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Sample records for primary embryonic fibroblasts

  1. Cerium oxide nanoparticles stimulate proliferation of primary mouse embryonic fibroblasts in vitro

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    Popov, Anton L., E-mail: antonpopovleonid@gmail.com [Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region (Russian Federation); Popova, Nelly R. [Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region (Russian Federation); Selezneva, Irina I. [Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region (Russian Federation); Pushchino State Institute of Natural sciences, Pushchino, Moscow region (Russian Federation); Akkizov, Azamat Y. [Kabardino-Balkarian State University, Nalchik (Russian Federation); Ivanov, Vladimir K. [Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences, Moscow (Russian Federation); National Research Tomsk State University, Tomsk (Russian Federation)

    2016-11-01

    The increasing application of cell therapy technologies in the treatment of various diseases requires the development of new effective methods for culturing primary cells. The major limitation for the efficient use of autologous cell material is the low rate of cell proliferation. Successful cell therapy requires sufficient amounts of cell material over a short period of time with the preservation of their differentiation and proliferative potential. In this regard, the development of novel, highly efficient stimulators of proliferative activity in stem cells is a truly urgent task. In this paper we have demonstrated that citrate-stabilized cerium oxide nanoparticles (nanoceria) enhance the proliferative activity of primary mouse embryonic fibroblasts in vitro. Cerium oxide nanoparticles stimulate cell proliferation in a wide range of concentrations (10{sup −3} M–10{sup −9} M) through reduction of intracellular levels of reactive oxygen species (ROS) during the lag phase of cell growth and by modulating the expression level of the major antioxidant enzymes. We found the optimal concentration of nanoceria, which provides the greatest acceleration of cell proliferation in vitro, while maintaining the levels of intracellular ROS and mRNA of antioxidant enzymes in the physiological range. Our results confirm that nanocrystalline ceria can be considered as a basis for effective and inexpensive supplements in cell culturing. - Highlights: • Citrate-stabilized cerium oxide nanoparticles are shown to stimulate proliferation of primary embryonic cells in vitro. • Some of mechanisms involved in stimulating of the proliferation by CeO{sub 2} have been uncovered. • The most effective (optimal) concentration of CeO{sub 2} nanoparticles for stimulation of proliferation was determined.

  2. Biocompatibility effects of biologically synthesized graphene in primary mouse embryonic fibroblast cells

    Science.gov (United States)

    Gurunathan, Sangiliyandi; Han, Jae Woong; Eppakayala, Vasuki; Dayem, Ahmed Abdal; Kwon, Deug-Nam; Kim, Jin-Hoi

    2013-09-01

    Due to unique properties and unlimited possible applications, graphene has attracted abundant interest in the areas of nanobiotechnology. Recently, much work has focused on the synthesis and properties of graphene. Here we show that a successful reduction of graphene oxide (GO) using spinach leaf extract (SLE) as a simultaneous reducing and stabilizing agent. The as-prepared SLE-reduced graphene oxide (S-rGO) was characterized by ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy. Dynamic light scattering technique was used to determine the average size of GO and S-rGO. Scanning electron microscopy and atomic force microscopy images provide clear surface morphological evidence for the formation of graphene. The resulting S-rGO has a mostly single-layer structure, is stable, and has significant water solubility. In addition, the biocompatibility of graphene was investigated using cell viability, leakage of lactate dehydrogenase and alkaline phosphatase activity in primary mouse embryonic fibroblast (PMEFs) cells. The results suggest that the biologically synthesized graphene has significant biocompatibility with PMEF cells, even at a higher concentration of 100 μg/mL. This method uses a `green', natural reductant and is free of additional stabilizing reagents; therefore, it is an environmentally friendly, simple, and cost-effective method for the fabrication of soluble graphene. This study could open up a promising view for substitution of hydrazine by a safe, biocompatible, and powerful reduction for the efficient deoxygenation of GO, especially in large-scale production and potential biomedical applications.

  3. Dual expression of Epstein-Barr virus, latent membrane protein-1 and human papillomavirus-16 E6 transform primary mouse embryonic fibroblasts through NF-κB signaling.

    Science.gov (United States)

    Shimabuku, Tetsuya; Tamanaha, Ayumi; Kitamura, Bunta; Tanabe, Yasuka; Tawata, Natsumi; Ikehara, Fukino; Arakaki, Kazunari; Kinjo, Takao

    2014-01-01

    The prevalence of Epstein-Barr virus (EBV) and high-risk human papilloma virus (HPV) infections in patients with oral cancer in Okinawa, southwest islands of Japan, has led to the hypothesis that carcinogenesis is related to EBV and HPV co-infection. To explore the mechanisms of transformation induced by EBV and HPV co-infection, we analyzed the transformation of primary mouse embryonic fibroblasts (MEFs) expressing EBV and HPV-16 genes, alone or in combination. Expression of EBV latent membrane protein-1 (LMP-1) alone or in combination with HPV-16 E6 increased cell proliferation and decreased apoptosis, whereas single expression of EBV nuclear antigen-1 (EBNA-1), or HPV-16 E6 did not. Co-expression of LMP-1 and E6 induced anchorage-independent growth and tumor formation in nude mice, whereas expression of LMP-1 alone did not. Although the singular expression of these viral genes showed increased DNA damage and DNA damage response (DDR), co-expression of LMP-1 and E6 did not induce DDR, which is frequently seen in cancer cells. Furthermore, co-expression of LMP-1 with E6 increased NF-κB signaling, and the knockdown of LMP-1 or E6 in co-expressing cells decreased cell proliferation, anchorage independent growth, and NF-κB activation. These data suggested that expression of individual viral genes is insufficient for inducing transformation and that co-expression of LMP-1 and E6, which is associated with suppression of DDR and increased NF-κB activity, lead to transformation. Our findings demonstrate the synergistic effect by the interaction of oncogenes from different viruses on the transformation of primary MEFs.

  4. Dehydrodiconiferyl Alcohol Isolated from Cucurbita moschata Shows Anti-adipogenic and Anti-lipogenic Effects in 3T3-L1 Cells and Primary Mouse Embryonic Fibroblasts*

    Science.gov (United States)

    Lee, Junghun; Kim, Donghyun; Choi, Jonghyun; Choi, Hyounjeong; Ryu, Jae-Ha; Jeong, Jinhyun; Park, Eun-Jin; Kim, Seon-Hee; Kim, Sunyoung

    2012-01-01

    A water-soluble extract from the stems of Cucurbita moschata, code named PG105, was previously found to contain strong anti-obesity activities in a high fat diet-induced obesity mouse model. One of its biological characteristics is that it inhibits 3T3-L1 adipocyte differentiation. To isolate the biologically active compound(s), conventional solvent fractionation was performed, and the various fractions were tested for anti-adipogenic activity using Oil Red O staining method. A single spot on thin layer chromatography of the chloroform fraction showed a potent anti-adipogenic activity. When purified, the structure of its major component was resolved as dehydrodiconiferyl alcohol (DHCA), a lignan, by NMR and mass spectrometry analysis. In 3T3-L1 cells, synthesized DHCA significantly reduced the expression of several adipocyte marker genes, including peroxisome proliferator-activated receptor γ (Pparg), CCAAT/enhancer-binding protein α (Cebpa), fatty acid-binding protein 4 (Fabp4), sterol response element-binding protein-1c (Srebp1c), and stearoyl-coenzyme A desaturase-1 (Scd), and decreased lipid accumulation without affecting cell viability. DHCA also suppressed the mitotic clonal expansion of preadipocytes (an early event of adipogenesis), probably by suppressing the DNA binding activity of C/EBPβ, and lowered the production level of cyclinA and cyclin-dependent kinase 2 (Cdk2), coinciding with the decrease in DNA synthesis and cell division. In addition, DHCA directly inhibited the expression of SREBP-1c and SCD-1. Similar observations were made, using primary mouse embryonic fibroblasts. Taken together, our data indicate that DHCA may contain dual activities, affecting both adipogenesis and lipogenesis. PMID:22262865

  5. Mitochondrial ribosomal protein S18-2 evokes chromosomal instability and transforms primary rat skin fibroblasts

    KAUST Repository

    Kashuba, Elena; Carbone, Ennio; Di Fabrizio, Enzo M.; Tirinato, Luca; Petruchek, Maria; Drummond, Catherine; Kovalevska, Larysa; Gurrapu, Sreeharsha; Mushtaq, Muhammad; Darekar, Suhas D.

    2015-01-01

    We have shown earlier that overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts. The derived cells expressed the embryonic stem cell markers, and cellular pathways

  6. Primary chondrocytes enhance cartilage tissue formation upon co-culture with expanded chondrocytes, dermal fibroblasts, 3T3 feeder cells and embryonic stem cells

    NARCIS (Netherlands)

    Hendriks, J.A.A.; Miclea, Razvan L.; Schotel, Roka; de Bruijn, Ewart; Moroni, Lorenzo; Karperien, Hermanus Bernardus Johannes; Riesle, J.U.; van Blitterswijk, Clemens

    2010-01-01

    Co-culture models have been increasingly used in tissue engineering applications to understand cell–cell interactions and consequently improve regenerative medicine strategies. Aiming at further elucidating cartilage tissue formation, we co-cultured bovine primary chondrocytes (BPCs) with human

  7. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension

    DEFF Research Database (Denmark)

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E

    2010-01-01

    Tendon fibroblasts synthesize collagen and form fibrils during embryonic development, but to what extent mature fibroblasts are able to recapitulate embryonic development and develop normal tendon structure is unknown. The present study examined the capability of mature human tendon fibroblasts t...

  8. Dioxin induces genomic instability in mouse embryonic fibroblasts.

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    Merja Korkalainen

    Full Text Available Ionizing radiation and certain other exposures have been shown to induce genomic instability (GI, i.e., delayed genetic damage observed many cell generations later in the progeny of the exposed cells. The aim of this study was to investigate induction of GI by a nongenotoxic carcinogen, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD. Mouse embryonic fibroblasts (C3H10T1/2 were exposed to 1, 10 or 100 nM TCDD for 2 days. Micronuclei (MN and expression of selected cancer-related genes were assayed both immediately and at a delayed point in time (8 days. For comparison, similar experiments were done with cadmium, a known genotoxic agent. TCDD treatment induced an elevated frequency of MN at 8 days, but not directly after the exposure. TCDD-induced alterations in gene expression were also mostly delayed, with more changes observed at 8 days than at 2 days. Exposure to cadmium produced an opposite pattern of responses, with pronounced effects immediately after exposure but no increase in MN and few gene expression changes at 8 days. Although all responses to TCDD alone were delayed, menadione-induced DNA damage (measured by the Comet assay, was found to be increased directly after a 2-day TCDD exposure, indicating that the stability of the genome was compromised already at this time point. The results suggested a flat dose-response relationship consistent with dose-response data reported for radiation-induced GI. These findings indicate that TCDD, although not directly genotoxic, induces GI, which is associated with impaired DNA damage response.

  9. Identification of SSEA-1 expressing enhanced reprogramming (SEER) cells in porcine embryonic fibroblasts

    DEFF Research Database (Denmark)

    Li, Dong; Secher, Jan Ole Bertelsen; Juhl, Morten

    2017-01-01

    Previous research has shown that a subpopulation of cells within cultured human dermal fibroblasts, termed multilineage-differentiating stress enduring (Muse) cells, are preferentially reprogrammed into induced pluripotent stem cells. However, controversy exists over whether these cells...... are the only cells capable of being reprogrammed from a heterogeneous population of fibroblasts. Similarly, there is little research to suggest such cells may exist in embryonic tissues or other species. To address if such a cell population exists in pigs, we investigated porcine embryonic fibroblast...... populations (pEFs) and identified heterogeneous expression of several key cell surface markers. Strikingly, we discovered a small population of stage-specific embryonic antigen 1 positive cells (SSEA-1+) in Danish Landrace and Göttingen minipig pEFs, which were absent in the Yucatan pEFs. Furthermore...

  10. Mitochondrial ribosomal protein S18-2 evokes chromosomal instability and transforms primary rat skin fibroblasts

    KAUST Repository

    Kashuba, Elena

    2015-05-12

    We have shown earlier that overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts. The derived cells expressed the embryonic stem cell markers, and cellular pathways that control cell proliferation, oxidative phosphorylation, cellular respiration, and other redox reactions were activated in the immortalized cells. Here we report that, upon overexpression of S18-2 protein, primary rat skin fibroblasts underwent cell transformation. Cells passed more than 300 population doublings, and two out of three tested clones gave rise to tumors in experimental animals. Transformed cells showed anchorage-independent growth and loss of contact inhibition; they expressed epithelial markers, such as E-cadherin and β-catenin. Transformed cells showed increased telomerase activity, disturbance of the cell cycle, and chromosomal instability. Taken together, our data suggest that S18-2 is a newly identified oncoprotein that may be involved in cancerogenesis.

  11. Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

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    Ahmed Kamel El-Sayed

    2014-11-01

    Full Text Available Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1 and Rex-1 (ZFP-42, zinc finger protein 42. Using embryonic stem cells (ESCs conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.

  12. FRS2α is Essential for the Fibroblast Growth Factor to Regulate the mTOR Pathway and Autophagy in Mouse Embryonic Fibroblasts

    OpenAIRE

    Xiang Lin, Yongyou Zhang, Leyuan Liu, Wallace L. McKeehan, Yuemao Shen, Siyang Song, Fen Wang

    2011-01-01

    Although the fibroblast growth factor (FGF) signaling axis plays important roles in cell survival, proliferation, and differentiation, the molecular mechanism underlying how the FGF elicits these diverse regulatory signals is not well understood. By using the Frs2α null mouse embryonic fibroblast (MEF) in conjunction with inhibitors to multiple signaling pathways, here we report that the FGF signaling axis activates mTOR via the FGF receptor substrate 2α (FRS2α)-mediated PI3K/A...

  13. Modulation of cellular phosphoprotein profiles in transformation and redifferentiation of murine and embryonic fibroblastic cells

    International Nuclear Information System (INIS)

    Chakrabarty, Subhas; Brattain, M.G.

    1985-01-01

    Cellular phosphoprotein profiles from normal mouse embryonic fibroblast AKR-2B cells were compared to those of their permanently, chemically transformed malignant counterparts AKR-MCA cells, and AKR-2B cells reversibly transformed by transforming growth factor (AKR-TGF). Similar 32 P-phosphorylation profiles were observed for both the AKR-TGF and AKR-MCA cells which were distinct from that of the normal AKR-2B cells. Dimethylformamide (DMF)-induced differentiation of the AKR-MCA cells resulted in restoration of the normal AKR-2B phosphorylation profile to the malignant AKR-MCA cells. (author)

  14. Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

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    Linta, Leonhard; Stockmann, Marianne; Kleinhans, Karin N; Böckers, Anja; Storch, Alexander; Zaehres, Holm; Lin, Qiong; Barbi, Gotthold; Böckers, Tobias M; Kleger, Alexander; Liebau, Stefan

    2012-04-10

    Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. © Mary Ann Liebert, Inc.

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Cellular phenotype-dependent and -independent effects of vitamin C on the renewal and gene expression of mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Shiu-Ming Kuo

    Full Text Available Vitamin C has been shown to delay the cellular senescence and was considered a candidate for chemoprevention and cancer therapy. To understand the reported contrasting roles of vitamin C: growth-promoting in the primary cells and growth-inhibiting in cancer cells, primary mouse embryonic fibroblasts (MEF and their isogenic spontaneously immortalized fibroblasts with unlimited cell division potential were used as the model pair. We used microarray gene expression profiling to show that the immortalized MEF possess human cancer gene expression fingerprints including a pattern of up-regulation of inflammatory response-related genes. Using the MEF model, we found that a physiological treatment level of vitamin C (10(-5 M, but not other unrelated antioxidants, enhanced cell growth. The growth-promoting effect was associated with a pattern of enhanced expression of cell cycle- and cell division-related genes in both primary and immortalized cells. In the immortalized MEF, physiological treatment levels of vitamin C also enhanced the expression of immortalization-associated genes including a down-regulation of genes in the extracellular matrix functional category. In contrast, confocal immunofluorescence imaging of the primary MEF suggested an increase in collagen IV protein upon vitamin C treatment. Similar to the cancer cells, the growth-inhibitory effect of the redox-active form of vitamin C was preferentially observed in immortalized MEF. All effects of vitamin C required its intracellular presence since the transporter-deficient SVCT2-/- MEF did not respond to vitamin C. SVCT2-/- MEF divided and became immortalized readily indicating little dependence on vitamin C for the cell division. Immortalized SVCT2-/- MEF required higher concentration of vitamin C for the growth inhibition compared to the immortalized wildtype MEF suggesting an intracellular vitamin C toxicity. The relevance of our observation in aging and human cancer prevention was

  17. Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Anisimov, Sergey V.; Christophersen, Nicolaj S.; Correia, Ana S.

    2011-01-01

    The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both...... the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early...... foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates....

  18. Effects of gentiopicroside, sweroside and swertiamarine, secoiridoids from gentian (Gentiana lutea ssp. symphyandra), on cultured chicken embryonic fibroblasts.

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    Oztürk, Nilgün; Korkmaz, Seval; Oztürk, Yusuf; Başer, K Hüsnü Can

    2006-03-01

    Wound healing properties of Gentian (Gentiana lutea ssp. symphyandra) extract and its main constituents, gentiopicroside, sweroside and swertiamarine (compounds 1-3, respectively) were evaluated by comparison with dexpanthenol on cultured chicken embryonic fibroblasts. The extract was also analyzed by HPLC to quantify its constituents. Chicken embryonic fibroblasts from fertilized eggs were incubated with the plant extract and its constituents, compounds 1-3. Using microscopy, mitotic ability, morphological changes and collagen production in the cultured fibroblasts were evaluated as parameters. Wound healing activity of Gentian seems to be mainly due to the increase in the stimulation of collagen production and the mitotic activity by compounds 2 and 3, respectively (p < 0.005 in all cases). All three compounds also exhibited cytoprotective effects, which may cause a synergism in terms of wound healing activity of Gentian. The findings demonstrated the wound healing activity of Gentian, which has previously been based only on ethnomedical data.

  19. [Isolation, purification and primary culture of adult mouse cardiac fibroblasts].

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    Li, Rujun; Gong, Kaizheng; Zhang, Zhengang

    2017-01-01

    Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0.8 g/L collagenase IV by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3 phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion, adherent fibroblasts formed spherical cell mass and after 3 days, cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly "S" shape. The positive expression rate of vimentin was 95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.

  20. Transfection of Primary Human Skin Fibroblasts for Peroxisomal Studies

    NARCIS (Netherlands)

    Koster, Janet; Waterham, Hans R.

    2017-01-01

    Functional studies with primary human skin fibroblasts from patients with a peroxisomal disorder often require efficient transfection with plasmids to correct the genetic defect or to express heterologous reporter proteins. Here, we describe a protocol we commonly use for efficient nonviral

  1. Correlation between DNA repair of embryonic fibroblasts and different life span of 3 inbred mouse strains

    Energy Technology Data Exchange (ETDEWEB)

    Paffenholz, V.

    1978-02-01

    Primary mouse fibroblast cultures were established from 10 day old embryos of 3 inbred strains with a genetically determined different life expectancy. The capacity for unscheduled DNA synthesis following uv irradiation was studied in these cells at various passage levels of the in vitro ageing process. The mouse fibroblasts show considerable repair synthesis corresponding to the duration of exposure time. The capacity for induction of unscheduled DNA synthesis was different in the cells of each strain and correlated to the natural life span of the animal. In each case, however, the ability to perform repair synthesis was subjected to an age-associated decline, although semiconservative DNA synthesis and proliferative potential of the cell was not changed until the cultures entered phase III passages.

  2. Overexpression of histone demethylase Fbxl10 leads to enhanced migration in mouse embryonic fibroblasts

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    Rohde, Magdalena; Sievers, Elisabeth; Janzer, Andreas [Institute of Pathology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn (Germany); Willmann, Dominica [Urologische Klinik/Frauenklinik und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, Breisacherstrasse 66, 79106 Freiburg (Germany); Egert, Angela; Schorle, Hubert [Department of Developmental Pathology, Institute of Pathology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn (Germany); Schüle, Roland [Urologische Klinik/Frauenklinik und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, Breisacherstrasse 66, 79106 Freiburg (Germany); Kirfel, Jutta, E-mail: Jutta.Kirfel@ukb.uni-bonn.de [Institute of Pathology, University of Bonn, Sigmund-Freud-Strasse 25, 53127 Bonn (Germany)

    2016-11-01

    Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing, immune responses and invasive tumors all require the orchestrated movement of cells to specific locations. Histone demethylase proteins alter transcription by regulating the chromatin state at specific gene loci. FBXL10 is a conserved and ubiquitously expressed member of the JmjC domain-containing histone demethylase family and is implicated in the demethylation of H3K4me3 and H3K36me2 and thereby removing active chromatin marks. However, the physiological role of FBXL10 in vivo remains largely unknown. Therefore, we established an inducible gain of function model to analyze the role of Fbxl10 and compared wild-type with Fbxl10 overexpressing mouse embryonic fibroblasts (MEFs). Our study shows that overexpression of Fbxl10 in MEFs doesn’t influence the proliferation capability but leads to an enhanced migration capacity in comparison to wild-type MEFs. Transcriptome and ChIP-seq experiments demonstrated that Fbxl10 binds to genes involved in migration like Areg, Mdk, Lmnb1, Thbs1, Mgp and Cxcl12. Taken together, our results strongly suggest that Fbxl10 plays a critical role in migration by binding to the promoter region of migration-associated genes and thereby might influences cell behaviour to a possibly more aggressive phenotype. - Highlights: • Migration capability of MEFs is enhanced after Fbxl10 upregulation. • Overexpression of Fbxl10 induced migration-associated genes. • Fbxl10 binds directly to migration-associated genes.

  3. Overexpression of histone demethylase Fbxl10 leads to enhanced migration in mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Rohde, Magdalena; Sievers, Elisabeth; Janzer, Andreas; Willmann, Dominica; Egert, Angela; Schorle, Hubert; Schüle, Roland; Kirfel, Jutta

    2016-01-01

    Cell migration is a central process in the development and maintenance of multicellular organisms. Tissue formation during embryonic development, wound healing, immune responses and invasive tumors all require the orchestrated movement of cells to specific locations. Histone demethylase proteins alter transcription by regulating the chromatin state at specific gene loci. FBXL10 is a conserved and ubiquitously expressed member of the JmjC domain-containing histone demethylase family and is implicated in the demethylation of H3K4me3 and H3K36me2 and thereby removing active chromatin marks. However, the physiological role of FBXL10 in vivo remains largely unknown. Therefore, we established an inducible gain of function model to analyze the role of Fbxl10 and compared wild-type with Fbxl10 overexpressing mouse embryonic fibroblasts (MEFs). Our study shows that overexpression of Fbxl10 in MEFs doesn’t influence the proliferation capability but leads to an enhanced migration capacity in comparison to wild-type MEFs. Transcriptome and ChIP-seq experiments demonstrated that Fbxl10 binds to genes involved in migration like Areg, Mdk, Lmnb1, Thbs1, Mgp and Cxcl12. Taken together, our results strongly suggest that Fbxl10 plays a critical role in migration by binding to the promoter region of migration-associated genes and thereby might influences cell behaviour to a possibly more aggressive phenotype. - Highlights: • Migration capability of MEFs is enhanced after Fbxl10 upregulation. • Overexpression of Fbxl10 induced migration-associated genes. • Fbxl10 binds directly to migration-associated genes.

