photoreceptor degeneration leading to blindness soon after birth. Previous microarray analysis revealed Ap3d down-regulation in the retina of mouse embryos specifically lacking cholinergic amacrine cells as a result of the absence of skeletal musculature. To investigate the role of Ap3d in the determination of retinal cell fate, the present study examined retinal morphology in newborn Ap3d-/- mice. The Ap3d-/- retina showed a complete absence of cholinergic amacrine cells and a decrease in parvalbumin-expressing amacrine cells and syntaxin- and VC1.1-expressing amacrine precursor cells, but had a normal number of cell layers and number of cells in each layer with no detectable difference in cell proliferation or apoptosis. These findings indicate that, despite having no apparent effect on the basic spatial organization of the retina at this stage of development, Ap3d could be involved in the regulation of progenitor cell competence and the eventual ratio of resulting differentiated cells. Finding the mouse mutant which phenocopies the eye defect seen in fetuses with no striated muscle was accomplished by the Systematic Subtractive Microarray Analysis Approach (SSMAA), explained in the discussion section. PMID:19631730

  16. The one-cell mouse embryo: cell cycle-dependent radiosensitivity and development of chromosomal anomalies in postradiation cell cycles

    International Nuclear Information System (INIS)

    One-cell mouse embryos were irradiated with X-rays at different cell cycle stages. Examination of structural chromosomal anomalies and micronucleus formation in postradiation mitoses and interphases demonstrated cell cycle-dependent radiosensitivities in the order: late G2 phase > G1 phase > S phase > early G2 phase > stage of decondensing nuclei. Comparison of the quality and quantity of chromosomal aberrations from the first to third mitosis led to the conclusion that new chromosomal anomalies were formed in the course of postirradiation cell cycles. This hypothesis was supported by an increasing number of micronuclei from 24 to 48 h post-conception. In addition to structural chromosomal aberrations, radiation-induced chromosome loss was observed with a frequency that was obviously independent of the exposed cell cycle phase. Loss of acentric chromosome fragments and of single chromosomes contributed to the micronucleus formation. (author)

  17. Interrogating a cell signalling network sensitively monitors cell fate transition during early differentiation of mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    LIU; Yi-Hsin; HO; Chih-ming

    2010-01-01

    The different cell types in an animal are often considered to be specified by combinations of transcription factors,and defined by marker gene expression.This paradigm is challenged,however,in stem cell research and application.Using a mouse embryonic stem cell(mESC) culture system,here we show that the expression level of many key stem cell marker genes/transcription factors such as Oct4,Sox2 and Nanog failed to monitor cell status transition during mESC differentiation.On the other hand,the response patterns of cell signalling network to external stimuli,as monitored by the dynamics of protein phosphorylation,changed dramatically.Our results also suggest that an irreversible alternation in the cell signalling network precedes the adjustment of transcription factor levels.This is consistent with the notion that signal transduction events regulate cell fate specification.We propose that interrogating a cell signalling network can assess the cell property more precisely,and provide a sensitive measurement for the early events in cell fate transition.We wish to bring attention to the potential problem of cell identification using a few marker genes,and suggest a novel methodology to address this issue.

  18. Induction of Functional Hair-Cell-Like Cells from Mouse Cochlear Multipotent Cells

    OpenAIRE

    Quanwen Liu; Yi Shen; Jiarong Chen; Jie Ding; Zihua Tang; Cui Zhang; Jianling Chen; Liang Li; Ping Chen; Jinfu Wang

    2016-01-01

    In this paper, we developed a two-step-induction method of generating functional hair cells from inner ear multipotent cells. Multipotent cells from the inner ear were established and induced initially into progenitor cells committed to the inner ear cell lineage on the poly-L-lysine substratum. Subsequently, the committed progenitor cells were cultured on the mitotically inactivated chicken utricle stromal cells and induced into hair-cell-like cells containing characteristic stereocilia bund...

  19. Six1 is required for mouse dental follicle cell and human periodontal ligament-derived cell proliferation.

    Science.gov (United States)

    Kawasaki, Tatsuki; Takahashi, Masanori; Yajima, Hiroshi; Mori, Yoshiyuki; Kawakami, Kiyoshi

    2016-08-01

    The periodontal ligament (PDL) is a connective tissue that attaches the tooth cementum to the alveolar bone and is derived from dental follicle cells (DFCs). The DFCs form fibroblasts, osteoblasts, cementoblasts, and PDL stem cells (PDLSCs). We previously reported homeobox transcription factor Six1 expression in mouse DFCs. However, the role of Six1 in periodontal tissue development is largely unknown. In this study, we analyzed SIX1 expression in mouse periodontal tissue cells during postnatal development and adulthood. We also addressed the role of SIX1 in mouse periodontium development and in human cultured PDL-derived cells (PDLCs). In mouse development, SIX1 production was abundant in DFCs and PDL cells by 2 weeks, but it was greatly diminished in the PDL at 4 weeks and in adults. Although the SIX1-positive cell distribution was sparse in the adult PDL, SIX1-positive cells were observed with low expression levels. We used 5-ethynyl-2'-deoxyuridine (EdU) for cell labeling to reveal numerous EdU/SIX1-double positive cells at 2 weeks; however, a few EdU-positive cells remained at 4 weeks. The proportion of DFCs that incorporated EdU was significantly lower in Six1-deficient mice compared with wild-type mice at E18.5. In human PDLCs, SIX1 was intensely expressed, and SIX1-knockdown using siRNA reduced proliferating PDLCs. Our results suggest that SIX1 is a key proliferation regulator in mouse DFCs and human PDLCs, which provides novel insight into Six family gene function in mammals. PMID:27241908

  20. Genetic and epigenetic control of early mouse development

    DEFF Research Database (Denmark)

    Albert, Mareike; Peters, Antoine H F M

    2009-01-01

    A decade after cloning the sheep Dolly, the induction of pluripotency by transcription factors has further revolutionized the possibilities of reprogramming a cell's identity, with exciting prospects for personalized medicine. Establishing totipotency during natural reproduction remains, however,...... extensive epigenetic reprogramming. This may underlie the efficient acquisition of totipotency during subsequent preimplantation development....

  1. Piperlongumine Inhibits LMP1/MYC-dependent Mouse B-Lymphoma Cells

    OpenAIRE

    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

    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/iMycEμ. PL inhibited tumor cell proliferation in a concentration-dependent manner and induced apoptosis of neoplasti...

  2. Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

    OpenAIRE

    Chavez, Miquella G.; Hu, Jimmy; SEIDEL, KERSTIN; Li, Chunying; Jheon, Andrew; Naveau, Adrien; Horst, Orapin; Klein, Ophir D.

    2014-01-01

    Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal's life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give...

  3. Genomic imprinting is variably lost during reprogramming of mouse iPS cells

    OpenAIRE

    Takikawa, Sachiko; Ray, Chelsea; Wang, Xin; Shamis, Yulia; Wu, Tien-Yuan; Li, Xiajun

    2013-01-01

    Derivation of induced pluripotent stem (iPS) cells is mainly an epigenetic reprogramming process. It is still quite controversial how genomic imprinting is reprogrammed in iPS cells. Thus, we derived multiple iPS clones from genetically identical mouse somatic cells. We found that parentally inherited imprint was variably lost among these iPS clones. Concurrent with the loss of DNA methylation imprint at the corresponding Snrpn and Peg3 imprinted regions, parental origin-specific expression o...

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

    OpenAIRE

    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 ...

  5. Differential adherence of hydrophobic and hydrophilic Candida albicans yeast cells to mouse tissues.

    OpenAIRE

    Hazen, K C; Brawner, D L; Riesselman, M H; Jutila, M A; Cutler, J E

    1991-01-01

    Using an ex vivo binding assay, we previously demonstrated that yeast cells grown at 37 degrees C display binding specificity in mouse spleen, lymph node, and kidney tissues. In spleen and lymph node tissues, binding was predominantly in regions rich in macrophages. Here, we tested the possibility that hydrophobic and hydrophilic cells bind differentially to host tissues. Hydrophobic and hydrophilic yeast cells of four Candida albicans strains were incubated for 15 min at 4 degrees C with cry...

  6. Transcriptional regulation induced by cAMP elevation in mouse Schwann cells

    OpenAIRE

    Schmid, Daniela; Zeis, Thomas; Schaeren-Wiemers, Nicole

    2014-01-01

    In peripheral nerves, Schwann cell development is regulated by a variety of signals. Some of the aspects of Schwann cell differentiation can be reproduced in vitro in response to forskolin, an adenylyl cyclase activator elevating intracellular cAMP levels. Herein, the effect of forskolin treatment was investigated by a comprehensive genome-wide expression study on primary mouse Schwann cell cultures. Additional to myelin-related genes, many so far unconsidered genes were ascertained to be mod...

  7. High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells

    OpenAIRE

    Luo, Zhongjun; Macris, Margaret A.; Faruqi, A. Fawad; Glazer, Peter M.

    2000-01-01

    To test the ability of triple helix-forming oligonucleotides (TFOs) to promote recombination within chromosomal sites in mammalian cells, a mouse LTK− cell line was established carrying two mutant copies of the herpes simplex virus thymidine kinase (TK) gene as direct repeats in a single chromosomal locus. Recombination between these repeats can produce a functional TK gene and occurs at a spontaneous frequency of 4 × 10−6 under standard culture conditions. When cells were microinjected with ...

  8. 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

  9. Skeletal myogenic potential of human and mouse neural stem cells.

    Science.gov (United States)

    Galli, R; Borello, U; Gritti, A; Minasi, M G; Bjornson, C; Coletta, M; Mora, M; De Angelis, M G; Fiocco, R; Cossu, G; Vescovi, A L

    2000-10-01

    Distinct cell lineages established early in development are usually maintained throughout adulthood. Thus, adult stem cells have been thought to generate differentiated cells specific to the tissue in which they reside. This view has been challenged; for example, neural stem cells can generate cells that normally originate from a different germ layer. Here we show that acutely isolated and clonally derived neural stem cells from mice and humans could produce skeletal myotubes in vitro and in vivo, the latter following transplantation into adult animals. Myogenic conversion in vitro required direct exposure to myoblasts, and was blocked if neural cells were clustered. Thus, a community effect between neural cells may override such myogenic induction. We conclude that neural stem cells, which generate neurons, glia and blood cells, can also produce skeletal muscle cells, and can undergo various patterns of differentiation depending on exposure to appropriate epigenetic signals in mature tissues. PMID:11017170

  10. Mesenchymal Stem Cells Induce Suppressive Macrophages Through Phagocytosis in a Mouse Model of Asthma.

    Science.gov (United States)

    Braza, Faouzi; Dirou, Stéphanie; Forest, Virginie; Sauzeau, Vincent; Hassoun, Dorian; Chesné, Julie; Cheminant-Muller, Marie-Aude; Sagan, Christine; Magnan, Antoine; Lemarchand, Patricia

    2016-07-01

    Mesenchymal stem cell (MSC) immunosuppressive functions make them attractive candidates for anti-inflammatory therapy in allergic asthma. However, the mechanisms by which they ensure therapeutic effects remain to be elucidated. In an acute mouse model of house dust mite (Der f)-induced asthma, one i.v. MSC injection was sufficient to normalize and stabilize lung function in Der f-sensitized mice as compared to control mice. MSC injection decreased in vivo airway responsiveness and decreased ex vivo carbachol-induced bronchial contraction, maintaining bronchial expression of the inhibitory type 2 muscarinic receptor. To evaluate in vivo MSC survival, MSCs were labeled with PKH26 fluorescent marker prior to i.v. injection, and 1 to 10 days later total lungs were digested to obtain single-cell suspensions. 91.5 ± 2.3% and 86.6 ± 6.3% of the recovered PKH26(+) lung cells expressed specific macrophage markers in control and Der f mice, respectively, suggesting that macrophages had phagocyted in vivo the injected MSCs. Interestingly, only PKH26(+) macrophages expressed M2 phenotype, while the innate PKH26(-) macrophages expressed M1 phenotype. Finally, the remaining 0.5% PKH26(+) MSCs expressed 10- to 100-fold more COX-2 than before injection, suggesting in vivo MSC phenotype modification. Together, the results of this study indicate that MSCs attenuate asthma by being phagocyted by lung macrophages, which in turn acquire a M2 suppressive phenotype. Stem Cells 2016;34:1836-1845. PMID:26891455

  11. Molecular mechanisms involved in casein gene expression and secretion in mouse mammary epithelial cells

    International Nuclear Information System (INIS)

    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

  12. Bioactivities of Culture Supernatants from Retroviral Packaging Cells Carrying the Mouse Fas Ligand Gene

    Institute of Scientific and Technical Information of China (English)

    LIU Lingbo; ZOU Ping; GUO Rong; XIAO Juan; XU Zhiliang

    2001-01-01

    The bioactivities of culture supernatants from retroviral packaging cells carrying the mouse Fas ligand (mFasL) gene was investigated. FasLcDNA was cloned into PLXIN with an internal ribosome entry site to link two cistrons through gene recombination technology, PLXIN and the recombinant vector PLFIN were separately transfected into PA317 retrovirus packing cell line by lipofectamine 2000, and the resistant clones were selected with G418 selective medium. The integration of genome DNA was assayed by genomic DNA PCR. NIH3T3 cells were transduced by the culture supernatants from PA317 carrying the mFasLcDNA gene, and were selected with G418 selective medium, so as to select the PLFIN-PA317 clone capable of producing higher titer of supernatants. The levels of mFasL protein on NIH3T3 cells membrane were assayed by flow cytometry (FCM). The biological activity of mFasL on NIH3T3 cells membrane was investigated by the inducing apoptosis of Fas+ Yac-1 cells co-cultured with NIH3T3 cells expressing Fas ligand. To explore the direct mFasL cytotoxicity of culture supernatants from retroviral packaging cells carrying the mFasL gene, the culture supernatants from PLFIN-PA317 and PLXIN-PA317 were separately co-cultured with Yac-1cells in parallel. The recombinant PLFIN was successfully constructed. The highest titer of supernatants from twelve resistant clones was 8. 5 × 105 colony-forming-unit (CFU)/ml. The NIH3T3cells transfected by above supernatants had a higher level of mFasL (53.81±6.9 %), and significantly induced the apoptosis of Fas+ Yac-1 cells (56. 78±4.5 %), as both were cocultured for 5 h at1 : 1 ratio, whereas it is 7. 08±3.4 % in control group (P<0. 01). Supernatant from PLFINPA317 could also directly induce the apoptosis of Yac-1 within 5 h of incubation. Thus, the culture supernatants from PLFIN-PA317 possessed both infectivity and cytotoxicity of mFasL.

  13. Cell cycle control in Alphaproteobacteria.

    Science.gov (United States)

    Collier, Justine

    2016-04-01

    Alphaproteobacteria include many medically and environmentally important organisms. Despite the diversity of their niches and lifestyles, from free-living to host-associated, they usually rely on very similar mechanisms to control their cell cycles. Studies on Caulobacter crescentus still lay the foundation for understanding the molecular details of pathways regulating DNA replication and cell division and coordinating these two processes with other events of the cell cycle. This review highlights recent discoveries on the regulation and the mode of action of conserved global regulators and small molecules like c-di-GMP and (p)ppGpp, which play key roles in cell cycle control. It also describes several newly identified mechanisms that modulate cell cycle progression in response to stresses or environmental conditions. PMID:26871482

  14. Ex vivo expansions and transplantations of mouse bone marrow-derived hematopoietic stem/progenitor cells

    Institute of Scientific and Technical Information of China (English)

    王金福; 吴亦凡; HARRINTONGJenny; McNIECEIanK.

    2004-01-01

    To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic tem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded ells, we expanded mononuclear cells (MNCs) and CD34+/c-kit+ cells from mouse bone marrow and transplanted the expanded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34+/c-kit+ cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34+/c-kit+ cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF 1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells, CD34+ cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34+/c-kit+ cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of expanded ceils by the co-culture with MSCs may result in more rapid engraftment ofneutrophils following infusion to transplant recipients.

  15. Mouse Brain PSA-NCAM Levels Are Altered by Graded-Controlled Cortical Impact Injury

    Directory of Open Access Journals (Sweden)

    Craig S. Budinich

    2012-01-01

    Full Text Available Traumatic brain injury (TBI is a worldwide endemic that results in unacceptably high morbidity and mortality. Secondary injury processes following primary injury are composed of intricate interactions between assorted molecules that ultimately dictate the degree of longer-term neurological deficits. One comparatively unexplored molecule that may contribute to exacerbation of injury or enhancement of recovery is the posttranslationally modified polysialic acid form of neural cell adhesion molecule, PSA-NCAM. This molecule is a critical modulator of central nervous system plasticity and reorganization after injury. In this study, we used controlled cortical impact (CCI to produce moderate or severe TBI in the mouse. Immunoblotting and immunohistochemical analysis were used to track the early (2, 24, and 48 hour and late (1 and 3 week time course and location of changes in the levels of PSA-NCAM after TBI. Variable and heterogeneous short- and long-term increases or decreases in expression were found. In general, alterations in PSA-NCAM levels were seen in the cerebral cortex immediately after injury, and these reductions persisted in brain regions distal to the primary injury site, especially after severe injury. This information provides a starting point to dissect the role of PSA-NCAM in TBI-related pathology and recovery.

  16. Short-term regulation of muscarinic acetylcholine receptors: An assessment utilizing mouse brain and mouse neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Cioffi, C.L.

    1988-01-01

    The effects of muscarinic agonists and diisopropylfluorophosphate (DFP) on muscarinic receptor density and muscarinic receptor-mediated responses was assessed in mouse brain and mouse neuroblastoma cells (clone N1E-115). Utilizing the antagonist ({sup 3}H)quinuclidinyl benzilate (({sup 3}H)QNB), there was no difference in the maximal binding capacity (B{sub max}) or equilibrium dissociation constant (K{sub d}) between untreated and 24 hour DFP-treated mice. However, one administration of DFP produced a 24% and 33% decrease in B{sub max} measured by ({sup 3}H)N-methylscopolamine (({sup 3}H)NMS) after 18 and 24 hours which was rapidly reversible within 36 hours after DFP treatment. The loss of ({sup 3}H)NMS binding sites following acute DFP treatment was not accompanied by a change in a particular muscarinic receptor binding conformation. Furthermore, the magnitude of muscarinic receptor-mediated phosphoinositide hydrolysis was unchanged following short-term DFP treatment.

  17. 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)

    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

  18. Hematopoietic cell transplantation in murine globoid cell leukodystrophy (the twitcher mouse): effects on levels of galactosylceramidase, psychosine, and galactocerebrosides

    International Nuclear Information System (INIS)

    Hematopoietic cell transplantation (HCT) prolongs survival in the twitcher mouse, an authentic animal model of human globoid cell leukodystrophy (Krabbe disease: galactosylceramidase deficiency), but the effects of HCT on levels of galactosylceramidase, psychosine, and cerebrosides in the tissues of twitcher mice have not been previously studied. Galactosylceramidase was less than 8% of control activity in tissues of untreated twitcher mice but reached normal values in brain and spleen and 20-30% of control kidney of 100-day-old twitchers that received HCT at age 10 days. Using a recently developed method for the simultaneous determination of psychosine and cerebrosides, the authors measured the tissue levels of these lipids in the above animals. The levels of psychosine in brain, sciatic nerve, and kidney of untreated twitcher mice were 44, 200, and 12 times control values, respectively, in 30-day-old animals and 69,500, and 14 times control levels in 40-day-old mice. On the other hand, levels of cerebroside were approximately 35% of control values in sciatic nerve, remained about the same in the brain, and were elevated 10-fold in the kidney of twitcher mice. After HCT, psychosine levels in the brains of 30-day-old twitchers were lowered to 30-35% of values in untreated twitchers, and the levels remained in that range during the post-HCT period. Similarly, brain cerebroside levels remained low in HCT-treated twitcher mice. It is not known whether the extremely high levels of psychosine in sciatic nerves ultimately contribute to the death of twitcher mice after HCT

  19. Preparative scale production of functional mouse aquaporin 4 using different cell-free expression modes.

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    Lei Kai

    Full Text Available The continuous progress in the structural and functional characterization of aquaporins increasingly attracts attention to study their roles in certain mammalian diseases. Although several structures of aquaporins have already been solved by crystallization, the challenge of producing sufficient amounts of functional proteins still remains. CF (cell free expression has emerged in recent times as a promising alternative option in order to synthesize large quantities of membrane proteins, and the focus of this report was to evaluate the potential of this technique for the production of eukaryotic aquaporins. We have selected the mouse aquaporin 4 as a representative of mammalian aquaporins. The protein was synthesized in an E. coli extract based cell-free system with two different expression modes, and the efficiencies of two modes were compared. In both, the P-CF (cell-free membrane protein expression as precipitate mode generating initial aquaporin precipitates as well as in the D-CF (cell-free membrane protein expression in presence of detergent mode, generating directly detergent solubilized samples, we were able to obtain mg amounts of protein per ml of cell-free reaction. Purified aquaporin samples solubilized in different detergents were reconstituted into liposomes, and analyzed for the water channel activity. The calculated P(f value of proteoliposome samples isolated from the D-CF mode was 133 µm/s at 10°C, which was 5 times higher as that of the control. A reversible inhibitory effect of mercury chloride was observed, which is consistent with previous observations of in vitro reconstituted aquaporin 4. In this study, a fast and convenient protocol was established for functional expression of aquaporins, which could serve as basis for further applications such as water filtration.