  4. A reliable and economical method for gaining mouse embryonic fibroblasts capable of preparing feeder layers.

    Science.gov (United States)

    Jiang, Guangming; Wan, Xiaoju; Wang, Ming; Zhou, Jianhua; Pan, Jian; Wang, Baolong

    2016-08-01

    Mouse embryonic fibroblasts (MEFs) are widely used to prepare feeder layers for culturing embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in vitro. Transportation lesions and exorbitant prices make the commercially obtained MEFs unsuitable for long term research. The aim of present study is to establish a method, which enables researchers to gain MEFs from mice and establish feeder layers by themselves in ordinary laboratories. MEFs were isolated from ICR mouse embryos at 12.5-17.5 day post-coitum (DPC) and cultured in vitro. At P2-P7, the cells were inactivated with mitomycin C or by X-ray irradiation. Then they were used to prepare feeder layers. The key factors of the whole protocol were analyzed to determine the optimal conditions for the method. The results revealed MEFs isolated at 12.5-13.5 DPC, and cultured to P3 were the best choice for feeder preparation, those P2 and P4-P5 MEFs were also suitable for the purpose. The P3-P5 MEFs treated with 10 μg/ml of mitomycin C for 3 h, or irradiated with X-ray at 1.5 Gy/min for 25 Gy were the most suitable feeder cells. Treating MEFs with 10 μg/ml of mitomycin C for 2.5 h, 15 μg/ml for 2.0 h, or irradiating the cells with 20 Gy of X-ray at 2.0 Gy/min could all serve as alternative methods for P3-P4 cells. Our study provides a reliable and economical way to obtain large amount of qualified MEFs for long term research of ESCs or iPSCs.

  5. Phosphatidylinositol 3,4,5-trisphosphate modulation in SHIP2-deficient mouse embryonic fibroblasts.

    Science.gov (United States)

    Blero, Daniel; Zhang, Jing; Pesesse, Xavier; Payrastre, Bernard; Dumont, Jacques E; Schurmans, Stéphane; Erneux, Christophe

    2005-05-01

    SHIP2, the ubiquitous SH2 domain containing inositol 5-phosphatase, includes a series of protein interacting domains and has the ability to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]in vitro. The present study, which was undertaken to evaluate the impact of SHIP2 on PtdIns(3,4,5)P(3) levels, was performed in a mouse embryonic fibroblast (MEF) model using SHIP2 deficient (-/-) MEF cells derived from knockout mice. PtdIns(3,4,5)P(3) was upregulated in serum stimulated -/- MEF cells as compared to +/+ MEF cells. Although the absence of SHIP2 had no effect on basal PtdIns(3,4,5)P(3) levels, we show here that this lipid was significantly upregulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P(3) levels in heterozygous fibroblast cells was intermediate between the +/+ and the -/- cells. In our model, insulin-like growth factor-1 stimulation did not show this upregulation. Serum stimulated phosphoinositide 3-kinase (PI 3-kinase) activity appeared to be comparable between +/+ and -/- cells. Moreover, protein kinase B, but not mitogen activated protein kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The upregulation of protein kinase B activity in serum stimulated cells was totally reversed in the presence of the PI 3-kinase inhibitor LY-294002, in both +/+ and -/- cells. Altogether, these data establish a link between SHIP2 and the acute control of PtdIns(3,4,5)P(3) levels in intact cells.

  6. Expression and loss of alleles in cultured mouse embryonic fibroblasts and stem cells carrying allelic fluorescent protein genes

    Directory of Open Access Journals (Sweden)

    Stringer Saundra L

    2006-10-01

    Full Text Available Abstract Background Loss of heterozygosity (LOH contributes to many cancers, but the rate at which these events occur in normal cells of the body is not clear. LOH would be detectable in diverse cell types in the body if this event were to confer an obvious cellular phenotype. Mice that carry two different fluorescent protein genes as alleles of a locus would seem to be a useful tool for addressing this issue because LOH would change a cell's phenotype from dichromatic to monochromatic. In addition, LOH caused by mitotic crossing over might be discernable in tissues because this event produces a pair of neighboring monochromatic cells that are different colors. Results As a step in assessing the utility of this approach, we derived primary embryonic fibroblast populations and embryonic stem cell lines from mice that carried two different fluorescent protein genes as alleles at the chromosome 6 locus, ROSA26. Fluorescence activated cell sorting (FACS showed that the vast majority of cells in each line expressed the two marker proteins at similar levels, and that populations exhibited expression noise similar to that seen in bacteria and yeast. Cells with a monochromatic phenotype were present at frequencies on the order of 10-4 and appeared to be produced at a rate of approximately 10-5 variant cells per mitosis. 45 of 45 stably monochromatic ES cell clones exhibited loss of the expected allele at the ROSA26 locus. More than half of these clones retained heterozygosity at a locus between ROSA26 and the centromere. Other clones exhibited LOH near the centromere, but were disomic for chromosome 6. Conclusion Allelic fluorescent markers allowed LOH at the ROSA26 locus to be detected by FACS. LOH at this locus was usually not accompanied by LOH near the centromere, suggesting that mitotic recombination was the major cause of ROSA26 LOH. Dichromatic mouse embryonic cells provide a novel system for studying genetic/karyotypic stability and factors

  7. Efficient transfection of primarily cultured porcine embryonic fibroblasts using the Amaxa Nucleofection system.

    Science.gov (United States)

    Nakayama, Asuka; Sato, Masahiro; Shinohara, Mariko; Matsubara, Shyuichiro; Yokomine, Takaaki; Akasaka, Eri; Yoshida, Mitsutoshi; Takao, Sonshin

    2007-01-01

    Porcine embryonic fibroblasts (PEF) are important as donor cells for nuclear transfer for generation of genetically modified pigs. In this study, we determined an optimal protocol for transfection of PEF with the Amaxa Nucleofection system, which directly transfers DNA into the nucleus of cells, and compared its efficiency with conventional lipofection and electroporation. Cell survival and transfection efficiency were assessed using dye-exclusion assay and a green fluorescent protein (GFP) reporter construct, respectively. Our optimized nucleofection parameters yielded survival rates above 60%. Under these conditions, FACS analysis demonstrated that 79% of surviving cells exhibited transgene expression 48 h after nucleofection when program U23 was used. This efficiency was higher than that of transfection of PEFs with electroporation (ca. 3-53%) or lipofection (ca. 3-8%). Transfected cells could be expanded as stably transgene-expressing clones over a month. When porcine nuclear transfer (NT) was performed using stable transformant expressing GFP as a donor cell, 5-6% of reconstituted embryos developed to blastocysts, from which 30-50% of embryos exhibited NT-embryo-derived green fluorescence. Under the conditions evaluated, nucleofection exhibited higher efficiency than conventional electroporation and lipofection, and may be a useful alternative for generation of genetically engineered pigs through nuclear transfer.

  8. Increasing mouse embryonic fibroblast cells adhesion on superhydrophilic vertically aligned carbon nanotube films

    Energy Technology Data Exchange (ETDEWEB)

    Lobo, A.O., E-mail: loboao@yahoo.com [Laboratory of Biomedical Nanotechnology (NanoBio), Instituto de Pesquisa e Desenvolvimento (IP and D), Universidade do Vale do Paraiba UniVap, Avenida Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, SP (Brazil) and Laboratory of Biomedical Vibrational Spectroscopy (LEVB), Instituto de Pesquisa e Desenvolvimento (IP and D), Universidade do Vale do Paraiba UniVap, Avenida Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, SP (Brazil); Marciano, F.R. [Laboratory of Biomedical Nanotechnology (NanoBio), Instituto de Pesquisa e Desenvolvimento (IP and D), Universidade do Vale do Paraiba UniVap, Avenida Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, SP (Brazil); Laboratory of Biomedical Vibrational Spectroscopy LEVB, Instituto de Pesquisa e Desenvolvimento (IP and D), Universidade do Vale do Paraiba (UniVap), Avenida Shishima Hifumi 2911, Sao Jose dos Campos, 12244-000, SP (Brazil); Ramos, S.C. [Laboratorio Associado de Sensores e Materiais (LAS), Instituto Nacional de Pesquisas Espaciais (INPE), Avenida dos Astronautas 1758, Sao Jose dos Campos, 12.245-970, SP (Brazil); Machado, M.M. [Centro Multidisciplinar para Investigacao Biologica na Area da Ciencia em Animais de Laboratorio (CEMIB), Universidade Estadual de Campinas (UNICAMP), Rua 05 de Junho s/no, Cidade Universitaria ' Zeferino Vaz' , 13083-877, Campinas (Brazil); Corat, E.J. [Laboratorio Associado de Sensores e Materiais (LAS), Instituto Nacional de Pesquisas Espaciais (INPE), Avenida dos Astronautas 1758, Sao Jose dos Campos, 12.245-970, SP (Brazil); Corat, M.A.F. [Centro Multidisciplinar para Investigacao Biologica na Area da Ciencia em Animais de Laboratorio (CEMIB), Universidade Estadual de Campinas (UNICAMP), Rua 05 de Junho s/no, Cidade Universitaria ' Zeferino Vaz' , 13083-877, Campinas (Brazil)

    2011-10-10

    We have analyzed the adhesion of mouse embryonic fibroblasts (MEFs) genetically modified by green fluorescence protein (GFP) gene cultured on vertically-aligned carbon nanotubes (VACNTs) after 6 days. The VACNTs films grown on Ti were obtained by microwave plasma chemical vapor deposition process using Fe catalyst and submitted to an oxygen plasma treatment, for 2 min, at 400 V and 80 mTorr, to convert them to superhydrophilic. Cellular adhesion and morphology were analyzed by scanning electron, fluorescence microscopy, and thermodynamics analysis. Characterizations of superhydrophilic VACNTs films were evaluated by contact angle and X-Ray Photoelectron Spectroscopy. Differences of crowd adhered cells, as well as their spreading on superhydrophilic VACNTs scaffolds, were evaluated using focal adhesion analysis. This study was the first to demonstrate, in real time, that the wettability of VACNTs scaffolds might have enhanced and differential adherence patterns to the MEF-GFP on VACNTs substrates. Highlights: {yields} A simple oxygen plasma treatment was used to obtain superhydrophilic CNT films. {yields} Superhydrophilic CNTs films were successfully produced by incorporation of carboxylic groups. {yields} Cellular adhesion on superhydrophilic VACNT films was analyzed in real time. {yields} Wettability of CNT films directly affects the cellular migration, proliferation and adhesion.

  9. Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Kukat, Alexandra; Edgar, Daniel; Bratic, Ivana; Maiti, Priyanka; Trifunovic, Aleksandra

    2011-01-01

    Highlights: → Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. → This process is independent of endogenous ROS production. → Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O 2 ) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

  10. Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

    International Nuclear Information System (INIS)

    Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

    2005-01-01

    In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of the TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian

  11. Comparing the mechanical influence of vinculin, focal adhesion kinase and p53 in mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Klemm, Anna H.; Diez, Gerold; Alonso, Jose-Luis; Goldmann, Wolfgang H.

    2009-01-01

    Cytoskeletal reorganization is an ongoing process when cells adhere, move or invade extracellular substrates. The cellular force generation and transmission are determined by the intactness of the actomyosin-(focal adhesion complex)-integrin connection. We investigated the intracellular course of action in mouse embryonic fibroblasts deficient in the focal adhesion proteins vinculin and focal adhesion kinase (FAK) and the nuclear matrix protein p53 using magnetic tweezer and nanoparticle tracking techniques. Results show that the lack of these proteins decrease cellular stiffness and affect cell rheological behavior. The decrease in cellular binding strength was higher in FAK- to vinculin-deficient cells, whilst p53-deficient cells showed no effect compared to wildtype cells. The intracellular cytoskeletal activity was lowest in wildtype cells, but increased in the following order when cells lacked FAK+p53 > p53 > vinculin. In summary, cell mechanical processes are differently affected by the focal adhesion proteins vinculin and FAK than by the nuclear matrix protein, p53.

  12. EB1 is required for primary cilia assembly in fibroblasts

    DEFF Research Database (Denmark)

    Schrøder, Jacob M; Schneider, Linda; Christensen, Søren T

    2007-01-01

    EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends and plays a role in regulating MT dynamics. EB1 also targets other MT-associated proteins to the plus end and thereby regulates interactions of MTs with the cell cortex, mitotic kinetochores, and diff...... that localization of EB1 at the centriole/basal body is required for primary cilia assembly in fibroblasts....

  13. Analysis of primary cilia in directional cell migration in fibroblasts

    DEFF Research Database (Denmark)

    Christensen, Søren Tvorup; Veland, Iben; Schwab, Albrecht

    2013-01-01

    summarize selected methods in analyzing ciliary function in directional cell migration, including immunofluorescence microscopy, scratch assay, and chemotaxis assay by micropipette addition of PDGFRα ligands to cultures of fibroblasts. These methods should be useful not only in studying cell migration....... In particular, platelet-derived growth factor receptor alpha (PDGFRα) is compartmentalized to the primary cilium to activate signaling pathways that regulate reorganization of the cytoskeleton required for lamellipodium formation and directional migration in the presence of a specific ligand gradient. We...

  14. Hydroxychloroquine susceptibility determination of Coxiella burnetii in human embryonic lung (HEL) fibroblast cells.

    Science.gov (United States)

    Angelakis, Emmanouil; Khalil, Jacques Bou; Le Bideau, Marion; Perreal, Celine; La Scola, Bernard; Raoult, Didier

    2017-07-01

    Coxiella burnetii, the causative agent of Q fever, survives and replicates in the acidic environment of monocytes/macrophages; hydroxychloroquine, through alkalinisation of the acidic vacuoles, is critical for the management of Q fever. In this study, a collection of C. burnetii strains isolated from human samples was tested to evaluate the in vitro minimum inhibitory concentrations (MICs) of doxycycline and hydroxychloroquine. Serial two-fold dilutions of doxycycline (0.25-8 mg/L) and hydroxychloroquine (0.25-4 mg/L) were added to C. burnetii-infected human embryonic lung (HEL) fibroblast cells after 48 h of incubation, in duplicate. DNA was detected by C. burnetii-specific semi-quantitative PCR with primers and probes designed for amplification of the IS1111 and IS30A spacers. A total of 29 C. burnetii isolates obtained from 29 patients were tested. Doxycycline MICs ranged from 0.25 mg/L to 0.5 mg/L and hydroxychloroquine MICs from 0.25 mg/L to >4 mg/L. Four C. burnetii stains had hydroxychloroquine MICs ≤ 1 mg/L. The concentration of hydroxychloroquine was associated with a significant decrease in C. burnetii DNA copies in HEL cells based on linear regression analysis (P= 0.01). Recommended serum concentrations of hydroxychloroquine significantly reduced the growth of C. burnetii. Moreover, some C. burnetii strains presented hydroxychloroquine MICs below the recommended serum concentrations, indicating that, for these cases, hydroxychloroquine treatment alone may even be effective. Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

  15. Self-recognition of high-mannose type glycans mediating adhesion of embryonal fibroblasts.

    Science.gov (United States)

    Yoon, Seon-Joo; Utkina, Natalia; Sadilek, Martin; Yagi, Hirokazu; Kato, Koichi; Hakomori, Sen-itiroh

    2013-07-01

    High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.

  16. Cellular radiosensitivity and DNA damage in primary human fibroblasts

    International Nuclear Information System (INIS)

    Wurm, R.; Burnet, N.G.; Duggal, N.

    1994-01-01

    To evaluate the relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts to decide whether the initial or residual DNA damage levels are more predictive of normal tissue cellular radiosensitivity. Five primary human nonsyndromic and two primary ataxia telangiectasia fibroblast strains grown in monolayer were studied. Cell survival was assessed by clonogenic assay. Irradiation was given at high dose rate (HDR) 1-2 Gy/min. DNA damage was measured in stationary phase cells and expressed as fraction released from the well by pulsed-field gel electrophoresis (PFGE). For initial damage, cells were embedded in agarose and irradiated at HDR on ice. Residual DNA damage was measured in monolayer by allowing a 4-h repair period after HDR irradiation. Following HDR irradiation, cell survival varied between SF 2 0.025 to 0.23. Measurement of initial DNA damage demonstrated linear induction up to 30 Gy, with small differences in the slope of the dose-response curve between strains. No correlation between cell survival and initial damage was found. Residual damage increased linearly up to 80 Gy with a variation in slope by a factor of 3.2. Cell survival correlated with the slope of the dose-response curves for residual damage of the different strains (p = 0.003). The relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts of differing radiosensitivity is closest with the amount of DNA damage remaining after repair. If assays of DNA damage are to be used as predictors of normal tissue response to radiation, residual DNA damage provides the most likely correlation with cell survival. 52 refs., 5 figs., 2 tabs

  17. Thy-1 attenuates TNF-alpha-activated gene expression in mouse embryonic fibroblasts via Src family kinase.

    Directory of Open Access Journals (Sweden)

    Bin Shan

    Full Text Available Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+ and negative (Thy-1- subsets of mouse embryonic fibroblasts (MEF. TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

  18. Mouse Embryonic Fibroblasts (MEF) Exhibit a Similar but not Identical Phenotype to Bone Marrow Stromal Stem Cells (BMSC)

    DEFF Research Database (Denmark)

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M

    2012-01-01

    Mouse embryonic fibroblasts have been utilized as a surrogate stem cell model for the postnatal bone marrow-derived stromal stem cells (BMSC) to study mesoderm-type cell differentiation e.g. osteoblasts, adipocytes and chondrocytes. However, no formal characterization of MEF phenotype has been...... by real-time PCR analysis. Compared to BMSC, MEF exhibited a more enhanced differentiation into adipocyte and chondrocyte lineages. Interestingly, both MEF and BMSC formed the same amount of heterotopic bone and bone marrow elements upon in vivo subcutaneous implantation with hydroxyapatite...... and differentiation to osteoblasts, adipocytes and chondrocytes....

  19. Interleukin-8 induces motile behavior and loss of focal adhesions in primary fibroblasts

    DEFF Research Database (Denmark)

    Dunlevy, J R; Couchman, J R

    1995-01-01

    Interleukin-8 (IL-8) is a proinflammatory cytokine that promotes neutrophil migration. Although fibroblasts are known to secrete IL-8, the actions of this cytokine on fibroblasts have not been previously reported. We have found that in subconfluent populations of cultured primary fibroblasts, IL-8...

  20. Fibronectin-synthesizing activity of free and membrane-bound polyribosomes from human embryonic fibroblasts and chick embryos

    International Nuclear Information System (INIS)

    Belkin, V.M.; Volodarskaya, S.M.

    1986-01-01

    The fibronectin-synthesizing activity of membrane-bound and free polyribosomes in a cell-free system was studied using immunochemical methods. It was found that fibronectin biosynthesis on membrane-bound polyribosomes from human embryonic fibroblasts accounts for 4.9% and those from 10-day-old chick embryos for 1.1% of the total amount of newly synthesized proteins, whereas on free polyribosomes it is 1.0 and 0.3%, respectively. Fibronectin monomers with a molecular weight of 220,000 were found only in the material of the cell-free system containing heavy fractions of membrane-bound polyribosomes newly synthesized in the presence of spermidine. Thus, it was shown that fibronectin is synthesized primarily on membrane-bound polyribosomes

  1. Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

    DEFF Research Database (Denmark)

    Hakim-Weber, Robab; Krogsdam, Anne-M; Jørgensen, Claus

    2011-01-01

    Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate...... this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs...... of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1.To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis...

  2. Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem Cell-Derived Fibroblasts and an L-929 Cell Line

    Directory of Open Access Journals (Sweden)

    Hui Peng

    2012-01-01

    Full Text Available Consensus about the toxicity of silver nanoparticles (Ag-NPs has not been reached, even though extensive attention has been paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety evaluation. In the present study, human embryonic stem cell-derived fibroblasts (EBFs, which have been considered a closer representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (~20 nm and ~100 nm in comparison with L-929 fibroblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles.

  3. Nonmuscle myosin IIA and IIB differentially contribute to intrinsic and directed migration of human embryonic lung fibroblasts.

    Science.gov (United States)

    Kuragano, Masahiro; Murakami, Yota; Takahashi, Masayuki

    2018-03-25

    Nonmuscle myosin II (NMII) plays an essential role in directional cell migration. In this study, we investigated the roles of NMII isoforms (NMIIA and NMIIB) in the migration of human embryonic lung fibroblasts, which exhibit directionally persistent migration in an intrinsic manner. NMIIA-knockdown (KD) cells migrated unsteadily, but their direction of migration was approximately maintained. By contrast, NMIIB-KD cells occasionally reversed their direction of migration. Lamellipodium-like protrusions formed in the posterior region of NMIIB-KD cells prior to reversal of the migration direction. Moreover, NMIIB KD led to elongation of the posterior region in migrating cells, probably due to the lack of load-bearing stress fibers in this area. These results suggest that NMIIA plays a role in steering migration by maintaining stable protrusions in the anterior region, whereas NMIIB plays a role in maintenance of front-rear polarity by preventing aberrant protrusion formation in the posterior region. These distinct functions of NMIIA and NMIIB might promote intrinsic and directed migration of normal human fibroblasts. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-β responsiveness

    International Nuclear Information System (INIS)

    Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua; Warner-Blankenship, Matthew; Lyons, Karen M.; Varga, John

    2008-01-01

    Background: The matricellular protein connective tissue growth factor (CCN2) has been implicated in pathological fibrosis, but its physiologic role remains elusive. In vitro, transforming growth factor-β (TGF-β) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-β, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-β. To explore this notion, we characterized TGF-β-induced activation of fibroblasts from CCN2-null (CCN2 -/- ) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-β signal transduction and regulation of collagen gene expression were examined in CCN2 -/- MEFs by immunohistochemistry, Northern, Western and RT-PCR analysis, immunocytochemistry and transient transfection assays. Results: Bleomycin-induced skin fibrosis in the mouse was associated with substantial CCN2 up-regulation in lesional fibroblasts. Whereas in vitro proliferation rate of CCN2 -/- MEFs was markedly reduced compared to wild type MEFs, TGF-β-induced activation of the Smad pathways, including Smad2 phosphorylation, Smad2/3 and Smad4 nuclear accumulation and Smad-dependent transcriptional responses, were unaffected by loss of CCN2. The stimulation of COL1A2 and fibronectin mRNA expression and promoter activity, and of corresponding protein levels, showed comparable time and dose-response in wild type and CCN2 -/- MEFs, whereas stimulation of alpha smooth muscle actin and myofibroblast transdifferentiation showed subtle impairment in MEFs lacking CCN2. Conclusion: Whereas endogenous CCN2 plays a role in regulation of proliferation and TGF-β-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts

  5. Chromosomal instability in mouse embryonic fibroblasts null for the transcriptional co-repressor Ski

    OpenAIRE

    Marcelain, Katherine; Armisen, Ricardo; Aguirre, Adam; Ueki, Nobuhide; Toro, Jessica; Colmenares, Clemencia; Hayman, Michael J

    2012-01-01

    Ski is a transcriptional regulator that has been considered an oncoprotein, given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski−/− mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time-lapse microscopy revealed lagging chromosomes and chroma...

  6. Induction of anchorage-independent growth of human embryonic fibroblasts with a deletion in the short arm of chromosome 11 by human papillomavirus type 16 DNA

    International Nuclear Information System (INIS)

    Smits, H.L.; Raadsheer, E.; Rood, I.; Mehendale, S.; Slater, R.M.; van der Noordaa, J.; Ter Schegget, J.

    1988-01-01

    Human embryonic fibroblasts with a large deletion (11p11.11p15.1) in the short arm of one chromosome 11 (del-11 cells) appeared to be susceptible to transformation by early human papillomavirus type 16 (HPV-16) DNA, whereas diploid human embryonic fibroblasts were not. This difference in susceptibility might be explained by the absence of a tumor suppressor gene located within the deleted part on the short arm of chromosome 11. The presence of abundant viral early-gene transcripts in transformed cells suggests that transformation was induced by an elevated level of an HPV-16 early-gene product(s). The low transcriptional activity of HPV-16 in diploid cells may indicate that cellular genes affect viral transcription. Interruption of the HPV-16 E2 early open reading frame is probably required for high-level HPV-16 early-gene expression driven from the homologous enhancer-promoter region

  7. Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Xin; Tian, Changhai; Liu, Miao; Wang, Yongxiang; Tolmachev, Aleksey V.; Sharma, Seema; Yu, Fang; Fu, Kai; Zheng, Jialin; Ding, Shi-Jian

    2012-04-06

    Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using this platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

  8. DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Elena K Braithwaite

    2010-08-01

    Full Text Available Base excision repair (BER is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol beta is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol lambda, was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases lambda and beta in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol beta and pol lambda. Neutral red viability assays demonstrated that pol lambda and pol beta double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol lambda to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol lambda and pol beta interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.

  9. Mutagenicity of ultraviolet A radiation in the lacI transgene in Big Blue mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Kim, Sang-in; Pfeifer, Gerd P.; Besaratinia, Ahmad

    2007-01-01

    Sunlight ultraviolet A (UVA) irradiation has been implicated in the etiology of human skin cancer. A genotoxic mode of action for UVA radiation has been suggested that involves photosensitization reactions giving rise to promutagenic DNA lesions. We investigated the mutagenicity of UVA in the lacI transgene in Big Blue mouse embryonic fibroblasts. UVA irradiation of these cells at a physiologically relevant dose of 18 J/cm 2 caused a 2.8-fold increase in the lacI mutant frequency relative to control, i.e., 12.12 ± 1.84 versus 4.39 ± 1.99 x 10 -5 (mean ± S.D.). DNA sequencing analysis showed that of 100 UVA-induced mutant plaques and 54 spontaneously arisen control plaques, 97 and 51, respectively, contained a minimum of one mutation along the lacI transgene. The vast majority of both induced- and spontaneous mutations were single base substitutions, although less frequently, there were also single and multiple base deletions and insertions, and tandem base substitutions. Detailed mutation spectrometry analysis revealed that G:C → T:A transversions, the signature mutations of oxidative DNA damage, were significantly induced by UVA irradiation (P -5 ; P < 0.00001). These findings are in complete agreement with those previously observed in the cII transgene of the same model system, and reaffirm the notion that intracellular photosensitization reactions causing promutagenic oxidative DNA damage are involved in UVA genotoxicity

  10. Mobile phone signal exposure triggers a hormesis-like effect in Atm+/+ and Atm-/- mouse embryonic fibroblasts.

    Science.gov (United States)

    Sun, Chuan; Wei, Xiaoxia; Fei, Yue; Su, Liling; Zhao, Xinyuan; Chen, Guangdi; Xu, Zhengping

    2016-11-18

    Radiofrequency electromagnetic fields (RF-EMFs) have been classified by the International Agency for Research on Cancer as possible carcinogens to humans; however, this conclusion is based on limited epidemiological findings and lacks solid support from experimental studies. In particular, there are no consistent data regarding the genotoxicity of RF-EMFs. Ataxia telangiectasia mutated (ATM) is recognised as a chief guardian of genomic stability. To address the debate on whether RF-EMFs are genotoxic, we compared the effects of 1,800 MHz RF-EMF exposure on genomic DNA in mouse embryonic fibroblasts (MEFs) with proficient (Atm +/+ ) or deficient (Atm -/- ) ATM. In Atm +/+ MEFs, RF-EMF exposure for 1 h at an average special absorption rate of 4.0 W/kg induced significant DNA single-strand breaks (SSBs) and activated the SSB repair mechanism. This effect reduced the DNA damage to less than that of the background level after 36 hours of exposure. In the Atm -/- MEFs, the same RF-EMF exposure for 12 h induced both SSBs and double-strand breaks and activated the two repair processes, which also reduced the DNA damage to less than the control level after prolonged exposure. The observed phenomenon is similar to the hormesis of a toxic substance at a low dose. To the best of our knowledge, this study is the first to report a hormesis-like effect of an RF-EMF.

  11. UV light induces premature senescence in Akt1-null mouse embryonic fibroblasts by increasing intracellular levels of ROS

    International Nuclear Information System (INIS)

    Jee, Hye Jin; Kim, Hyun-Ju; Kim, Ae Jeong; Bae, Yoe-Sik; Bae, Sun Sik; Yun, Jeanho

    2009-01-01

    Akt/PKB plays a pivotal role in cell survival and proliferation. Previously, we reported that UV-irradiation induces extensive cell death in Akt2 -/- mouse embryonic fibroblasts (MEFs) while Akt1 -/- MEFs show cell cycle arrest. Here, we find that Akt1 -/- MEFs exhibit phenotypic changes characteristics of senescence upon UV-irradiation. An enlarged and flattened morphology, a reduced cell proliferation and an increased senescence-associated β-galactosidase (SA β-gal) staining indicate that Akt1 -/- MEFs undergo premature senescence after UV-irradiation. Restoring Akt1 expression in Akt1 -/- MEFs suppressed SA β-gal activity, indicating that UV-induced senescence is due to the absence of Akt1 function. Notably, levels of ROS were rapidly increased upon UV-irradiation and the ROS scavenger NAC inhibits UV-induced senescence of Akt1 -/- MEFs, suggesting that UV light induces premature senescence in Akt1 -/- MEFs by modulating intracellular levels of ROS. In conjunction with our previous work, this indicates that different isoforms of Akt have distinct function in response to UV-irradiation.

  12. Phospholipase C-related catalytically inactive protein participates in the autophagic elimination of Staphylococcus aureus infecting mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Kae Harada-Hada

    Full Text Available Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule-associated protein 1 light chain 3 (LC3, a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP that was originally identified as an inositol trisphosphate-binding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of Staphylococcus aureus in infected mouse embryonic fibroblasts (MEFs. We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of S. aureus cells in PRIP-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that S. aureus proliferates in LC3-positive autophagosome-like vacuoles in PRIP-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in PRIP-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in PRIP-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of S. aureus-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells.

  13. Low-level laser therapy activates NF-kB via generation of reactive oxygen species in mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Aaron C-H Chen

    Full Text Available Despite over forty years of investigation on low-level light therapy (LLLT, the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear.In this study, we isolated murine embryonic fibroblasts (MEF from transgenic NF-kB luciferase reporter mice and studied their response to 810 nm laser radiation. Significant activation of NF-kB was observed at fluences higher than 0.003 J/cm(2 and was confirmed by Western blot analysis. NF-kB was activated earlier (1 hour by LLLT compared to conventional lipopolysaccharide treatment. We also observed that LLLT induced intracellular reactive oxygen species (ROS production similar to mitochondrial inhibitors, such as antimycin A, rotenone and paraquat. Furthermore, we observed similar NF-kB activation with these mitochondrial inhibitors. These results, together with inhibition of laser induced NF-kB activation by antioxidants, suggests that ROS play an important role in the laser induced NF-kB signaling pathways. However, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that LLLT also upregulates mitochondrial respiration.We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive NFkB signaling via generation of ROS. Expression of anti-apoptosis and pro-survival genes responsive to NFkB could explain many clinical effects of LLLT.

  14. Primary skin fibroblasts as a model of Parkinson's disease

    NARCIS (Netherlands)

    Auburger, G.; Klinkenberg, M.; Droste, J.A.H.; Marcus, K.; Morales-Gordo, B.; Kunz, W.S.; Brandt, U.; Broccoli, V.; Reichmann, H.; Gispert, S.; Jendrach, M.

    2012-01-01

    Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts

  15. Chromosomal instability in mouse embryonic fibroblasts null for the transcriptional co-repressor Ski.

    Science.gov (United States)

    Marcelain, Katherine; Armisen, Ricardo; Aguirre, Adam; Ueki, Nobuhide; Toro, Jessica; Colmenares, Clemencia; Hayman, Michael J

    2012-01-01

    Ski is a transcriptional regulator that has been considered an oncoprotein given its ability to induce oncogenic transformation in avian model systems. However, studies in mouse and in some human tumor cells have also indicated a tumor suppressor activity for this protein. We found that Ski-/- mouse embryo fibroblasts exhibit high levels of genome instability, namely aneuploidy, consistent with a tumor suppressor function for Ski. Time-lapse microscopy revealed lagging chromosomes and chromatin/chromosome bridges as the major cause of micronuclei (MN) formation and the subsequent aneuploidy. Although these cells arrested in mitosis after treatment with spindle disrupting drugs and exhibited a delayed metaphase/anaphase transition, spindle assembly checkpoint (SAC) was not sufficient to prevent chromosome missegregation, consistent with a weakened SAC. Our in vivo analysis also showed dynamic metaphase plate rearrangements with switches in polarity in cells arrested in metaphase. Importantly, after ectopic expression of Ski the cells that displayed this metaphase arrest died directly during metaphase or after aberrant cell division, relating SAC activation and mitotic cell death. This increased susceptibility to undergo mitosis-associated cell death reduced the number of MN-containing cells. The presented data support a new role for Ski in the mitotic process and in maintenance of genetic stability, providing insights into the mechanism of tumor suppression mediated by this protein. Copyright © 2011 Wiley Periodicals, Inc.

  16. Isolation and culture of primary adult skin fibroblasts from the Asian elephant (Elephas maximus

    Directory of Open Access Journals (Sweden)

    Puntita Siengdee

    2018-01-01

    Full Text Available Background Primary cultures from Asian elephants (Elephas maximus allow scientists to obtain representative cells that have conserved most of their original characteristics, function, physiology and biochemistry. This technique has thus gained significant importance as a foundation for further cellular, cell biology and molecular research. Therefore, the aim of this study was to describe conditions for the successful establishment of primary adult fibroblasts from Asian elephant carcasses. Methods Ear tissue sample collection from Asian elephant carcasses and our recommendations are given. We describe here a simple modified protocol for successful isolation and maintenance of primary adult fibroblasts from elephant ear skin. Ear samples from each individual (five 3 × 3 cm2 pieces were brought to the laboratory within 3 h after collection, kept in transportation medium at 0–4 °C. The ear tissues were prepared by a combination of 10% collagenase type II digestion procedure together with a simple explant procedure. Primary fibroblasts were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM with 20% fetal calf serum (FCS in a humidified atmosphere containing 5% CO2. After the third passage, fibroblasts were routinely trypsinized with 0.25% trypsin/EDTA and cultured in DMEM with 10% FCS at 37 °C and 5% CO2. Traditional cell counting method was used to measure cell viability and growth curve. Long-term storage of cells used freezing medium consisting of 40% FCS (v/v. Results We explored the most suitable conditions during sample collection (post-mortem storage time and sample storage temperature, which is the most important step in determining primary outgrowth. Our study successfully established and cultured primary adult skin fibroblasts obtained from post-mortem E. maximus ear skin tissues from six carcasses, with a success rate of around 83.3%. Outgrowth could be seen 4–12 days after explantation, and epithelial

  17. Cellular Mechanics of Primary Human Cervical Fibroblasts: Influence of Progesterone and a Pro-inflammatory Cytokine.

    Science.gov (United States)

    Shukla, Vasudha; Barnhouse, Victoria; Ackerman, William E; Summerfield, Taryn L; Powell, Heather M; Leight, Jennifer L; Kniss, Douglas A; Ghadiali, Samir N

    2018-01-01

    The leading cause of neonatal mortality, pre-term birth, is often caused by pre-mature ripening/opening of the uterine cervix. Although cervical fibroblasts play an important role in modulating the cervix's extracellular matrix (ECM) and mechanical properties, it is not known how hormones, i.e., progesterone, and pro-inflammatory insults alter fibroblast mechanics, fibroblast-ECM interactions and the resulting changes in tissue mechanics. Here we investigate how progesterone and a pro-inflammatory cytokine, IL-1β, alter the biomechanical properties of human cervical fibroblasts and the fibroblast-ECM interactions that govern tissue-scale mechanics. Primary human fibroblasts were isolated from non-pregnant cervix and treated with estrogen/progesterone, IL-1β or both. The resulting changes in ECM gene expression, matrix remodeling, traction force generation, cell-ECM adhesion and tissue contractility were monitored. Results indicate that IL-1β induces a significant reduction in traction force and ECM adhesion independent of pre-treatment with progesterone. These cell level effects altered tissue-scale mechanics where IL-1β inhibited the contraction of a collagen gel over 6 days. Interestingly, progesterone treatment alone did not modulate traction forces or gel contraction but did result in a dramatic increase in cell-ECM adhesion. Therefore, the protective effect of progesterone may be due to altered adhesion dynamics as opposed to altered ECM remodeling.

  18. Human embryonic stem cells in culture possess primary cilia with hedgehog signaling machinery

    DEFF Research Database (Denmark)

    Kiprilov, Enko N; Awan, Aashir; Desprat, Romain

    2008-01-01

    Human embryonic stem cells (hESCs) are potential therapeutic tools and models of human development. With a growing interest in primary cilia in signal transduction pathways that are crucial for embryological development and tissue differentiation and interest in mechanisms regulating human hESC d...

  19. Chemosensitivity of primary human fibroblasts with defective unhooking of DNA interstrand cross-links

    International Nuclear Information System (INIS)

    Clingen, Peter H.; Arlett, Colin F.; Hartley, John A.; Parris, Christopher N.

    2007-01-01

    Xeroderma pigmentosum (XP) is characterised by defects in nucleotide excision repair, ultraviolet (UV) radiation sensitivity and increased skin carcinoma. Compared to other complementation groups, XP-F patients show relatively mild cutaneous symptoms. DNA interstrand cross-linking agents are a highly cytotoxic class of DNA damage induced by common cancer chemotherapeutics such as cisplatin and nitrogen mustards. Although the XPF-ERCC1 structure-specific endonuclease is required for the repair of ICLs cellular sensitivity of primary human XP-F cells has not been established. In clonogenic survival assays, primary fibroblasts from XP-F patients were moderately sensitive to both UVC and HN2 compared to normal cells (2- to 3-fold and 3- to 5-fold, respectively). XP-A fibroblasts were considerably more sensitive to UVC (10- to 12-fold) but not sensitive to HN2. The sensitivity of XP-F fibroblasts to HN2 correlated with the defective incision or 'unhooking' step of ICL repair. Using the comet assay, XP-F cells exhibited only 20% residual unhooking activity over 24 h. Over the same time, normal and XP-A cells unhooked greater than 95% and 62% of ICLs, respectively. After HN2 treatment, ICL-associated DNA double-strand breaks (DSBs) are detected by pulse field gel electrophoresis in dividing cells. Induction and repair of DNA DSBs was normal in XP-F fibroblasts. These findings demonstrate that in primary human fibroblasts, XPF is required for the unhooking of ICLs and not for the induction or repair of ICL-associated DNA DSBs induced by HN2. In terms of cancer chemotherapy, people with mild DNA repair defects affecting ICL repair may be more prevalent in the general population than expected. Since cellular sensitivity of primary human fibroblasts usually reflects clinical sensitivity such patients with cancer would be at risk of increased toxicity

  20. Evaluating Electroporation and Lipofectamine Approaches for Transient and Stable Transgene Expressions in Human Fibroblasts and Embryonic Stem Cells

    Science.gov (United States)

    Sharifi Tabar, Mehdi; Hesaraki, Mahdi; Esfandiari, Fereshteh; Sahraneshin Samani, Fazel; Vakilian, Haghighat; Baharvand, Hossein

    2015-01-01

    Objective Genetic modification of human embryonic stem cells (hESCs) is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI) into the target cell line, however, there are few re- ports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor. Materials and Methods In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for tran- sient expression of green fluorescent protein in two genetically different hESC lines, Roy- an H5 (XX) and Royan H6 (XY), as well as human foreskin fibroblasts (hFF). For long-term EGFP expression VASA and OLIG2 promoters (germ cell and motoneuron specific genes, respectively), were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after trans- fection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells (PGCs) was conducted to confirm stable integration of the transgene. Results Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation ef- ficiency revealed that the vector concentrations from 20-60 μg and the density of the sub- jected cells (5×105and 1×106cells) were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies. Conclusion

  1. Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles

    Directory of Open Access Journals (Sweden)

    Líbalová Helena

    2012-01-01

    Full Text Available Abstract Background Recently, we used cell-free assays to demonstrate the toxic effects of complex mixtures of organic extracts from urban air particles (PM2.5 collected in four localities of the Czech Republic (Ostrava-Bartovice, Ostrava-Poruba, Karvina and Trebon which differed in the extent and sources of air pollution. To obtain further insight into the biological mechanisms of action of the extractable organic matter (EOM from ambient air particles, human embryonic lung fibroblasts (HEL12469 were treated with the same four EOMs to assess changes in the genome-wide expression profiles compared to DMSO treated controls. Method For this purpose, HEL cells were incubated with subtoxic EOM concentrations of 10, 30, and 60 μg EOM/ml for 24 hours and global gene expression changes were analyzed using human whole genome microarrays (Illumina. The expression of selected genes was verified by quantitative real-time PCR. Results Dose-dependent increases in the number of significantly deregulated transcripts as well as dose-response relationships in the levels of individual transcripts were observed. The transcriptomic data did not differ substantially between the localities, suggesting that the air pollution originating mainly from various sources may have similar biological effects. This was further confirmed by the analysis of deregulated pathways and by identification of the most contributing gene modulations. The number of significantly deregulated KEGG pathways, as identified by Goeman's global test, varied, depending on the locality, between 12 to 29. The Metabolism of xenobiotics by cytochrome P450 exhibited the strongest upregulation in all 4 localities and CYP1B1 had a major contribution to the upregulation of this pathway. Other important deregulated pathways in all 4 localities were ABC transporters (involved in the translocation of exogenous and endogenous metabolites across membranes and DNA repair, the Wnt and TGF-β signaling pathways

  2. Elevated nuclear sphingoid base-1-phosphates and decreased histone deacetylase activity after fumonisin B1 treatment in mouse embryonic fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, Nicole M., E-mail: nicolegardner@creighton.edu [Department of Pharmacology, Creighton University School of Medicine, Omaha, NE 68178 (United States); Riley, Ronald T.; Showker, Jency L.; Voss, Kenneth A. [USDA-ARS, Toxicology and Mycotoxin Research Unit, Athens, GA 30605 (United States); Sachs, Andrew J.; Maddox, Joyce R.; Gelineau-van Waes, Janee B. [Department of Pharmacology, Creighton University School of Medicine, Omaha, NE 68178 (United States)

    2016-05-01

    Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. FB1 inhibits ceramide synthase enzymes in sphingolipid biosynthesis, causing sphinganine (Sa) and bioactive sphinganine-1-phosphate (Sa1P) accumulation in blood, cells, and tissues. Sphingosine kinases (Sphk) phosphorylate Sa to form Sa1P. Upon activation, Sphk1 associates primarily with the plasma membrane, while Sphk2 is found predominantly in the nucleus. In cells over-expressing Sphk2, accumulation of Sa1P in the nuclear compartment inhibits histone deacetylase (HDAC) activity, causing increased acetylation of histone lysine residues. In this study, FB1 treatment in LM/Bc mouse embryonic fibroblasts (MEFs) resulted in significant accumulation of Sa1P in nuclear extracts relative to cytoplasmic extracts. Elevated nuclear Sa1P corresponded to decreased histone deacetylase (HDAC) activity and increased histone acetylation at H2BK12, H3K9, H3K18, and H3K23. Treatment of LM/Bc MEFs with a selective Sphk1 inhibitor, PF-543, or with ABC294640, a selective Sphk2 inhibitor, significantly reduced nuclear Sa1P accumulation after FB1, although Sa1P levels remained significantly increased relative to basal levels. Concurrent treatment with both PF-543 and ABC294640 prevented nuclear accumulation of Sa1P in response to FB1. Other HDAC inhibitors are known to cause NTDs, so these results suggest that FB1-induced disruption of sphingolipid metabolism leading to nuclear Sa1P accumulation, HDAC inhibition, and histone hyperacetylation is a potential mechanism for FB1-induced NTDs. - Highlights: • FB1 treatment results in accumulation of Sa1P primarily in the nucleus of MEFs. • FB1 treatment and elevated nuclear Sa1P are associated with HDAC inhibition. • Sphk2 inhibition alone

  3. Elevated nuclear sphingoid base-1-phosphates and decreased histone deacetylase activity after fumonisin B1 treatment in mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Gardner, Nicole M.; Riley, Ronald T.; Showker, Jency L.; Voss, Kenneth A.; Sachs, Andrew J.; Maddox, Joyce R.; Gelineau-van Waes, Janee B.

    2016-01-01

    Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. FB1 inhibits ceramide synthase enzymes in sphingolipid biosynthesis, causing sphinganine (Sa) and bioactive sphinganine-1-phosphate (Sa1P) accumulation in blood, cells, and tissues. Sphingosine kinases (Sphk) phosphorylate Sa to form Sa1P. Upon activation, Sphk1 associates primarily with the plasma membrane, while Sphk2 is found predominantly in the nucleus. In cells over-expressing Sphk2, accumulation of Sa1P in the nuclear compartment inhibits histone deacetylase (HDAC) activity, causing increased acetylation of histone lysine residues. In this study, FB1 treatment in LM/Bc mouse embryonic fibroblasts (MEFs) resulted in significant accumulation of Sa1P in nuclear extracts relative to cytoplasmic extracts. Elevated nuclear Sa1P corresponded to decreased histone deacetylase (HDAC) activity and increased histone acetylation at H2BK12, H3K9, H3K18, and H3K23. Treatment of LM/Bc MEFs with a selective Sphk1 inhibitor, PF-543, or with ABC294640, a selective Sphk2 inhibitor, significantly reduced nuclear Sa1P accumulation after FB1, although Sa1P levels remained significantly increased relative to basal levels. Concurrent treatment with both PF-543 and ABC294640 prevented nuclear accumulation of Sa1P in response to FB1. Other HDAC inhibitors are known to cause NTDs, so these results suggest that FB1-induced disruption of sphingolipid metabolism leading to nuclear Sa1P accumulation, HDAC inhibition, and histone hyperacetylation is a potential mechanism for FB1-induced NTDs. - Highlights: • FB1 treatment results in accumulation of Sa1P primarily in the nucleus of MEFs. • FB1 treatment and elevated nuclear Sa1P are associated with HDAC inhibition. • Sphk2 inhibition alone

  4. Low-Dose Radiation Induces Cell Proliferation in Human Embryonic Lung Fibroblasts but not in Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Xinyue Liang

    2016-01-01

    Full Text Available Hormesis and adaptive responses are 2 important biological effects of low-dose ionizing radiation (LDR. In normal tissue, LDR induces hormesis as evinced by increased cell proliferation; however, whether LDR also increases tumor cell proliferation needs to be investigated. In this study, cell proliferation was assayed by total cell numbers and the Cell Counting Kit 8 assay. Mitogen-activated protein kinases (MAPK/extracellular signal-regulated kinase (ERK and phosphatidylinositol 3′ -kinase(PI3K-Akt (PI3K/AKT phosphorylation were determined by Western blot analysis. Human embryonic lung fibroblast 2BS and lung cancer NCI-H446 cell lines were irradiated with LDR at different doses (20-100 mGy. In response to 20 to 75 mGy X-rays, cell proliferation was significantly increased in 2BS but not in NCI-H446 cells. In 2BS cells, LDR at 20 to 75 mGy also stimulated phosphorylation of MAPK/ERK pathway proteins including ERK, MEK, and Raf and of the PI3K/AKT pathway protein AKT. To test whether ERK1/2 and AKT pathway activation was involved in the stimulation of cell proliferation in 2BS cells, the MAPK/ERK and PI3K/AKT pathways were inhibited using their specific inhibitors, U0126 and LY294002. U0126 decreased the phosphorylation of ERK1/2, and LY294002 decreased the phosphorylation of AKT; each could significantly inhibit LDR-induced 2BS cell proliferation. However, LDR did not stimulate these kinases, and kinase inhibitors also did not affect cell proliferation in the NCI-H446 cells. These results suggest that LDR stimulates cell proliferation via the activation of both MAPK/ERK and PI3K/AKT signaling pathways in 2BS but not in NCI-H446 cells. This finding implies the potential for applying LDR to protect normal tissues from radiotherapy without diminishing the efficacy of tumor therapy.