  20. IFN-γ induces lipogenesis in mouse mesangial cells via the JAK2/STAT1 pathway.

    Science.gov (United States)

    Hao, Jun; Zhang, Yu-jun; Lv, Xin; Xu, Ning; Liu, Qin-juan; Zhao, Song; Feng, Xiao-juan; Xing, Ling-ling; Kang, Peng-peng; Li, Gui-ying; Liu, Shu-xia

    2013-04-15

    The objective is to investigate the effect of high mobility group box-1 (HMGB1) on lipid deposition in γ-interferon (IFN-γ)-stimulated mouse mesangial cell line (MMC) and to determine whether the Janus kinase 2 and signal transducer and activator of transcription 1 (JAK2/STAT1) signaling pathway plays an important role in this process. We employed a control group, an IFN-γ stimulation group, and an IFN-γ + AG490 (JAK2 inhibitor) group. RNA interference aimed at sterol regulatory element-binding protein-1 (SREBP-1) or HMGB1 was used to investigate the effect of these proteins on IFN-γ-induced lipid deposition. Western blotting was used to detect phospho (p)-JAK2, JAK2, p-STAT1, STAT1, SREBP-1, fatty acid synthase (FAS), and HMGB1 protein expression. RT-PCR was used to detect SREBP-1, FAS, and HMGB1 mRNA. Oil Red O staining and the triglyceride assay were used to detect lipid deposition and triglyceride content. Results were as follows: 1) IFN-γ increased MMC cell lipid deposition, triglyceride content, and p-JAK2, p-STAT1, SREBP-1, and FAS expression; 2) SREBP-1 inhibition prevented FAS upregulation and attenuated IFN-γ-induced MMC cell lipid deposition and triglyceride content; 3) HMGB1 upregulated SREBP-1 and FAS mRNA and protein levels, which increased lipid deposition in MMC cells. Small interfering RNA-mediated inhibition of HMGB1 decreased SREBP-1 and FAS expression and lipid accumulation; 4) AG490 decreased upregulation of HMGB1 and p-JAK2/p-STAT1, as well as IFN-γ-induced lipogenesis. In conclusion, the JAK2/STAT1 pathway mediates IFN-γ-induced lipogenesis in MMC cells through regulation of HMGB1/SREBP-1/FAS. PMID:23407880

  1. A therapeutic anti-CD4 monoclonal antibody inhibits T cell receptor signal transduction in mouse autoimmune cardiomyopathy

    Institute of Scientific and Technical Information of China (English)

    WANG Zhao-hui; LIAO Yu-hua; YUAN Jing; ZHANG Li; WANG Min; ZHANG Jing-hui; LIU Zhong-ping; DONG Ji-hua

    2007-01-01

    Background T cell immune abnormalities in patients with dilated cardiomyopathy (DCM) has been intensively studied over the past 10 years. Our previous study has suggested that immunization of mice with the peptides derived from human adenine nucleotide translocator (ANT) result in the production of autoantibodies against the ANT and histopathological changes similar to those in human DCM. The ANT peptides can induce autoimmune cardiomyopathy like DCM in Balb/c mice. In this study we aimed to focus on the molecular mechanism of T cells in the autoimmune cardiomyopathy mouse model by detecting the expression of the two T cell signaling molecules.Methods The ANT peptides were used to cause autoimmune cardiomyopathy in Balb/c mice. Anti-L3T4 or rat anti-mouse IgG was administered to the mice (n=6 in each group) simultaneously immunized with ANT. ELISA analysis was used to detect autoantibodies against the ANT peptides and the percentages of interferon-Y and interleukin-4 producing cells among splenic CD4+ lymphocytes was determined by using flow cytometry analysis. The expression of CD45 in spleen T cells was determined by immunohistochemistry and the mRNAs of T cell signaling molecules were detected by real-time PCR.Results Treatment of ANT immunized Balb/c mice with anti-CD4 mAb caused a reduction in the gene expression of P56lck and Zap-70 and a lower level of CD45 expression by spleen T cells. Aiso, a reverse of the Th1/Th2 ratio that results in the reduced production of antibodies against ANT was found in the anti-CD4 monoclonal antibodies (mAb)group. Whereas irrelevant antibody (rat anti-mouse IgG) did not suppress T cell signaling molecules nor inhibit CD45 expression, and control-antibody mice did not show any significant differences compared with the DCM group.Conclusion The results show that anti-CD4 mAb is a powerful inhibitor of the early initiating events of T cell receptor(TCR) signal transduction in mouse autoimmune dilated cardiomyopathy.

  2. Oncogenes in X-ray-transformed C3H 10T1/2 mouse cells and in X-ray-induced mouse fibrosarcoma (RIF-1) cells

    International Nuclear Information System (INIS)

    The authors have investigated RNA levels of oncogenes in an X-ray transformed C3H 10T1/2 fibroblast line (XTD) and RIF-1 cells isolated from an X-ray-induced fibrosarcoma in a C3H mouse. Steady-state levels of K-ras, H-ras, N-ras, abl, sis, src, and fos were unchanged in the X-ray-transformed cells compared with non-transformed C3H 10T1/2 cells. However, myc and raf mRNA levels were increased dramatically in the transformed cells. Data further suggests a possible alteration in processing of raf RNA in the XTD cells. Southern blot analysis of secondary transfectants induced with XTD DNA indicated that the oncogenic phenotype did not segregate with the myc or raf loci; nor with nine other oncogenes analysed. (author)

  3. Gene expression profiles during early differentiation of mouse embryonic stem cells

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    Wride Michael A

    2009-01-01

    Full Text Available Abstract Background Understanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES cell differentiation can be triggered by embryoid body (EB formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1–4 EBs Results An initial array study identified 4 gene expression changes between 3 undifferentiated ES cell lines. Tissue culture conditions for ES differentiation were then optimized to give the maximum range of gene expression and growth. -Undifferentiated ES cells and EBs cultured with and without LIF at each day for 4 days were subjected to microarray analysis. -Differential expression of 23 genes was identified. 13 of these were also differentially regulated in a separate array comparison between undifferentiated ES cells and compartments of very early embryos. A high degree of inter-replicate variability was noted when confirming array results. Using a panel of marker genes, RNA amplification and RT-PCR, we examined expression pattern variation between individual -D4-Lif EBs. We found that individual EBs selected from the same dish were highly variable in gene expression profile. Conclusion ES cell lines derived from different mouse strains and carrying different genetic modifications are almost invariant in gene expression profile under conditions used to maintain pluripotency. Tissue culture conditions that give the widest range of gene expression and maximise EB growth involve the use of 20% serum and starting cell numbers of 1000 per EB. 23 genes of importance to early development have been

  4. Delayed type hypersensitivity to allogeneic mouse epidermal cell antigens, 2

    International Nuclear Information System (INIS)

    A low dose of ultraviolet B radiation impairs the effectiveness of epidermal cell antigens. We studied the effect of ultraviolet B radiation on the delayed type hypersensitivity induced by allogeneic epidermal cell antigen. The delayed type hypersensitivity response was assayed by footpad swelling in mice. When epidermal cells were exposed to ultraviolet B radiation (660 J/m2), their ability to induce T cells of delayed type hypersensitivity activation was markedly inhibited in any combination of recipient mice and allogeneic epidermal cells. The effect of ultraviolet B radiation on epidermal cells was observed before immunization and challenge. Ultraviolet B treated epidermal cells did not induce suppressor T cells in mice. These results indicate that ultraviolet B radiation destroys the antigenicity of epidermal cells. (author)

  5. 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.

  6. MicroRNAs and Induced Pluripotent Stem Cells for Human Disease Mouse Modeling

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    Chingiz Underbayev

    2012-01-01

    Full Text Available Human disease animal models are absolutely invaluable tools for our understanding of mechanisms involved in both physiological and pathological processes. By studying various genetic abnormalities in these organisms we can get a better insight into potential candidate genes responsible for human disease development. To this point a mouse represents one of the most used and convenient species for human disease modeling. Hundreds if not thousands of inbred, congenic, and transgenic mouse models have been created and are now extensively utilized in the research labs worldwide. Importantly, pluripotent stem cells play a significant role in developing new genetically engineered mice with the desired human disease-like phenotype. Induced pluripotent stem (iPS cells which represent reprogramming of somatic cells into pluripotent stem cells represent a significant advancement in research armament. The novel application of microRNA manipulation both in the generation of iPS cells and subsequent lineage-directed differentiation is discussed. Potential applications of induced pluripotent stem cell—a relatively new type of pluripotent stem cells—for human disease modeling by employing human iPS cells derived from normal and diseased somatic cells and iPS cells derived from mouse models of human disease may lead to uncovering of disease mechanisms and novel therapies.

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

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    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.

  8. Molecular Mechanisms Controlling the Early Mouse Embryo Development

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    Alexandra Ivan

    2010-05-01

    Full Text Available Few are known about the molecular mechanism controlling the early embryo development. The reduce dimension of the embryos, only a few μm, the small quantities of proteins synthesized and the artificial environment influence makes difficult to decode the mechanisms controlling early embryonic stages of development. Although, in the last few years many genes have been showed to be active in the early embryonic stages of development, only a few have been characterized and found to be implicated in the molecular mechanism responsible of preimplantational embryos development. Ped gene (Preimplantational embryo development is considered to be involved in regulation of embryonic cleavage division and subsequent embryo survival. This review presents, based on a rich documentation, the main mechanisms involved in early embryo development.

  9. Adenosine transport systems on dissociated brain cells from mouse, guinea-pig, and rat

    Energy Technology Data Exchange (ETDEWEB)

    Johnston, M.E.; Geiger, J.D. (Univ. of Manitoba, Winnipeg (Canada))

    1990-09-01

    The kinetics and sodium dependence of adenosine transport were determined using an inhibitor-stop method on dissociated cell body preparations obtained from mouse, guinea-pig and rat brain. Transport affinity (KT) values for the high affinity adenosine transport systems KT(H) were significantly different between these three species; mean +/- SEM values were 0.34 +/- 0.1 in mouse, 0.9 +/- 0.2 in rat, and 1.5 +/- 0.5 microM in guinea-pig. The KT values for the low affinity transport system KT(L) were not different between the three species. Brain cells from rat displayed a significantly greater maximal capacity to accumulate (3H)adenosine (Vmax) than did mouse or guinea-pig for the high affinity system, or than did mouse for the low affinity system. When sodium chloride was replaced in the transport medium with choline chloride, the KT(H) values for guinea-pig and rat were both increased by approximately 100%; only in rat did the change reach statistical significance. The sodium-dependence of adenosine transport in mouse brain was clearly absent. The differences between KT(H) values in mouse and those in guinea-pig or rat were accentuated in the absence of sodium. The differences in kinetic values, ionic requirements, and pharmacological characteristics between adenosine transporters in CNS tissues of mouse, guinea-pig and rat may help account for some of the variability noted among species in terms of their physiological responses to adenosine.

  10. Delayed BMP4 exposure increases germ cell differentiation in mouse embryonic stem cells.

    Science.gov (United States)

    Talaei-Khozani, Tahereh; Zarei Fard, Nehleh; Bahmanpour, Soghra; Jaberipour, Mansoureh; Hosseini, Ahmah; Esmaeilpour, Tahereh

    2014-01-01

    Fate mapping studies have revealed that bone morphogenetic protein 4 (BMP4) signaling has a key role in segregation of primordial germ cells from proximal epiblast. Adding BMP4 to the culture media of embryonic stem (ES) cells could induce expression of germ cell markers; however, to provide a desired number of germ cells has remained a challenge. In the current study, we intended to establish an in vitro system to obtain reliable germ cells derived from ES cells. Differentiation was induced in ES cells via embryoid body (EB) and monolayer culture system. Cells were cultured with BMP4 from the beginning (++BMP4) or after 48 hours (+BMP4) of culturing for five days. The cultures were assessed for alkaline phosphatase (ALP) activity, expression of Oct4, Mvh and c-kit. In EB culture protocol, the expression of Mvh, Oct4 and ALP activity significantly increased in +BMP4 culture condition, but a significant down-regulation in the expression of germ cell markers was shown in ++BMP4 condition compared with the control group. Parallel differentiation experiments using monolayer culture system indicated the number of putative germ cells did not change. In the current study, we compared two differentiation methods (EB and monolayer) to achieve an optimal germ cell production. The EBs with a short exposure time period to BMP4, showing typical characteristics of germ cells. Therefore, our approach provides a strategy for the production of germline cells from ES cells. PMID:24969978

  11. DBA-Lectin Reactivity Defines Mouse Uterine Natural Killer Cell Subsets with Biased Gene Expression 1

    Science.gov (United States)

    Chen, Zhilin; Zhang, Jianhong; Hatta, Kota; Lima, Patricia D. A.; Yadi, Hakim; Colucci, Francesco; Yamada, Aureo T.; Croy, B. Anne

    2012-01-01

    Endometrial decidualization, a process essential for blastocyst implantation in species with hemochorial placentation, is accompanied by an enormous but transient influx of Natural Killer (NK) cells. Mouse uterine (u)NK cell subsets have been defined by diameter and cytoplasmic granule number, reflecting stage of maturity and by histochemical reactivity with Periodic Acid Schiff’s (PAS) reagent, with or without co-reactivity with Dolichos biflorus agglutinin (DBA) lectin. We asked whether DBA− and DBA+ mouse uNK cells were equivalent using quantitative (q)RT-PCR analyses of flow separated, midpregnancy (gestation day (gd)10) cells and using immunohistochemistry. CD3E (CD3)-IL2RB (CD122)+DBA− cells were identified as the dominant Ifng transcript source. Skewed IFNG production by uNK cell subsets was confirmed by analysis of uNK cells from eYFP-tagged IFNG-reporter mice. In contrast, CD3E-IL2RB+DBA+ uNK cells expressed genes compatible with significantly greater potential for IL22 synthesis, angiogenesis and participation in regulation mediated by the renin-angiotensin system (RAS). CD3E-IL2RB+DBA+ cells were further divided into VEGFA+ and VEGFA− subsets. CD3E-IL2RB+DBA+ uNK cells but not CD3E-IL2RB+DBA− uNK cells arose from circulating, bone marrow-derived progenitor cells by gd6. These findings indicate the heterogeneous nature of mouse uNK cells and suggest that studies using only DBA + uNK cells will give biased data that does not fully represent the uNK cell population. PMID:22875907

  12. In vivo phenotypic characterisation of nucleoside label-retaining cells in mouse periosteum

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    HM Cherry

    2014-03-01

    Full Text Available Periosteum is known to contain cells that, after isolation and culture-expansion, display properties of mesenchymal stromal/stem cells (MSCs. However, the equivalent cells have not been identified in situ mainly due to the lack of specific markers. Postnatally, stem cells are slow-cycling, long-term nucleoside-label-retaining cells. This study aimed to identify and characterise label-retaining cells in mouse periosteum in vivo. Mice received iodo-deoxy-uridine (IdU via the drinking water for 30 days, followed by a 40-day washout period. IdU+ cells were identified by immunostaining in conjunction with MSC and lineage markers. IdU-labelled cells were detected throughout the periosteum with no apparent focal concentration, and were negative for the endothelial marker von Willebrand factor and the pan-haematopoietic marker CD45. Subsets of IdU+ cells were positive for the mesenchymal/stromal markers vimentin and cadherin-11. IdU+ cells expressed stem cell antigen-1, CD44, CD73, CD105, platelet-derived growth factor receptor-α and p75, thereby displaying an MSC-like phonotype. Co-localisation was not detectable between IdU and the pericyte markers CD146, alpha smooth muscle actin or NG2, nor did IdU co-localise with β-galactosidase in a transgenic mouse expressing this reporter gene in pericytes and smooth muscle cells. Subsets of IdU+ cells expressed the osteoblast-lineage markers Runx2 and osteocalcin. The IdU+ cells expressing osteocalcin were lining the bone and were negative for the MSC marker p75. In conclusion, mouse periosteum contains nucleoside-label-retaining cells with a phenotype compatible with MSCs that are distinct from pericytes and osteoblasts. Future studies characterising the MSC niche in vivo could reveal novel therapeutic targets for promoting bone regeneration/repair.

  13. Prohibitin 2 regulates the proliferation and lineage-specific differentiation of mouse embryonic stem cells in mitochondria.

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    Megumi Kowno

    Full Text Available BACKGROUND: The pluripotent state of embryonic stem (ES cells is controlled by a network of specific transcription factors. Recent studies also suggested the significant contribution of mitochondria on the regulation of pluripotent stem cells. However, the molecules involved in these regulations are still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we found that prohibitin 2 (PHB2, a pleiotrophic factor mainly localized in mitochondria, is a crucial regulatory factor for the homeostasis and differentiation of ES cells. PHB2 was highly expressed in undifferentiated mouse ES cells, and the expression was decreased during the differentiation of ES cells. Knockdown of PHB2 induced significant apoptosis in pluripotent ES cells, whereas enhanced expression of PHB2 contributed to the proliferation of ES cells. However, enhanced expression of PHB2 strongly inhibited ES cell differentiation into neuronal and endodermal cells. Interestingly, only PHB2 with intact mitochondrial targeting signal showed these specific effects on ES cells. Moreover, overexpression of PHB2 enhanced the processing of a dynamin-like GTPase (OPA1 that regulates mitochondrial fusion and cristae remodeling, which could induce partial dysfunction of mitochondria. CONCLUSIONS/SIGNIFICANCE: Our results suggest that PHB2 is a crucial mitochondrial regulator for homeostasis and lineage-specific differentiation of ES cells.

  14. Langerhans' cells, veiled cells, and interdigitating cells in the mouse recognized by a monoclonal antibody

    OpenAIRE

    1986-01-01

    An mAb, NLDC-145, is described that specifically reacts with a group of nonlymphoid dendritic cells including Langerhans cells (LC), veiled cells (VC), and interdigitating cells (IDC). The antibody does not react with precursor cells in bone marrow and blood. Macrophages are not stained by the antibody, but a subpopulation of Ia+ peritoneal exudate cells is recognized. Possible relationships of the various nonlymphoid dendritic cell (NLDC) types are discussed.

  15. T-Cell Mediated Immune Responses Induced in ret Transgenic Mouse Model of Malignant Melanoma

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    Dirk Schadendorf

    2012-04-01

    Full Text Available Poor response of human malignant melanoma to currently available treatments requires a development of innovative therapeutic strategies. Their evaluation should be based on animal models that resemble human melanoma with respect to genetics, histopathology and clinical features. Here we used a transgenic mouse model of spontaneous skin melanoma, in which the ret transgene is expressed in melanocytes under the control of metallothionein-I promoter. After a short latency, around 25% mice develop macroscopic skin melanoma metastasizing to lymph nodes, bone marrow, lungs and brain, whereas other transgenic mice showed only metastatic lesions without visible skin tumors. We found that tumor lesions expressed melanoma associated antigens (MAA tyrosinase, tyrosinase related protein (TRP-1, TRP-2 and gp100, which could be applied as targets for the immunotherapy. Upon peptide vaccination, ret transgenic mice without macroscopic melanomas were able to generate T cell responses not only against a strong model antigen ovalbumin but also against typical MAA TRP-2. Although mice bearing macroscopic primary tumors could also display an antigen-specific T cell reactivity, it was significantly down-regulated as compared to tumor-free transgenic mice or non-transgenic littermates. We suggest that ret transgenic mice could be used as a pre-clinical model for the evaluation of novel strategies of melanoma immunotherapy.