  5. Highly Efficient Stable Expression of Indoleamine 2,3 Dioxygenase Gene in Primary Fibroblasts

    Directory of Open Access Journals (Sweden)

    Rezakhanlou Alireza

    2010-03-01

    Full Text Available Abstract Indoleamine 2,3 dioxygenase (IDO is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.

  6. The initiation of embryonic-like collagen fibrillogenesis by adult human tendon fibroblasts when cultured under tension

    DEFF Research Database (Denmark)

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E

    2010-01-01

    to initiate collagen fibrillogenesis when cultured in fixed-length fibrin gels. Fibroblasts were dissected from semitendinosus and gracilis tendons from healthy humans and cultured in 3D linear fibrin gels. The fibroblasts synthesized an extracellular matrix of parallel collagen fibrils that were aligned...

  7. Fabrication and evaluation of electrohydrodynamic jet 3D printed polycaprolactone/chitosan cell carriers using human embryonic stem cell-derived fibroblasts.

    Science.gov (United States)

    Wu, Yang; Sriram, Gopu; Fawzy, Amr S; Fuh, Jerry Yh; Rosa, Vinicius; Cao, Tong; Wong, Yoke San

    2016-08-01

    Biological function of adherent cells depends on the cell-cell and cell-matrix interactions in three-dimensional space. To understand the behavior of cells in 3D environment and their interactions with neighboring cells and matrix requires 3D culture systems. Here, we present a novel 3D cell carrier scaffold that provides an environment for routine 3D cell growth in vitro We have developed thin, mechanically stable electrohydrodynamic jet (E-jet) 3D printed polycaprolactone and polycaprolactone/Chitosan macroporous scaffolds with precise fiber orientation for basic 3D cell culture application. We have evaluated the application of this technology by growing human embryonic stem cell-derived fibroblasts within these 3D scaffolds. Assessment of cell viability and proliferation of cells seeded on polycaprolactone and polycaprolactone/Chitosan 3D-scaffolds show that the human embryonic stem cell-derived fibroblasts could adhere and proliferate on the scaffolds over time. Further, using confocal microscopy we demonstrate the ability to use fluorescence-labelled cells that could be microscopically monitored in real-time. Hence, these 3D printed polycaprolactone and polycaprolactone/Chitosan scaffolds could be used as a cell carrier for in vitro 3D cell culture-, bioreactor- and tissue engineering-related applications in the future. © The Author(s) 2016.

  8. Differential Gene Expression in Primary Human Skin Keratinocytes and Fibroblasts in Response to Ionizing Radiation

    Science.gov (United States)

    Warters, Raymond L.; Packard, Ann T.; Kramer, Gwen F.; Gaffney, David K.; Moos, Philip J.

    2009-01-01

    Although skin is usually exposed during human exposures to ionizing radiation, there have been no thorough examinations of the transcriptional response of skin fibroblasts and keratinocytes to radiation. The transcriptional response of quiescent primary fibroblasts and keratinocytes exposed to from 10 cGy to 5 Gy and collected 4 h after treatment was examined. RNA was isolated and examined by microarray analysis for changes in the levels of gene expression. Exposure to ionizing radiation altered the expression of 279 genes across both cell types. Changes in RNA expression could be arranged into three main categories: (1) changes in keratinocytes but not in fibroblasts, (2) changes in fibroblasts but not in keratinocytes, and (3) changes in both. All of these changes were primarily of p53 target genes. Similar radiation-induced changes were induced in immortalized fibroblasts or keratinocytes. In separate experiments, protein was collected and analyzed by Western blotting for expression of proteins observed in microarray experiments to be overexpressed at the mRNA level. Both Q-PCR and Western blot analysis experiments validated these transcription changes. Our results are consistent with changes in the expression of p53 target genes as indicating the magnitude of cell responses to ionizing radiation. PMID:19580510

  9. Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

    Directory of Open Access Journals (Sweden)

    Paola Brun

    Full Text Available Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and

  10. Piper betle induces phase I & II genes through Nrf2/ARE signaling pathway in mouse embryonic fibroblasts derived from wild type and Nrf2 knockout cells.

    Science.gov (United States)

    Wan Hasan, Wan Nuraini; Kwak, Mi-Kyoung; Makpol, Suzana; Wan Ngah, Wan Zurinah; Mohd Yusof, Yasmin Anum

    2014-02-23

    Nuclear factor-erythroid 2 p45 related factor 2 (Nrf2) is a primary transcription factor, protecting cells from oxidative stress by regulating a number of antioxidants and phase II detoxifying enzymes. Dietary components such as sulforaphane in broccoli and quercetin in onions have been shown to be inducers of Nrf2. Piper betle (PB) grows well in tropical climate and the leaves are used in a number of traditional remedies for the treatment of stomach ailments and infections among Asians. The aim of this study was to elucidate the effect of Piper betle (PB) leaves extract in Nrf2 signaling pathway by using 2 types of cells; mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) and Nrf2 knockout (N0) mice. WT and N0 cells were treated with 5 and 10 μg/ml of PB for 10 and 12-h for the determination of nuclear translocation of Nrf2 protein. Luciferase reporter gene activity was performed to evaluate the antioxidant response element (ARE)-induction by PB. Real-time PCR and Western blot were conducted on both WT and N0 cells after PB treatment for the determination of antioxidant enzymes [superoxide dismutase (SOD1) and heme-oxygenase (HO-1)], phase I oxidoreductase enzymes [ quinone oxidoreductase (NQO1)] and phase II detoxifying enzyme [glutathione S-transferase (GST)]. Nuclear translocation of Nrf2 by PB in WT cells was better after 10 h incubation compared to 12 h. Real time PCR and Western blot analysis showed increased expressions of Nrf2, NQO1 and GSTA1 genes with corresponding increases in glutathione, NQO1 and HO-1 proteins in WT cells. Reporter gene ARE was stimulated by PB as shown by ARE/luciferase assay. Interestingly, PB induced SOD1 gene and protein expressions in N0 cells but not in WT cells. The results of this study confirmed that PB activated Nrf2-ARE signaling pathway which subsequently induced some phase I oxidoreductase, phase II detoxifying and antioxidant genes expression via ARE reporter gene involved in the Nrf2 pathway with the

  11. Expression of the small T antigen of Lymphotropic Papovavirus is sufficient to transform primary mouse embryo fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Tushar; Robles, Maria Teresa Sáenz [Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260 (United States); Schowalter, Rachel M.; Buck, Christopher B. [Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-4263 (United States); Pipas, James M., E-mail: pipas@pitt.edu [Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260 (United States)

    2016-01-15

    Polyomaviruses induce cell proliferation and transformation through different oncoproteins encoded within the early region (ER): large T antigen (LT), small T antigen (sT) and, in some cases, additional components. Each virus utilizes different mechanisms to achieve transformation. For instance, the LTs of Simian virus 40 (SV40), BK and/or JC virus can induce transformation; but Merkel Cell Polyomavirus (MCPyV) requires expression of sT. Lymphotropic Papovavirus (LPV) is closely related to Human Polyomavirus 9 (HuPyV9) and, under similar conditions, mice expressing LPV.ER exhibit higher rates of tumor formation than mice expressing SV40.ER. We have investigated the contributions of individual LPV.ER components to cell transformation. In contrast to SV40, LPV.ER transforms mouse embryonic fibroblasts (MEFs), but expression of LPV LT is insufficient to transform MEFs. Furthermore, LPV sT induces immortalization and transformation of MEFs. Thus, in the case of LPV, sT is the main mediator of oncogenesis. - Highlights: • Characterization of early region products from the Lymphotropic Polyomavirus (LPV). • On its own, sT immortalizes and transforms mouse primary cells, and is able to block p53 activation. • Combined LT and sT expression induces a greater rate of proliferation than either LT or sT alone.

  12. Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone.

    Science.gov (United States)

    Yamamoto, Akito; Kumakura, Shin-ichi; Uchida, Minoru; Barrett, J Carl; Tsutsui, Takeki

    2003-09-01

    The ability of the human papillomavirus type 16 (HPV-16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE-7 cells was examined. WHE-7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV-16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X-ray irradiation. The expression of INK4a protein (p16(INK4a)) was undetectable in the E6-infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16(INK4a) protein was overexpressed in all the E7-infected immortal clones and their clones in the pre-crisis period as well as all the E6/E7-infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV-16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16(INK4a)/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for

  13. Alpha-Tocopherol Counteracts the Cytotoxicity Induced by Ochratoxin A in Primary Porcine Fibroblasts

    DEFF Research Database (Denmark)

    Fusi, Elenora; Rebucci, Raffaella; Pecorini, Chiara

    2010-01-01

    The aims of the current study were to determine the half-lethal concentration of ochratoxin A (OTA) as well as the levels of lactate dehydrogenase release and DNA fragmentation induced by OTA in primary porcine fibroblasts, and to examine the role of α-tocopherol in counteracting its toxicity....... Cells showed a dose-, time- and origin-dependent (ear vs. embryo) sensitivity to ochratoxin A. Pre-incubation for 3 h with 1 nM α-tocopherol significantly (P tocopherol...

  14. Primary fibroblasts from CSP? mutation carriers recapitulate hallmarks of the adult onset neuronal ceroid lipofuscinosis

    OpenAIRE

    Benitez, Bruno A.; Sands, Mark S.

    2017-01-01

    Mutations in the co- chaperone protein, CSP?, cause an autosomal dominant, adult-neuronal ceroid lipofuscinosis (AD-ANCL). The current understanding of CSP? function exclusively at the synapse fails to explain the autophagy-lysosome pathway (ALP) dysfunction in cells from AD-ANCL patients. Here, we demonstrate unexpectedly that primary dermal fibroblasts from pre-symptomatic mutation carriers recapitulate in vitro features found in the brains of AD-ANCL patients including auto-fluorescent sto...

  15. Comparison of frailty of primary neurons, embryonic, and aging mouse cortical layers.

    Science.gov (United States)

    Fugistier, Patrick; Vallet, Philippe G; Leuba, Geneviève; Piotton, Françoise; Marin, Pascale; Bouras, Constantin; Savioz, Armand

    2014-02-01

    Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aβ peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aβ(1-42), okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aβ and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aβ and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells

    Czech Academy of Sciences Publication Activity Database

    Dvořák, Petr; Dvořáková, D.; Košková, S.; Vidinská, M.; Najvirtová, M.; Krekáč, D.; Hampl, Aleš

    2005-01-01

    Roč. 23, č. 8 (2005), s. 1200-1211 ISSN 1066-5099 R&D Projects: GA ČR(CZ) GA301/03/1122; GA ČR(CZ) GA305/05/0434; GA MŠk(CZ) LN00A065 Institutional research plan: CEZ:AV0Z50390512 Keywords : growth factor * human embryonic stem cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.094, year: 2005

  17. Cellular metabolic rates from primary dermal fibroblast cells isolated from birds of different body masses.

    Science.gov (United States)

    Jimenez, Ana Gabriela; Williams, Joseph B

    2014-10-01

    The rate of metabolism is the speed at which organisms use energy, an integration of energy transformations within the body; it governs biological processes that influence rates of growth and reproduction. Progress at understanding functional linkages between whole organism metabolic rate and underlying mechanisms that influence its magnitude has been slow despite the central role this issue plays in evolutionary and physiological ecology. Previous studies that have attempted to relate how cellular processes translate into whole-organism physiology have done so over a range of body masses of subjects. However, the data still remains controversial when observing metabolic rates at the cellular level. To bridge the gap between these ideas, we examined cellular metabolic rate of primary dermal fibroblasts isolated from 49 species of birds representing a 32,000-fold range in body masses to test the hypothesis that metabolic rate of cultured cells scales with body size. We used a Seahorse XF-96 Extracellular flux analyzer to measure cellular respiration in fibroblasts. Additionally, we measured fibroblast size and mitochondrial content. We found no significant correlation between cellular metabolic rate, cell size, or mitochondrial content and body mass. Additionally, there was a significant relationship between cellular basal metabolic rate and proton leak in these cells. We conclude that metabolic rate of cells isolated in culture does not scale with body mass, but cellular metabolic rate is correlated to growth rate in birds. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Characteristics of human infant primary fibroblast cultures from Achilles tendons removed post-mortem

    DEFF Research Database (Denmark)

    Rohde, Marianne Cathrine; Corydon, Thomas Juhl; Hansen, Jakob

    2014-01-01

    Primary cell cultures were investigated as a tool for molecular diagnostics in a forensic setting. Fibroblast cultures had been established from human Achilles tendon resected at autopsies, from cases of sudden infant death syndrome and control infants who died in traumatic events (n=41). After...... established from post-mortem tissue are renewable sources of biological material; they can be the foundation for genetic, metabolic and other functional studies and thus constitute a valuable tool for molecular and pathophysiological investigations in biomedical and forensic sciences....

  19. 99Tcm-N(NOEt2 Uptake Kinetics Difference among KMB17 Human Embryonic Lung Diploid Fibroblast and Different Human Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Wei JIA

    2010-04-01

    Full Text Available Background and objective PET/CT imaging is expensive, so searching the tumor imaging agent for SPECT/CT is necessary. 99Tcm-N(NOEt2 [bis (N-ethoxy-N-ethyl dithiocarbamato nitrido99Tcm (V] can be uptaken by lung cancer cells and other cells alike. The aim of this study is to evaluate the distinctive value in lung tumor with 99Tcm-N(NOEt2, the difference in its uptake kinetics in human embryonic lung diploid fibroblasts KMB17 and several kinds of lung cancer cells lines. Methods Firstly, six different cell culture medium which contained YTMLC Gejiu human lung squamous carcinoma cell, SPC-A1 human lung adenocarcinoma cell, AGZY low metastatic human lung adenocarcinoma, 973 high metastatic human lung adenocarcinoma cell, GLC-82 Gejiu human lung adenocarcinoma cell, and KMB17 human embryonic lung diploid fibroblast, respectively with equal cell density of 1×106/mL and the same volume were prepared; secondly, the same radioactive dose of 99Tcm-N(NOEt2 was added into each sample and then 300 μL mixed sample was taken out respectively and cultured in 37 oC culture box; Finally, 5 min, 15 min, 30 min, 45 min, 60 min, 75 min, 90 min after cultivation, centrifuged each cultured sample and determined the intracellular radiocounts of each sample, calculated each cell sample’s uptake rate of 99Tcm-N(NOEt2 at different time. Results Statistical difference was found among six cell samples, and the uptake rate sequence from high to low is 973 and SPC-A1>YTMLC>GLC-82>AGZY>KMB17 respectively; furthermore, 30 min-45 min after culture, the uptake rate reached stability, and the 45 min uptake rate of each sample was higher than its 96.7% uptake peak. Conclusion Based on the results above mentioned, it is supposed that there are discriminative clinical value when using 99Tcm-N(NOEt2 as a tumor targeting imaging agent, and 30 min or so after injection may be the best imaging time in the early imaging stage.

  20. Hydrogen-rich medium protects mouse embryonic fibroblasts from oxidative stress by activating LKB1-AMPK-FoxO1 signal pathway.

    Science.gov (United States)

    Lee, Jihyun; Yang, Goowon; Kim, Young-Joo; Tran, Quynh Hoa; Choe, Wonchae; Kang, Insug; Kim, Sung Soo; Ha, Joohun

    2017-09-23

    Persistent oxidative stress is recognized as a major cause of many pathological conditions as well as ageing. However, most clinical trials of dietary antioxidants have failed to produce successful outcomes in treating oxidative stress-induced diseases. Molecular hydrogen (H 2 ) has recently received considerable attention as a therapeutic agent owing to its novel antioxidant properties, a selective scavenger of hydroxyl and peroxynitrite radicals. Beyond this, numerous reports support that H 2 can modulate the activity of various cellular signal pathways. However, its effect on AMP-activated protein kinase (AMPK) signal pathway, a central regulator of energy hemostasis, has remained almost elusive. Here, we report that hydrogen-rich medium activated LKB1-AMPK signal pathway without ATP depletion, which in turn induced FoxO1-dependent transcription of manganese superoxide dismutase and catalase in mouse embryonic fibroblasts. Moreover, hydrogen-rich media effectively reduced the level of reactive oxygen species in cells treated with hydrogen peroxide and protected these cells from apoptosis in an AMPK-dependent manner. These results suggest that the LKB1-AMPK-FoxO1 signaling pathway is a critical mediator of the antioxidant properties of H 2 , further supporting the idea that H 2 acts as a signaling molecule to serve various physiological functions. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3.

    Science.gov (United States)

    Phan, Chia-Wei; David, Pamela; Naidu, Murali; Wong, Kah-Hui; Sabaratnam, Vikineswary

    2013-10-11

    Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health.

  2. Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells

    Directory of Open Access Journals (Sweden)

    Tsutomu Motohashi

    2016-03-01

    Full Text Available Neural crest cells (NC cells are multipotent cells that emerge from the edge of the neural folds and migrate throughout the developing embryo. Although the gene regulatory network for generation of NC cells has been elucidated in detail, it has not been revealed which of the factors in the network are pivotal to directing NC identity. In this study we analyzed the gene expression profile of a pure NC subpopulation isolated from Sox10-IRES-Venus mice and investigated whether these genes played a key role in the direct conversion of Sox10-IRES-Venus mouse embryonic fibroblasts (MEFs into NC cells. The comparative molecular profiles of NC cells and neural tube cells in 9.5-day embryos revealed genes including transcription factors selectively expressed in developing trunk NC cells. Among 25 NC cell-specific transcription factor genes tested, SOX10 and SOX9 were capable of converting MEFs into SOX10-positive (SOX10+ cells. The SOX10+ cells were then shown to differentiate into neurons, glial cells, smooth muscle cells, adipocytes and osteoblasts. These SOX10+ cells also showed limited self-renewal ability, suggesting that SOX10 and SOX9 directly converted MEFs into NC cells. Conversely, the remaining transcription factors, including well-known NC cell specifiers, were unable to convert MEFs into SOX10+ NC cells. These results suggest that SOX10 and SOX9 are the key factors necessary for the direct conversion of MEFs into NC cells.

  3. SORBS2 and TLR3 induce premature senescence in primary human fibroblasts and keratinocytes

    International Nuclear Information System (INIS)

    Liesenfeld, Melanie; Mosig, Sandy; Funke, Harald; Jansen, Lars; Runnebaum, Ingo B; Dürst, Matthias; Backsch, Claudia

    2013-01-01

    Genetic aberrations are required for the progression of HPV-induced cervical precancers. A prerequisite for clonal expansion of cancer cells is unlimited proliferative capacity. In a cell culture model for cervical carcinogenesis loss of genes located on chromosome 4q35→qter and chromosome 10p14-p15 were found to be associated with escape from senescence. Moreover, by LOH and I-FISH analyses a higher frequency of allele loss of these regions was also observed in cervical carcinomas as compared to CIN3. The aim of this study was to identify candidate senescence-related genes located on chromosome 4q35→qter and chromosome 10p14-p15 which may contribute to clonal expansion at the transition of CIN3 to cancer. Microarray expression analyses were used to identify candidate genes down-regulated in cervical carcinomas as compared to CIN3. In order to relate these genes with the process of senescence their respective cDNAs were overexpressed in HPV16-immortalized keratinocytes as well as in primary human fibroblasts and keratinocytes using lentivirus mediated gene transduction. Overall fifteen genes located on chromosome 4q35→qter and chromosome 10p14-p15 were identified. Ten of these genes could be validated in biopsies by RT-PCR. Of interest is the novel finding that SORBS2 and TLR3 can induce senescence in primary human fibroblasts and keratinocytes but not in HPV-immortalized cell lines. Intriguingly, the endogenous expression of both genes increases during finite passaging of primary keratinocytes in vitro. The relevance of the genes SORBS2 and TLR3 in the process of cellular senescence warrants further investigation. In ongoing experiments we are investigating whether this increase in gene expression is also characteristic of replicative senescence

  4. The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells.

    Science.gov (United States)

    Fujita, Naonobu; Tamura, Ayako; Higashidani, Aya; Tonozuka, Takashi; Freeze, Hudson H; Nishikawa, Atsushi

    2008-02-01

    Mannose for mammalian glycan biosynthesis can be imported directly from the medium, derived from glucose or salvaged from endogenous or external glycans. All pathways must generate mannose 6-phosphate, the activated form of mannose. Imported or salvaged mannose is directly phosphorylated by hexokinase, whereas fructose 6-phosphate from glucose is converted to mannose 6-phosphate by phosphomannose isomerase (PMI). Normally, PMI provides the majority of mannose for glycan synthesis. To assess the contribution of PMI-independent pathways, we used PMI-null fibroblasts to study N-glycosylation of DNase I, a highly sensitive indicator protein. In PMI-null cells, imported mannose and salvaged mannose make a significant contribution to N-glycosylation. When these cells were grown in mannose-free medium along with the mannosidase inhibitor, swainsonine, to block the salvage pathways, N-glycosylation of DNase I was almost completely eliminated. Adding approximately 13 microm mannose to the medium completely restored normal glycosylation. Treatment with bafilomycin A(1), an inhibitor of lysosomal acidification, also markedly reduced N-glycosylation of DNase I, but in this case only 8 microm mannose was required to restore full glycosylation, indicating that a nonlysosomal source of mannose made a significant contribution. Glycosylation levels were greatly also reduced in glycoconjugate-free medium, when endosomal membrane trafficking was blocked by expression of a mutant SKD1. From these data, we conclude that PMI-null cells can salvage mannose from both endogenous and external glycoconjugates via lysosomal and nonlysosomal degradation pathways.

  5. Primary cell culture and morphological characterization of canine dermal papilla cells and dermal fibroblasts.

    Science.gov (United States)

    Bratka-Robia, Christine B; Mitteregger, Gerda; Aichinger, Amanda; Egerbacher, Monika; Helmreich, Magdalena; Bamberg, Elmar

    2002-02-01

    Skin biopsies were taken from female dogs, the primary hair follicles isolated and the dermal papilla dissected. After incubation in supplemented Amniomax complete C100 medium in 24-well culture plates, the dermal papilla cells (DPC) grew to confluence within 3 weeks. Thereafter, they were subcultivated every 7 days. Dermal fibroblast (DFB) cultures were established by explant culture of interfollicular dermis in serum-free medium, where they reached confluence in 10 days. They were subcultivated every 5 days. For immunohistochemistry, cells were grown on cover slips for 24 h, fixed and stained with antibodies against collagen IV and laminin. DPC showed an aggregative growth pattern and formation of pseudopapillae. Intensive staining for collagen IV and laminin could be observed until the sixth passage. DFB grew as branching, parallel lines and showed only weak staining for collagen IV and laminin.

  6. Induced pluripotent stem cells show metabolomic differences to embryonic stem cells in polyunsaturated phosphatidylcholines and primary metabolism.

    Directory of Open Access Journals (Sweden)

    John K Meissen

    Full Text Available Induced pluripotent stem cells are different from embryonic stem cells as shown by epigenetic and genomics analyses. Depending on cell types and culture conditions, such genetic alterations can lead to different metabolic phenotypes which may impact replication rates, membrane properties and cell differentiation. We here applied a comprehensive metabolomics strategy incorporating nanoelectrospray ion trap mass spectrometry (MS, gas chromatography-time of flight MS, and hydrophilic interaction- and reversed phase-liquid chromatography-quadrupole time-of-flight MS to examine the metabolome of induced pluripotent stem cells (iPSCs compared to parental fibroblasts as well as to reference embryonic stem cells (ESCs. With over 250 identified metabolites and a range of structurally unknown compounds, quantitative and statistical metabolome data were mapped onto a metabolite networks describing the metabolic state of iPSCs relative to other cell types. Overall iPSCs exhibited a striking shift metabolically away from parental fibroblasts and toward ESCs, suggestive of near complete metabolic reprogramming. Differences between pluripotent cell types were not observed in carbohydrate or hydroxyl acid metabolism, pentose phosphate pathway metabolites, or free fatty acids. However, significant differences between iPSCs and ESCs were evident in phosphatidylcholine and phosphatidylethanolamine lipid structures, essential and non-essential amino acids, and metabolites involved in polyamine biosynthesis. Together our findings demonstrate that during cellular reprogramming, the metabolome of fibroblasts is also reprogrammed to take on an ESC-like profile, but there are select unique differences apparent in iPSCs. The identified metabolomics signatures of iPSCs and ESCs may have important implications for functional regulation of maintenance and induction of pluripotency.

  7. Tumor suppressors TSC1 and TSC2 differentially modulate actin cytoskeleton and motility of mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Elena A Goncharova

    Full Text Available TSC1 and TSC2 mutations cause neoplasms in rare disease pulmonary LAM and neuronal pathfinding in hamartoma syndrome TSC. The specific roles of TSC1 and TSC2 in actin remodeling and the modulation of cell motility, however, are not well understood. Previously, we demonstrated that TSC1 and TSC2 regulate the activity of small GTPases RhoA and Rac1, stress fiber formation and cell adhesion in a reciprocal manner. Here, we show that Tsc1(-/- MEFs have decreased migration compared to littermate-derived Tsc1(+/+ MEFs. Migration of Tsc1(-/- MEFs with re-expressed TSC1 was comparable to Tsc1(+/+ MEF migration. In contrast, Tsc2(-/- MEFs showed an increased migration compared to Tsc2(+/+ MEFs that were abrogated by TSC2 re-expression. Depletion of TSC1 and TSC2 using specific siRNAs in wild type MEFs and NIH 3T3 fibroblasts also showed that TSC1 loss attenuates cell migration while TSC2 loss promotes cell migration. Morphological and immunochemical analysis demonstrated that Tsc1(-/- MEFs have a thin protracted shape with a few stress fibers; in contrast, Tsc2(-/- MEFs showed a rounded morphology and abundant stress fibers. Expression of TSC1 in either Tsc1(-/- or Tsc2(-/- MEFs promoted stress fiber formation, while TSC2 re-expression induced stress fiber disassembly and the formation of cortical actin. To assess the mechanism(s by which TSC2 loss promotes actin re-arrangement and cell migration, we explored the role of known downstream effectors of TSC2, mTORC1 and mTORC2. Increased migration of Tsc2(-/- MEFs is inhibited by siRNA mTOR and siRNA Rictor, but not siRNA Raptor. siRNA mTOR or siRNA Rictor promoted stress fiber disassembly in TSC2-null cells, while siRNA Raptor had little effect. Overexpression of kinase-dead mTOR induced actin stress fiber disassembly and suppressed TSC2-deficient cell migration. Our data demonstrate that TSC1 and TSC2 differentially regulate actin stress fiber formation and cell migration, and that only TSC2 loss promotes

  8. ­Glial and stem cell expression of murine Fibroblast Growth Factor Receptor 1 in the embryonic and perinatal nervous system

    Directory of Open Access Journals (Sweden)

    Jantzen C. Collette

    2017-06-01

    Full Text Available Background Fibroblast growth factors (FGFs and their receptors (FGFRs are involved in the development and function of multiple organs and organ systems, including the central nervous system (CNS. FGF signaling via FGFR1, one of the three FGFRs expressed in the CNS, stimulates proliferation of stem cells during prenatal and postnatal neurogenesis and participates in regulating cell-type ratios in many developing regions of the brain. Anomalies in FGFR1 signaling have been implicated in certain neuropsychiatric disorders. Fgfr1 expression has been shown, via in situ hybridization, to vary spatially and temporally throughout embryonic and postnatal development of the brain. However, in situ hybridization lacks sufficient resolution to identify which cell-types directly participate in FGF signaling. Furthermore, because antibodies raised against FGFR1 commonly cross-react with other members of the FGFR family, immunocytochemistry is not alone sufficient to accurately document Fgfr1 expression. Here, we elucidate the identity of Fgfr1 expressing cells in both the embryonic and perinatal mouse brain. Methods To do this, we utilized a tgFGFR1-EGFPGP338Gsat BAC line (tgFgfr1-EGFP+ obtained from the GENSAT project. The tgFgfr1-EGFP+ line expresses EGFP under the control of a Fgfr1 promoter, thereby causing cells endogenously expressing Fgfr1 to also present a positive GFP signal. Through simple immunostaining using GFP antibodies and cell-type specific antibodies, we were able to accurately determine the cell-type of Fgfr1 expressing cells. Results This technique revealed Fgfr1 expression in proliferative zones containing BLBP+ radial glial stem cells, such as the cortical and hippocampal ventricular zones, and cerebellar anlage of E14.5 mice, in addition to DCX+ neuroblasts. Furthermore, our data reveal Fgfr1 expression in proliferative zones containing BLBP+ cells of the anterior midline, hippocampus, cortex, hypothalamus, and cerebellum of P0.5 mice

  9. Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

    OpenAIRE

    Jin, Muzi; Wu, Asga; Dorzhin, Sergei; Yue, Qunhua; Ma, Yuzhen; Liu, Dongjun

    2012-01-01

    Although isolation and characterization of embryonic stem cells have been successful in cattle, maintenance of bovine embryonic stem cells in culture remains difficult. In this study, we compared different methods of cell passaging, feeder cell layers and medium conditions for bovine embryonic stem cell-like cells. We found that a murine embryonic fibroblast feeder layer is more suitable for embryonic stem cell-like cells than bovine embryonic fibroblasts. When murine embryonic fibroblasts we...

  10. Embryonic Testicular Regression Syndrome Presenting as Primary Amenorrhea: A Case Report and Review of Disorders of Sexual Development.

    Science.gov (United States)

    Hunter, J D; Pierce, S R; Calikoglu, A S; Howell, J O

    2016-08-01

    Sex development depends on the synchronous interaction of complicated genetic and hormonal events. Sex differentiation begins with sex determination, which is the assignment of the embryonic bipotential gonads as either testes or ovaries on the basis of transcriptional regulation. Hormonal regulation then directs the development of the male or female phenotype. Disruptions of this intricate cascade of events result in disorders of sexual development. A 16-year-old female adolescent presented with primary amenorrhea. Evaluation revealed female external genitalia, XY karyotype, absent gonadal tissue, and rudimentary Müllerian structures. On the basis of her constellation of findings, the most logical diagnosis was the rare embryonic testicular regression syndrome. A careful understanding of embryonic sexual development is critical to the evaluation of patients with disorders of sexual development. Copyright © 2016 North American Society for Pediatric and Adolescent Gynecology. Published by Elsevier Inc. All rights reserved.

  11. Photodynamic therapy induces antifibrotic alterations in primary human vocal fold fibroblasts.

    Science.gov (United States)

    Zhang, Chi; Wang, Jiajia; Chou, Adriana; Gong, Ting; Devine, Erin E; Jiang, Jack J

    2018-04-18

    Photodynamic therapy (PDT) is a promising treatment modality for laryngeal dysplasia, early-stage carcinoma, and papilloma, and was reported to have the ability to preserve laryngeal function and voice quality without clinical fibrotic response. We aimed to investigate the mechanism behind the antifibrotic effects of PDT on primary human vocal fold fibroblasts (VFFs) in vitro. In vitro analysis from one human donor. Cell viability of VFFs in response to varying doses of PDT was investigated by the Cell Counting Kit-8 method. Sublethal-dose PDT (SL-PDT) was used for the following experiments. Expression of genes related to vocal fold extracellular matrix formation was analyzed by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting. Effects of PDT on cell migration, collagen contraction, and transforming growth factor β-1 (TGF-β1)-induced myofibroblast differentiation were also analyzed. PDT affects the viability of VFFs in a dose-dependent manner. SL-PDT significantly changed the expression profile of VFFs with antifibrotic effects. It also inhibited cell migration, reduced collagen contraction, and reversed the fibroblast-myofibroblast differentiation induced by TGF-β1. SL-PDT induces antifibrotic alterations in VFFs. This could explain the low incidence of vocal fold scar associated with PDT. Moreover, PDT may be useful in treating existing vocal fold scars. Further studies should focus on the in vivo effect of PDT on vocal fold wound healing and scar remodeling. NA Laryngoscope, 2018. © 2018 The American Laryngological, Rhinological and Otological Society, Inc.

  12. Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3

    Science.gov (United States)

    2013-01-01

    Background Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity. Methods The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively. Results Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p < 0.05) promoted the neurite outgrowth in N2a cells by 38.4 ± 4.2%, 38.1 ± 2.6%, 33.4 ± 4.6%, 33.7 ± 1.5%, and 35.8 ± 3.4%; respectively. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts. Conclusion Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health. PMID:24119256

  13. The primary role of zebrafish nanog is in extra-embryonic tissue.

    Science.gov (United States)

    Gagnon, James A; Obbad, Kamal; Schier, Alexander F

    2018-01-09

    The role of the zebrafish transcription factor Nanog has been controversial. It has been suggested that Nanog is primarily required for the proper formation of the extra-embryonic yolk syncytial layer (YSL) and only indirectly regulates gene expression in embryonic cells. In an alternative scenario, Nanog has been proposed to directly regulate transcription in embryonic cells during zygotic genome activation. To clarify the roles of Nanog, we performed a detailed analysis of zebrafish nanog mutants. Whereas zygotic nanog mutants survive to adulthood, maternal-zygotic (MZ nanog ) and maternal mutants exhibit developmental arrest at the blastula stage. In the absence of Nanog, YSL formation and epiboly are abnormal, embryonic tissue detaches from the yolk, and the expression of dozens of YSL and embryonic genes is reduced. Epiboly defects can be rescued by generating chimeric embryos of MZ nanog embryonic tissue with wild-type vegetal tissue that includes the YSL and yolk cell. Notably, cells lacking Nanog readily respond to Nodal signals and when transplanted into wild-type hosts proliferate and contribute to embryonic tissues and adult organs from all germ layers. These results indicate that zebrafish Nanog is necessary for proper YSL development but is not directly required for embryonic cell differentiation. © 2018. Published by The Company of Biologists Ltd.

  14. Inhibition of aromatase activity by methyl sulfonyl PCB metabolites in primary culture of human mammary fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Berg, M. van den; Heneweer, M.; Geest, M. de; Sanderson, T. [Inst. for Risk Assessment Sciences and Utrecht Univ. (Netherlands); Jong, P. de [St. Antonius Hospital, Nieuwegein (Netherlands); Bergman, A. [Stockholm Univ., Stockholm (Sweden)

    2004-09-15

    Methyl sulfonyl PCB metabolites (MeSO2-PCBs) are persistent contaminants and are ubiquitously present in humans and the environment. Lipophilicity of MeSO2- PCB metabolites is similar to the parent compounds and they have been detected in human milk, adipose, liver and lung tissue. 4- MeSO2-PCB-149 is the most abundant PCB metabolite in human adipose tissue and milk at a level of 1.5 ng/g lipids. Human blood concentration of 4-MeSO2-PCB-149 is approximately 0.03 nM. 3- MeSO2-PCB-101 is the predominant PCB metabolite in muscle and blubber in wildlife, such as otter, mink and grey seal. In the environment, they have been linked to chronic and reproductive toxicity in exposed mink. Additionaly, some MeSO{sub 2}-PCBs have been shown to be glucocorticoid receptor (GR) antagonists. Since approximately 60% of all breast tumors are estrogen responsive, exposure to compounds that are able to alter estrogen synthesis through interference with the aromatase enzyme, can lead to changes in estrogen levels and possibly to accelerated or inhibit breast tumor growth. Therefore, it is important to identify exogenous compounds that can alter aromatase activity in addition to those compounds which have direct interaction with the estrogen receptor (ER). Aromatase (CYP19) comprises the ubiquitous flavoprotein, NADPH-cytochrome P450 reductase, and a unique cytochrome P450 that is exclusively expressed in estrogen producing cells. Previous studies have revealed that expression of the aromatase gene is regulated in a species- and tissue specific manner. In healthy breast tissue, the predominantly active aromatase promoter region I.4 is regulated by glucocorticoids and class I cytokines. Therefore, it is important to investigate possible aromatase inhibiting properties of MeSO{sub 2}-PCBs (as anti glucocorticoids?) in relevant human tissues. We used primary human mammary fibroblasts because of their role in breast cancer development. We compared the results in primary fibroblasts with

  15. Regional differences in actomyosin contraction shape the primary vesicles in the embryonic chicken brain

    International Nuclear Information System (INIS)

    Filas, Benjamen A; Oltean, Alina; Majidi, Shabnam; Bayly, Philip V; Taber, Larry A; Beebe, David C

    2012-01-01

    In the early embryo, the brain initially forms as a relatively straight, cylindrical epithelial tube composed of neural stem cells. The brain tube then divides into three primary vesicles (forebrain, midbrain, hindbrain), as well as a series of bulges (rhombomeres) in the hindbrain. The boundaries between these subdivisions have been well studied as regions of differential gene expression, but the morphogenetic mechanisms that generate these constrictions are not well understood. Here, we show that regional variations in actomyosin-based contractility play a major role in vesicle formation in the embryonic chicken brain. In particular, boundaries did not form in brains exposed to the nonmuscle myosin II inhibitor blebbistatin, whereas increasing contractile force using calyculin or ATP deepened boundaries considerably. Tissue staining showed that contraction likely occurs at the inner part of the wall, as F-actin and phosphorylated myosin are concentrated at the apical side. However, relatively little actin and myosin was found in rhombomere boundaries. To determine the specific physical mechanisms that drive vesicle formation, we developed a finite-element model for the brain tube. Regional apical contraction was simulated in the model, with contractile anisotropy and strength estimated from contractile protein distributions and measurements of cell shapes. The model shows that a combination of circumferential contraction in the boundary regions and relatively isotropic contraction between boundaries can generate realistic morphologies for the primary vesicles. In contrast, rhombomere formation likely involves longitudinal contraction between boundaries. Further simulations suggest that these different mechanisms are dictated by regional differences in initial morphology and the need to withstand cerebrospinal fluid pressure. This study provides a new understanding of early brain morphogenesis. (paper)

  16. An altered redox balance and increased genetic instability characterize primary fibroblasts derived from xeroderma pigmentosum group A patients

    International Nuclear Information System (INIS)

    Parlanti, Eleonora; Pietraforte, Donatella; Iorio, Egidio; Visentin, Sergio; De Nuccio, Chiara; Zijno, Andrea; D’Errico, Mariarosaria; Simonelli, Valeria; Sanchez, Massimo; Fattibene, Paola; Falchi, Mario; Dogliotti, Eugenia

    2015-01-01

    Highlights: • Increased levels and different types of intracellular radical species as well as an altered glutathione redox state characterize XP-A human cells when compared to normal. • A more glycolytic metabolism and higher ATP levels are associated with alteration of mitochondrial morphology and response to mitochondrial toxicants when XPA is defective. • XP-A human cells show increased spontaneous micronuclei frequency, a hallmark of cancer risk. - Abstract: Xeroderma pigmentosum (XP)-A patients are characterized by increased solar skin carcinogenesis and present also neurodegeneration. XPA deficiency is associated with defective nucleotide excision repair (NER) and increased basal levels of oxidatively induced DNA damage. In this study we search for the origin of increased levels of oxidatively generated DNA lesions in XP-A cell genome and then address the question of whether increased oxidative stress might drive genetic instability. We show that XP-A human primary fibroblasts present increased levels and different types of intracellular reactive oxygen species (ROS) as compared to normal fibroblasts, with O_2_−· and H_2O_2 being the major reactive species. Moreover, XP-A cells are characterized by decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios as compared to normal fibroblasts. The significant increase of ROS levels and the alteration of the glutathione redox state following silencing of XPA confirmed the causal relationship between a functional XPA and the control of redox balance. Proton nuclear magnetic resonance ("1H NMR) analysis of the metabolic profile revealed a more glycolytic metabolism and higher ATP levels in XP-A than in normal primary fibroblasts. This perturbation of bioenergetics is associated with different morphology and response of mitochondria to targeted toxicants. In line with cancer susceptibility, XP-A primary fibroblasts showed increased spontaneous micronuclei (MN) frequency, a hallmark of cancer

  17. An altered redox balance and increased genetic instability characterize primary fibroblasts derived from xeroderma pigmentosum group A patients

    Energy Technology Data Exchange (ETDEWEB)

    Parlanti, Eleonora [Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Pietraforte, Donatella; Iorio, Egidio; Visentin, Sergio; De Nuccio, Chiara [Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Zijno, Andrea; D’Errico, Mariarosaria; Simonelli, Valeria [Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Sanchez, Massimo [Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Fattibene, Paola [Department of Technology and Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Falchi, Mario [National AIDS Center, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Dogliotti, Eugenia, E-mail: dogliotti@iss.it [Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy)

    2015-12-15

    Highlights: • Increased levels and different types of intracellular radical species as well as an altered glutathione redox state characterize XP-A human cells when compared to normal. • A more glycolytic metabolism and higher ATP levels are associated with alteration of mitochondrial morphology and response to mitochondrial toxicants when XPA is defective. • XP-A human cells show increased spontaneous micronuclei frequency, a hallmark of cancer risk. - Abstract: Xeroderma pigmentosum (XP)-A patients are characterized by increased solar skin carcinogenesis and present also neurodegeneration. XPA deficiency is associated with defective nucleotide excision repair (NER) and increased basal levels of oxidatively induced DNA damage. In this study we search for the origin of increased levels of oxidatively generated DNA lesions in XP-A cell genome and then address the question of whether increased oxidative stress might drive genetic instability. We show that XP-A human primary fibroblasts present increased levels and different types of intracellular reactive oxygen species (ROS) as compared to normal fibroblasts, with O{sub 2−}· and H{sub 2}O{sub 2} being the major reactive species. Moreover, XP-A cells are characterized by decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios as compared to normal fibroblasts. The significant increase of ROS levels and the alteration of the glutathione redox state following silencing of XPA confirmed the causal relationship between a functional XPA and the control of redox balance. Proton nuclear magnetic resonance ({sup 1}H NMR) analysis of the metabolic profile revealed a more glycolytic metabolism and higher ATP levels in XP-A than in normal primary fibroblasts. This perturbation of bioenergetics is associated with different morphology and response of mitochondria to targeted toxicants. In line with cancer susceptibility, XP-A primary fibroblasts showed increased spontaneous micronuclei (MN) frequency, a

  18. An altered redox balance and increased genetic instability characterize primary fibroblasts derived from xeroderma pigmentosum group A patients.

    Science.gov (United States)

    Parlanti, Eleonora; Pietraforte, Donatella; Iorio, Egidio; Visentin, Sergio; De Nuccio, Chiara; Zijno, Andrea; D'Errico, Mariarosaria; Simonelli, Valeria; Sanchez, Massimo; Fattibene, Paola; Falchi, Mario; Dogliotti, Eugenia

    2015-12-01

    Xeroderma pigmentosum (XP)-A patients are characterized by increased solar skin carcinogenesis and present also neurodegeneration. XPA deficiency is associated with defective nucleotide excision repair (NER) and increased basal levels of oxidatively induced DNA damage. In this study we search for the origin of increased levels of oxidatively generated DNA lesions in XP-A cell genome and then address the question of whether increased oxidative stress might drive genetic instability. We show that XP-A human primary fibroblasts present increased levels and different types of intracellular reactive oxygen species (ROS) as compared to normal fibroblasts, with O₂₋• and H₂O₂ being the major reactive species. Moreover, XP-A cells are characterized by decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios as compared to normal fibroblasts. The significant increase of ROS levels and the alteration of the glutathione redox state following silencing of XPA confirmed the causal relationship between a functional XPA and the control of redox balance. Proton nuclear magnetic resonance (¹H NMR) analysis of the metabolic profile revealed a more glycolytic metabolism and higher ATP levels in XP-A than in normal primary fibroblasts. This perturbation of bioenergetics is associated with different morphology and response of mitochondria to targeted toxicants. In line with cancer susceptibility, XP-A primary fibroblasts showed increased spontaneous micronuclei (MN) frequency, a hallmark of cancer risk. The increased MN frequency was not affected by inhibition of ROS to normal levels by N-acetyl-L-cysteine. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Primary fibroblasts from CSPα mutation carriers recapitulate hallmarks of the adult onset neuronal ceroid lipofuscinosis.

    Science.gov (United States)

    Benitez, Bruno A; Sands, Mark S

    2017-07-24

    Mutations in the co- chaperone protein, CSPα, cause an autosomal dominant, adult-neuronal ceroid lipofuscinosis (AD-ANCL). The current understanding of CSPα function exclusively at the synapse fails to explain the autophagy-lysosome pathway (ALP) dysfunction in cells from AD-ANCL patients. Here, we demonstrate unexpectedly that primary dermal fibroblasts from pre-symptomatic mutation carriers recapitulate in vitro features found in the brains of AD-ANCL patients including auto-fluorescent storage material (AFSM) accumulation, CSPα aggregates, increased levels of lysosomal proteins and lysosome enzyme activities. AFSM accumulation correlates with CSPα aggregation and both are susceptible to pharmacological modulation of ALP function. In addition, we demonstrate that endogenous CSPα is present in the lysosome-enriched fractions and co-localizes with lysosome markers in soma, neurites and synaptic boutons. Overexpression of CSPα wild-type (WT) decreases lysotracker signal, secreted lysosomal enzymes and SNAP23-mediated lysosome exocytosis. CSPα WT, mutant and aggregated CSPα are degraded mainly by the ALP but this disease-causing mutation exhibits a faster rate of degradation. Co-expression of both WT and mutant CSPα cause a block in the fusion of autophagosomes/lysosomes. Our data suggest that aggregation-dependent perturbation of ALP function is a relevant pathogenic mechanism for AD-ANCL and supports the use of AFSM or CSPα aggregation as biomarkers for drug screening purposes.