  16. T-Cell Mediated Immune Responses Induced in ret Transgenic Mouse Model of Malignant Melanoma

    International Nuclear Information System (INIS)

    Poor response of human malignant melanoma to currently available treatments requires a development of innovative therapeutic strategies. Their evaluation should be based on animal models that resemble human melanoma with respect to genetics, histopathology and clinical features. Here we used a transgenic mouse model of spontaneous skin melanoma, in which the ret transgene is expressed in melanocytes under the control of metallothionein-I promoter. After a short latency, around 25% mice develop macroscopic skin melanoma metastasizing to lymph nodes, bone marrow, lungs and brain, whereas other transgenic mice showed only metastatic lesions without visible skin tumors. We found that tumor lesions expressed melanoma associated antigens (MAA) tyrosinase, tyrosinase related protein (TRP)-1, TRP-2 and gp100, which could be applied as targets for the immunotherapy. Upon peptide vaccination, ret transgenic mice without macroscopic melanomas were able to generate T cell responses not only against a strong model antigen ovalbumin but also against typical MAA TRP-2. Although mice bearing macroscopic primary tumors could also display an antigen-specific T cell reactivity, it was significantly down-regulated as compared to tumor-free transgenic mice or non-transgenic littermates. We suggest that ret transgenic mice could be used as a pre-clinical model for the evaluation of novel strategies of melanoma immunotherapy

  17. Comparative genomics as a tool to reveal functional equivalences between human and mouse dendritic cell subsets.

    Science.gov (United States)

    Crozat, Karine; Guiton, Rachel; Guilliams, Martin; Henri, Sandrine; Baranek, Thomas; Schwartz-Cornil, Isabelle; Malissen, Bernard; Dalod, Marc

    2010-03-01

    During evolution, vertebrates have developed an adaptive immune system able to cope with a variety of pathogens. Dendritic cells (DCs) are central to this process. DCs integrate information derived from pathogens or endogenous danger signals and convey them to T lymphocytes. Most of the present knowledge on DCs was generated in mice or by using human DCs differentiated in vitro from monocytes. In both species, several DC subsets have been identified in vivo based on differences in their phenotypes, anatomical locations or functions. In mice, protective immunity against intracellular pathogens or tumors can be induced most efficiently by targeting antigens to the CD8 alpha(+) DCs, a subset of DCs which resides in lymphoid tissues and is especially efficient at cross-presenting exogenous antigens to CD8(+) T lymphocytes. In contrary, harnessing human DC subsets for medical purposes is currently hampered by insufficient knowledge about these cells. To overcome this cognitive gap, we are using comparative genomics as a tool for designing hypotheses and experiments to further characterize DC subset functions and their molecular control, including the investigation of the functional equivalences that might exist between human and mouse DC subsets. PMID:20193019

  18. T-Cell Mediated Immune Responses Induced in ret Transgenic Mouse Model of Malignant Melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Abschuetz, Oliver [Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg and Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Mannheim , Heidelberg 69120 (Germany); Osen, Wolfram [Division of Translational Immunology, German Cancer Center, Heidelberg 69120 (Germany); Frank, Kathrin [Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg and Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Mannheim , Heidelberg 69120 (Germany); Kato, Masashi [Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Aichi 487-8501 (Japan); Schadendorf, Dirk [Department of Dermatology, University Hospital Essen, Essen 45122 (Germany); Umansky, Viktor, E-mail: v.umansky@dkfz.de [Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg and Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl University of Heidelberg, Mannheim , Heidelberg 69120 (Germany)

    2012-04-26

    Poor response of human malignant melanoma to currently available treatments requires a development of innovative therapeutic strategies. Their evaluation should be based on animal models that resemble human melanoma with respect to genetics, histopathology and clinical features. Here we used a transgenic mouse model of spontaneous skin melanoma, in which the ret transgene is expressed in melanocytes under the control of metallothionein-I promoter. After a short latency, around 25% mice develop macroscopic skin melanoma metastasizing to lymph nodes, bone marrow, lungs and brain, whereas other transgenic mice showed only metastatic lesions without visible skin tumors. We found that tumor lesions expressed melanoma associated antigens (MAA) tyrosinase, tyrosinase related protein (TRP)-1, TRP-2 and gp100, which could be applied as targets for the immunotherapy. Upon peptide vaccination, ret transgenic mice without macroscopic melanomas were able to generate T cell responses not only against a strong model antigen ovalbumin but also against typical MAA TRP-2. Although mice bearing macroscopic primary tumors could also display an antigen-specific T cell reactivity, it was significantly down-regulated as compared to tumor-free transgenic mice or non-transgenic littermates. We suggest that ret transgenic mice could be used as a pre-clinical model for the evaluation of novel strategies of melanoma immunotherapy.

  19. Cardiomyocyte marker expression in a human lymphocyte cell line using mouse cardiomyocyte extract.

    Science.gov (United States)

    Vojdani, Zahra; Tavakolinejad, Sima; Talaei-Khozani, Tahereh; Esmaeilpour, Tahereh; Rasooli, Manuchehr

    2011-03-01

    Cell transplantation shows potential for the treatment of cardiac diseases. Embryonic stem cells, cord blood and mesenchymal stem cells have been suggested as sources for transplantation therapy. Because of some technical limitations with the use of stem cells, transdifferentiation of fully differentiated cells is a potentially useful alternative. We investigated whether human peripheral blood cells could transdifferentiate into cardiomyocyte. Transdifferentiation was induced in a human B lymphocyte cell line (Raji). Cardiomyocyte extract was prepared from adult mouse cardiomyocytes. The cells were treated with 5-aza-2-deoxycytidine and trichostatin A, permeabilized with streptolysin O, and exposed to the mouse cardiomyocyte extract. They were cultured for 10 days, 3 weeks and 4 weeks. Cardiomyocyte markers were detected with immunohistochemistry and flow cytometry. Immunocytochemistry revealed that some cells expressed myosin heavy chain, α-actinin and cardiac troponin T after 3 and 4 weeks. Flow cytometry confirmed these data. In cells exposed to trichostatin A and 5-aza-2-deoxycytidine and permeabilized in the presence of the cardiomyocyte extract, troponin T expression was seen in 3.53% of the cells and 3.11% of them expressed α-actinin. After exposure to the cardiomyocyte extract, some permeabilized cells adhered to the plate loosely; however, the morphology did not change significantly, and they continued to show a rounded shape after 4 weeks. Our treated lymphocytes expressed cardiomyocyte markers. Our results suggest that lymphocytes may be useful in future research as a source of cells for reprogramming procedures. PMID:21547694

  20. Ciliated epithelial cell lifespan in the mouse trachea and lung

    OpenAIRE

    Rawlins, Emma L.; Brigid L M Hogan

    2008-01-01

    The steady-state turnover of epithelial cells in the lung and trachea is highly relevant to investigators who are studying endogenous stem cells, manipulating gene expression in vivo, or using viral vectors for gene therapy. However, the average lifetime of different airway epithelial cell types has not previously been assessed using currently available genetic techniques. Here, we use Cre/loxP genetic technology to indelibly label a random fraction of ciliated cells throughout the airways of...

  1. Synchrony in human, mouse and bacterial cell cultures--a comparison

    Science.gov (United States)

    Helmstetter, Charles E.; Thornton, Maureen; Romero, Ana; Eward, K. Leigh

    2003-01-01

    Growth characteristics of synchronous human MOLT-4, human U-937 and mouse L1210 cultures produced with a new minimally-disturbing technology were compared to each other and to synchronous Escherichia coli B/r. Based on measurements of cell concentrations during synchronous growth, synchrony persisted in similar fashion for all cells. Cell size and DNA distributions in the mammalian cultures also progressed synchronously and reproducibly for multiple cell cycles. The results demonstrate that unambiguous multi-cycle synchrony, critical for verifying the absence of significant growth imbalances induced by the synchronization procedure, is feasible with these cell lines, and possibly others.

  2. Localization of vitamin A-storing cell in mouse uterus. A radioautographic study

    International Nuclear Information System (INIS)

    Localization of vitamin A in mouse uterus was studied with both light and electron microscopic radioautography after oral administration of tritiated vitamin A. Radioactivity was detected in the lipid droplets of particular cells in the connective tissue space of the uterus. The cells showed morphological features common to the vitamin A-storing cells in other organs, and in addition, the intimate relationship with collagenous fibrils was noticed. The distribution of the cells was most numerous in the subserosal space and decreased towards luminal side. The functional significance of these cells in uterus was still uncertain

  3. Strategies for Profiling Single Mouse Intestinal Epithelial Cells by Targeted Gene Expression

    OpenAIRE

    McDowell, W.; Box, A. (Antonio); Staehling, K.; Wang, F.; Li, L.; Zueckert-Gaudenz, K.

    2014-01-01

    Targeted gene expression profiling of single cells permits the study of heterogeneity in cell populations. Here, a pool of mouse intestinal crypt-base CD44+/GRP78- cells was collected by fluorescence activated cell sorting. Aliquots were either loaded onto Fluidigm's C1 System for microfluidic cell capture and cDNA synthesis in nanoliter volumes, or flow-sorted directly into individual PCR plate wells for cDNA synthesis in microliter volumes. The pre-amplified cDNAs were transferred to the Bi...

  4. Kinetic Analysis of CpG-Induced Mouse B Cell Growth and Ig Production

    OpenAIRE

    Kim, Young-Ha; Lee, Sang-Hoon; Yoo, Yung-Choon; Lee, Junglim; Park, Jong-Hwan; Park, Seok-Rae

    2012-01-01

    Immune cells express toll-like receptors (TLRs) and respond to molecular patterns of various pathogens. CpG motif in bacterial DNA activates innate and acquired immune systems through binding to TLR9 of immune cells. Several studies reported that CpG can directly regulate B cell activation, differentiation, and Ig production. However, the role of CpG in B cell growth and Ig production is not fully understood. In this study, we analyzed the effect of CpG on the kinetics of mouse B cell viabili...

  5. Generation of L-cells in mouse and human small intestine organoids

    OpenAIRE

    Petersen, Natalia; Reimann, Frank; Bartfeld, Sina; Farin, Henner F.; Ringnalda, Femke C.; Vries, Robert G J; van den Brink, Stieneke; Clevers, Hans; Gribble, Fiona M.; de Koning, Eelco J. P.

    2013-01-01

    Upon a nutrient challenge, L-cells produce glucagon-like peptide 1 (GLP-1), a powerful stimulant of insulin release. Strategies to augment endogenous GLP-1 production include promoting L-cell differentiation and increasing L-cell number. Here we present a novel in vitro platform to generate functional L-cells from 3D cultures of mouse and human intestinal crypts. We show that short-chain fatty acids (SCFAs) selectively increase the number of L-cells resulting in an elevation of GLP-1 release....

  6. In vitro generation of motor neuron precursors from mouse embryonic stem cells using mesoporous nanoparticles

    DEFF Research Database (Denmark)

    Garcia-Bennett, Alfonso E; König, Niclas; Abrahamsson, Ninnie;

    2014-01-01

    Aim: Stem cell-derived motor neurons (MNs) are utilized to develop replacement strategies for spinal cord disorders. Differentiation of embryonic stem cells into MN precursors involves factors and their repeated administration. We investigated if delivery of factors loaded into mesoporous...... nanoparticles could be effective for stem cell differentiation in vitro. Materials & methods: We used a mouse embryonic stem cell line expressing green fluorescent protein under the promoter for the MN-specific gene Hb9 to visualize the level of MN differentiation. The differentiation of stem cells was...

  7. 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...

  8. BLM has early and late functions in homologous recombination repair in mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Chu, W K; Hanada, K; Kanaar, R;

    2010-01-01

    ' roles, such as dissolution of double Holliday junctions. However, most of the evidence for these putative roles comes from in vitro biochemical data. In this study, we report the characterization of mouse embryonic stem cells with disruption of Blm and/or Rad54 genes. We show that Blm has roles both...

  9. 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...

  10. 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...

  11. CDC25A phosphatase controls meiosis I progression in mouse oocytes

    Czech Academy of Sciences Publication Activity Database

    Šolc, Petr; Šašková, Adéla; Baran, V.; Kubelka, Michal; Schultz, R. M.; Motlík, Jan

    Lausanne : EPF , ISREC, 2008. s. 148-148. [ EPF Life Sciences Symposium Isrec Cancer and the Cell Cycle. 21.08.2008-23.08.2008, Lausanne] R&D Projects: GA ČR GA305/06/1413; GA MŠk ME08030; GA ČR(CZ) GD204/05/H023 Institutional research plan: CEZ:AV0Z50450515 Keywords : mouse oocytes Subject RIV: EB - Genetics ; Molecular Biology

  12. Radioprotective effects of citrus extract against γ-irradiation in mouse bone marrow cells

    International Nuclear Information System (INIS)

    The radioprotective effects of citrus extract were investigated by using the micronucleus test for anti-clastogenic and cell proliferation activity. A single intraperitoneal (i.p.) injection of citrus extract (Citrus aurantium var. amara) at 250, 500, 1,000 mg/kg body weight 1 h prior to γ-ray irradiation (1.5 Gy) reduced the frequencies of micronucleated polychromatic erythrocytes (MnPCEs) and normochromatic erythrocytes (MnNCEs). All three doses of citrus extract significantly reduced the frequencies of MnPCEs and MnNCEs in mice bone marrow compared to non-drug-treated irradiated control (p<0.005-0.05). The optimum dose for protection in mouse was 250 mg/kg to protect mice bone marrow 2.2-fold against the side effects of γ-irradiation with respect to the non-drug-treated irradiated control. The flavonoids were contained in citrus extract, probably to show protective activity, and reduced the clastogenic effect of radiation on mice bone marrow. Therefore fruits and vegetables contain flavonoids to be useful as protective effects under such stress conditions as irradiation. (author)

  13. Effects of feeder layer and BRL conditioned medium on mouse embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    TsungHsiaochien; christine,L.Mummery

    1990-01-01

    In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice,the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF).Compared with the feeder layer of MEF cells,medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyotypic normality of ES cells only in short term cell propagation.Besides,ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras.Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more converient and efficient than the conventional microdrop method.

  14. Immunologic glycosphingolipidomics and NKT cell development in mouse thymus

    DEFF Research Database (Denmark)

    Li, Yunsen; Thapa, Prakash; Hawke, David;

    2009-01-01

    Invariant NKT cells are a hybrid cell type of Natural Killer cells and T cells, whose development is dependent on thymic positive selection mediated by double positive thymocytes through their recognition of natural ligands presented by CD1d, a nonpolymorphic, non-MHC, MHC-like antigen presenting...... molecule. Genetic evidence suggested that beta-glucosylceramide derived glycosphingolipids (GSLs) are natural ligands for NKT cells. N-butyldeoxygalactonojirimycin (NB-DGJ), a drug that specifically inhibits the glucosylceramide synthase, inhibits the endogenous ligands for NKT cells. Furthermore, we and...... for identifying additional ligands for NKT cells. Our results also provide early insights into cellular lipidomics studies, with a specific focus on the important immunological functions of glycosphingolipids....

  15. Follicles were reconstituted from dissociated mouse fetal ovarian cells in vitro

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Early folliculogenesis involved in the interaction of germ cellsand somatic cells is a complicated physiological event. Female germ cells are committed to differentiate into oocytes and finish complete development in the functional units of follicles. Thus there will be great significance in basal research and practices to evaluate the possibility of ovarian cells to reconstitute into follicles in vitro. In the present research, 12—16 dpc (days post coitum) mouse fetal ovarian cells were respectively isolated using collagenase digestion and cultured in droplets in vitro. The results revealed that the fetal ovarian cells of 12—16 dpc appeared to form multiple cell aggregates and tissue-like pieces in vitro. However, 12—13 dpc ovarian cells failed to form the follicles. 14—15 dpc ovarian cells were competent to form a few follicle-like complexes. Furthermore many small typical follicles were reconstituted from 16 dpc ovarian cells in vitro. The results showed for the first time that mouse embryonic ovarian cells were able to form the follicles in vitro. It was a gradual progression for the female germ cells to achieve the ability to induce somatic cells differentiation and reconstitution into follicles, which may directly lead to the success in reorganization and transplantation of genetically modified ovary in vitro.

  16. Rate equation model of phototransduction into the membranous disks of mouse rod cells

    CERN Document Server

    Takamoto, Rei; Awazu, Akinori

    2015-01-01

    A theoretical model was developed to investigate the rod phototransduction process in the mouse. In particular, we explored the biochemical reactions of several chemical components that contribute to the signaling process into/around the membranous disks in the outer segments of the rod cells. We constructed a rate equation model incorporating the molecular crowding effects of rhodopsin according to experimental results, which may hinder the diffusion of molecules on the disk mem- brane. The present model could effectively reproduce and explain the mechanisms of the following phenomena observed in experiments. First, the activations and relaxation of the wild-type mouse rod cell progressed more slowly than those of mutant cells containing half the amount of rhodopsin on the disk membrane. Second, the strong photoactivated state of the cell was sustained for a longer period when the light stimuli were strong. Finally, the lifetime of photoactivation exhibited a logarithmic increase with increasing light streng...

  17. Immune-deficient mouse models for analysis of human stem cells.

    Science.gov (United States)

    Meyerrose, Todd E; Herrbrich, Phillip; Hess, David A; Nolta, Jan A

    2003-12-01

    The field of murine models of xenotransplantation has grown immensely over the past two decades. The explosive growth in this field is in part due to the fact that good in vitro methods do not exist yet to allow examination of human stem cell homing into the bone marrow compartment versus other tissues, long-term survival of human stem cells, or differentiation into tissues outside of the hematopoietic system. Since these important aspects of human stem cell biology can be examined in vivo using immune-deficient mice, the number of different strains and models is constantly increasing. The current review discusses the merits and drawbacks of each immune-deficient mouse xenograft system as it stands to date and reviews how each immune-deficient mouse model has been used to further our knowledge of human hematopoietic stem cell biology. PMID:14682062

  18. ATM Kinase Is Required for Telomere Elongation in Mouse and Human Cells

    Directory of Open Access Journals (Sweden)

    Stella Suyong Lee

    2015-11-01

    Full Text Available Short telomeres induce a DNA damage response, senescence, and apoptosis, thus maintaining telomere length equilibrium is essential for cell viability. Telomerase addition of telomere repeats is tightly regulated in cells. To probe pathways that regulate telomere addition, we developed the ADDIT assay to measure new telomere addition at a single telomere in vivo. Sequence analysis showed telomerase-specific addition of repeats onto a new telomere occurred in just 48 hr. Using the ADDIT assay, we found that ATM is required for addition of new repeats onto telomeres in mouse cells. Evaluation of bulk telomeres, in both human and mouse cells, showed that blocking ATM inhibited telomere elongation. Finally, the activation of ATM through the inhibition of PARP1 resulted in increased telomere elongation, supporting the central role of the ATM pathway in regulating telomere addition. Understanding this role of ATM may yield new areas for possible therapeutic intervention in telomere-mediated disease.

  19. 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)

    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 CYP1A1CYP1A2 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(+/+)severe-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.

  20. Multicolor karyotype analyses of mouse embryonic stem cells.

    Science.gov (United States)

    Guo, Jianli; Jauch, Anna; Heidi, Holtgreve-Grez; Schoell, Brigitte; Erz, Dorothee; Schrank, Martina; Janssen, Johannes W G

    2005-01-01

    The manipulation of embryonic stem (ES) cells to introduce directional genetic changes into the genome of mice has become an important tool in biomedical research. Monitoring of cell morphology before and after DNA manipulation and special culture conditions are a prerequisite to preserve the pluripotent properties of ES cells and thus their ability to generate chimera and effective germline transmission (GLT). It has been reported that prolonged cell culturing may affect the diploid chromosomal composition of cells and therefore the percentage of chimerism and GLT. Herein, we report multicolor-fluorescence in situ hybridization (M-FISH) analysis of four different ES cell lines/clones. Although the morphology of all four ES cell lines/clones appeared normal and all four expressed the early markers Oct-3/4 and Nanog, two cell lines presented consistent numerical and structural chromosome aberrations. We demonstrate that M-FISH is a sensitive and accurate method for a comprehensive karyotype analysis of ES cells and may minimize time, costs, and disappointments due to inadequate ES cell sources. PMID:16409114

  1. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model*

    OpenAIRE

    Tong, Guo-qing; Heng, Boon-chin; Ng, Soon-chye

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 µmol/L strontium chloride for 5 h and sub...

  2. Role of TRPV1 in the Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes

    OpenAIRE

    Qi, Yan; Qi, Zenghua; Li, Zhichao; Wong, Chun-kit, Arthur; So, Chun; Lo, Iek-Chi; Yu HUANG; Yao, Xiaoqiang; Tsang, Suk-Ying

    2015-01-01

    Cytosolic Ca2+ ([Ca2+]i) is an important signal that regulates cardiomyocyte differentiation during cardiogenesis. TRPV1 is a Ca2+-permeable channel that is expressed in cardiomyocytes. In the present study, we utilized mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) as a model to investigate the functional role of TRPV1 in cardiomyocyte differentiation. Induction of embryonic stem cells into cardiomyocytes was achieved using embryoid body (EB)-based differentiation method. Quanti...