  20. MicroRNA transcriptome analysis identifies miR-365 as a novel negative regulator of cell proliferation in Zmpste24-deficient mouse embryonic fibroblasts

    International Nuclear Information System (INIS)

    Xiong, Xing-dong; Jung, Hwa Jin; Gombar, Saurabh; Park, Jung Yoon; Zhang, Chun-long; Zheng, Huiling; Ruan, Jie; Li, Jiang-bin; Kaeberlein, Matt

    2015-01-01

    Highlights: • A comprehensive miRNA transcriptome of MEFs from Zmpste24 −/− and control mice. • Identification of miR-365 as a down-regulated miRNA in Zmpste24 −/− MEFs. • Characterization of miR-365 as a modulator of cellular growth in part by targeting Rasd1. - Abstract: Zmpste24 is a metalloproteinase responsible for the posttranslational processing and cleavage of prelamin A into mature laminA. Zmpste24 −/− mice display a range of progeroid phenotypes overlapping with mice expressing progerin, an altered version of lamin A associated with Hutchinson-Gilford progeria syndrome (HGPS). Increasing evidence has demonstrated that miRNAs contribute to the regulation of normal aging process, but their roles in progeroid disorders remain poorly understood. Here we report the miRNA transcriptomes of mouse embryonic fibroblasts (MEFs) established from wild type (WT) and Zmpste24 −/− progeroid mice using a massively parallel sequencing technology. With data from 19.5 × 10 6 reads from WT MEFs and 16.5 × 10 6 reads from Zmpste24 −/− MEFs, we discovered a total of 306 known miRNAs expressed in MEFs with a wide dynamic range of read counts ranging from 10 to over 1 million. A total of 8 miRNAs were found to be significantly down-regulated, with only 2 miRNAs upregulated, in Zmpste24 −/− MEFs as compared to WT MEFs. Functional studies revealed that miR-365, a significantly down-regulated miRNA in Zmpste24 −/− MEFs, modulates cellular growth phenotypes in MEFs. Overexpression of miR-365 in Zmpste24 −/− MEFs increased cellular proliferation and decreased the percentage of SA-β-gal-positive cells, while inhibition of miR-365 function led to an increase of SA-β-gal-positive cells in WT MEFs. Furthermore, we identified Rasd1, a member of the Ras superfamily of small GTPases, as a functional target of miR-365. While expression of miR-365 suppressed Rasd1 3′ UTR luciferase-reporter activity, this effect was lost with mutations in the

  1. The oncogenic properties of EWS/WT1 of desmoplastic small round cell tumors are unmasked by loss of p53 in murine embryonic fibroblasts

    International Nuclear Information System (INIS)

    Bandopadhayay, Pratiti; Thomas, David M; Algar, Elizabeth; Ekert, Paul G; Jabbour, Anissa M; Riffkin, Christopher; Salmanidis, Marika; Gordon, Lavinia; Popovski, Dean; Rigby, Lin; Ashley, David M; Watkins, David N

    2013-01-01

    Desmoplastic small round cell tumor (DSRCT) is characterized by the presence of a fusion protein EWS/WT1, arising from the t (11;22) (p13;q12) translocation. Here we examine the oncogenic properties of two splice variants of EWS/WT1, EWS/WT1-KTS and EWS/WT1 + KTS. We over-expressed both EWS/WT1 variants in murine embryonic fibroblasts (MEFs) of wild-type, p53 +/- and p53 -/- backgrounds and measured effects on cell-proliferation, anchorage-independent growth, clonogenicity after serum withdrawal, and sensitivity to cytotoxic drugs and gamma irradiation in comparison to control cells. We examined gene expression profiles in cells expressing EWS/WT1. Finally we validated our key findings in a small series of DSRCT. Neither isoform of EWS/WT1 was sufficient to transform wild-type MEFs however the oncogenic potential of both was unmasked by p53 loss. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 enhanced cell-proliferation, clonogenic survival and anchorage-independent growth. EWS/WT1 expression in wild-type MEFs conferred resistance to cell-cycle arrest after irradiation and daunorubicin induced apoptosis. We show DSRCT commonly have nuclear localization of p53, and copy-number amplification of MDM2/MDMX. Expression of either isoform of EWS/WT1 induced characteristic mRNA expression profiles. Gene-set enrichment analysis demonstrated enrichment of WNT pathway signatures in MEFs expressing EWS/WT1 + KTS. Wnt-activation was validated in cell lines with over-expression of EWS/WT1 and in DSRCT. In conclusion, we show both isoforms of EWS/WT1 have oncogenic potential in MEFs with loss of p53. In addition we provide the first link between EWS/WT1 and Wnt-pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterize DSRCT

  2. Gold Nanoparticle-Mediated Delivery of Molecules into Primary Human Gingival Fibroblasts Using ns-Laser Pulses: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Judith Krawinkel

    2016-05-01

    Full Text Available Interaction of gold nanoparticles (AuNPs in the vicinity of cells’ membrane with a pulsed laser (λ = 532 nm, τ = 1 ns leads to perforation of the cell membrane, thereby allowing extracellular molecules to diffuse into the cell. The objective of this study was to develop an experimental setting to deliver molecules into primary human gingival fibroblasts (pHFIB-G by using ns-laser pulses interacting with AuNPs (study group. To compare the parameters required for manipulation of pHFIB-G with those needed for cell lines, a canine pleomorphic adenoma cell line (ZMTH3 was used (control group. Non-laser-treated cells incubated with AuNPs and the delivery molecules served as negative control. Laser irradiation (up to 35 mJ/cm2 resulted in a significant proportion of manipulated fibroblasts (up to 85%, compared to non-irradiated cells: p < 0.05, while cell viability (97% was not reduced significantly. pHFIB-G were perforated as efficiently as ZMTH3. No significant decrease of metabolic cell activity was observed up to 72 h after laser treatment. The fibroblasts took up dextrans with molecular weights up to 500 kDa. Interaction of AuNPs and a pulsed laser beam yields a spatially selective technique for manipulation of even primary cells such as pHFIB-G in high throughput.

  3. Cytotoxic effects of denture adhesives on primary human oral keratinocytes, fibroblasts and permanent L929 cell lines.

    Science.gov (United States)

    Chen, Fengying; Wu, Tianfu; Cheng, Xiangrong

    2014-03-01

    To date, there have been very little data on the cytotoxic responses of different cell lines to denture adhesives. To determine the cytotoxicity of three denture adhesives on primary human oral keratinocytes (HOKs), fibroblasts (HOFs) and permanent mouse fibroblasts cell lines (L929). Three commercial denture adhesives (two creams and one powder) were prepared for indirect contact using the agar diffusion test, as well as extracts in MTT assay. The results of the MTT assay were statistically analysed by one-way anova and Tukey's test (p adhesives showed mild to moderate cytotoxicity to primary HOKs (p  0.05) in both assays. For primary HOFs cultures, slight cytotoxicity was observed for one of the products from the agar diffusion test and undiluted eluates of all tested adhesives with MTT assay (p adhesives are toxic to the primary HOKs and HOFs cultures, whereas non-toxic to L929 cells. The results suggest that primary human oral mucosal cells may provide more valuable information in toxicity screening of denture adhesives. © 2012 John Wiley & Sons A/S and The Gerodontology Association. Published by John Wiley & Sons Ltd.

  4. Embryonic origin of the Arabidopsis primary root and root meristem initials

    NARCIS (Netherlands)

    Scheres, B.J.G.; Wolkenfelt, H.; Willemsen, V.; Terlouw, M.; Lawson, E.; Dean, C.; Weisbeek, P.

    1994-01-01

    The embryonic origin of the Arabidopsis root and hypocotyl region has been investigated using histological techniques and clonal analysis. Our data reveal the pattern of cell division in the embryo giving rise to the various initials within the root promeristem. A small region of the root at its

  5. MicroRNA transcriptome analysis identifies miR-365 as a novel negative regulator of cell proliferation in Zmpste24-deficient mouse embryonic fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Xing-dong [Institute of Aging Research, Guangdong Medical College, Xin Cheng Avenue 1#, Songshan Lake, Dongguan, Guangdong 523808 (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical College, Zhanjiang 524023 (China); Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan 523808 (China); Institute of Laboratory Medicine, Guangdong Medical College, Dongguan, Guangdong 523808 (China); Jung, Hwa Jin [Departments of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461 (United States); Gombar, Saurabh [Departments of Systems Biology, Albert Einstein College of Medicine, Bronx, NY 10461 (United States); Park, Jung Yoon [Departments of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461 (United States); Zhang, Chun-long; Zheng, Huiling [Institute of Aging Research, Guangdong Medical College, Xin Cheng Avenue 1#, Songshan Lake, Dongguan, Guangdong 523808 (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical College, Zhanjiang 524023 (China); Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan 523808 (China); Ruan, Jie; Li, Jiang-bin [Institute of Aging Research, Guangdong Medical College, Xin Cheng Avenue 1#, Songshan Lake, Dongguan, Guangdong 523808 (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical College, Zhanjiang 524023 (China); Key Laboratory for Medical Molecular Diagnostics of Guangdong Province, Dongguan 523808 (China); Institute of Laboratory Medicine, Guangdong Medical College, Dongguan, Guangdong 523808 (China); Kaeberlein, Matt [Institute of Aging Research, Guangdong Medical College, Xin Cheng Avenue 1#, Songshan Lake, Dongguan, Guangdong 523808 (China); Department of Pathology, University of Washington, Seattle, WA 98195 (United States); and others

    2015-07-15

    Highlights: • A comprehensive miRNA transcriptome of MEFs from Zmpste24{sup −/−} and control mice. • Identification of miR-365 as a down-regulated miRNA in Zmpste24{sup −/−} MEFs. • Characterization of miR-365 as a modulator of cellular growth in part by targeting Rasd1. - Abstract: Zmpste24 is a metalloproteinase responsible for the posttranslational processing and cleavage of prelamin A into mature laminA. Zmpste24{sup −/−} mice display a range of progeroid phenotypes overlapping with mice expressing progerin, an altered version of lamin A associated with Hutchinson-Gilford progeria syndrome (HGPS). Increasing evidence has demonstrated that miRNAs contribute to the regulation of normal aging process, but their roles in progeroid disorders remain poorly understood. Here we report the miRNA transcriptomes of mouse embryonic fibroblasts (MEFs) established from wild type (WT) and Zmpste24{sup −/−} progeroid mice using a massively parallel sequencing technology. With data from 19.5 × 10{sup 6} reads from WT MEFs and 16.5 × 10{sup 6} reads from Zmpste24{sup −/−} MEFs, we discovered a total of 306 known miRNAs expressed in MEFs with a wide dynamic range of read counts ranging from 10 to over 1 million. A total of 8 miRNAs were found to be significantly down-regulated, with only 2 miRNAs upregulated, in Zmpste24{sup −/−} MEFs as compared to WT MEFs. Functional studies revealed that miR-365, a significantly down-regulated miRNA in Zmpste24{sup −/−} MEFs, modulates cellular growth phenotypes in MEFs. Overexpression of miR-365 in Zmpste24{sup −/−} MEFs increased cellular proliferation and decreased the percentage of SA-β-gal-positive cells, while inhibition of miR-365 function led to an increase of SA-β-gal-positive cells in WT MEFs. Furthermore, we identified Rasd1, a member of the Ras superfamily of small GTPases, as a functional target of miR-365. While expression of miR-365 suppressed Rasd1 3′ UTR luciferase-reporter activity

  6. Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

    Directory of Open Access Journals (Sweden)

    Coppock Donald L

    2008-12-01

    Full Text Available Abstract Background Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging. The objective of this study was 1 To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF on these cells. 2 To describe a methodology to create fibroblast cell lines from patients with chronic wounds. Methods Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing. Results Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA Cell Repository. Conclusion We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their in vivo phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators in vitro.

  7. Transcriptional profile of fibroblasts obtained from the primary site, lymph node and bone marrow of breast cancer patients

    Directory of Open Access Journals (Sweden)

    Paulo Roberto Del Valle

    2014-09-01

    Full Text Available Cancer-associated fibroblasts (CAF influence tumor development at primary as well as in metastatic sites, but there have been no direct comparisons of the transcriptional profiles of stromal cells from different tumor sites. In this study, we used customized cDNA microarrays to compare the gene expression profile of stromal cells from primary tumor (CAF, n = 4, lymph node metastasis (N+, n = 3 and bone marrow (BM, n = 4 obtained from breast cancer patients. Biological validation was done in another 16 samples by RT-qPCR. Differences between CAF vs N+, CAF vs BM and N+ vs BM were represented by 20, 235 and 245 genes, respectively (SAM test, FDR < 0.01. Functional analysis revealed that genes related to development and morphogenesis were overrepresented. In a biological validation set, NOTCH2 was confirmed to be more expressed in N+ (vs CAF and ADCY2, HECTD1, HNMT, LOX, MACF1, SLC1A3 and USP16 more expressed in BM (vs CAF. Only small differences were observed in the transcriptional profiles of fibroblasts from the primary tumor and lymph node of breast cancer patients, whereas greater differences were observed between bone marrow stromal cells and the other two sites. These differences may reflect the activities of distinct differentiation programs.

  8. Induction of chromosomal aberrations in human primary fibroblasts and immortalized cancer cells exposed to extremely-low-frequency electromagnetic fields

    International Nuclear Information System (INIS)

    Seyyedi, S. S.; Mozdarani, H.; Rezaei Tavirani, M.; Heydari, S.

    2010-01-01

    Rapidly increasing possibilities of exposure to environmental extremely low-frequency electromagnetic fields have become a topic of worldwide investigation. Epidemiological and laboratory studies suggest that exposure to extremely low-frequency electromagnetic fields may increase cancer risk therefore assessment of chromosomal damage in various cell lines might be of predictive value for future risk estimation. Materials and Methods: Primary cultures of fibroblasts from human skin biopsy were exposed to continuous extremely low-frequency electromagnetic fields (3, 50 and 60 Hz, sinusoidal, 3h, and 4 m T). Also immortalized cell lines, SW480, MCF-7 and 1321N1 were exposed to continuous extremely low-frequency electromagnetic fields (50 Hz, sinusoidal, 3 h, 4 m T). Metaphase plates Were prepared according to standard methods and stained in 5% Giemsa solution. Chromosomal aberrations of both chromosome and chromatid types were scored to evaluate the effects of extremely low-frequency electromagnetic fields on primary or established cell lines. Results: Results indicate that by increasing the frequency of extremely low-frequency electromagnetic fields, chromosomal aberrations were increased up to 7-fold above background levels in primary human fibroblast cells. In addition, continuous exposure to a 50 Hz electromagnetic field led to a significant increase in chromosomal aberrations in SW480, MCF-7 and 1321N1 cell lines compared to sham control. Conclusion: Results obtained indicate that extremely low-frequency electromagnetic fields has the potential for induction of chromosomal aberrations in all cell types.

  9. Background Levels of Neomorphic 2-hydroxyglutarate Facilitate Proliferation of Primary Fibroblasts

    Czech Academy of Sciences Publication Activity Database

    Dvořák, Aleš; Zelenka, Jaroslav; Smolková, Katarína; Vítek, L.; Ježek, Petr

    2017-01-01

    Roč. 66, č. 2 (2017), s. 293-304 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GPP301/12/P381; GA ČR(CZ) GA16-04788S Institutional support: RVO:67985823 Keywords : 2-hydroxyglutarate * fibroblast proliferation * SH-SY5Y neuroblastoma cells Subject RIV: CE - Biochemistry OBOR OECD: Oncology Impact factor: 1.461, year: 2016

  10. Determining the pharmacological activity of Physalis peruviana fruit juice on rabbit eyes and fibroblast primary cultures.

    Science.gov (United States)

    Pardo, Juan Manuel; Fontanilla, Marta Raquel; Ospina, Luis Fernando; Espinosa, Lady

    2008-07-01

    The pharmacologic activity of compounds isolated from Physalis peruviana has been demonstrated. The use of this fruit juice for treating pterygium has been reported in Colombian traditional medicine. However, studies demonstrating the fruit juice's pharmacologic activity when used in this disease have not been published to date. In the present study the anti-inflammatory and cytostatic activities of P. peruviana fruit juice in a rabbit eye inflammatory model were investigated. A novel rabbit eye inflammation model was developed for studying the juice's anti-inflammatory activity (based on an adaptation of the Draize test). Cytostatic activity was evaluated by measuring and comparing growth rates of cultured fibroblasts exposed and not exposed to various fruit juice concentrations. P. peruviana fruit juice exhibited a mild anti-inflammatory activity compared with methylprednisolone, a known anti-inflammatory drug. An interesting dose-dependent cytostatic effect on cultured fibroblasts was also established. The data found suggest that the P. peruviana fruit juice anti-pterygium effect described in traditional medicine may be related to its inhibiting fibroblast growth. The present study contributes to the pharmacologic knowledge regarding a remedy commonly used in Colombian traditional medicine.

  11. Prostaglandin E synthase interacts with inducible heat shock protein 70 after heat stress in bovine primary dermal fibroblast cells.

    Science.gov (United States)

    Richter, Constanze; Viergutz, Torsten; Schwerin, Manfred; Weitzel, Joachim M

    2015-01-01

    Exposure to heat stress in dairy cows leads to undesired side effects that are reflected by complex alterations in endocrine parameters, such as reduced progesterone, estradiol, and thyroid hormone concentrations. These endocrine maladaptation leads to failure to resume cyclicity, a poor uterine environment and inappropriate immune responses in postpartum dairy cows. Prostaglandins (PG's) are lipid mediators, which serve as signal molecules in response to various external stimuli as well as to cell-specific internal signal molecules. A central role in PG synthesis plays prostaglandin E synthase (PGES) that catalyzes the isomerization of PGH2 to PGE2 .The present study was conducted to investigate heat stress associated PGES expression. Expression of PGES and inducible heat shock protein 70 (HSP70), as a putative chaperonic protein, was studied in bovine primary fibroblasts under different heat shock conditions. Bovine primary fibroblasts produce PGE2 at homoiothermical norm temperature (38.5°C in bovine), but reduce PGE2 production rates under extreme heat stress (at 45°C for 6 h). By contrast, PGE2 production rates are maintained after a milder heat stress (at 41.5°C for 6 h). PGE2 synthesis is abolished by application of cyclooxygenase inhibitor indomethacin, indicating de novo synthesis. Heat stress increases HSP70 but not PGES protein concentrations. HSP70 physically interacts with PGES and the PGES-HSP70 complex did not dissociate upon heat stress at 45°C even after returning the cells to 37°C. The PGE2 production negatively correlates with the portion of PGES-HSP70 complex. These results suggest a protein interaction between HSP70 and PGES in dermal fibroblast cells. Blockage of PGES protein by HSP70 seems to interfere with the regulatory processes essential for cellular adaptive protection. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

  12. Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts.

    Science.gov (United States)

    Kathirvel, Poonkodi; Ravi, Subban

    2012-01-01

    This study examines the chemical composition and in vitro anticancer activity of the essential oil from Ocimum basilicum Linn. (Lamiaceae), cultivated in the Western Ghats of South India. The chemical compositions of basil fresh leaves were identified by GC-MS: 11 components were identified. The major constituents were found to be methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%) and camphor (1.52%). The results revealed that this plant may belong to the methyl cinnamate and linalool chemotype. A methyl thiazol tetrazolium assay was used for in vitro cytotoxicity screening against the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cell line (HEp-2) and NIH 3T3 mouse embryonic fibroblasts. The IC(50) values obtained were 90.5 and 96.3 µg mL(-1), respectively, and the results revealed that basil oil has potent cytotoxicity.

  13. Geometry and mechanics of teleost gastrulation and the formation of primary embryonic axes.

    Science.gov (United States)

    Cherdantseva, Elena M; Cherdantsev, Vladimir G

    2006-01-01

    Examination of normal shaping dynamics and immediate and long-term responses to blastoderm cutting in zebrafish and loach embryos prior to the onset of gastrulation and during the course of epiboly revealed that anteroposterior (AP) and dorsoventral (DV) polarity formation is connected with shaping of the blastoderm circumferential region, which stretches along and shrinks across its movement axes and originates the non-isotropic fields of tensile stresses. Based on data from cutting experiments and quantitative morphology, we reconstructed the movement-shaping patterns of epiboly and embryonic shield formation. We revealed that AP and DV axes originate as a mass cell movement subject to the movement-shaping equivalence principle, which means the spatial series of differently shaped areas corresponding to the time succession of the same area shaping. Maintenance of the main body axes in orthogonal orientation depends on the mechanical equilibrium principle allowing for converting shape asymmetry into that of tensile stresses and vice versa. The causal relationship between the main movement-shaping axes and that of embryonic polarity was proved in cutting experiments in which the DV axis direction was subject to rearrangement so as to adjust to the new direction of mass cell movement axes induced by healing the wound in the blastoderm circumferential region.

  14. Modulation of mitochondrial morphology by bioenergetics defects in primary human fibroblasts

    DEFF Research Database (Denmark)

    Guillery, O.; Malka, F.; Frachon, P.

    2008-01-01

    induced partial but significant mitochondrial fragmentation, whereas dissipation of mitochondrial membrane potential (D Psi m) provoked complete fragmentation, and glycolysis inhibition had no effect. Oxidative phosphorylation defective fibroblasts had essentially normal filamentous mitochondria under...... basal conditions, although when challenged some of them presented with mild alteration of fission or fusion efficacy. Severely defective cells disclosed complete mitochondrial fragmentation under glycolysis inhibition. In conclusion, mitochondrial morphology is modulated by D Psi m but loosely linked...... to mitochondrial oxidative phosphorylation. Its alteration by glycolysis, inhibition points to a severe oxidative phosphorylation defect. (C) 2008 Elsevier B.V. All rights reserved Udgivelsesdato: 2008/4...

  15. Establishment and Maintenance of Primary Fibroblast Repositories for Rare Diseases—Friedreich's Ataxia Example

    Science.gov (United States)

    Li, Yanjie; Polak, Urszula; Clark, Amanda D.; Bhalla, Angela D.; Chen, Yu-Yun; Li, Jixue; Farmer, Jennifer; Seyer, Lauren; Lynch, David

    2016-01-01

    Friedreich's ataxia (FRDA) represents a rare neurodegenerative disease caused by expansion of GAA trinucleotide repeats in the first intron of the FXN gene. The number of GAA repeats in FRDA patients varies from approximately 60 to repositories, especially in the context of rare and heterogeneous disorders, are presented. Although the economic aspect of creating and maintaining such repositories is important, the benefits of easy access to a collection of well-characterized cell lines for the purpose of drug discovery or disease mechanism studies overshadow the associated costs. Importantly, all FRDA fibroblast cell lines collected in our repository are available to the scientific community. PMID:27002638

  16. Establishment and Maintenance of Primary Fibroblast Repositories for Rare Diseases-Friedreich's Ataxia Example.

    Science.gov (United States)

    Li, Yanjie; Polak, Urszula; Clark, Amanda D; Bhalla, Angela D; Chen, Yu-Yun; Li, Jixue; Farmer, Jennifer; Seyer, Lauren; Lynch, David; Butler, Jill S; Napierala, Marek

    2016-08-01

    Friedreich's ataxia (FRDA) represents a rare neurodegenerative disease caused by expansion of GAA trinucleotide repeats in the first intron of the FXN gene. The number of GAA repeats in FRDA patients varies from approximately 60 to repositories, especially in the context of rare and heterogeneous disorders, are presented. Although the economic aspect of creating and maintaining such repositories is important, the benefits of easy access to a collection of well-characterized cell lines for the purpose of drug discovery or disease mechanism studies overshadow the associated costs. Importantly, all FRDA fibroblast cell lines collected in our repository are available to the scientific community.

  17. Model-based investigation of the circadian clock and cell cycle coupling in mouse embryonic fibroblasts: Prediction of RevErb-α up-regulation during mitosis

    OpenAIRE

    Traynard , Pauline; Feillet , Céline; Soliman , Sylvain; Delaunay , Franck; Fages , François

    2016-01-01

    International audience; Experimental observations have put in evidence autonomous self-sustained circadian oscillators in most mammalian cells, and proved the existence of molecular links between the circadian clock and the cell cycle. Some mathematical models have also been built to assess conditions of control of the cell cycle by the circadian clock. However, recent studies in individual NIH3T3 fibroblasts have shown an unexpected acceleration of the circadian clock together with the cell ...

  18. Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities.

    Science.gov (United States)

    Sakai, Yusuke; Koike, Makiko; Kawahara, Daisuke; Hasegawa, Hideko; Murai, Tomomi; Yamanouchi, Kosho; Soyama, Akihiko; Hidaka, Masaaki; Takatsuki, Mitsuhisa; Fujita, Fumihiko; Kuroki, Tamotsu; Eguchi, Susumu

    2018-03-05

    Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1×10 3  cells/cm 2 . Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. No significant level of inheritable interchromosomal aberrations in the progeny of bystander primary human fibroblasts after alpha particle irradiation

    Science.gov (United States)

    Hu, Burong; Zhu, Jiayun; Zhou, Hongning; Hei, Tom K.