  3. Keratin filaments of mouse epithelial cells are rapidly affected by epidermal growth factor

    OpenAIRE

    1981-01-01

    The effects of epidermal growth factor (EGF) on the cytokeratin filaments of cultured murine epithelial cells were studied by the indirect immunofluorescence technique with affinity-purified antibodies. Mouse epithelial cells (MMC-E), grown on glass cover slips, and viewed by immunofluorescence microscopy, showed keratin-specific fluorescence as typical bright perinuclear aggregates corresponding to dense paracrystalline granules seen in electron microscopy. Within minutes after an exposure t...

  4. Molecular structure and evolution of double-minute chromosomes in methotrexate-resistant cultured mouse cells.

    OpenAIRE

    Hahn, P J; Nevaldine, B; Longo, J A

    1992-01-01

    To determine whether microscopically visible double-minute chromosomes (DMs) are derived from submicroscopic precursors, we monitored the amplification of the dihydrofolate reductase (DHFR) gene in 10 independent isolates of methotrexate (MTX)-resistant mouse cells. At every other doubling in MTX concentration, the cells were examined both microscopically, to detect the presence of microscopically visible DMs, and by pulsed-field gel electrophoresis and hybridization to a DHFR-specific probe,...

  5. Correlation between electrical activity and ACTH/beta-endorphin secretion in mouse pituitary tumor cells

    OpenAIRE

    1982-01-01

    The electrical and secretory activities of mouse pituitary tumor cells (AtT-20/D-16v), which contain and release the ACTH/beta-endorphin family of peptides, were studied by means of intracellular recordings and radioimmunoassays. Injection of depolarizing current pulses evoked action potentials in all cells and the majority (82%) displayed spontaneous action potential activity. Action potentials were found to be calcium-dependent. Barium increased membrane resistance, action potential amplitu...

  6. Adipose-derived Stromal Cells Overexpressing Vascular Endothelial Growth Factor Accelerate Mouse Excisional Wound Healing

    OpenAIRE

    Nauta, Allison; Seidel, Catharina; Deveza, Lorenzo; Montoro, Daniel; Grova, Monica; Ko, Sae Hee; Hyun, Jeong; Geoffrey C Gurtner; Longaker, Michael T.; Yang, Fan

    2012-01-01

    Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a c...

  7. DBA-Lectin Reactivity Defines Mouse Uterine Natural Killer Cell Subsets with Biased Gene Expression 1

    OpenAIRE

    Chen, Zhilin; Zhang, Jianhong; Hatta, Kota; Lima, Patricia D.A.; Yadi, Hakim; Colucci, Francesco; Yamada, Aureo T.; Croy, B. Anne

    2012-01-01

    Endometrial decidualization, a process essential for blastocyst implantation in species with hemochorial placentation, is accompanied by an enormous but transient influx of Natural Killer (NK) cells. Mouse uterine (u)NK cell subsets have been defined by diameter and cytoplasmic granule number, reflecting stage of maturity and by histochemical reactivity with Periodic Acid Schiff’s (PAS) reagent, with or without co-reactivity with Dolichos biflorus agglutinin (DBA) lectin. We asked whether DBA...

  8. Estimation of mean exocytic vesicle capacitance in mouse adrenal chromaffin cells

    OpenAIRE

    Moser, Tobias; Neher, Erwin

    1997-01-01

    Whole-cell membrane capacitance measurements are frequently used to monitor neuronal and nonneuronal secretory activity. However, unless individual fusion events can be resolved, the type of the fusing vesicles cannot be identified in these experiments. Here we apply statistical analysis of trial-to-trial variations between depolarization-induced capacitance increases of mouse adrenal chromaffin cells and obtain estimates for the capacitance contribution of individual exocytic vesicles betwee...

  9. Regulatory T Cells Prevent Liver Fibrosis During HIV Type 1 Infection in a Humanized Mouse Model

    OpenAIRE

    Nunoya, Jun-ichi; Washburn, Michael L.; Kovalev, Grigoriy I; Su, Lishan

    2013-01-01

    Human immunodeficiency virus type 1 (HIV-1) disease is associated with aberrant immune activation, and coinfection with hepatitis C virus (HCV) exacerbates hepatic inflammation and fibrosis. However, the role of HIV-1 infection or host immune modulation in liver pathogenesis is not clearly defined. Here, we report that regulatory T (Treg) cells prevent liver immunopathogenesis during HIV-1 infection in a humanized mouse model. In the absence of Treg cells, HIV-1 infection induced liver fibros...

  10. Intrinsic bursting of AII amacrine cells underlies oscillations in the rd1 mouse retina

    OpenAIRE

    Choi, Hannah; Zhang, Lei; Cembrowski, Mark S.; Sabottke, Carl F.; Markowitz, Alexander L.; Butts, Daniel A.; Kath, William L.; Joshua H Singer; Riecke, Hermann

    2014-01-01

    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...

  11. The influence of He-Ne laser radiation on chemiluminescence of mouse spleen cells

    International Nuclear Information System (INIS)

    A study was made of He-Ne laser radiation (λ=632.8 nm) on spontaneous chemiluminescence of mouse splenic cells and that stimulated by addition of Candida albicans. Irradiation with low-intensity red light was shown to stimulate cell chemiluminescence and to intensify that stimulated by C. albicans within the dose range from 100 to 300 J/m2 with a maximum at about 200 J/m2

  12. Umami Responses in Mouse Taste Cells Indicate More than One Receptor

    OpenAIRE

    MARUYAMA, Yutaka; Pereira, Elizabeth; Margolskee, Robert F.; Chaudhari, Nirupa; Roper, Stephen D.

    2006-01-01

    A number of gustatory receptors have been proposed to underlie umami, the taste of L-glutamate, and certain other amino acids and nucleotides. However, the response profiles of these cloned receptors have not been validated against responses recorded from taste receptor cells that are the native detectors of umami taste. We investigated umami taste responses in mouse circumvallate taste buds in an intact slice preparation, using confocal calcium imaging. Approximately 5% of taste cells select...

  13. [6]-Shogaol inhibits melanogenesis in B16 mouse melanoma cells through activation of the ERK pathway

    OpenAIRE

    Yao, Cheng; Oh, Jang-Hee; Oh, Inn Gyung; Park, Chi-Hyun; Chung, Jin Ho

    2012-01-01

    Aim: To investigate the effect of [6]-shogaol, an active ingredient in ginger, on melanogenesis and the underlying mechanisms. Methods: B16F10 mouse melanoma cells were tested. Cell viability was determined with the MTT assay. Melanin content and tyrosinase activity were analyzed with a spectrophotometer. The protein expression of tyrosinase and microphthalmia associated transcription factor (MITF), as well as phosphorylated or total ERK1/2 and Akt were measured using Western blot. Results: T...

  14. Adapted cytokinesis-block micronucleus assay (CBMn) for mouse embryonic stem cells

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Hamid Kalantari, Hamid Gourabi & Hossein Baharvand ### Abstract Our observation showed the addition of cytochalasin-B to mouse embryonic stem cells (mESC) culture for CBMn analysis led to the induction of apoptosis in these cells. On the other hand, addition of cyt-B is the most critical part of the cytokinesis-block micronucleus assay (CBMn) technique that cannot be omitted. Thus, modification of the traditional CBMn assay seems to be necessary. In this paper, we attempt...

  15. Expression of the protein product of the mouse mammary tumor virus long terminal repeat gene in phorbol ester-treated mouse T-cell-leukemia cells

    International Nuclear Information System (INIS)

    Exposure of C57BL/6 mouse EL-4 T-cell leukemia cells to phorbol ester (12-O-tetradecanoylphorbol-13-acetate) (TPA) induced the synthesis of protein products encoded by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) region. Analysis of TPA-treated EL-4 cells with antiserum raised against a synthetic peptide predicted by the MMTV LTR open reading frame sequence detected a polypeptide migrating in gels with an apparent molecular weight of 37,000 M/sub r/, as well as three less prominent proteins with apparent molecular weights of 31,000, 34,000, and 39,000. Tryptic peptide analysis established the identity of the immunoprecipitated cellular proteins with the LTR proteins obtained from in vitro translation of MMTV genomic RNA. All four proteins were glycosylated and were derived from one initial nonglycosylated translation product of 21,000 M/sub r/. At least 10 acquired MMTV proviruses are present in the EL-4 genome, and examination of the degree of proviral methylation revealed extensive demethylation. However, no qualitative differences in the state of proviral methylation were apparent between TPA-treated and untreated cells

  16. Cell adhesion geometry regulates non-random DNA segregation and asymmetric cell fates in mouse skeletal muscle stem cells.

    Science.gov (United States)

    Yennek, Siham; Burute, Mithila; Théry, Manuel; Tajbakhsh, Shahragim

    2014-05-22

    Cells of several metazoan species have been shown to non-randomly segregate their DNA such that older template DNA strands segregate to one daughter cell. The mechanisms that regulate this asymmetry remain undefined. Determinants of cell fate are polarized during mitosis and partitioned asymmetrically as the spindle pole orients during cell division. Chromatids align along the pole axis; therefore, it is unclear whether extrinsic cues that determine spindle pole position also promote non-random DNA segregation. To mimic the asymmetric divisions seen in the mouse skeletal stem cell niche, we used micropatterns coated with extracellular matrix in asymmetric and symmetric motifs. We show that the frequency of non-random DNA segregation and transcription factor asymmetry correlates with the shape of the motif and that these events can be uncoupled. Furthermore, regulation of DNA segregation by cell adhesion occurs within a defined time interval. Thus, cell adhesion cues have a major impact on determining both DNA segregation patterns and cell fates. PMID:24836002

  17. Cell Adhesion Geometry Regulates Non-Random DNA Segregation and Asymmetric Cell Fates in Mouse Skeletal Muscle Stem Cells

    Directory of Open Access Journals (Sweden)

    Siham Yennek

    2014-05-01

    Full Text Available Cells of several metazoan species have been shown to non-randomly segregate their DNA such that older template DNA strands segregate to one daughter cell. The mechanisms that regulate this asymmetry remain undefined. Determinants of cell fate are polarized during mitosis and partitioned asymmetrically as the spindle pole orients during cell division. Chromatids align along the pole axis; therefore, it is unclear whether extrinsic cues that determine spindle pole position also promote non-random DNA segregation. To mimic the asymmetric divisions seen in the mouse skeletal stem cell niche, we used micropatterns coated with extracellular matrix in asymmetric and symmetric motifs. We show that the frequency of non-random DNA segregation and transcription factor asymmetry correlates with the shape of the motif and that these events can be uncoupled. Furthermore, regulation of DNA segregation by cell adhesion occurs within a defined time interval. Thus, cell adhesion cues have a major impact on determining both DNA segregation patterns and cell fates.

  18. In vitro effect of X radiation on respiration and glycolysis of Ehrlich ascites carcinoma cells of the mouse - an experimental comparison with the mouse tumortetanus assay

    International Nuclear Information System (INIS)

    Depending on the dose of X-rays, in vitro irradiation of Ehrlich ascites carcinoma cells of the mouse affected both respiration and glycolysis. 38.7 C/kg irradiation suppressed the aerobic and anaerobic energy metabolism rather strongly followed by a reduction of the 'take' and growth of the subcutaneously injected tumour cells, as opposed to the growth behavior of non-irradiated cells. In analogy, tetanus mortality rates were reduced in the mouse tumor-tetanus assay with 38.7 C/kg irradiated cells. On the other hand, irradiation with 5.16 C/kg of Ehrlich carcinoma cells resulted in unchanged rates of respiration and glycolysis, in spite of the strongly limited growth capacity of the tumor cells. The tumor-tetanus assay of the mouse showed good correlation with subcutaneous tumor growth; no such correlation was found in the tetanus assay and the manometric values of respiration and glycolysis with 5.16 C/kg irradiated tumor cells. After subcutaneous injection of mixed cell suspensions consisting of 1 x 105 viable and 1 x 106 38.7 C/kg irradiated Ehrlich ascites carcinoma cells as well as of 3 x 102 tetanus spores per single dose, we observed similar rates of tumor growth and tetanus mortality, respectively, if 1 x 105 viable tumor cells alone were administered together with 3 x 102 tetanus spores, without addition of irradiated tumor cells. (author)

  19. Cholesterol esterification during differentiation of mouse erythroleukemia (Friend) cells

    OpenAIRE

    Maria Franca Mulas; Antonella Mandas; Claudia Abete; Sandra Dessì; Alessandra Mocali; Francesco Paoletti

    2011-01-01

    Cholesterol is an essential constituent of all mammalian cell membranes, and its availability is therefore a prerequisite for cellular growth and other functions. Several lines of evidence are now indicating an association between alterations of cholesterol homeostasis and cell cycle progression. However, the role of cholesterol in cell differentiation is still largely unknown. To begin to address this issue, in this study we examined changes in cholesterol metabolism and in the mRNA levels o...

  20. p53 regulation and activity in mouse embryonic stem cells

    OpenAIRE

    Solozobova, Valeriya

    2010-01-01

    P53 is a tumour development p53. The aim of this work was to study the regulation of p53 in embryonic stem cells and its activation in response to DNA damage. p53 was found that p53 becomes transcriptionally active in ES cells after DNA damage. Embryonic stem cells contain a relatively high amount of p53 protein and p53 RNA. After differentiation p53 level is rapidly downregulated. The high abundance of p53 in undifferentiated ES cells is a result of enhanced translation.

  1. Left-half-body-irradiation induced mouse bone marrow hematopoietic cells DNA damage in non-irradiation area

    International Nuclear Information System (INIS)

    Objective: To investigate the DNA damage of mouse bone marrow hematopoietic cells in-non-irradiation area after being irradiated by way of left-half-body. Methods: 6-8 weeks male Kunming strain mice were randomly divided into 4 groups i.e. normal control (NC), total-body-irradiated (TBI), left-half-body-irradiated (LHBI), and total -body-shield-irradiated (TBSI). Half-body-irradiated model were made with two pieces of 5 cm x 8 cm x 16 cm over- lapped lead bricks shielding right-side body and irradiated with 8.0 Gy 60Co γ-ray. The TNF-α, SOD, MDA in mouse serum were measured and the DNA damages of bone marrow hematopoietic cells were observed by comet assay and the frequency of polychromatic erythrocytes micronucleated(fMPCE). Results: In the left-half-body-irradiated condition, The TNF-α and MDA were increased and the SOD was decreased in serum remarkably(compared with NC, P<0.01); In non-irradiation area, the fMPCE and the percentage of bone marrow hematopoietic cells with comet-like tail, were aggravated significantly. Conclusions: Our study suggest that the local irradiation result in the DNA damage of bone marrow hematopoietic cells in non- irradiation area, and the increasing of TNF-α and reactive oxygen or free radicals may play an important role in the damages. (authors)

  2. Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines

    Institute of Scientific and Technical Information of China (English)

    Xue-Jun Dong; Guo-Rong Zhang; Qing-Jun Zhou; Ruo-Lang Pan; Ye Chen; Li-Xin Xiang; Jian-Zhong Shao

    2009-01-01

    AIM: To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes. METHODS: Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells. RESULTS: Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages. CONCLUSION: Hepatic cells

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

    International Nuclear Information System (INIS)

    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 [AT1R] 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 of

  4. A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

    Science.gov (United States)

    Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

    2016-08-25

    Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. PMID:27365425

  5. Comparative studies on the induction of mutation and malignant transformation in mouse and human cells

    International Nuclear Information System (INIS)

    Somatic cell mutation has long been considered as a possible mechanism of the carcinogenesis by physical and chemical agents. A system with which the frequency of mutation and transformation being assayed simultaneously was developed, using human diploid fibroblasts and the subclones of BALB/3T3 mouse cell line. These 2 target cells were chosen because of their availability and their difference in the species. The induced frequency of mutation seems significantly lower than transformation frequency, if the alteration of clonal morphology is used as a criterion of transformation. Among many speculations proposed to explain the difficulty of transformation of human diploid cells, it is emphasized that they have very low probability to acquire the immortality. The conversion of normal diploid cells into malignant cells in culture is a stepwise development through qualitatively different stages. This multistep development may correspond to the progression of in vivo tumors. Carcinogens will not be directly responsible for this entire multistep process. The direct action of carcinogens on diploid cells will not be manifested rapidly and effectively as tumorigenic conversion as compared to that on the established cell lines. There is marked difference in the probability of acquisition of immortality between human and hamster or mouse cells. The low frequency of the malignant transformation of human diploid cells is ascribed to the low probability of immortal conversion of human diploid cells. (Yamashita, S.)

  6. Mouse Spermatogenic Stem Cells Continually Interconvert between Equipotent Singly Isolated and Syncytial States

    Science.gov (United States)

    Hara, Kenshiro; Nakagawa, Toshinori; Enomoto, Hideki; Suzuki, Mikiko; Yamamoto, Masayuki; Simons, Benjamin D.; Yoshida, Shosei

    2014-01-01

    Summary The identity and behavior of mouse spermatogenic stem cells have been a long-standing focus of interest. In the prevailing “As model,” stem cell function is restricted to singly isolated (As) spermatogonia. By examining single-cell dynamics of GFRα1+ stem cells in vivo, we evaluate an alternative hypothesis that, through fragmentation, syncytial spermatogonia also contribute to stem cell function in homeostasis. We use live imaging and pulse labeling to quantitatively determine the fates of individual GFRα1+ cells and find that, during steady-state spermatogenesis, the entire GFRα1+ population comprises a single stem cell pool, in which cells continually interconvert between As and syncytial states. A minimal biophysical model, relying only on the rates of incomplete cell division and syncytial fragmentation, precisely predicts the stochastic fates of GFRα1+ cells during steady state and postinsult regeneration. Thus, our results define an alternative and dynamic model for spermatogenic stem cell function in the mouse testis. PMID:24792118

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

    International Nuclear Information System (INIS)

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

    International Nuclear Information System (INIS)

    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 (H2O2) sensitivity. Hypersensitivity to H2O2 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

  9. Nicotinamide-Induced Apoptosis Can Be Enhanced by Melatonin in Mouse Myeloma Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Guiyou; SHENG Hongzhi; LIU Jia

    2006-01-01

    The mechanism of apoptosis induced by nicotinamide was investigated by treating mouse myeloma cells (Sp2/0) with various concentrations of nicotinamide. The typical hallmarks of apoptosis, including chromatin condensation and DNA fragmentation, were detected when cells were treated with nicotinamide at concentrations of 30, 40, 50, and 60 mmol/L. The apoptosis percentage increased with increasing nicotinamide concentration. Interestingly, the strong antioxidant melatonin did not restrain the apoptosis induced by nicotinamide in mouse myeloma cells but greatly increased the induction of nicotinamide on apoptosis. When cells were preincubated with 0.1, 1, and 10 mmol/L melatonin before nicotinamide induction, the percentage of apoptosis induced by 50 mmol/L nicotinamide markedly increased with increasing melatonin concentration. These results suggest that apoptosis induced by nicotinamide has no relationship with oxidative stress and melatonin could enhance nicotinamide-induced apoptosis in mouse myeloma cells by stimulating cell division in a certain manner. Nicotinamide may provide a new method to treat some kinds of tumors with no damage to normal tissues.

  10. Ionizing radiation is a potent inducer of mitotic recombination in mouse embryonic stem cells

    International Nuclear Information System (INIS)

    Highlights: → Embryonic stem cells have a distinct mutational response to X-rays. → X-rays induce more mutations in embryonic stem cells than in somatic cells. → Mitotic recombination is more readily induced by X-rays in embryonic stem cells. → Radiation hazards may have different consequences on different types of cells. - Abstract: Maintenance of genomic integrity in embryonic cells is pivotal to proper embryogenesis, organogenesis and to the continuity of species. Cultured mouse embryonic stem cells (mESCs), a model for early embryonic cells, differ from cultured somatic cells in their capacity to remodel chromatin, in their repertoire of DNA repair enzymes, and in the regulation of cell cycle checkpoints. Using 129XC3HF1 mESCs heterozygous for Aprt, we characterized loss of Aprt heterozygosity after exposure to ionizing radiation. We report here that the frequency of loss of heterozygosity mutants in mESCs can be induced several hundred-fold by exposure to 5-10 Gy of X-rays. This induction is 50-100-fold higher than the induction reported for mouse adult or embryonic fibroblasts. The primary mechanism underlying the elevated loss of heterozygosity after irradiation is mitotic recombination, with lesser contributions from deletions and gene conversions that span Aprt. Aprt point mutations and epigenetic inactivation are very rare in mESCs compared to fibroblasts. Mouse ESCs, therefore, are distinctive in their response to ionizing radiation and studies of differentiated cells may underestimate the mutagenic effects of ionizing radiation on ESC or other stem cells. Our findings are important to understanding the biological effects of ionizing radiation on early development and carcinogenesis.

  11. Ionizing radiation is a potent inducer of mitotic recombination in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Denissova, Natalia G.; Tereshchenko, Irina V.; Cui, Eric [Department of Genetics, Rutgers University, Piscataway, 145 Bevier Rd, NJ 08854 (United States); Stambrook, Peter J. [Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, OH 45267 (United States); Shao, Changshun [Department of Genetics, Rutgers University, Piscataway, 145 Bevier Rd, NJ 08854 (United States); Tischfield, Jay A., E-mail: jay@biology.rutgers.edu [Department of Genetics, Rutgers University, Piscataway, 145 Bevier Rd, NJ 08854 (United States)

    2011-10-01

    Highlights: {yields} Embryonic stem cells have a distinct mutational response to X-rays. {yields} X-rays induce more mutations in embryonic stem cells than in somatic cells. {yields} Mitotic recombination is more readily induced by X-rays in embryonic stem cells. {yields} Radiation hazards may have different consequences on different types of cells. - Abstract: Maintenance of genomic integrity in embryonic cells is pivotal to proper embryogenesis, organogenesis and to the continuity of species. Cultured mouse embryonic stem cells (mESCs), a model for early embryonic cells, differ from cultured somatic cells in their capacity to remodel chromatin, in their repertoire of DNA repair enzymes, and in the regulation of cell cycle checkpoints. Using 129XC3HF1 mESCs heterozygous for Aprt, we characterized loss of Aprt heterozygosity after exposure to ionizing radiation. We report here that the frequency of loss of heterozygosity mutants in mESCs can be induced several hundred-fold by exposure to 5-10 Gy of X-rays. This induction is 50-100-fold higher than the induction reported for mouse adult or embryonic fibroblasts. The primary mechanism underlying the elevated loss of heterozygosity after irradiation is mitotic recombination, with lesser contributions from deletions and gene conversions that span Aprt. Aprt point mutations and epigenetic inactivation are very rare in mESCs compared to fibroblasts. Mouse ESCs, therefore, are distinctive in their response to ionizing radiation and studies of differentiated cells may underestimate the mutagenic effects of ionizing radiation on ESC or other stem cells. Our findings are important to understanding the biological effects of ionizing radiation on early development and carcinogenesis.

  12. 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.

  13. Evaluation of genetic potential of Bacopa monnieri extract in Mouse bone marrow cells by chromosomal analysis test

    Directory of Open Access Journals (Sweden)

    Shilki Vishnoi

    2013-06-01

    Full Text Available Herbs have always been used as a common source of medicines, the Bacopa monnieri is an important herb used in Aruveda as a traditional medicinal system of India. In the present investigations, the genotoxic potential of Bacopa monnieri Hydromethanolic extract (BMH was evaluated employing Chromosomal analysis assay invivo. BMH was administered to Swiss Albino mice as i.p. dose of 80mg/kg, 160mg/kg, 240mg/kg body wt., 24 hours prior the administration of cyclophosphamide (CP (positive control at the dose of 50 mg/kg body wt. A dose-dependent, significant decrease in chromosome aberration was observed with respect to control. Result suggested that BMHhave a preventive potential against CP induced chromosomal aberration in Swiss albino mouse bone marrow cells at the dose tested. Therefore seems to have a preventive potential against Chromosomal aberrations in Swiss Albinomouse bone marrow cells.

  14. Pointright: a system to redirect mouse and keyboard control among multiple machines

    Science.gov (United States)

    Johanson, Bradley E.; Winograd, Terry A.; Hutchins, Gregory M.

    2008-09-30

    The present invention provides a software system, PointRight, that allows for smooth and effortless control of pointing and input devices among multiple displays. With PointRight, a single free-floating mouse and keyboard can be used to control multiple screens. When the cursor reaches the edge of a screen it seamlessly moves to the adjacent screen and keyboard control is simultaneously redirected to the appropriate machine. Laptops may also redirect their keyboard and pointing device, and multiple pointers are supported simultaneously. The system automatically reconfigures itself as displays go on, go off, or change the machine they display.

  15. Different mouse mast cell populations express various combinations of at least six distinct mast cell serine proteases.

    OpenAIRE

    Reynolds, D. S.; Stevens, R L; Lane, W S; Carr, M H; Austen, K. F.; Serafin, W E

    1990-01-01

    Mouse serosal mast cells (SMCs) and Kirsten sarcoma virus-immortalized mast cells store large amounts of mast cell carboxypeptidase A and serine proteases in their secretory granules. Secretory granule proteins from 2.6 x 10(6) purified SMCs were separated by NaDodSO4/PAGE, trans-blotted to poly(vinylidine difluoride) membranes, and subjected to amino-terminal amino acid sequencing. Four distinct mast cell serine proteases were identified. With mast cell carboxypeptidase A, these serine prote...

  16. A new immuno-, dystrophin-deficient model, the NSG-mdx(4Cv) 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

    2013-08-01

    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-mdx(4Cv) 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-mdx(4Cv) 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-mdx(4Cv) 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-mdx(4Cv) model for studies on muscle regeneration and Duchenne muscular dystrophy therapy. PMID:23606600

  17. Mouse protein arrays from a TH1 cell cDNA library for antibody screening and serum profiling

    OpenAIRE

    Gutjahr, C.; Murphy, D.; Lueking, A.; Koenig, A.; Janitz, M; O'Brien, J.; Korn, B. (Bernhard); S. Horn; Lehrach, H; Cahill, D.

    2005-01-01

    The mouse is the premier genetic model organism for the study of disease and development. We describe the establishment of a mouse T helper cell type 1 (TH1) protein expression library that provides direct access to thousands of recombinant mouse proteins, in particular those associated with immune responses. The advantage of a system based on the combination of large cDNA expression libraries with microarray technology is the direct connection of the DNA sequence information from a particula...

  18. Mouse Incisor Stem Cell Niche and Myb Transcription Factors

    Czech Academy of Sciences Publication Activity Database

    Švandová, Eva; Veselá, Barbora; Šmarda, J.; Hampl, A.; Radlanski, R.J.; Matalová, Eva

    2015-01-01

    Roč. 44, č. 5 (2015), s. 338-344. ISSN 0340-2096 R&D Projects: GA ČR GAP304/11/1418; GA ČR GCP302/12/J059 Institutional support: RVO:67985904 Keywords : c-Myb * stem cell niches Subject RIV: EA - Cell Biology Impact factor: 0.672, year: 2014

  19. Divergence of zebrafish and mouse lymphatic cell fate specification pathways

    DEFF Research Database (Denmark)

    van Impel, Andreas; Zhao, Zhonghua; Hermkens, Dorien M A;

    2014-01-01

    In mammals, the homeodomain transcription factor Prox1 acts as the central regulator of lymphatic cell fate. Its restricted expression in a subset of cardinal vein cells leads to a switch towards lymphatic specification and hence represents a prerequisite for the initiation of lymphangiogenesis. ...

  20. Action of DTPA on hepatic plutonium. II. DTPA-induced removal of monomeric plutonium from mouse liver parenchymal cells

    International Nuclear Information System (INIS)

    Liver parenchymal cells were isolated 6 and 24 hr following the administration of diethylenetriaminepentaacetic acid (DTPA, 0.25 mmole/kg) to mice previously injected with 239Pu-citrate (4.4 μCi/kg). Isolated parenchymal cells contained 440 dpm Pu/106 cells at 24 hr after Pu injection, just prior to DTPA administration. The PU content decreased to 330 dpm/106 cells at 6 hr and 140 dpm/106 cells at 24 hr after DTPA administration. Thus DTPA induced a striking decrease in the Pu content of isolated liver parenchymal cells. Parenchymal cells isolated from control mice not treated with DTPA changed little in Pu content from 24 to 48 hr after Pu injection. By 24 hr after DTPA treatment, the decrease in the Pu content of isolated liver parenchymal cells could account for the DTPA-induced release of Pu from the intact liver. Thus in the liver DTPA appears to act preferentially on the Pu associated with parenchymal cells. Liver parenchymal cells isolated 6 hr after DTPA administration and containing 330 dpm Pu/106 cells were incubated in vitro in the absence of added DTPA. After 18 hr of incubation the cells contained 130 dpm Pu/106 cells. This level corresponds to the level observed in cells isolated 24 hr after DTPA administration. Cells isolated from untreated mice lost only 15% of their Pu content during a similar in vitro incubation. Thus, by 6 hr after DTPA administration to the mouse, isolated liver parenchymal cells appeared to retain their ability to release Pu in vitro with no need for additional exposure to DTPA. The physiological significance of this finding is discussed

  1. Contrasting quiescent G0 phase with mitotic cell cycling in the mouse immune system.

    Directory of Open Access Journals (Sweden)

    Michio Tomura

    Full Text Available A transgenic mouse line expressing Fucci (fluorescent ubiquitination-based cell-cycle indicator probes allows us to monitor the cell cycle in the hematopoietic system. Two populations with high and low intensities of Fucci signals for Cdt1(30/120 accumulation were identified by FACS analysis, and these correspond to quiescent G0 and cycling G1 cells, respectively. We observed the transition of immune cells between quiescent and proliferative phases in lymphoid organs during differentiation and immune responses.

  2. CD13 is dispensable for normal hematopoiesis and myeloid cell functions in the mouse

    OpenAIRE

    Winnicka, Beata; O'Conor, Catherine; Schacke, Wolfgang; Vernier, Kaitlyn; Grant, Christina L.; Fenteany, Fiona Hall; Pereira, Flavia E.; Liang, Brannen; Kaur, Anupinder; Zhao, Ran; Montrose, David C.; Rosenberg, Daniel W; Aguila, Hector L.; Shapiro, Linda H.

    2010-01-01

    The robust and consistent expression of the CD13 cell surface marker on very early as well as differentiated myeloid hematopoietic cells has prompted numerous investigations seeking to define roles for CD13 in myeloid cells. To address the function of myeloid CD13 directly, we created a CD13 null mouse and assessed the responses of purified primary macrophages or DCs from WT and CD13 null animals in cell assays and inflammatory disease models, where CD13 has been implicated previously. We fin...

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Snail1 controls epithelial–mesenchymal lineage commitment in focal adhesion kinase–null embryonic cells

    OpenAIRE

    Li, Xiao-Yan; Zhou, Xiaoming; Rowe, R. Grant; Hu, Yuexian; Schlaepfer, David D.; Ilić, Dusko; Dressler, Gregory; Park, Ann; Guan, Jun-Lin; Weiss, Stephen J.

    2011-01-01

    Mouse embryonic cells isolated from focal adhesion kinase (FAK)–null animals at embryonic day 7.5 display multiple defects in focal adhesion remodeling, microtubule dynamics, mechanotransduction, proliferation, directional motility, and invasion. To date, the ability of FAK to modulate cell function has been ascribed largely to its control of posttranscriptional signaling cascades in this embryonic cell population. In this paper, we demonstrate that FAK unexpectedly exerts control over an epi...

  5. Mouse DRG Cell Line with Properties of Nociceptors.

    Directory of Open Access Journals (Sweden)

    Ciara Doran

    Full Text Available In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons.

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

    International Nuclear Information System (INIS)

    Research highlights: → Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2rγnull mice. → Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. → The islet β cells were selectively destroyed by infiltrated human T cells. → 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 β 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 IL2rγnull mice. The selective destruction of pancreatic islet β 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 β-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the β 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 β cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4+ T cell infiltration and clonal expansion, and the mouse islet β-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet β 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.

  7. 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.

  8. Accurate cell counts in live mouse embryos using optical quadrature and differential interference contrast microscopy

    Science.gov (United States)

    Warger, William C., II; Newmark, Judith A.; Zhao, Bing; Warner, Carol M.; DiMarzio, Charles A.

    2006-02-01

    Present imaging techniques used in in vitro fertilization (IVF) clinics are unable to produce accurate cell counts in developing embryos past the eight-cell stage. We have developed a method that has produced accurate cell counts in live mouse embryos ranging from 13-25 cells by combining Differential Interference Contrast (DIC) and Optical Quadrature Microscopy. Optical Quadrature Microscopy is an interferometric imaging modality that measures the amplitude and phase of the signal beam that travels through the embryo. The phase is transformed into an image of optical path length difference, which is used to determine the maximum optical path length deviation of a single cell. DIC microscopy gives distinct cell boundaries for cells within the focal plane when other cells do not lie in the path to the objective. Fitting an ellipse to the boundary of a single cell in the DIC image and combining it with the maximum optical path length deviation of a single cell creates an ellipsoidal model cell of optical path length deviation. Subtracting the model cell from the Optical Quadrature image will either show the optical path length deviation of the culture medium or reveal another cell underneath. Once all the boundaries are used in the DIC image, the subtracted Optical Quadrature image is analyzed to determine the cell boundaries of the remaining cells. The final cell count is produced when no more cells can be subtracted. We have produced exact cell counts on 5 samples, which have been validated by Epi-Fluorescence images of Hoechst stained nuclei.

  9. Nek11 regulates asymmetric cell division during mouse oocyte meiotic maturation.

    Science.gov (United States)

    Guo, Lei; Wang, Zhen-Bo; Wang, Hong-Hui; Zhang, Teng; Qi, Shu-Tao; Ouyang, Ying-Chun; Hou, Yi; Sun, Qing-Yuan

    2016-06-10

    Nek11, a member of the never in mitosis gene A (NIMA) family, is activated in somatic cells associated with G1/S or G2/M arrest. However, its function in meiosis is unknown. In this research, the expression, localization and functions of NEK11 in the mouse oocyte meiotic maturation were examined. Western blotting indicated that NEK11S was the major NEK11 protein in mouse oocyte. MYC-tagged Nek11 mRNA microinjection and immunofluorescent staining showed that NEK11 was localized to the meiotic spindles at MI and MII stage. Knockdown of Nek11 by microinjection of siRNA did not affect germinal vesicle breakdown (GVBD) and the first polar body extrusion, but caused formation of 2-cell-like eggs. These results demonstrate that Nek11 regulates asymmetric cell division during oocyte meiotic maturation. PMID:27150633

  10. Humanized Mouse Models to Study Cell-Mediated Immune Responses to Liver-Stage Malaria Vaccines.

    Science.gov (United States)

    Good, Michael F; Hawkes, Michael T; Yanow, Stephanie K

    2015-11-01

    Malaria vaccine development is hampered by the lack of small animal models that recapitulate human immune responses to Plasmodium falciparum. We review the burgeoning literature on humanized mice for P. falciparum infection, including challenges in engraftment of human immune cells, hepatocytes, and erythrocytes. Recent advances in immune-compromised mouse models and stem cell technology have already enabled proof of concept that the entire parasite life cycle can be sustained in a murine model and that adaptive human immune responses to several parasite stages can be measured. Nonetheless, optimization is needed to achieve a reproducible and relevant murine model for malaria vaccine development. This review is focused on the complexities of T cell development in a mouse humanized with both a lymphoid system and hepatocytes. An understanding of this will facilitate the use of humanized mice in the development of liver-stage vaccines. PMID:26458783

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

    International Nuclear Information System (INIS)

    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 137Cs 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)

  12. Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq.

    Science.gov (United States)

    Gokce, Ozgun; Stanley, Geoffrey M; Treutlein, Barbara; Neff, Norma F; Camp, J Gray; Malenka, Robert C; Rothwell, Patrick E; Fuccillo, Marc V; Südhof, Thomas C; Quake, Stephen R

    2016-07-26

    The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs) that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states. PMID:27425622

  13. Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq

    Directory of Open Access Journals (Sweden)

    Ozgun Gokce

    2016-07-01

    Full Text Available The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs that have specific markers and that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states.

  14. 1-cell embryo transfer into pseudopregnant recipient female mouse

    OpenAIRE

    sprotocols

    2014-01-01

    ### Abstract 1-cell embryo transfer is best performed after allowing injected embryos a little recovery time in culture. This allows better evaluation of the cells' survival - those that have been damaged during the injection process will undergo cytoplasmic condensation, causing the cellular material to become less glossy and darker in color as the cytoplasm shrinks away from the zona pellucida. This should be balanced against the increased survival rate with decreased in vitro exposure....

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

    International Nuclear Information System (INIS)

    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.)

  16. Species-specific metastasis of human tumor cells in the severe combined immunodeficiency mouse engrafted with human tissue.

    OpenAIRE

    Shtivelman, E; Namikawa, R

    1995-01-01

    We have attempted to model human metastatic disease by implanting human target organs into the immunodeficient C.B-17 scid/scid (severe combined immunodeficiency; SCID) mouse, creating SCID-hu mice. Preferential metastasis to implants of human fetal lung and human fetal bone marrow occurred after i.v. injection of human small cell lung cancer (SCLC) cells into SCID-hu mice; the homologous mouse organs were spared. Clinically more aggressive variant SCLC cells metastasized more efficiently to ...

  17. Production of proinflammatory mediators by indoor air bacteria and fungal spores in mouse and human cell lines.

    OpenAIRE

    Huttunen, Kati; Hyvärinen, Anne; Nevalainen, Aino; Komulainen, Hannu; Hirvonen, Maija-Riitta

    2003-01-01

    We compared the inflammatory and cytotoxic responses caused by household mold and bacteria in human and mouse cell lines. We studied the fungi Aspergillus versicolor, Penicillium spinulosum, and Stachybotrys chartarum and the bacteria Bacillus cereus, Pseudomonas fluorescens, and Streptomyces californicus for their cytotoxicity and ability to stimulate the production of inflammatory mediators in mouse RAW264.7 and human 28SC macrophage cell lines and in the human A549 lung epithelial cell lin...

  18. 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.

  19. 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.

  20. Immunohistochemical analysis of Clara cell secretory protein expression in a transgenic model of mouse lung carcinogenesis

    International Nuclear Information System (INIS)

    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

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

    International Nuclear Information System (INIS)

    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

  2. Tetrandrine protects mouse retinal ganglion cells from ischemic injury

    Directory of Open Access Journals (Sweden)

    Li WY

    2014-03-01

    Full Text Available Weiyi Li,1,2 Chen Yang,2 Jing Lu,2 Ping Huang,1 Colin J Barnstable,2 Chun Zhang,1 Samuel S Zhang2,3 1Department of Ophthalmology, Peking University Third Hospital, Peking University Eye Center, Beijing, People's Republic of China; 2Department of Neural and Behavioral Sciences, Penn State University, Hershey, PA, USA; 3Singapore Eye Research Institute, Singapore National Eye Centre, Singapore Abstract: This study aimed to determine the protective effects of tetrandrine (Tet on murine ischemia-injured retinal ganglion cells (RGCs. For this, we used serum deprivation cell model, glutamate and hydrogen peroxide (H2O2-induced RGC-5 cell death models, and staurosporine-differentiated neuron-like RGC-5 in vitro. We also investigated cell survival of purified primary-cultured RGCs treated with Tet. An in vivo retinal ischemia/reperfusion model was used to examine RGC survival after Tet administration 1 day before ischemia. We found that Tet affected RGC-5 survival in a dose- and time-dependent manner. Compared to dimethyl sulfoxide treatment, Tet increased the numbers of RGC-5 cells by 30% at 72 hours. After 48 hours, Tet protected staurosporine-induced RGC-5 cells from serum deprivation-induced cell death and significantly increased the relative number of cells cultured with 1 mM H2O2 (P<0.01. Several concentrations of Tet significantly prevented 25-mM-glutamate-induced cell death in a dose-dependent manner. Tet also increased primary RGC survival after 72 and 96 hours. Tet administration (10 µM, 2 µL 1 day before retinal ischemia showed RGC layer loss (greater survival, which was less than those in groups with phosphate-buffered saline intravitreal injection plus ischemia in the central (P=0.005, n=6, middle (P=0.018, n=6, and peripheral (P=0.017, n=6 parts of the retina. Thus, Tet conferred protective effects on serum deprivation models of staurosporine-differentiated neuron-like RGC-5 cells and primary cultured murine RGCs. Furthermore, Tet showed

  3. Cell-type-specific neuroanatomy of cliques of autism-related genes in the mouse brain

    Directory of Open Access Journals (Sweden)

    Pascal eGrange

    2015-05-01

    Full Text Available Two cliques of genes identified computationally for their high co-expression in the mouse brain according to the Allen Brain Atlas, and for their enrichment in genes related to autism spectrum disorder, have recently been shown to be highly co-expressed in the cerebellar cortex, compared to what could be expected by chance. Moreover, the expression of these cliques of genes is not homogeneous across the cerebellar cortex, and it has been noted that their expression pattern seems to highlight the granular layer. However, this observation was only made by eye, and recent advances in computational neuroanatomy allow to rank cell types in the mouse brain (characterized by their transcriptome profiles according to the similarity between their spatial density profiles and the expression profiles of the cliques. We establish by Monte Carlo simulation that with probability at least 99%, the expression profiles of the two cliques are more similar to the density profile of granule cells than 99% of the expression of cliques containing the same number of genes (Purkinje cells also score above 99% in one of the cliques. Thresholding the expression profiles shows that the signal is more intense in the granular layer. Finally, we work out pairs of cell types whose combined expression profiles are more similar to the expression profiles of the cliquesthan any single cell type. These pairs predominantly consist of one cortical pyramidal cell and one cerebellar cell (whichcan be either a granule cell or a Purkinje cell.