    2013-02-01

    A major concern for bystander effects is the probability that normal healthy cells adjacent to the irradiated cells become genomically unstable and undergo further carcinogenesis after therapeutic irradiation or space mission where astronauts are exposed to low dose of heavy ions. Genomic instability is a hallmark of cancer cells. In the present study, two irradiation protocols were performed in order to ensure pure populations of bystander cells and the genomic instability in their progeny were investigated. After irradiation, chromosomal aberrations of cells were analyzed at designated time points using G2 phase premature chromosome condensation (G2-PCC) coupled with Giemsa staining and with multiplex fluorescent in situ hybridization (mFISH). Our Giemsa staining assay demonstrated that elevated yields of chromatid breaks were induced in the progeny of pure bystander primary fibroblasts up to 20 days after irradiation. mFISH assay showed no significant level of inheritable interchromosomal aberrations were induced in the progeny of the bystander cell groups, while the fractions of gross aberrations (chromatid breaks or chromosomal breaks) significantly increased in some bystander cell groups. These results suggest that genomic instability occurred in the progeny of the irradiation associated bystander normal fibroblasts exclude the inheritable interchromosomal aberration.

  20. Differential gene expression in primary fibroblasts induced by proton and cobalt-60 beam irradiation

    DEFF Research Database (Denmark)

    Nielsen, Steffen; Bassler, Niels; Grzanka, Leszek

    2017-01-01

    profile: entrance, mid-SOBP and at the SOBP distal edge. Dose was delivered in three fractions × 3.5 Gy(RBE) (RBE 1.1). Cobalt-60 (Co-60) irradiation was used as reference. Real-time qPCR was performed to determine gene expression levels for 17 genes associated with inflammation response, fibrosis...... and angiogenesis. RESULTS: Differences in median gene expression levels were observed for multiple genes such as IL6, IL8 and CXCL12. Median IL6 expression was 30%, 24% and 47% lower in entrance, mid-SOBP and SOBP distal edge groups than in Co-60 irradiated cells. No genes were found to be oppositely regulated...... fibroblast cultures. Inflammatory factors were generally less extensively upregulated by proton irradiation compared with Co-60 photon irradiation. These effects may possibly influence the development of normal tissue damage in patients treated with proton beam therapy....

  1. High-throughput screening of microscale pitted substrate topographies for enhanced nonviral transfection efficiency in primary human fibroblasts

    DEFF Research Database (Denmark)

    Adler, Andrew F; Speidel, Alessondra T; Christoforou, Nicolas

    2011-01-01

    of microscale topographies, we have demonstrated an improvement in nonviral transfection efficiency for cells cultured on dense micropit patterns compared to smooth substrates, as verified with flow cytometry. A 25% increase in GFP(+) cells was observed independent of proliferation rate, accompanied by SEM....... Emerging literature has highlighted the influence of cell-topography interactions on modulation of many cell phenotypes, including protein expression and cytoskeletal behaviors implicated in endocytosis. Using high-throughput screening of primary human dermal fibroblasts cultured on a combinatorial library...... and confocal microscopy characterization to help explain the phenomenon qualitatively. This finding encourages researchers to investigate substrate topography as a new design consideration for the optimization of nonviral transfection systems....

  2. Graphene oxide improves the biocompatibility of collagen membranes in an in vitro model of human primary gingival fibroblasts.

    Science.gov (United States)

    De Marco, Patrizia; Zara, Susi; De Colli, Marianna; Radunovic, Milena; Lazović, Vladimir; Ettorre, Valeria; Di Crescenzo, Antonello; Piattelli, Adriano; Cataldi, Amelia; Fontana, Antonella

    2017-09-13

    Commercial collagen membranes are used in oral surgical procedures as scaffolds for bone deposition in guided bone regeneration. Here, we have enriched them with graphene oxide (GO) via a simple non-covalent functionalization, exploiting the capacity of oxygenated carbon functional moieties of GO to interact through hydrogen bonding with collagen. In the present paper, the GO-coated membranes have been characterized in terms of stability, nano-roughness, biocompatibility and induction of inflammatory response in human primary gingival fibroblast cells. The obtained coated membranes are demonstrated not to leak GO in the bulk solution, and to change some features of the membrane, such as stiffness and adhesion between the membrane and the atomic force microscopy (AFM) tip. Moreover, the presence of GO increases the roughness and the total surface exposed to the cells, as demonstrated by AFM analyses. The obtained material is biocompatible, and does not induce inflammation in the tested cells.

  3. A modified fluorimetric host cell reactivation assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts

    Directory of Open Access Journals (Sweden)

    Gebhard Daniel

    2010-06-01

    Full Text Available Abstract Background The Host Cell Reactivation Assay (HCRA is widely used to identify circumstances and substances affecting the repair capacity of cells, however, it is restricted by the transfection procedure used and the sensitivity of the detection method. Primary skin cells are particularly difficult to transfect, and therefore sensitive methods are needed to detect any variations due to the cell-type or inter-individual differences or changes induced by diverse substances. A sensitive and repeatable method to detect the repair capacity of skin cells would be useful in two different aspects: On the one hand, to identify substances influencing the repair capacity in a positive manner (these substances could be promising ingredients for cosmetic products and on the other hand, to exclude the negative effects of substances on the repair capacity (this could serve as one step further towards replacing or at least reducing animal testing. Results In this paper, we present a rapid and sensitive assay to determine the repair capacity of primary keratinocytes, melanocytes and fibroblasts based on two wave-length Green Fluorescent Protein (GFP and DsRed reporter technology in order to test different substances and their potential to influence the DNA repair capacity. For the detection of plasmid restoration, we used FACS technology, which, in comparison to luminometer technology, is highly sensitive and allows single cell based analysis. The usefulness of this assay and studying the repair capacity is demonstrated by the evidence that DNA repair is repressed by Cyclosporin A in fibroblasts. Conclusions The methodology described in this paper determines the DNA repair capacity in different types of human skin cells. The described transfection protocol is suitable for the transfection of melanocytes, keratinocytes and fibroblasts, reaching efficacies suitable for the detection of the restored plasmids by FACS technology. Therefore the repair capacity

  4. mTOR complex 2 phosphorylates IMP1 cotranslationally to promote IGF2 production and the proliferation of mouse embryonic fibroblasts

    DEFF Research Database (Denmark)

    Dai, Ning; Christiansen, Jan; Nielsen, Finn

    2013-01-01

    uncover a new mechanism by which mTOR regulates organismal growth by promoting IGF2 production in the mouse embryo through mTORC2-catalyzed cotranslational IMP1/IMP3 phosphorylation. Inasmuch as TORC2 is activated by association with ribosomes, the present results indicate that mTORC2-catalyzed...... production, and diminished proliferation. The proliferation of the IMP1-null fibroblasts can be restored to wild-type levels by IGF2 in vitro or by re-expression of IMP1, which corrects the defects in IGF2 RNA splicing and translation. The ability of IMP1 to correct these defects is dependent on IMP1...

  5. Ablation of the mitochondrial complex IV assembly protein Surf1 leads to increased expression of the UPRMT and increased resistance to oxidative stress in primary cultures of fibroblasts

    Directory of Open Access Journals (Sweden)

    Gavin Pharaoh

    2016-08-01

    Full Text Available Mice deficient in the electron transport chain (ETC complex IV assembly protein SURF1 have reduced assembly and activity of cytochrome c oxidase that is associated with an upregulation of components of the mitochondrial unfolded protein response (UPRMT and increased mitochondrial number. We hypothesized that the upregulation of proteins associated with the UPRMT in response to reduced cytochrome c oxidase activity in Surf1−/− mice might contribute to increased stress resistance. To test this hypothesis we asked whether primary cultures of fibroblasts from Surf1−/− mice exhibit enhanced resistance to stressors compared to wild-type fibroblasts. Here we show that primary dermal fibroblasts isolated from Surf1−/− mice have increased expression of UPRMT components ClpP and Hsp60, and increased expression of Lon protease. Fibroblasts from Surf1−/− mice are significantly more resistant to cell death caused by oxidative stress induced by paraquat or tert-Butyl hydroperoxide compared to cells from wild-type mice. In contrast, Surf1−/− fibroblasts show no difference in sensitivity to hydrogen peroxide stress. The enhanced cell survival in response to paraquat or tert-Butyl hydroperoxide in Surf1−/− fibroblasts compared to wild-type fibroblasts is associated with induced expression of Lon, ClpP, and Hsp60, increased maximal respiration, and increased reserve capacity as measured using the Seahorse Extracellular Flux Analyzer. Overall these data support a protective role for the activation of the UPRMT in cell survival.

  6. In vitro radiosensitivity of primary human fibroblasts. Lack of correlation with acute radiation toxicity in patients with head and neck cancer

    International Nuclear Information System (INIS)

    Rudat, Volker; Dietz, Andreas; Conradt, Christian; Weber, Klaus-Josef; Flentje, Michael

    1997-01-01

    Background and purpose: There is a considerable hope among clinicians and radiobiologists to detect genetically radiosensitive patients prior to radiotherapy. A predictive assay would enable adjustment of the total irradiation dose to the individual at a constant risk of normal tissue complications. In this prospective study, the clonogenic survival assay for primary human fibroblasts to determine radiosensitivity in vitro was evaluated and then correlated with clinically observed acute radiation reactions. Materials and methods: One hundred twenty-five independent survival experiments with primary fibroblasts derived from 63 biopsies from 55 cancer and non-cancer patients were performed. Results: A wide variation of cell survival between biopsies was detected. Statistical analysis revealed a highly significantly larger interindividual than intraindividual variation of SF2 values. However, a considerable scatter of SF2 values in repeated experiments was observed in individual cases. Age, gender, disease status (cancer patient, non-cancer patient) and origin of fibroblasts (skin, periodontal tissue) were demonstrated not to be statistically significant confounding factors on the intrinsic radiosensitivity in vitro. In a prospective study, no correlation of the SF2 and acute reactions in 25 patients with head and neck cancer treated with a primary accelerated radiochemotherapy was detected. Conclusion: Our data show that the clonogenic assay is able to distinguish between intrinsic radiosensitivities of primary human fibroblasts if a statistical approach is used but does not predict acute radiation toxicity

  7. Primary cilia function regulates the length of the embryonic trunk axis and urogenital field in mice

    DEFF Research Database (Denmark)

    Wainwright, Elanor N.; Svingen, Terje; Ting Ng, Ee

    2014-01-01

    The issues of whether and how some organs coordinate their size and shape with the blueprint of the embryo axis, while others appear to regulate their morphogenesis autonomously, remain poorly understood. Mutations in Ift144, encoding a component of the trafficking machinery of primary cilia asse...

  8. Directional cell migration and chemotaxis in wound healing response to PDGF-AA are coordinated by the primary cilium in fibroblasts

    DEFF Research Database (Denmark)

    Schneider, Linda; Cammer, Michael; Lehman, Jonathan

    2010-01-01

    Cell motility and migration play pivotal roles in numerous physiological and pathophysiological processes including development and tissue repair. Cell migration is regulated through external stimuli such as platelet-derived growth factor-AA (PDGF-AA), a key regulator in directional cell migration....... Here we used micropipette analysis to show that a normal chemosensory response to PDGF-AA in fibroblasts requires the primary cilium. In vitro and in vivo wound healing assays revealed that in ORPK mouse (IFT88(Tg737Rpw)) fibroblasts, where ciliary assembly is defective, chemotaxis towards PDGF-AA...

  9. Comparison of gene expression profile in embryonic mesencephalon and neuronal primary cultures.

    Directory of Open Access Journals (Sweden)

    Dario Greco

    Full Text Available In the mammalian central nervous system (CNS an important contingent of dopaminergic neurons are localized in the substantia nigra and in the ventral tegmental area of the ventral midbrain. They constitute an anatomically and functionally heterogeneous group of cells involved in a variety of regulatory mechanisms, from locomotion to emotional/motivational behavior. Midbrain dopaminergic neuron (mDA primary cultures represent a useful tool to study molecular mechanisms involved in their development and maintenance. Considerable information has been gathered on the mDA neurons development and maturation in vivo, as well as on the molecular features of mDA primary cultures. Here we investigated in detail the gene expression differences between the tissue of origin and ventral midbrain primary cultures enriched in mDA neurons, using microarray technique. We integrated the results based on different re-annotations of the microarray probes. By using knowledge-based gene network techniques and promoter sequence analysis, we also uncovered mechanisms that might regulate the expression of CNS genes involved in the definition of the identity of specific cell types in the ventral midbrain. We integrate bioinformatics and functional genomics, together with developmental neurobiology. Moreover, we propose guidelines for the computational analysis of microarray gene expression data. Our findings help to clarify some molecular aspects of the development and differentiation of DA neurons within the midbrain.

  10. Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector

    International Nuclear Information System (INIS)

    Shitara, Shingo; Kakeda, Minoru; Nagata, Keiko; Hiratsuka, Masaharu; Sano, Akiko; Osawa, Kanako; Okazaki, Akiyo; Katoh, Motonobu; Kazuki, Yasuhiro; Oshimura, Mitsuo; Tomizuka, Kazuma

    2008-01-01

    Telomerase-mediated life-span extension enables the expansion of normal cells without malignant transformation, and thus has been thought to be useful in cell therapies. Currently, integrating vectors including the retrovirus are used for human telomerase reverse transcriptase (hTERT)-mediated expansion of normal cells; however, the use of these vectors potentially causes unexpected insertional mutagenesis and/or activation of oncogenes. Here, we established normal human fibroblast (hPF) clones retaining non-integrating human artificial chromosome (HAC) vectors harboring the hTERT expression cassette. In hTERT-HAC/hPF clones, we observed the telomerase activity and the suppression of senescent-associated SA-β-galactosidase activity. Furthermore, the hTERT-HAC/hPF clones continued growing beyond 120 days after cloning, whereas the hPF clones retaining the silent hTERT-HAC senesced within 70 days. Thus, hTERT-HAC-mediated episomal expression of hTERT allows the extension of the life-span of human primary cells, implying that gene delivery by non-integrating HAC vectors can be used to control cellular proliferative capacity of primary cultured cells

  11. A co-culture system with three different primary human cell populations reveals that biomaterials and MSC modulate macrophage-driven fibroblast recruitment.

    Science.gov (United States)

    Caires, Hugo R; Barros da Silva, Patrícia; Barbosa, Mário A; Almeida, Catarina R

    2018-03-01

    The biological response to implanted biomaterials is a complex and highly coordinated phenomenon involving many different cell types that interact within 3D microenvironments. Here, we increased the complexity of a 3D platform to include at least 3 cell types that play a role in the host response upon scaffold implantation. With this system, it was possible to address how immune responses triggered by 3D biomaterials mediate recruitment of stromal cells that promote tissue regeneration, mesenchymal stromal/stem cells (MSC), or a foreign body response, fibroblasts. Primary human macrophages yielded the highest fibroblast recruitment when interacting with chitosan scaffolds but not polylactic acid. Interestingly, when there were MSC and fibroblasts in the same environment, macrophages in chitosan scaffolds again promoted a significant increase on fibroblast recruitment, but not of MSC. However, macrophages that were firstly allowed to interact with MSC within the scaffolds were no longer able to recruit fibroblasts. This study illustrates the potential to use different scaffolds to regulate the dynamics of recruitment of proregenerative or fibrotic cell types through immunomodulation. Overall, this work strengths the idea that ex vivo predictive systems need to consider the different players involved in the biological response to biomaterials and that timing of arrival of specific cell types will affect the outcome. Copyright © 2017 John Wiley & Sons, Ltd.

  12. Optimizing bone morphogenic protein 4-mediated human embryonic stem cell differentiation into trophoblast-like cells using fibroblast growth factor 2 and transforming growth factor-β/activin/nodal signalling inhibition.

    Science.gov (United States)

    Koel, Mariann; Võsa, Urmo; Krjutškov, Kaarel; Einarsdottir, Elisabet; Kere, Juha; Tapanainen, Juha; Katayama, Shintaro; Ingerpuu, Sulev; Jaks, Viljar; Stenman, Ulf-Hakan; Lundin, Karolina; Tuuri, Timo; Salumets, Andres

    2017-09-01

    Several studies have demonstrated that human embryonic stem cells (hESC) can be differentiated into trophoblast-like cells if exposed to bone morphogenic protein 4 (BMP4) and/or inhibitors of fibroblast growth factor 2 (FGF2) and the transforming growth factor beta (TGF-β)/activin/nodal signalling pathways. The goal of this study was to investigate how the inhibitors of these pathways improve the efficiency of hESC differentiation when compared with basic BMP4 treatment. RNA sequencing was used to analyse the effects of all possible inhibitor combinations on the differentiation of hESC into trophoblast-like cells over 12 days. Genes differentially expressed compared with untreated cells were identified at seven time points. Additionally, expression of total human chorionic gonadotrophin (HCG) and its hyperglycosylated form (HCG-H) were determined by immunoassay from cell culture media. We showed that FGF2 inhibition with BMP4 activation up-regulates syncytiotrophoblast-specific genes (CGA, CGB and LGALS16), induces several molecular pathways involved in embryo implantation and triggers HCG-H production. In contrast, inhibition of the TGF-β/activin/nodal pathway decreases the ability of hESC to form trophoblast-like cells. Information about the conditions needed for hESC differentiation toward trophoblast-like cells helps us to find an optimal model for studying the early development of human trophoblasts in normal and in complicated pregnancy. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  13. Model-based investigation of the circadian clock and cell cycle coupling in mouse embryonic fibroblasts: Prediction of RevErb-α up-regulation during mitosis.

    Science.gov (United States)

    Traynard, Pauline; Feillet, Céline; Soliman, Sylvain; Delaunay, Franck; Fages, François

    2016-11-01

    Experimental observations have put in evidence autonomous self-sustained circadian oscillators in most mammalian cells, and proved the existence of molecular links between the circadian clock and the cell cycle. Some mathematical models have also been built to assess conditions of control of the cell cycle by the circadian clock. However, recent studies in individual NIH3T3 fibroblasts have shown an unexpected acceleration of the circadian clock together with the cell cycle when the culture medium is enriched with growth factors, and the absence of such acceleration in confluent cells. In order to explain these observations, we study a possible entrainment of the circadian clock by the cell cycle through a regulation of clock genes around the mitosis phase. We develop a computational model and a formal specification of the observed behavior to investigate the conditions of entrainment in period and phase. We show that either the selective activation of RevErb-α or the selective inhibition of Bmal1 transcription during the mitosis phase, allow us to fit the experimental data on both period and phase, while a uniform inhibition of transcription during mitosis seems incompatible with the phase data. We conclude on the arguments favoring the RevErb-α up-regulation hypothesis and on some further predictions of the model. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Cell surface response of chemically transformed, malignant mouse embryonal fibroblasts and human colon cancer cells to the maturation-promoting agent, N,N-dimethylformamide

    International Nuclear Information System (INIS)

    Marks, M.E.

    1985-01-01

    The lactoperoxidase/ 125 I radioiodination procedure was used to probe the cell surface of normal, nontransformed AKR-2B mouse embryo fibroblasts and malignant, permanently methylcholanthrene-transformed AKR-2B (AKR-MCA) cells to establish the relationship between cell surface changes and transformation/differentiation in this call system. AKR-MCA cells displayed surface alterations secondary to N,N-dimethylformamide (DFM)-promoted differentiation. Growth of AKR-MCA cells in DMF virtually eliminated the 85,000 and 63,000 molecular weight surface proteins susceptible to radioiodination and increased surface material of ∼200,000 molecular weight. Thus, surface profiles of DFM-treated AKR-MCA cells were essentially identical to those of nontransformed AKR-2B cells. Experimentation was extended to a cultured human colon cancer cell line (HCT MOSER). HCT MOSER cells exposed to DMF manifested marked, reversible morphological and surface changes which occurred as a function of time of growth in DMF and DMF concentration. Interestingly, material reactive with anti-fibronectin was found on the surfaces and in the culture medium of DFM-treated HCT MOSER cells

  15. [Phenotype-based primary screening for drugs promoting neuronal subtype differentiation in embryonic stem cells with light microscope].

    Science.gov (United States)

    Gao, Yi-ning; Wang, Dan-ying; Pan, Zong-fu; Mei, Yu-qin; Wang, Zhi-qiang; Zhu, Dan-yan; Lou, Yi-jia

    2012-07-01

    To set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope. Hanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10(-6)mol/L) and Isobavachin (IBA, 10(-7)mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes. The cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons. Phenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.

  16. RABL6A, a Novel RAB-Like Protein, Controls Centrosome Amplification and Chromosome Instability in Primary Fibroblasts

    Science.gov (United States)

    Zhang, Xuefeng; Hagen, Jussara; Muniz, Viviane P.; Smith, Tarik; Coombs, Gary S.; Eischen, Christine M.; Mackie, Duncan I.; Roman, David L.; Van Rheeden, Richard; Darbro, Benjamin; Tompkins, Van S.; Quelle, Dawn E.

    2013-01-01

    RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the Alternative Reading Frame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53−/− MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis. PMID:24282525

  17. Miltefosine inhibits Chikungunya virus replication in human primary dermal fibroblasts [version 1; referees: 2 approved, 1 approved with reservations

    Directory of Open Access Journals (Sweden)

    Anuj Sharma

    2018-01-01

    Full Text Available Background: Chikungunya virus (CHIKV is a re-emerging pathogen that has caused widespread outbreaks affecting millions of people around the globe. Currently, there is no specific therapeutic drug against CHIKV, with symptomatic treatment only to manage the disease. Pi3-akt signaling has been implicated in infection of several viruses including that of CHIKV. Effect of Pi3-akt signaling inhibitors on CHIKV replication was evaluated in this study. Methods: Human primary dermal fibroblast cells were treated with inhibitors of the Pi3-akt signaling pathway. Suppression of CHIKV replication was evaluated as reduction in virus titer in cell supernatants. Effect of miltefosine (MF on CHIKV replication was evaluated in pre and post treatment regimen. Inhibition of virus replication was determined by cell growth, virus titer and western blot. Results: Inhibition of Akt-phosphorylation significantly inhibited CHIKV replication. No effect on CHIKV replication was observed after treatment with Pi3-kinase and mTOR activation inhibitors. Further, MF, an FDA-approved Akt-inhibitor, inhibited CHIKV replication in pre- and post-infection treatment regimens. Conclusion: Data suggests that Akt-phosphorylation can be an amenable target of therapy against CHIKV infection. This is the first study to show inhibition of CHIKV replication by MF, and presents a case for further development of MF as an anti-CHIKV drug.

  18. In vitro effects of preservative-free and preserved prostaglandin analogs on primary cultured human conjunctival fibroblast cells.