  4. Cell-type-specific neuroanatomy of cliques of autism-related genes in the mouse brain.

    Science.gov (United States)

    Grange, Pascal; Menashe, Idan; Hawrylycz, Michael

    2015-01-01

    Two cliques of genes identified computationally for their high co-expression in the mouse brain according to the Allen Brain Atlas, and for their enrichment in genes related to autism spectrum disorder (ASD), have recently been shown to be highly co-expressed in the cerebellar cortex, compared to what could be expected by chance. Moreover, the expression of these cliques of genes is not homogeneous across the cerebellar cortex, and it has been noted that their expression pattern seems to highlight the granular layer. However, this observation was only made by eye, and recent advances in computational neuroanatomy allow to rank cell types in the mouse brain (characterized by their transcriptome profiles) according to the similarity between their spatial density profiles and the spatial expression profiles of the cliques. We establish by Monte Carlo simulation that with probability at least 99%, the expression profiles of the two cliques are more similar to the density profile of granule cells than 99% of the expression of cliques containing the same number of genes (Purkinje cells also score above 99% in one of the cliques). Thresholding the expression profiles shows that the signal is more intense in the granular layer. Finally, we work out pairs of cell types whose combined expression profiles are more similar to the expression profiles of the cliques than any single cell type. These pairs predominantly consist of one cortical pyramidal cell and one cerebellar cell (which can be either a granule cell or a Purkinje cell). PMID:26074809

  5. Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

  6. Hhex Is Necessary for the Hepatic Differentiation of Mouse ES Cells and Acts via Vegf Signaling.

    Science.gov (United States)

    Arterbery, Adam S; Bogue, Clifford W

    2016-01-01

    Elucidating the molecular mechanisms involved in the differentiation of stem cells to hepatic cells is critical for both understanding normal developmental processes as well as for optimizing the generation of functional hepatic cells for therapy. We performed in vitro differentiation of mouse embryonic stem cells (mESCs) with a null mutation in the homeobox gene Hhex and show that Hhex(-/-) mESCs fail to differentiate from definitive endoderm (Sox17(+/)Foxa2(+)) to hepatic endoderm (Alb(+)/Dlk(+)). In addition, hepatic culture elicited a >7-fold increase in Vegfa mRNA expression in Hhex(-/-) cells compared to Hhex(+/+) cells. Furthermore, we identified VEGFR2(+)/ALB(+/)CD34(-) in early Hhex(+/+) hepatic cultures. These cells were absent in Hhex(-/-) cultures. Finally, through manipulation of Hhex and Vegfa expression, gain and loss of expression experiments revealed that Hhex shares an inverse relationship with the activity of the Vegf signaling pathway in supporting hepatic differentiation. In summary, our results suggest that Hhex represses Vegf signaling during hepatic differentiation of mouse ESCs allowing for cell-type autonomous regulation of Vegfr2 activity independent of endothelial cells. PMID:26784346

  7. Cardiomyocyte Marker Expression in Mouse Embryonic Fibroblasts by Cell-Free Cardiomyocyte Extract and Epigenetic Manipulation

    Directory of Open Access Journals (Sweden)

    Tahereh Talaei-Khozani

    2014-03-01

    Full Text Available Background: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium. Methods: Mouse embryonic fibroblasts were treated with Trichostatin A (TSA and 5-Aza-2-Deoxycytidine (5-aza-dC. The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21 days. Cardiomyocyte markers were detected by immunohistochemistry. Alkaline phosphatase activity and OCT4 were also detected in cells treated by chromatin-modifying agents. Results: The cells exposed to a combination of 5-aza-dC and TSA and permeabilized in the presence of the cardiomyocyte extract showed morphological changes. The cells were unable to express cardiomyocyte markers after 24 h. Immunocytochemical assays showed a notable degree of myosin heavy chain and α-actinin expressions after 10 days. The expression of the natriuretic factor and troponin T occurred after 21 days in these cells. The cells exposed to chromatin-modifying agents also expressed cardiomyocyte markers; however, the proportion of reprogrammed cells was clearly smaller than that in the cultures exposed to 5-aza-dC , TSA, and extract. Conclusion: It seems that the fibroblasts were able to eliminate the previous epigenetic markers and form new ones according to the factors existing in the extract. Since no beating was observed, at least up to 21 days, the cells may need an appropriate extracellular matrix for their function.

  8. Evaluation of direct and indirect effects of the PPARγ agonist troglitazone on mouse endothelial cell proliferation.

    Science.gov (United States)

    Kakiuchi-Kiyota, Satoko; Arnold, Lora L; Yokohira, Masanao; Koza-Taylor, Petra; Suzuki, Shugo; Varney, Michelle; Pennington, Karen L; Cohen, Samuel M

    2011-12-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) agonists and PPARγ/α dual agonists are used in the treatment of type 2 diabetes mellitus and hyperlipidemias. In carcinogenicity studies, some of these agonists induced hemangiomas/hemangiosarcomas in mice, but not in rats. We hypothesized that increased endothelial cell (EC) proliferation may be involved in the mechanism of PPAR agonist-induced vascular tumors in mice. We previously showed that the sarcomagenic PPARγ agonist troglitazone (TG) increased EC proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses (400 and 800 mg/kg) after four weeks of treatment. In vitro, TG had a mitogenic effect on mouse microvascular mouse ECs by increasing cell proliferation and survival. The current studies showed that treatment of mouse ECs in vitro induced alterations in proliferation pathway gene expression, especially the expression of insulin-like growth factor-1, but had no effect on mouse oxidative stress pathways. In vivo, treatment with vitamin E did not inhibit TG-induced EC proliferation in liver and adipose tissue. In addition, no hypoxic effect was detected in adipose tissue of TG-treated mice; however, TG had a minor effect on hepatocellular hypoxia. These results provide additional evidence supporting a direct mitogenic effect in the mode of action of TG-induced hemangiosarcomas in mice. PMID:21937740

  9. Tritiated thymidine and deoxycytidine suicide of mouse hemopoietic colony forming cells (CFC)

    International Nuclear Information System (INIS)

    Significant enhancement of tritiated dCyd suicide occurred when unlabelled dThd was added to cultures of mouse monocytic colony-forming cells. Incorporation experiments supported the suicide experiments in that incorporation of tritiated dCyd into DNA was significantly increased. One hundred micromolar dCyd significantly reduced the radiotoxicity of 0.3 μCi of tritiated dThd; incorporation experiments indicated a dose-related reduction in the incorporation of tritiated dThd into DNA with the addition of 1-100 μM unlabelled dCyd. The addition of 1 μM aminopterin reversed the effect of 100 μM deoxycytidine; viz., incorporation of dThd into DNA was 90% of controls. Aminopterin had a similar effect on deoxyuridine reversal of tritiated dThd incorporation into DNA. Aminopterin had no effect on the reduction of tritiated dThd incorporation into DNA due to the addition of 100 μM unlabelled thymidine. Unlabelled ribonucleosides, Urd and Cyd, did not significantly affect the suicide pattern of tritiated dThd or dCyd when they were added to CFC cultures. Unlabelled deoxyribonucleosides, dThd or dCyd, did not significantly affect the suicide pattern of either tritiated Cyd or Urd when they were added to cultures containing tritiated ribonucleosides. Unlabelled Urd or Cyd was effective in reversing the suicide due to tritiated Urd or Cyd. (author)

  10. The Late Stage of T Cell Development within Mouse Thymus

    Institute of Scientific and Technical Information of China (English)

    Weifeng Chen

    2004-01-01

    After positive selection and lineage commitment, the TCRαβ+CD4/CD8 SP medullary thymocytes migrate into and reside in thymic medulla, where they undergo an ordered program of late stage of T cell functional maturation and negative selection to delete self-reactive clones by apoptosis. Accomplishment of this final differentiation pathway, a physiological T cell repertoire is formed : T cells acquire immunocompetence to respond to foreign antigens and tolerance to self-antigens, ready for the emigration to homing to the T cell regions of peripheral lymphoid organs and tissues. In this review, emphases are put on introducing the approaches applied in this area and our own observations. Basically, we have analyzed the late stage of medullary thymocyte phenotypic differentiation pathways of both CD4 SP and CD8 SP medullary thymocytes and the concomitant functional maturation pathway, in particular, of CD4 SP thymocytes. It is to provide a standard to compare the functional capacity of the cells at the developmental stages induced by different conditions. The cellular and molecular basis of this differentiation process has been partially described. Cellular & Molecular Immunology. 2004;1(1):3-11.

  11. The Late Stage of T Cell Development within Mouse Thymus

    Institute of Scientific and Technical Information of China (English)

    WeifengChen

    2004-01-01

    After positive selection and lineage commitment, the TCRαβ+CD4/CD8 SP medullary thymocytes migrate into and reside in thymic medulla, where they undergo an ordered program of late stage of T cell functional maturation and negative selection to delete self-reactive clones by apoptosis. Accomplishment of this final differentiation pathway, a physiological T cell repertoire is formed: T cells acquire immunocompetence to respond to foreign antigens and tolerance to self-antigens, ready for the emigration to homing to the T cell regions of peripheral lymphoid organs and tissues. In this review, emphases are put on introducing the approaches applied in this area and our own observations. Basically, we have analyzed the late stage of medullary thymocyte phenotypic differentiation pathways of both CD4 SP and CD8 SP medullary thymocytes and the concomitant functional maturation pathway, in particular, of CD4 SP thymocytes. It is to provide a standard to compare the functional capacity of the cells at the developmental stages induced by different conditions. The cellular and molecular basis of this differentiation process has been partially described. Cellular & Molecular Immunology. 2004;1(1):3-11.

  12. Functional integration of human neural precursor cells in mouse cortex.

    Directory of Open Access Journals (Sweden)

    Fu-Wen Zhou

    Full Text Available This study investigates the electrophysiological properties and functional integration of different phenotypes of transplanted human neural precursor cells (hNPCs in immunodeficient NSG mice. Postnatal day 2 mice received unilateral injections of 100,000 GFP+ hNPCs into the right parietal cortex. Eight weeks after transplantation, 1.21% of transplanted hNPCs survived. In these hNPCs, parvalbumin (PV-, calretinin (CR-, somatostatin (SS-positive inhibitory interneurons and excitatory pyramidal neurons were confirmed electrophysiologically and histologically. All GFP+ hNPCs were immunoreactive with anti-human specific nuclear protein. The proportions of PV-, CR-, and SS-positive cells among GFP+ cells were 35.5%, 15.7%, and 17.1%, respectively; around 15% of GFP+ cells were identified as pyramidal neurons. Those electrophysiologically and histological identified GFP+ hNPCs were shown to fire action potentials with the appropriate firing patterns for different classes of neurons and to display spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs. The amplitude, frequency and kinetic properties of sEPSCs and sIPSCs in different types of hNPCs were comparable to host cells of the same type. In conclusion, GFP+ hNPCs produce neurons that are competent to integrate functionally into host neocortical neuronal networks. This provides promising data on the potential for hNPCs to serve as therapeutic agents in neurological diseases with abnormal neuronal circuitry such as epilepsy.

  13. Repair of ultraviolet irradiation damage in mouse neuroblasts cells

    International Nuclear Information System (INIS)

    It was demonstrated, using hydroxyurea inhibition of DNA replication and CsCl density centrifugation, that excision repair occurs both in the differentiated state, and when cells have been restored to growing conditions. since the ability to remove photodamage is present, we postulated that sensitivity was due to failure to remove damage from critical regions of the genome or alternatively a deficiency in another mechanism of repair, such as post replication repair, which has been demonstrated in rodent cells. The first possibility was examined by comparing excision repair in pyrmidine tracts, in which preferential formation of dimers occurs at lower UV doses, and nontract regions. Excision repair was also determined in satellite and main band DNA. The results indicate that in the particular regions of the genome examined no preference for excision repair is detected. Post replication repair (i.e. the ability to elongate DNA which is synthesized in low molecular weight pieces after UV irradiation) was determined in growing and differentiated cells. The results show that postreplication repair is normal in differentiated cells which have entered the first S phase after serum return. since excision repair and postreplication repair appear to be normal it is possible that an additional repair process is involved or that some other cell function is irreversible damage. (author)

  14. Abnormal sperm morphology in mouse germ cells after short-term exposures to acetamiprid, propineb, and their mixture.

    Science.gov (United States)

    Rasgele, Pinar Göç

    2014-03-01

    Pesticides are one of the most potent environmental contaminants, which accumulate in biotic and abiotic components of ecosystems. Acetamiprid (Acm), a neonicotinoid insecticide, and Propineb (Pro), a dithiocarbamate fungicide, are widely used to control sucking insects and fungal infections on crops, respectively. The present study was undertaken to investigate the genotoxic effects of these compounds, individually and in mixtures, in mouse germ cells by using the sperm morphology assay. Mice were injected intraperitoneally with 0.625, 1.25, and 2.50 μg mL⁻¹ of Acm, 12.5, 25, and 50 μg mL⁻¹ of Pro, and their mixture at the same concentrations over 24 and 48 h. Acm did not significantly increase the percentage of abnormal sperm at any concentration. The frequency of abnormal sperm significantly increased after 24 and 48 h of exposure to 50 μg mL⁻¹ of Pro. The mixtures of 2.50 μg mL⁻¹ of Acm and 50 μg mL⁻¹ of Pro induced sperm abnormalities antagonistically both after 24 and 48 h of exposure. Results suggest that Acm was non-genotoxic for mouse germ cells, while Pro may have been a germ cell mutagen due to the observed increase in the frequency of sperm abnormalities. However, to gain better insight into the mutagenicity and DNA damaging potential of both of these pesticides, further studies at molecular level should be done. PMID:24535295

  15. 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.

  16. Cellular response to DNA damage is enhanced by the pR plasmid in mouse cells and in Escherichia coli

    International Nuclear Information System (INIS)

    The pR plasmid, which enhances the survival of Escherichia coli C600 exposed to UV light by induction of the SOS regulatory mechanism, showed the same effect when it transformed mouse LTA cells (tk-, aprt-). With Tn5 insertion mutagenesis which inactivates UV functions in the pR plasmid, we recognized two different regions of the plasmid, uvp1 and uvp2. These pR UVR- mutants exhibited the same effect in LTA transformed cells, demonstrating that resistance to UV light, carried by the pR plasmid, was really due to the expression of these two regions, which were also in the mouse cells. Statistical analysis showed that the expression of the uvp1 and uvp2 regions significantly increased (P less than 0.01) the survival upon exposure to UV light in mouse cells and bacteria. These results might suggest the presence of an inducible repair response to DNA damage in mouse LTA cells

  17. Study on the therapeutic role of transplantation of human umbilical cord mesenchymal stem cells in NOD-SCID mouse model of gastrointestinal radiation damage

    International Nuclear Information System (INIS)

    Objective: To explore the therapeutic role of transplantation of human umbilical cord mesenchymal stem cells (MSC) in NOD-SCID mouse model of gastrointestinal radiation damage and search for the reliable donator cell resource for the treatment of acute enter radiation disease. Methods: To observe the potential of multi-differentiation of huma umbilical cord MSC in vitro, transplant human umbilical cord MSC which were labeled with CM-DiI to the mouse model and study not only the pathological changed of entero-mucosa using histochemical stain but also the location of MSC in the entero-mucosa of mouse model using confocal microscopy. Results: The lesions in the mice treated with MSC were improved obviously compared with that of control mice.Many CM-DiI + MSCs derived cells were distributed in intestinal mucosa and submucosa of the recipient mice, which indicated that MSCs-derived cells were involved in regeneration of the gastrointestinal radiation damage. Conclusion: The data indicate that systemically administrated MSCs maybe accelerated tissue repair in mouse model and it is hopeful in the treatment of human acute enter radiation sickness in the future. (authors)

  18. Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds.

    Science.gov (United States)

    Shojaie, Sharareh; Lee, Joyce; Wang, Jinxia; Ackerley, Cameron; Post, Martin

    2016-01-01

    Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis. PMID:27214388

  19. Return to quiescence of mouse neural stem cells by degradation of a proactivation protein.

    Science.gov (United States)

    Urbán, Noelia; van den Berg, Debbie L C; Forget, Antoine; Andersen, Jimena; Demmers, Jeroen A A; Hunt, Charles; Ayrault, Olivier; Guillemot, François

    2016-07-15

    Quiescence is essential for long-term maintenance of adult stem cells. Niche signals regulate the transit of stem cells from dormant to activated states. Here, we show that the E3-ubiquitin ligase Huwe1 (HECT, UBA, and WWE domain-containing 1) is required for proliferating stem cells of the adult mouse hippocampus to return to quiescence. Huwe1 destabilizes proactivation protein Ascl1 (achaete-scute family bHLH transcription factor 1) in proliferating hippocampal stem cells, which prevents accumulation of cyclin Ds and promotes the return to a resting state. When stem cells fail to return to quiescence, the proliferative stem cell pool becomes depleted. Thus, long-term maintenance of hippocampal neurogenesis depends on the return of stem cells to a transient quiescent state through the rapid degradation of a key proactivation factor. PMID:27418510

  20. A transgenic mouse marking live replicating cells reveals in vivo transcriptional program of proliferation

    DEFF Research Database (Denmark)

    Klochendler, Agnes; Weinberg-Corem, Noa; Moran, Maya;

    2012-01-01

    biological material. We describe a transgenic mouse strain, expressing a CyclinB1-GFP fusion reporter, that marks replicating cells in the S/G2/M phases of the cell cycle. Using flow cytometry, we isolate live replicating cells from the liver and compare their transcriptome to that of quiescent cells to......Most adult mammalian tissues are quiescent, with rare cell divisions serving to maintain homeostasis. At present, the isolation and study of replicating cells from their in vivo niche typically involves immunostaining for intracellular markers of proliferation, causing the loss of sensitive...... reveal gene expression programs associated with cell proliferation in vivo. We find that replicating hepatocytes have reduced expression of genes characteristic of liver differentiation. This reporter system provides a powerful platform for gene expression and metabolic and functional studies of...

  1. Localization of tritiated vitamin A in lymph nodes of the mouse: an autoradiographic study of vitamin A-storing cells

    International Nuclear Information System (INIS)

    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

  2. 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.

  3. A transgenic mouse model of sickle cell disorder.

    NARCIS (Netherlands)

    D.R. Greaves; P.J. Fraser (Peter); M.A. Vidal; M.J. Hedges; D. Ropers; L. Luzzatto; F.G. Grosveld (Frank)

    1990-01-01

    textabstractA single base-pair mutation (beta s) in codon 6 of the human beta-globin gene, causing a single amino-acid substitution, is the cause of sickle cell anaemia. The mutant haemoglobin molecule, HbS, polymerizes when deoxygenated and causes deformation of the erythrocytes to a characteristic

  4. Identification of a population of cells with hematopoietic stem cell properties in mouse aorta-gonad-mesonephros cultures

    International Nuclear Information System (INIS)

    The aorta-gonad-mesonephros (AGM) region is a primary source of definitive hematopoietic cells in the midgestation mouse embryo. In cultures of dispersed AGM regions, adherent cells containing endothelial cells are observed first, and then non-adherent hematopoietic cells are produced. Here we report on the characterization of hematopoietic cells that emerge in the AGM culture. Based on the expression profiles of CD45 and c-Kit, we defined three cell populations: CD45low c-Kit+ cells that had the ability to form hematopoietic cell colonies in methylcellulose media and in co-cultures with stromal cells; CD45low c-Kit- cells that showed a granulocyte morphology; CD45high c-Kitlow/- that exhibited a macrophage morphology. In co-cultures of OP9 stromal cells and freshly prepared AGM cultures, CD45low c-Kit+ cells from the AGM culture had the abilities to reproduce CD45low c-Kit+ cells and differentiate into CD45low c-Kit- and CD45high c-Kitlow/- cells, whereas CD45low c-Kit- and CD45high c-Kitlow/- did not produce CD45low c-Kit+ cells. Furthermore, CD45low c-Kit+ cells displayed a long-term repopulating activity in adult hematopoietic tissue when transplanted into the liver of irradiated newborn mice. These results indicate that CD45low c-Kit+ cells from the AGM culture have the potential to reconstitute multi-lineage hematopoietic cells

  5. Characterization of angiotensin-converting enzyme 2 ectodomain shedding from mouse proximal tubular cells.

    Directory of Open Access Journals (Sweden)

    Fengxia Xiao

    Full Text Available Angiotensin-converting enzyme 2 (ACE2 is highly expressed in the kidney proximal tubule, where it cleaves angiotensin (Ang II to Ang-(1-7. Urinary ACE2 levels increase in diabetes, suggesting that ACE2 may be shed from tubular cells. The aim of this study was to determine if ACE2 is shed from proximal tubular cells, to characterize ACE2 fragments, and to study pathways for shedding. Studies involved primary cultures of mouse proximal tubular cells, with ACE2 activity measured using a synthetic substrate, and analysis of ACE2 fragments by immunoblots and mass spectrometry. The culture media from mouse proximal tubular cells demonstrated a time-dependent increase in ACE2 activity, suggesting constitutive ACE2 shedding. ACE2 was detected in media as two bands at ∼ 90 kDa and ∼ 70 kDa on immunoblots. By contrast, full-length ACE2 appeared at ∼ 100 kDa in cell lysates or mouse kidney cortex. Mass spectrometry of the two deglycosylated fragments identified peptides matching mouse ACE2 at positions 18-706 and 18-577, respectively. The C-terminus of the 18-706 peptide fragment contained a non-tryptic site, suggesting that Met(706 is a candidate ACE2 cleavage site. Incubation of cells in high D-glucose (25 mM (and to a lesser extent Ang II for 48-72 h increased ACE2 activity in the media (p<0.001, an effect blocked by inhibition of a disintegrin and metalloproteinase (ADAM17. High D-glucose increased ADAM17 activity in cell lysates (p<0.05. These data indicate that two glycosylated ACE2 fragments are constitutively shed from mouse proximal tubular cells. ACE2 shedding is stimulated by high D-glucose, at least partly via an ADAM17-mediated pathway. The results suggest that proximal tubular shedding of ACE2 may increase in diabetes, which could enhance degradation of Ang II in the tubular lumen, and increase levels of Ang-(1-7.

  6. Characterizations of individual mouse red blood cells parasitized by Babesia microti using 3-D holographic microscopy

    CERN Document Server

    Park, HyunJoo; Kim, Kyoohyun; Cho, Shin-Hyeong; Lee, Won-Ja; Kim, Youngchan; Lee, SangEun; Park, YongKeun

    2015-01-01

    Babesia microti causes emergency human babesiosis. However, little is known about the alterations in B. microti invaded red blood cells (Bm-RBCs) at the individual cell level. Through quantitative phase imaging techniques based on laser interferometry, we present the simultaneous measurements of structural, chemical, and mechanical modifications in individual mouse Bm-RBCs. 3-D refractive index maps of individual RBCs and in situ parasite vacuoles are imaged, from which total contents and concentration of dry mass are also precisely quantified. In addition, we examine the dynamic membrane fluctuation of Bm-RBCs, which provide information on cell membrane deformability.

  7. Amniotic Epithelial Cells from the Human Placenta Potently Suppress a Mouse Model of Multiple Sclerosis

    OpenAIRE

    Yu Han Liu; Vijesh Vaghjiani; Jing Yang Tee; Kelly To; Peng Cui; Ding Yuan Oh; Ursula Manuelpillai; Ban-Hock Toh; James Chan

    2012-01-01

    Human amniotic epithelial cells (hAEC) have stem cell-like features and immunomodulatory properties. Here we show that hAEC significantly suppressed splenocyte proliferation in vitro and potently attenuated a mouse model of multiple sclerosis (MS). Central nervous system (CNS) CD3(+) T cell and F4/80(+) monocyte/macrophage infiltration and demyelination were significantly reduced with hAEC treatment. Besides the known secretion of prostaglandin E2 (PGE2), we report the novel finding that hAEC...

  8. Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells

    OpenAIRE

    1993-01-01

    Ba2+ currents through L-type Ca2+ channels were recorded from cell- attached patches on mouse pancreatic beta cells. In 10 mM Ba2+, single- channel currents were recorded at -70 mV, the beta cell resting membrane potential. This suggests that Ca2+ influx at negative membrane potentials may contribute to the resting intracellular Ca2+ concentration and thus to basal insulin release. Increasing external Ba2+ increased the single-channel current amplitude and shifted the current-voltage relation...

  9. Brucella beta 1,2 cyclic glucan is an activator of human and mouse dendritic cells

    OpenAIRE

    A Martirosyan; Perez-Gutierrez, C. (Camino); Banchereau, R; Dutartre, H; Lecine, P.; Dullaers, M. (Melissa); Mello, M.; Pinto, S; Muller, A; Leserman, L; Levy, Y.; Zurawski, G; Zurawski, S; Moreno, E; Moriyon, I

    2012-01-01

    Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella beta 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella beta 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glu...

  10. Modulation of secreted proteins of mouse mammary epithelial cells by the collagenous substrata

    OpenAIRE

    1984-01-01

    It has been shown previously that cultures of mouse mammary epithelial cells retain their characteristic morphology and their ability to produce gamma-casein, a member of the casein gene family, only if they are maintained on floating collagen gels (Emerman, J.T., and D.R. Pitelka, 1977, In Vitro, 13:316-328). In this paper we show: (a) Cells on floating collagen gels secrete not only gamma-casein but also alpha 1-, alpha 2-, and beta-caseins. These are not secreted by cells on plastic and ar...

  11. A novel preadipocyte cell line established from mouse adult mature adipocytes

    International Nuclear Information System (INIS)

    We have established a novel preadipocyte cell line from mouse adult mature adipocytes. The mature adipocytes were isolated from fat tissues by taking only the floating population of mature fat cells. The isolated mature adipocytes were de-differentiated into fibroblast-like cells. The in vitro studies showed that the cells could re-differentiate into mature adipocytes after over 20 passages. The in vivo transplantation study also demonstrated that the cells had the full potential to differentiate into mature adipocytes, which has not been shown for the 3T3-L1 preadipocyte cell line derived from mouse embryo. We have further analyzed the expression profile of key fat regulatory genes such as the peroxisome proliferator-activated receptorγ or CCAAT/enhancer-binding protein gene families. We conclude that our cell line could be used as a preferred alternative to 3T3-L1, potentially reflecting the characteristics of mature adipocytes more, since the cell line is actually derived from adult mature adipocytes

  12. Cholesterol esterification during differentiation of mouse erythroleukemia (Friend) cells.

    Science.gov (United States)

    Mulas, Maria Franca; Mandas, Antonella; Abete, Claudia; Dessì, Sandra; Mocali, Alessandra; Paoletti, Francesco

    2011-08-31

    Cholesterol is an essential constituent of all mammalian cell membranes and its availability is therefore a prerequisite for cellular growth and other functions. Several lines of evidence are now indicating an association between alterations of cholesterol homeostasis and cell cycle progression. However, the role of cholesterol in cell differentiation is still largely unknown. To begin to address this issue, in this study we examined changes in cholesterol metabolism and in the mRNA levels of proteins involved in cholesterol import and esterification (multi-drug resistance, MDR-3) and acylCoA: cholesterol acyltransferase (ACAT) and cholesterol export (caveolin-1) in Friend virus-induced erythroleukemia cells (MELC), in the absence or in the presence of the chemical inducer of differentiation, hexamethylene bisacetamide (HMBA). FBS-stimulated growth of MELC was accompanied by an immediate elevation of cholesterol synthesis and cholesterol esterification, and by an increase in the levels of MDR-3 and ACAT mRNAs. A decrease in caveolin-1 expression was also observed. However, when MELC were treated with HMBA, the inhibition of DNA synthesis caused by HMBA treatment, was associated with a decrease in cholesterol esterification and in ACAT and MDR-3 mRNA levels and an increase in caveolin-1 mRNA. Detection of cytoplasmic neutral lipids by staining MELC with oil red O, a dye able to evidence CE but not FC, revealed that HMBA-treatment also reduced growth-stimulated accumulation of cholesterol ester to approximately the same extent as the ACAT inhibitor, SaH. Overall, these results indicate for the first time a role of cholesterol esterification and of some related genes in differentiation of erythroid cells. PMID:22184540

  13. Cholesterol esterification during differentiation of mouse erythroleukemia (Friend) cells

    Science.gov (United States)

    Mulas, Maria Franca; Mandas, Antonella; Abete, Claudia; Dessì, Sandra; Mocali, Alessandra; Paoletti, Francesco

    2011-01-01

    Cholesterol is an essential constituent of all mammalian cell membranes and its availability is therefore a prerequisite for cellular growth and other functions. Several lines of evidence are now indicating an association between alterations of cholesterol homeostasis and cell cycle progression. However, the role of cholesterol in cell differentiation is still largely unknown. To begin to address this issue, in this study we examined changes in cholesterol metabolism and in the mRNA levels of proteins involved in cholesterol import and esterification (multi-drug resistance, MDR-3) and acylCoA: cholesterol acyltransferase (ACAT) and cholesterol export (caveolin-1) in Friend virus-induced erythroleukemia cells (MELC), in the absence or in the presence of the chemical inducer of differentiation, hexamethylene bisacetamide (HMBA). FBS-stimulated growth of MELC was accompanied by an immediate elevation of cholesterol synthesis and cholesterol esterification, and by an increase in the levels of MDR-3 and ACAT mRNAs. A decrease in caveolin-1 expression was also observed. However, when MELC were treated with HMBA, the inhibition of DNA synthesis caused by HMBA treatment, was associated with a decrease in cholesterol esterification and in ACAT and MDR-3 mRNA levels and an increase in caveolin-1 mRNA. Detection of cytoplasmic neutral lipids by staining MELC with oil red O, a dye able to evidence CE but not FC, revealed that HMBA-treatment also reduced growth-stimulated accumulation of cholesterol ester to approximately the same extent as the ACAT inhibitor, SaH. Overall, these results indicate for the first time a role of cholesterol esterification and of some related genes in differentiation of erythroid cells. PMID:22184540

  14. Cholesterol esterification during differentiation of mouse erythroleukemia (Friend cells

    Directory of Open Access Journals (Sweden)

    Maria Franca Mulas

    2011-10-01

    Full Text Available Cholesterol is an essential constituent of all mammalian cell membranes, and its availability is therefore a prerequisite for cellular growth and other functions. Several lines of evidence are now indicating an association between alterations of cholesterol homeostasis and cell cycle progression. However, the role of cholesterol in cell differentiation is still largely unknown. To begin to address this issue, in this study we examined changes in cholesterol metabolism and in the mRNA levels of proteins involved in cholesterol import and esterification (multi-drug resistance, MDR-3 and acylCoA:cholesterol acyltransferase (ACAT and cholesterol export (caveolin-1 in Friend virus-induced erythroleukemia cells (MELC, in the absence or in the presence of the chemical inducer of differentiation, hexamethylene bisacetamide (HMBA. FBS-stimulated growth of MELC was accompanied by an immediate elevation of cholesterol synthesis and cholesterol esterification, and by an increase in the levels of MDR-3 and ACAT mRNAs. A decrease in caveolin-1 expression was also observed. However, when MELC were treated with HMBA, the inhibition of DNA synthesis caused by HMBA treatment, was associated with a decrease in cholesterol esterification and in ACAT and MDR-3 mRNA levels and an increase in caveolin-1 mRNA. Detection of cytoplasmic neutral lipids by staining MELC with oil red O, a dye able to evidence CE but not FC, revealed that HMBA-treatment also reduced growth-stimulated accumulation of cholesterol ester to approximately the same extent as the ACAT inhibitor, SaH. Overall, these results indicate for the first time a role of cholesterol esterification and of some related genes in differentiation of erythroid cells.

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

    International Nuclear Information System (INIS)

    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

  16. Hypoxia enhances differentiation of mouse embryonic stem cells into definitive endoderm and distal lung cells.

    Science.gov (United States)

    Pimton, Pimchanok; Lecht, Shimon; Stabler, Collin T; Johannes, Gregg; Schulman, Edward S; Lelkes, Peter I

    2015-03-01

    We investigated the effects of hypoxia on spontaneous (SP)- and activin A (AA)-induced definitive endoderm (DE) differentiation of mouse embryonic stem cells (mESCs) and their subsequent differentiation into distal pulmonary epithelial cells. SP differentiation for 6 days of mESCs toward endoderm at hypoxia of 1% O2, but not at 3% or 21% (normoxia), increased the expression of Sox17 and Foxa2 by 31- and 63-fold above maintenance culture, respectively. Treatment of mESCs with 20 ng/mL AA for 6 days under hypoxia further increased the expression of DE marker genes Sox17, Foxa2, and Cxcr4 by 501-, 1,483-, and 126-fold above maintenance cultures, respectively. Transient exposure to hypoxia, as short as 24 h, was sufficient to enhance AA-induced endoderm formation. The involvement of hypoxia-inducible factor (HIF)-1α and reactive oxygen species (ROS) in the AA-induced endoderm enrichment was assessed using HIF-1α(-/-) mESCs and the ROS scavenger N-acetylcysteine (NAC). Under SP conditions, HIF-1α(-/-) mESCs failed to increase the expression of endodermal marker genes but rather shifted toward ectoderm. Hypoxia induced only a marginal potentiation of AA-induced endoderm differentiation in HIF-1α(-/-) mESCs. Treatment of mESCs with AA and NAC led to a dose-dependent decrease in Sox17 and Foxa2 expression. In addition, the duration of exposure to hypoxia in the course of a recently reported lung differentiation protocol resulted in differentially enhanced expression of distal lung epithelial cell marker genes aquaporin 5 (Aqp5), surfactant protein C (Sftpc), and secretoglobin 1a1 (Scgb1a1) for alveolar epithelium type I, type II, and club cells, respectively. Our study is the first to show the effects of in vitro hypoxia on efficient formation of DE and lung lineages. We suggest that the extent of hypoxia and careful timing may be important components of in vitro differentiation bioprocesses for the differential generation of distal lung epithelial cells from

  17. Use of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells.

    Science.gov (United States)

    Xin, Yurong; Kim, Jinrang; Ni, Min; Wei, Yi; Okamoto, Haruka; Lee, Joseph; Adler, Christina; Cavino, Katie; Murphy, Andrew J; Yancopoulos, George D; Lin, Hsin Chieh; Gromada, Jesper

    2016-03-22

    This study provides an assessment of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells. The system combines microfluidic technology and nanoliter-scale reactions. We sequenced 622 cells, allowing identification of 341 islet cells with high-quality gene expression profiles. The cells clustered into populations of α-cells (5%), β-cells (92%), δ-cells (1%), and pancreatic polypeptide cells (2%). We identified cell-type-specific transcription factors and pathways primarily involved in nutrient sensing and oxidation and cell signaling. Unexpectedly, 281 cells had to be removed from the analysis due to low viability, low sequencing quality, or contamination resulting in the detection of more than one islet hormone. Collectively, we provide a resource for identification of high-quality gene expression datasets to help expand insights into genes and pathways characterizing islet cell types. We reveal limitations in the C1 Fluidigm cell capture process resulting in contaminated cells with altered gene expression patterns. This calls for caution when interpreting single-cell transcriptomics data using the C1 Fluidigm system. PMID:26951663

  18. A transgenic mouse model of neuroepithelial cell specific inducible overexpression of dopamine D1-receptor.

    Science.gov (United States)

    Fujimoto, K; Araki, K; McCarthy, D M; Sims, J R; Ren, J Q; Zhang, X; Bhide, P G

    2010-10-27

    Dopamine and its receptors appear in the brain during early embryonic period suggesting a role for dopamine in brain development. In fact, dopamine receptor imbalance resulting from impaired physiological balance between D1- and D2-receptor activities can perturb brain development and lead to persisting changes in brain structure and function. Dopamine receptor imbalance can be produced experimentally using pharmacological or genetic methods. Pharmacological methods tend to activate or antagonize the receptors in all cell types. In the traditional gene knockout models the receptor imbalance occurs during development and also at maturity. Therefore, assaying the effects of dopamine imbalance on specific cell types (e.g. precursor versus postmitotic cells) or at specific periods of brain development (e.g. pre- or postnatal periods) is not feasible in these models. We describe a novel transgenic mouse model based on the tetracycline dependent inducible gene expression system in which dopamine D1-receptor transgene expression is induced selectively in neuroepithelial cells of the embryonic brain at experimenter-chosen intervals of brain development. In this model, doxycycline-induced expression of the transgene causes significant overexpression of the D1-receptor and significant reductions in the incorporation of the S-phase marker bromodeoxyuridine into neuroepithelial cells of the basal and dorsal telencephalon indicating marked effects on telencephalic neurogenesis. The D1-receptor overexpression occurs at higher levels in the medial ganglionic eminence (MGE) than the lateral ganglionic eminence (LGE) or cerebral wall (CW). Moreover, although the transgene is induced selectively in the neuroepithelium, D1-receptor protein overexpression appears to persist in postmitotic cells. The mouse model can be modified for neuroepithelial cell-specific inducible expression of other transgenes or induction of the D1-receptor transgene in other cells in specific brain regions by

  19. Mouse retinal adaptive response to proton irradiation: Correlation with DNA repair and photoreceptor cell death

    Science.gov (United States)

    Tronov, V. A.; Vinogradova, Yu. V.; Poplinskaya, V. A.; Nekrasova, E. I.; Ostrovsky, M. A.

    2015-01-01

    Emerging body of data indicate protecting effect of low level of stress (preconditioning) on retina. Our previous study revealed non-linear dose-response relationship for cytotoxicity of both ionizing radiation and N-methyl-N-nitrosourea (MNU) on mouse retina. Moreover, non cytotoxic dose of MNU increased tolerance of retina to following challenge dose of MNU. This result displays protection of retina through mechanism of recovery. In present study we used the mouse model for MNU-induced retinal degeneration to evaluate adaptive response of retina to proton irradiation and implication in it of glial Muller cells. The data showed that the recovery of retina after genotoxic agents has been associated with increased efficacy of DNA damage repair and lowered death of retinal photoreceptor cells.

  20. Proliferation of Cultured Mouse Choroid Plexus Epithelial Cells

    OpenAIRE

    Barkho, Basam Z.; Monuki, Edwin S.

    2015-01-01

    The choroid plexus (ChP) epithelium is a multifunctional tissue found in the ventricles of the brain. The major function of the ChP epithelium is to produce cerebrospinal fluid (CSF) that bathes and nourishes the central nervous system (CNS). In addition to the CSF, ChP epithelial cells (CPECs) produce and secrete numerous neurotrophic factors that support brain homeostasis, such as adult hippocampal neurogenesis. Accordingly, damage and dysfunction to CPECs are thought to accelerate and inte...

  1. Tumourigenicity and Immunogenicity of Induced Neural Stem Cell Grafts Versus Induced Pluripotent Stem Cell Grafts in Syngeneic Mouse Brain

    Science.gov (United States)

    Gao, Mou; Yao, Hui; Dong, Qin; Zhang, Hongtian; Yang, Zhijun; Yang, Yang; Zhu, Jianwei; Xu, Minhui; Xu, Ruxiang

    2016-01-01

    Along with the development of stem cell-based therapies for central nervous system (CNS) disease, the safety of stem cell grafts in the CNS, such as induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs), should be of primary concern. To provide scientific basis for evaluating the safety of these stem cells, we determined their tumourigenicity and immunogenicity in syngeneic mouse brain. Both iPSCs and embryonic stem cells (ESCs) were able to form tumours in the mouse brain, leading to tissue destruction along with immune cell infiltration. In contrast, no evidence of tumour formation, brain injury or immune rejection was observed with iNSCs, neural stem cells (NSCs) or mesenchymal stem cells (MSCs). With the help of gene ontology (GO) enrichment analysis, we detected significantly elevated levels of chemokines in the brain tissue and serum of mice that developed tumours after ESC or iPSC transplantation. Moreover, we also investigated the interactions between chemokines and NF-κB signalling and found that NF-κB activation was positively correlated with the constantly rising levels of chemokines, and vice versa. In short, iNSC grafts, which lacked any resulting tumourigenicity or immunogenicity, are safer than iPSC grafts. PMID:27417157

  2. DMPD: The p110delta subunit of phosphoinositide 3-kinase is required for thelipopolysaccharide response of mouse B cells. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available polysaccharide response of mouse B cells. Hebeis BJ, Vigorito E, Turner M. Biochem Soc Trans. 2004 Nov;32(Pt...d for thelipopolysaccharide response of mouse B cells. PubmedID 15494016 Title Th...e p110delta subunit of phosphoinositide 3-kinase is required for thelipopolysaccharide response of mouse B c

  3. LET dependent recovery of mouse hematopoietic stem cells from particle irradiation

    International Nuclear Information System (INIS)

    Radiation induced acute changes of mouse hematopoietic functions and its recovery were studied in different LET of heavy ions, carbon (13keV/∝m and 29 keV/∝m), neon (32keV/∝m and 55 keV/∝m), silicon (56keV/∝m), argon (85keV/∝m), iron (200keV/∝m). After whole body irradiation was applied up to 3 Gy, the subsequent hematopoetic parameters were followed by the blood analysis for 3 weeks. All functions, spleen weight, WBC, hemoglobin concentration, granulocyte monocyte colony forming unit (GM-CFU) in bone marrow and spleen indicated dose dependent reduction within 2 days after the irradiation and followed by a slow recovery. Complete recovery took approximately 3 weeks for all functions. GM-CFU in spleen showed the highest sensitivity to all particles indicating a large reduction down to less than 1 % of control at 3 Gy exposure. Of the particles exposed Si (55keV/∝m) induced the largest effects and the slowest recovery, whereas Fe (200keV/∝m) indicated significantly the least effects and quick recovery. The results suggested a strong LET dependency of the hematopoietic progenitor cells which may have a RBE maximum less than 100 keV/∝m. The recovery kinetics indicated non-monotonic increase within 10 days suggested an involvement of time dependent releases of cytokines (IL-1b and IL-3) which may modify the proliferation of hematopoietic cells

  4. Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate

    Science.gov (United States)

    Mederacke, Ingmar; Komatsu, Yoshihiro; Stice, Steve; Schwabe, Robert F.; Mistretta, Charlotte M.; Mishina, Yuji; Liu, Hong-Xiang

    2016-01-01

    Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC. PMID:26741369

  5. Induction of Chromosomal Translocations in Mouse and Human Cells Using Site-Specific Endonucleases

    OpenAIRE

    Weinstock, David M.; Brunet, Erika; Jasin, Maria

    2008-01-01

    Reciprocal chromosomal translocations are early and essential events in the malignant transformation of several tumor types, yet the precise mechanisms that mediate translocation formation are poorly understood. We review here the development of approaches to induce and recover translocations between two targeted DNA double-strand breaks (DSBs) in mammalian chromosomes. Using mouse cells, we find that nonhomologous end-joining readily mediates translocation formation between two DSBs generate...

  6. Fingerprinting of neurotoxic compounds using a mouse embryonic stem cell dual luminescence reporter assay

    OpenAIRE

    Colaianna, Marilena; Ilmjarv, Sten; Peterson, Hedi; Kern, Ilse; Sgroi, Stéphanie; Baquie, Mathurin; Pallocca, Giorgia; Bosgra, Sieto; Sachinidis, Agapios; Hengstler, Jan G.; Leist, Marcel; Krause, Karl-Heinz

    2016-01-01

    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 line CGR8 (ESCs). The test system uses ESCs at two differentiation stages: undifferentiated ESCs and ESC-derived neurons. Under each condition, concentration-response curves were obtained for three ...

  7. 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.

  8. Repair of radiation damages to haemopoietic stem cells of mouse embryonal liver: kinetic aspects

    International Nuclear Information System (INIS)

    The method of fractionated irradiation was used to study kinetic aspects of repair of sublethal radiation damages in precursor cells from mouse embryonal liver that form in vivo colonies on 8th and 11th days. It was shown that 11-day CFUs had a lesser ability to repair sublethal radiation damages than 8-day ones at different time-intervals between radiation fractions (from 2 to 6 h). These two CFUs subpopulations differed also in the repair kinetics

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

    OpenAIRE

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

    2008-01-01

    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 dise...

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

    International Nuclear Information System (INIS)

    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

  11. Expression of Trypsin by Epithelial Cells of Various Tissues, Leukocytes, and Neurons in Human and Mouse

    OpenAIRE

    Koshikawa, Naohiko; Hasegawa, Satoshi; Nagashima, Yoji; Mitsuhashi, Keisuke; Tsubota, Yoshiaki; Miyata, Satoshi; Miyagi, Yohei; Yasumitsu, Hidetaro; Miyazaki, Kaoru

    1998-01-01

    It has long been believed that trypsin is normally synthesized only in the pancreas. In the present study, expression of trypsin in human and mouse nonpancreatic tissues was examined. Northern blot analysis of normal human tissues indicated that the trypsin gene is expressed at high levels in the pancreas and spleen and considerably in the small intestine. However, in situ hybridization and immunohistochemistry demonstrated that trypsin is widely expressed in epithelial cells of the skin, eso...

  12. Human endometrial cell coculture reduces the endocrine disruptor toxicity on mouse embryo development

    OpenAIRE

    Lee Myeong-Seop; Lee Young-Sang; Lee Hae-Hyeog; Song Ho-Yeon

    2012-01-01

    Abstract Backgrounds Previous studies suggested that endocrine disruptors (ED) are toxic on preimplantation embryos and inhibit development of embryos in vitro culture. However, information about the toxicity of endocrine disruptors on preimplantation development of embryo in human reproductive environment is lacking. Methods Bisphenol A (BPA) and Aroclor 1254 (polychlorinated biphenyls) were used as endocrine disruptors in this study. Mouse 2-cell embryos were cultured in medium alone or veh...

  13. HDAC3 controls gap 2/mitosis progression in adult neural stem/progenitor cells by regulating CDK1 levels

    OpenAIRE

    Jiang, Yindi; Hsieh, Jenny

    2014-01-01

    Cell cycle regulation is one of the most fundamental mechanisms to control various biological processes, including the proliferation of neural stem/progenitor cells (NSPCs) in adult mouse brain. This study shows that histone deacetylase 3 (HDAC3), a well-studied epigenetic factor, is required for the proliferation of neural stem cells. We also demonstrate that HDAC3 controls gap 2 and mitosis phase of cell cycle through stabilization of cell cycle protein cyclin-dependent kinase 1. These find...

  14. 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. PMID:25008417

  15. The protein PprI provides protection against radiation injury in human and mouse cells.

    Science.gov (United States)

    Shi, Yi; Wu, Wei; Qiao, Huiping; Yue, Ling; Ren, Lili; Zhang, Shuyu; Yang, Wei; Yang, Zhanshan

    2016-01-01

    Severe acute radiation injuries are both very lethal and exceptionally difficult to treat. Though the radioresistant bacterium D. radiodurans was first characterized in 1956, genes and proteins key to its radioprotection have not yet to be applied in radiation injury therapy for humans. In this work, we express the D. radiodurans protein PprI in Pichia pastoris yeast cells transfected with the designed vector plasmid pHBM905A-pprI. We then treat human umbilical endothelial vein cells and BALB/c mouse cells with the yeast-derived PprI and elucidate the radioprotective effects the protein provides upon gamma irradiation. We see that PprI significantly increases the survival rate, antioxidant viability, and DNA-repair capacity in irradiated cells and decreases concomitant apoptosis rates and counts of damage-indicative γH2AX foci. Furthermore, we find that PprI reduces mortality and enhances bone marrow cell clone formation and white blood cell and platelet counts in irradiated mice. PprI also seems to alleviate pathological injuries to multiple organs and improve antioxidant viability in some tissues. Our results thus suggest that PprI has crucial radioprotective effects on irradiated human and mouse cells. PMID:27222438

  16. An investigation of the shedding of macromolecules from the Ehrlich mouse ascites tumor cell

    International Nuclear Information System (INIS)

    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 ([3H]H2DIDS) 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 [1-14C]-D-glucosamine hydrochloride, D-[2-3H]-mannose and various [3H]-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. Transplantation of Adipose Derived Stromal Cells into the Developing Mouse Eye

    International Nuclear Information System (INIS)

    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

  18. Effects of C-reactive protein on the expression of OX40 ligand in mouse aorta endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Fei Wang; Shushu Zhu; Xuefu Han; Jindan Xu; Jinnan Zhang; Di Yang

    2009-01-01

    Objective: To investigate the expression of OX40 ligand(OX40L) on C-reactive protein(CRP)-triggered mouse aorta endothelial cells (MAECs) in vitro. Methods: MAECs from aorta were isolated by digestion with collagenase type Ⅱ. The cell growth was confirmed by morphological characteristics and the immunological marker, factor Ⅷ(or Willebrand factor, vWF). The expression of OX40L by MAECs was detected by RT-PCR and western blot after incubating with 100 μg/ml CRP for 48 hours. Results: Twenty-day cultures of MAECs formed confluent monolayer of a cobblestone pattern. RT-PCR and western blot assay showed that the level of OX40L expression in MAECs receiving CRP treatment was higher than control. Conclusion: A reliable method is described to isolate and propagate MAECs. CRP upregulates OX40L expression in MAECs.

  19. The Chymase, Mouse Mast Cell Protease 4, Constitutes the Major Chymotrypsin-like Activity in Peritoneum and Ear Tissue. A Role for Mouse Mast Cell Protease 4 in Thrombin Regulation and Fibronectin Turnover

    OpenAIRE

    Tchougounova, Elena; Pejler, Gunnar; Åbrink, Magnus

    2003-01-01

    To gain insight into the biological role of mast cell chymase we have generated a mouse strain with a targeted deletion in the gene for mast cell protease 4 (mMCP-4), the mouse chymase that has the closest relationship to the human chymase in terms of tissue localization and functional properties. The inactivation of mMCP-4 did not affect the storage of other mast cell proteases and did not affect the number of mast cells or the mast cell morphology. However, mMCP-4 inactivation resulted in c...

  20. RNA-binding proteins in mouse male germline stem cells: a mammalian perspective.

    Science.gov (United States)

    Qi, Huayu

    2016-01-01

    Adult stem cells that reside in particular types of tissues are responsible for tissue homeostasis and regeneration. Cellular functions of adult stem cells are intricately related to the gene expression programs in those cells. Past research has demonstrated that regulation of gene expression at the transcriptional level can decisively alter cell fate of stem cells. However, cellular contents of mRNAs are sometimes not equivalent to proteins, the functional units of cells. It is increasingly realized that post-transcriptional and translational regulation of gene expression are also fundamental for stem cell functions. Compared to differentiated somatic cells, effects on cellular status manifested by varied expression of RNA-binding proteins and global protein synthesis have been demonstrated in several stem cell systems. Through the cooperation of both cis-elements of mRNAs and trans-acting RNA-binding proteins that are intimately associated with them, regulation of localization, stability, and translational status of mRNAs directly influences the self-renewal and differentiation of stem cells. Previous studies have uncovered some of the molecular mechanisms that underlie the functions of RNA-binding proteins in stem cells in invertebrate species. However, their roles in adult stem cells in mammals are just beginning to be unveiled. This review highlights some of the RNA-binding proteins that play important functions during the maintenance and differentiation of mouse male germline stem cells, the adult stem cells in the male reproductive organ. PMID:26839690

  1. The developmental fate of green fluorescent mouse embryonic germ cells in chimeric embryos

    Institute of Scientific and Technical Information of China (English)

    XUXIN; SUMIOSUGANO; 等

    1999-01-01

    Primordial germ cells (PGCs),as precursors of mammalian germ lineage,have been gaining more attention as a new resource of pluripotent stem cells,which bring a great possibility to study developmental events of germ cell in vitro and at animal level.EG4 cells derived from 10.5 days post coitum (dpc) PGCs of 129/svJ strain mouse were established and maintained in an undifferentiated state.With an attempt to study the differentiation capability of EG4 cells with a reporter protein:green fluorescence protein,and the possible application of EG4 cells in the research of germ cell development,we have generated several EG4-GFP cell lines expressing enhanced green fluorescence protein (EGFP) and still maintaining typical characteristics of pluripotent stem cells.Then,the differentiation of EG4-GFP cells in vitro as well as their developmental fate in chimeric embryos which were produced by aggregating EG4-GFP cells to 8-cell stage embryos were studied.The results showed that EG4 cells carrying green fluorescence have a potential use in the research of germ cell development and other related studies.

  2. 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.

  3. Phage ΦPan70, a Putative Temperate Phage, Controls Pseudomonas aeruginosa in Planktonic, Biofilm and Burn Mouse Model Assays

    Directory of Open Access Journals (Sweden)

    Angela V. Holguín

    2015-08-01

    Full Text Available Pseudomonas aeruginosa is one of the Multi-Drug-Resistant organisms most frequently isolated worldwide and, because of a shortage of new antibiotics, bacteriophages are considered an alternative for its treatment. Previously, P. aeruginosa phages were isolated and best candidates were chosen based on their ability to form clear plaques and their host range. This work aimed to characterize one of those phages, ΦPan70, preliminarily identified as a good candidate for phage-therapy. We performed infection curves, biofilm removal assays, transmission-electron-microscopy, pulsed-field-gel-electrophoresis, and studied the in vivo ΦPan70 biological activity in the burned mouse model. ΦPan70 was classified as a member of the Myoviridae family and, in both planktonic cells and biofilms, was responsible for a significant reduction in the bacterial population. The burned mouse model showed an animal survival between 80% and 100%, significantly different from the control animals (0%. However, analysis of the ΦPan70 genome revealed that it was 64% identical to F10, a temperate P. aeruginosa phage. Gene annotation indicated ΦPan70 as a new, but possible temperate phage, therefore not ideal for phage-therapy. Based on this, we recommend genome sequence analysis as an early step to select candidate phages for potential application in phage-therapy, before entering into a more intensive characterization.

  4. ERα and ERβ in mouse testicular cells and sperm

    Czech Academy of Sciences Publication Activity Database

    Dostálová, Pavla; Děd, Lukáš; Pěknicová, Jana

    Praha : Biotechnologický ústav, 2013 - (Pěknicová, J.). s. 22-23 [XIX. Symposium imunologie a biologie reprodukce s mezinárodní účastí. 23.05.2013-25.05.2013, Třešť] R&D Projects: GA ČR(CZ) GAP503/12/1834 Institutional research plan: CEZ:AV0Z50520701 Keywords : Estrogen receptor α β * Estrogen receptor β * Testicular cells * Spermatogenesis Subject RIV: EB - Genetics ; Molecular Biology

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

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    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

  6. CD103+ Dendritic Cells Control Th17 Cell Function in the Lung

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    Teresa Zelante

    2015-09-01

    Full Text Available Th17 cells express diverse functional programs while retaining their Th17 identity, in some cases exhibiting a stem-cell-like phenotype. Whereas the importance of Th17 cell regulation in autoimmune and infectious diseases is firmly established, the signaling pathways controlling their plasticity are undefined. Using a mouse model of invasive pulmonary aspergillosis, we found that lung CD103+ dendritic cells (DCs would produce IL-2, dependent on NFAT signaling, leading to an optimally protective Th17 response. The absence of IL-2 in DCs caused unrestrained production of IL-23 and fatal hyperinflammation, which was characterized by strong Th17 polarization and the emergence of a Th17 stem-cell-like population. Although several cell types may be affected by deficient IL-2 production in DCs, our findings identify the balance between IL-2 and IL-23 productions by lung DCs as an important regulator of the local inflammatory response to infection.

  7. A stringent dual control system overseeing transcription and activity of the Cre recombinase for the liver-specific conditional gene knock-out mouse model

    Institute of Scientific and Technical Information of China (English)

    Yu Wu; Yinghua He; Hongyu Zhang; Xinlan Dai; Xiaoyu Zhou; Jun Gu; Guan Wang; Jingde Zhu

    2008-01-01

    Liver cancer is one of the most threatening diseases in Chinese population. Just like in other tissues, tumor initiation and development in liver involve multiple steps of genetic and epigenetic alterations with several unknown details. However, unlike in other tissues, a tis- sue specific inducible Cre recombinase system that allows temporal and spatial deletion of a target DNA fragment is still not available for in vivo functional gene annotation in hepatocytes. In our pursuit to establish such a mouse model, we designed a dual inducible Cre transgene system and tested it in cultured cells. By combining a CCAAT/enhancer binding protein β (C/EBP β) promoter derived Tet-off expression system and the estrogen receptor (ER) mediated functional control, we show a desirable profile of both hepatocyte-specificity and regulability of the Cre expression in a series of critical assessments in the cell culture system, which provides confidence in continua- tion of our ongoing pursuit in mouse.

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

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    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.

  9. A maladaptive role for EP4 receptors in mouse mesangial cells.

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    Guang-xia Yang

    Full Text Available Roles of the prostaglandin E2 E-prostanoid 4 receptor (EP4 on extracellular matrix (ECM accumulation induced by TGF-β1 in mouse glomerular mesangial cells (GMCs remain unknown. Previously, we have identified that TGF-β1 stimulates the expression of FN and Col I in mouse GMCs. Here we asked whether stimulation of EP4 receptors would exacerbate renal fibrosis associated with enhanced glomerular ECM accumulation. We generated EP4(Flox/Flox and EP4(+/- mice, cultured primary WT, EP4(Flox/Flox and EP4(+/- GMCs, AD-EP4 transfected WT GMCs (EP4 overexpression and AD-Cre transfected EP4(Flox/Flox GMCs (EP4 deleted. We found that TGF-β1-induced cAMP and PGE2 synthesis decreased in EP4 deleted GMCs and increased in EP4 overexpressed GMCs. Elevated EP4 expression in GMCs augmented the coupling of TGF-β1 to FN, Col I expression and COX2/PGE2 signaling, while TGF-β1 induced FN, Col I expression and COX2/PGE2 signaling were down-regulated in EP4 deficiency GMCs. 8 weeks after 5/6 nephrectomy (Nx, WT and EP4(+/- mice exhibited markedly increased accumulation of ECM compared with sham-operated controls. Albuminuria, blood urea nitrogen and creatinine (BUN and Cr concentrations were significantly increased in WT mice as compared to those of EP4(+/- mice. Urine osmotic pressure was dramatically decreased after 5/6 Nx surgery in WT mice as compared to EP4(+/- mice. The pathological changes in kidney of EP4(+/- mice was markedly alleviated compared with WT mice. Immunohistochemical analysis showed significant reductions of Col I and FN in the kidney of EP4(+/- mice compared with WT mice. Collectively, this investigation established EP4 as a potent mediator of the pro-TGF-β1 activities elicited by COX2/PGE2 in mice GMCs. Our findings suggested that prostaglandin E2, acting via EP4 receptors contributed to accumulation of ECM in GMCs and promoted renal fibrosis.

  10. A neonatal mouse spinal cord injury model for assessing post-injury adaptive plasticity and human stem cell integration.

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    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

  11. Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells.

    Science.gov (United States)

    Liu, Li; Liu, Ying-Feng; Zhang, Jing; Duan, Yin-Zhong; Jin, Yan

    2016-06-01

    Induced pluripotent stem (iPS) cells possess the ability of self-renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum-free conditioned medium (ASF-CM), we successfully generated ameloblast-like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF-CM supplemented with BMP4 (ASF-BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast-specific as well as odontoblast-specific genes. On the other hand, culturing mouse iPS cells in ASF-CM supplemented with noggin (ASF-noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF-BMP4 to differentiate into ameloblast-like and odontoblast-like cells. The results of our study raise the possibility of using patient-specific iPS cells for tooth regeneration in the future. Copyright © 2016 John Wiley & Sons, Ltd. PMID:23606575

  12. Relief of preintegration inhibition and characterization of additional blocks for HIV replication in primary mouse T cells.

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    Jing-xin Zhang

    Full Text Available Development of a small animal model to study HIV replication and pathogenesis has been hampered by the failure of the virus to replicate in non-primate cells. Most studies aimed at achieving replication in murine cells have been limited to fibroblast cell lines, but generating an appropriate model requires overcoming blocks to viral replication in primary T cells. We have studied HIV-1 replication in CD4(+ T cells from human CD4/CCR5/Cyclin T1 transgenic mice. Expression of hCD4 and hCCR5 in mouse CD4(+ T cells enabled efficient entry of R5 strain HIV-1. In mouse T cells, HIV-1 underwent reverse transcription and nuclear import as efficiently as in human T cells. In contrast, chromosomal integration of HIV-1 proviral DNA was inefficient in activated mouse T cells. This process was greatly enhanced by providing a secondary T cell receptor (TCR signal after HIV-1 infection, especially between 12 to 24 h post infection. This effect was specific for primary mouse T cells. The pathways involved in HIV replication appear to be PKCtheta-, CARMA1-, and WASp-independent. Treatment with Cyclosporin A (CsA further relieved the pre-integration block. However, transcription of HIV-1 RNA was still reduced in mouse CD4(+ T cells despite expression of the hCyclin T1 transgene. Additional post-transcriptional defects were observed at the levels of Gag expression, Gag processing, Gag release and virus infectivity. Together, these post-integration defects resulted in a dramatically reduced yield of infectious virus (300-500 fold after a single cycle of HIV-1 replication. This study implies the existence of host factors, in addition to those already identified, that are critical for HIV-1 replication in mouse cells. This study also highlights the differences between primary T cells and cell lines regarding pre-integration steps in the HIV-1 replication cycle.

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