    Science.gov (United States)

    Kim, Eun Joo; Kim, Yeoun-Hee; Kang, Sun-Hee; Lee, Kyoo Won; Park, Young Jeung

    2013-12-01

    Long-term use of topical medication is needed for glaucoma treatment. One of the most commonly prescribed classes of hypotensive agents are prostaglandin analogs (PGs) used as both first-line monotherapy; as well as in combination therapy with other hypotensive agents. Several side effects of eye drops can be caused by preservatives. The purpose of this study was to evaluate the effects of PGs with varying concentrations of benzalkonium chloride (BAC), alternative preservatives, or no preservatives on human conjunctival fibroblast cells. Primary human conjunctival fibroblast cells were used in these experiments. Cells were exposed to the following drugs: BAC at different concentrations, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), tafluprost 0.0015% with/without 0.001% BAC and travoprost 0.004% (with 0.001% Polyquad) for 15 and 30 minutes. Cell cytotoxicity was evaluated by phase-contrast microscopy to monitor morphological changes of cells, Counting Kit-8 (CCK-8) assay to cell viability, and fluorescent activated cell sorting (FACS) analysis to measure apoptosis. BAC caused cell shrinkage and detachment from the plate in a dose-dependent manner. Morphological changes were observed in cells treated with bimatoprost 0.01% and latanoprost 0.005%. However, mild cell shrinkage was noted in cells treated with tafluprost 0.0015%, while a non-toxic effect was noted with travoprost 0.004% and preservative-free tafluprost 0.0015%. CCK-8 assay and FACS analysis showed all groups had a significantly decreased cell viability and higher apoptosis rate compared with the control group. However, travoprost 0.004% and preservative-free tafluprost 0.0015% showed lower cytotoxicity and apoptosis rate than other drugs. This in vitro study revealed that BAC-induced cytotoxicity is dose-dependent, although it is important to emphasize that the clinical significance of toxicity differences observed among the different PGs formulations has not yet been firmly

  19. Surface modified superparamagnetic nanoparticles: Interaction with fibroblasts in primary cell culture

    Energy Technology Data Exchange (ETDEWEB)

    Chapa Gonzalez, Christian; Roacho Pérez, Jorge A.; Martínez Pérez, Carlos A.; Olivas Armendáriz, Imelda [Instituto de Ingeniería y Tecnología, Universidad Autónoma de Ciudad Juárez, Ave. Del Charro #610 norte, Col. Partido Romero, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico); Jimenez Vega, Florinda [Instituto de Ciencias Biomédicas, Universidad Autónoma de Ciudad Juárez, Anillo envolvente del PRONAF y Estocolmo, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico); Castrejon Parga, Karen Y. [Instituto de Ingeniería y Tecnología, Universidad Autónoma de Ciudad Juárez, Ave. Del Charro #610 norte, Col. Partido Romero, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico); Garcia Casillas, Perla E., E-mail: pegarcia@uacj.mx [Instituto de Ingeniería y Tecnología, Universidad Autónoma de Ciudad Juárez, Ave. Del Charro #610 norte, Col. Partido Romero, C.P. 32320 Cd. Juárez, Chihuahua, México (Mexico)

    2014-12-05

    Highlights: • An inorganic layer before an organic material shell onto MNPs improves cell viability. • The coating type and the concentration of nanoparticles directly affect cell viability. • Modified magnetite nanoparticles with organic and inorganic materials was developed. - Abstract: The development of a variety of medical applications such as drug delivery, cell labeling, and medical imaging have been possible owing to the unique features exhibited by magnetic nanoparticles. Nanoparticle–cell interaction is related to the surface aspects of nanoparticle, which may be described based on their chemistry or inorganic/organic characteristics. The coating on particle surface reduces the inter-particle interactions and provides properties such as biocompatibility. Among the coating materials used for nanoparticles employed in biomedical applications, oleic acid is one of the most utilized due to its biocompatibility. However, a major drawback with this naturally occurring fatty acid is that it is easily oxidized by cells and this reduces their performance in biomedical applications. In order to avoid the direct contact of the cell with the magnetite particle, coating with an inorganic material prior to the oleic acid shell would be effective. This would retard the magnetite dissociation thereby improve the cell viability. Here we report our investigation on the effect of surface modified magnetite nanoparticles (MNPs) on the cell viability using primary cultures incubated with those particles. We prepared magnetite nanoparticles by chemical co-precipitation method; nanoparticle surface was first modified by silanol condensation followed by chemisorption of oleic acid. All nanostructures have a particle size less than 100 nm, depending on the material coating and superparamagnetic behavior. The saturated magnetizations (M{sub s}) of the magnetite samples coated with oleic acid (MAO; 49.15 emu/g) and double shell silica-oleic acid (MSAO; 46.16 emu/g) are

  20. AMP-activated protein kinase α2 and E2F1 transcription factor mediate doxorubicin-induced cytotoxicity by forming a positive signal loop in mouse embryonic fibroblasts and non-carcinoma cells.

    Science.gov (United States)

    Yang, Wookyeom; Park, In-Ja; Yun, Hee; Im, Dong-Uk; Ock, Sangmi; Kim, Jaetaek; Seo, Seon-Mi; Shin, Ha-Yeon; Viollet, Benoit; Kang, Insug; Choe, Wonchae; Kim, Sung-Soo; Ha, Joohun

    2014-02-21

    Doxorubicin is one of the most widely used anti-cancer drugs, but its clinical application is compromised by severe adverse effects in different organs including cardiotoxicity. In the present study we explored mechanisms of doxorubicin-induced cytotoxicity by revealing a novel role for the AMP-activated protein kinase α2 (AMPKα2) in mouse embryonic fibroblasts (MEFs). Doxorubicin robustly induced the expression of AMPKα2 in MEFs but slightly reduced AMPKα1 expression. Our data support the previous notion that AMPKα1 harbors survival properties under doxorubicin treatment. In contrast, analyses of Ampkα2(-/-) MEFs, gene knockdown of AMPKα2 by shRNA, and inhibition of AMPKα2 activity with an AMPK inhibitor indicated that AMPKα2 functions as a pro-apoptotic molecule under doxorubicin treatment. Doxorubicin induced AMPKα2 at the transcription level via E2F1, a transcription factor that regulates apoptosis in response to DNA damage. E2F1 directly transactivated the Ampkα2 gene promoter. In turn, AMPKα2 significantly contributed to stabilization and activation of E2F1 by doxorubicin, forming a positive signal amplification loop. AMPKα2 directly interacted with and phosphorylated E2F1. This signal loop was also detected in H9c2, C2C12, and ECV (human epithelial cells) cells as well as mouse liver under doxorubicin treatment. Resveratrol, which has been suggested to attenuate doxorubicin-induced cytotoxicity, significantly blocked induction of AMPKα2 and E2F1 by doxorubicin, leading to protection of these cells. This signal loop appears to be non-carcinoma-specific because AMPKα2 was not induced by doxorubicin in five different tested cancer cell lines. These results suggest that AMPKα2 may serve as a novel target for alleviating the cytotoxicity of doxorubicin.

  1. Immunization against Capillaria hepatica: The effects of primary infections, X-irradiated stages, non-embryonated eggs and soluble egg extracts

    Energy Technology Data Exchange (ETDEWEB)

    Zahner, H.; Schmidt, H.; Laemmler, G.; Geyer, E.

    1980-01-01

    The worm reproductivity was significantly suppressed in a sublethal challenge infection given 11 days after a primary infection of Mastomys natalensis with 50, 150, 400, and 800 eggs per animal. The administration of 600 X-irradiated (2.2 Krd) embryonated eggs 36 days before challenge as well as an intraperitoneal injection of non-embryonated eggs 12, 10, 8, 6, 4, and 2 days before challenge also reduced significantly the egg production of a weak (50 eggs/animal) infection. No effect was observed on a moderate challenge (300 eggs/animal). The effect was not markedly enhanced by the repeated administration of X-irradiated eggs or by the combination of X-irradiated infective eggs and non-embryonated eggs. Immunization of mice with soluble egg extracts resulted in significant reduction of egg production determined 60 days after challenge. Two hundred and thirty eggs of C. hepatica/g bodyweight proved to be a lethal infection dose for M. natalensis. The animals died between 20 and 35 days after infection. After single infections with 50, 150, 400, or 800 eggs per animal the mortality of Mastomys challenged 36 or 52 days later was reduced to 0-30%. Using X-irradiated embryonated eggs for immunization only repeated administration led to protection in 70 to 80%, of the animals. About 40% of the animals could be protected by the intraperitoneal injection of non-embryonated eggs. If death occurred it was delayed. The combination of X-irradiated stages and eggs did not enhance the protection.

  2. Dextran sulphate crowding and sodium deoxycholate lysis of primary breast fibroblast cells achieve extracellular matrix deposition and decellularization for breast cancer stem cell culture

    Directory of Open Access Journals (Sweden)

    Aroem Naruni

    2016-01-01

    .Extracellular matrix provides tissue integrity, acts as a native scaffold for cell attachment and interaction and alsoserves as reservoir for growth factors. The aim of this experiment w asto achieve the deposition and decellularizationof ECM.Methods: Cells crowder have been developed to increase ECM deposit in the primary breast fibroblast cells layerobtained from isolation of single cell from breast mammoplasty specimen. Five hundred kDa dextran was addedinto DMEM medium containing 0.5% fetal bovine serum (FBS and 100 μm of L-ascorbic acid 2-phosphate. Afterseven days, cells were lysed by sodium deoxycholate (DOC. Results were observed in a fluorescence microscope.Results: Extracellular matrix deposition and decellularization of primary breast fibroblast cells were detected byusing extracellular matrix protein, fibronectin via rabbit anti human fibronectin and anti-rabbit IgG conjugated withAlexa Fluor 488.Conclusion: Dextran sulphate increased extracellular matrix deposit in primary breast fibroblast cell layer and thetreatment with sodium deoxycolate lysed cells resulted in extracellular matrix decellularization. (Health ScienceJournal of Indonesia 2015;6:43-7Keywords: extracellular matrix, breast cancer stem cell, breast fibroblast cell

  3. Plasma rich in growth factors (PRGF-Endoret) stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-beta1-Induced myodifferentiation.

    Science.gov (United States)

    Anitua, Eduardo; Sanchez, Mikel; Merayo-Lloves, Jesus; De la Fuente, Maria; Muruzabal, Francisco; Orive, Gorka

    2011-08-01

    Plasma rich in growth factors (PRGF-Endoret) technology is an autologous platelet-enriched plasma obtained from patient's own blood, which after activation with calcium chloride allows the release of a pool of biologically active proteins that influence and promote a range of biological processes including cell recruitment, and growth and differentiation. Because ocular surface wound healing is mediated by different growth factors, we decided to explore the potential of PRGF-Endoret technology in stimulating the biological processes related with fibroblast-induced tissue repair. Furthermore, the anti-fibrotic properties of this technology were also studied. Blood from healthy donors was collected, centrifuged and, whole plasma column (WP) and the plasma fraction with the highest platelet concentration (F3) were drawn off, avoiding the buffy coat. Primary human cells including keratocytes and conjunctival fibroblasts were used to perform the "in vitro" investigations. The potential of PRGF-Endoret in promoting wound healing was evaluated by means of a proliferation and migration assays. Fibroblast cells were induced to myofibroblast differentiation after the treatment with 2.5 ng/mL of TGF-β1. The capability of WP and F3 to prevent and inhibit TGF-β1-induced differentiation was evaluated. Results show that this autologous approach significantly enhances proliferation and migration of both keratocytes and conjunctival fibroblasts. In addition, plasma rich in growth factors prevents and inhibits TGF-β1-induced myofibroblast differentiation. No differences were found between WP and F3 plasma fractions. These results suggest that PRGF-Endoret could reduce scarring while stimulating wound healing in ocular surface. F3 or whole plasma column show similar biological effects in keratocytes and conjunctival fibroblast cells.

  4. Rat primary embryo fibroblast cells suppress transformation by the E6 and E7 genes of human papillomavirus type 16 in somatic hybrid cells.

    OpenAIRE

    Miyasaka, M; Takami, Y; Inoue, H; Hakura, A

    1991-01-01

    The E6 and E7 genes of human papillomavirus type 16 (HPV-16) transform established lines of rat cells but not rat cells in primary culture irrespective of the expression of the two genes. The reason for this difference between the susceptibilities of cell lines and primary cells was examined by using hybrid cells obtained by somatic cell fusion of rat cell lines transformed by the E6 and E7 genes of HPV-16 and freshly isolated rat embryo fibroblast cells. In these hybrid cells, transformed ph...

  5. Simultaneous Expression of Cancer Stem Cell-Like Properties and Cancer-Associated Fibroblast-Like Properties in a Primary Culture of Breast Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Ishikawa, Mami; Inoue, Takahiro; Shirai, Takuma; Takamatsu, Kazuhiko; Kunihiro, Shiori; Ishii, Hirokazu [Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe 650-0047 (Japan); Nishikata, Takahito, E-mail: nisikata@konan-u.ac.jp [Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, Kobe 650-0047 (Japan); Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, Kobe 650-0047 (Japan)

    2014-07-31

    The importance of cancer-associated fibroblasts (CAFs) in cancer biology has been recently highlighted owing to their critical roles in cancer growth, progression, metastasis, and therapeutic resistance. We have previously established a primary culture of breast cancer cells, which showed epithelial-mesenchymal transition and cancer stem cell-like properties. In this study, we found that the primary culture also showed CAF-like properties. For example, hypoxia inducible factor 1α (HIF1A) and its downstream genes, nuclear factor-kappa B2 (NF-κB2) and BCL2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), and many enzymes involved in glycolysis, such as GAPDH, LDH, PGAM1, and PKM2, were highly overexpressed in the primary culture. Moreover, media conditioned with the primary culture cells enhanced the growth of breast cancer cells. Similar to previous CAF studies, this enhancement suggested to be occurred through fibroblast growth factor signaling. This MCKH primary culture cell, which showed simultaneous expression of tumorigenic and CAF properties, offers a unique experimental system for studying the biology of CAFs.

  6. Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

    Science.gov (United States)

    Cong, Shan; Cao, Guifang; Liu, Dongjun

    2014-12-01

    To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1-5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

  7. Immunofluorescence Microscopy and mRNA Analysis of Human Embryonic Stem Cells (hESCs) Including Primary Cilia Associated Signaling Pathways

    DEFF Research Database (Denmark)

    Vestergaard, Maj Linea; Awan, Aashir; Warzecha, Caroline Becker

    2016-01-01

    onto 16-well glass chambers, and continuing with the general IFM and qPCR anlysis. The techniques are illustrated with results on cellular localization of transcriptional factors and components of the Hedgehog, Wnt, PDGF, and TGFβ signaling pathways to primary cilia in stem cell maintenance......This chapter describes the procedures for immunofluorescence microscopy (IFM) and quantitative PCR (qPCR) analyses of human embryonic stem cells (hESCs) grown specifically under feeder-free conditions. A detailed protocol is provided outlining the steps from initially growing the cells, passaging...

  8. Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts

    Directory of Open Access Journals (Sweden)

    María Questa

    2016-03-01

    Full Text Available Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls.

  9. Growth of primary embryo cells in a microculture system.

    Science.gov (United States)

    Villa, Max; Pope, Sara; Conover, Joanne; Fan, Tai-Hsi

    2010-04-01

    We present optimal perfusion conditions for the growth of primary mouse embryonic fibroblasts (mEFs) and mouse embryonic stem cells (mESCs) using a microfluidic perfusion culture system. In an effort to balance nutrient renewal while ensuring the presence of cell secreted factors, we found that the optimal perfusion rate for culturing primary embryonic fibroblasts (mEFs) in our experimental setting is 10 nL/min with an average flow velocity 0.55 microm/s in the microchannel. Primary mEFs may have a greater dependence on cell secreted factors when compared to their immortalized counterpart 3T3 fibroblasts cultured under similar conditions. Both the seeding density and the perfusion rate are critical for the proliferation of primary cells. A week long cultivation of mEFs and mESCs using the microculture system exhibited similar morphology and viability to those grown in a petri dish. Both mEFs and mESCs were analyzed using fluorescence immunoassays to determine their proliferative status and protein expression. Our results demonstrate that a perfusion-based microculture environment is capable of supporting the highly proliferative status of pluripotent embryonic stem cells.

  10. Identification of transcripts regulated by CUG-BP, Elav-like family member 1 (CELF1 in primary embryonic cardiomyocytes by RNA-seq

    Directory of Open Access Journals (Sweden)

    Yotam Blech-Hermoni

    2015-12-01

    Full Text Available CUG-BP, Elav-like family member 1 (CELF1 is a multi-functional RNA binding protein that regulates pre-mRNA alternative splicing in the nucleus, as well as polyadenylation status, mRNA stability, and translation in the cytoplasm [1]. Dysregulation of CELF1 has been implicated in cardiomyopathies in myotonic dystrophy type 1 and diabetes [2–5], but the targets of CELF1 regulation in the heart have not been systematically investigated. We previously demonstrated that in the developing heart CELF1 expression is restricted to the myocardium and peaks during embryogenesis [6–8]. To identify transcripts regulated by CELF1 in the embryonic myocardium, RNA-seq was used to compare the transcriptome of primary embryonic cardiomyocytes following siRNA-mediated knockdown of CELF1 to that of controls. Raw data files of the RNA-seq reads have been deposited in NCBI's Gene Expression Omnibus [9] under the GEO Series accession number GSE67360. These data can be used to identify transcripts whose levels or alternative processing (i.e., alternative splicing or polyadenylation site usage are regulated by CELF1, and should provide insight into the pathways and processes modulated by this important RNA binding protein during normal heart development and during cardiac pathogenesis.

  11. Fibroblast Growth Factor Receptor 3 (FGFR3–Analyses of the S249C Mutation and Protein Expression in Primary Cervical Carcinomas

    Directory of Open Access Journals (Sweden)

    Haiyan Dai

    2001-01-01

    Full Text Available Fibroblast growth factor receptor 3 (FGFR3 seems to play an inhibitory role in bone development, as activating mutations in the gene underlie disorders such as achondroplasia and thanatophoric dysplasia. Findings from multiple myeloma (MM indicate that FGFR3 also can act as an oncogene, and mutation of codon 249 in the fibroblast growth factor receptor 3 (FGFR3 gene was recently detected in 3/12 primary cervical carcinomas. We have analysed 91 cervical carcinomas for this specific S249C mutation using amplification created restriction site methodology (ACRS, and detected no mutations. Immunohistochemistry was performed on 73 of the tumours. Reduced protein staining was seen in 43 (58.8% samples. Six of the tumours (8.2% revealed increased protein staining compared with normal cervical tissue. These patients had a better prognosis than those with reduced or normal levels, although not statistically significant. This report weakens the hypothesis of FGFR3 as an oncogene of importance in cervical carcinomas.

  12. Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix.

    Science.gov (United States)

    Müller, Stephan A; van der Smissen, Anja; von Feilitzsch, Margarete; Anderegg, Ulf; Kalkhof, Stefan; von Bergen, Martin

    2012-12-01

    Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.

  13. The influence of glancing angle deposited nano-rough platinum surfaces on the adsorption of fibrinogen and the proliferation of primary human fibroblasts

    International Nuclear Information System (INIS)

    Dolatshahi-Pirouz, A; Foss, M; Chevallier, J; Besenbacher, F; Pennisi, C P; Yoshida, K; Skeldal, S; Andreasen, P; Zachar, V

    2009-01-01

    We have used the glancing angle deposition (GLAD) method as a simple and fast method to generate nano-rough surfaces for protein adsorption experiments and cell assays. The surface roughness and the detailed geometrical surface morphology of the thin films were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). As the GLAD deposition angle approaches grazing incidence, sharp and whisker-like columnar protrusions are formed. Smaller and less sharp surface features appear for the thin films synthesized at higher deposition angles. By changing the GLAD deposition angle together with the total amount of mass deposited per area on the respective surfaces, the size of the surface features can be varied on the nanoscale. Using the GLAD topographies as model surfaces, we have investigated the influence of the nano-roughness on fibrinogen adsorption and on the proliferation of primary human fibroblasts. It is found that fibrinogen, an important blood protein, preferentially adheres on the whisker-like nano-rough substrates in comparison to a flat surface. Furthermore, the proliferation of the human fibroblasts is significantly reduced on the nano-rough substrates. These results demonstrate that the GLAD technique can be used to fabricate nano-rough surface morphologies that significantly influence both protein and cellular adhesion to surfaces and are therefore well suited for biological assays.

  14. A feeder- and xeno-free human induced pluripotent stem cell line obtained from primary human dermal fibroblasts with epigenetic repression of reprogramming factors expression: GPCCi001-A

    Directory of Open Access Journals (Sweden)

    Michał Stefan Lach

    2017-04-01

    Full Text Available The primary human dermal fibroblasts (PHDFs from breast cancer patient were obtained to generate the human induced pluripotent stem cell line GPCCi001-A via lentiviral transfection. Thus, a modified EF1a-hSTEMCCA-loxP with tetO operator which regulates transgene expression was used. This method takes advantage of epigenetic regulation of transcription and allows for stable silencing of the reprogramming factors in obtained hiPS cells. To increase the potential utility of hiPSCs for clinical applications, they were adapted to feeder- and xeno-free conditions. The pluripotency of GPCCi001-A cell line and ability to differentiate into three germ layers was confirmed.

  15. ATP-dependent paracrine communication between enteric neurons and glia in a primary cell culture derived from embryonic mice.

    Science.gov (United States)

    Gomes, P; Chevalier, J; Boesmans, W; Roosen, L; van den Abbeel, V; Neunlist, M; Tack, J; Vanden Berghe, P

    2009-08-01

    The importance of dynamic interactions between glia and neurons is increasingly recognized, both in the central and enteric nervous system. However, apart from their protective role, little is known about enteric neuro-glia interaction. The aim was to investigate neuro-glia intercellular communication in a mouse culture model using optical techniques. Complete embryonic (E13) guts were enzymatically dissociated, seeded on coverslips and studied with immunohistochemistry and Ca(2+)-imaging. Putative progenitor-like cells (expressing both PGP9.5 and S-100) differentiated over approximately 5 days into glia or neurons expressing typical cell-specific markers. The glia-neuron ratio could be manipulated by specific supplements (N2, G5). Neurons and glia were functionally identified both by their Ca(2+)-response to either depolarization (high K(+)) or lysophosphatidic acid and by the expression of typical markers. Neurons responded to ACh, DMPP, 5-HT, ATP and electrical stimulation, while glia responded to ATP and ADPbetas. Inhibition of glial responses by MRS2179 suggests involvement of P2Y1 receptors. Neuronal stimulation also caused delayed glial responses, which were reduced by suramin and by exogenous apyrases that catalyse nucleotide breakdown. Conversely, glial responses were enhanced by ARL-67156, an ecto-ATPase inhibitor. In this mouse enteric co-culture, functional glia and neurons can be easily monitored using optical techniques. Glial cells can be activated directly by ATP or ADPbetas. Activation of neuronal cells (DMPP, K(+)) causes secondary responses in glial cells, which can be modulated by tuning ATP and ADP breakdown. This strongly supports the involvement of paracrine purinergic communication between enteric neurons and glia.

  16. Tumor-Induced Osteomalacia Caused by Primary Fibroblast Growth Factor 23 Secreting Neoplasm in Axial Skeleton: A Case Report

    Directory of Open Access Journals (Sweden)

    Gunjan Y. Gandhi

    2012-01-01

    Full Text Available We report the case of a 66-year-old woman with tumor-induced osteomalacia (TIO caused by fibroblast growth factor 23 (FGF-23 secreting mesenchymal tumor localized in a lumbar vertebra and review other cases localized to the axial skeleton. She presented with nontraumatic low back pain and spontaneous bilateral femur fractures. Laboratory testing was remarkable for low serum phosphorus, phosphaturia, and significantly elevated serum FGF-23 level. Magnetic resonance imaging (MRI of the lumbar spine showed a focal lesion in the L-4 vertebra which was hypermetabolic on positron emission tomography (PET scan. A computed tomography (CT guided needle biopsy showed a low grade spindle cell neoplasm with positive FGF-23 mRNA expression by reverse transcriptase polymerase chain reaction (RT-PCR, confirming the diagnosis of a phosphaturic mesenchymal tumor mixed connective tissue variant (PMTMCT. The patient elected to have surgery involving anterior resection of L-4 vertebra with subsequent normalization of serum phosphorus. Including the present case, we identified 12 cases of neoplasms localized to spine causing TIO. To our knowledge, this paper represents the first documented case of lumbar vertebra PMT causing TIO. TIO is a rare metabolic bone disorder that carries a favorable prognosis. When a lesion is identifiable, surgical intervention is typically curative.

  17. PDGFR alpha signaling in the primary cilium regulates NHE1-dependent fibroblast migration via coordinated differential activity of MEK1/2-ERK1/2-p90(RSK) and AKT signaling pathways

    DEFF Research Database (Denmark)

    Clement, Ditte L.; Mally, Sabine; Stock, Christian

    2013-01-01

    In fibroblasts, platelet-derived growth factor receptor alpha (PDGFR alpha) is upregulated during growth arrest and compartmentalized to the primary cilium. PDGF-AA mediated activation of the dimerized ciliary receptor produces a phosphorylation cascade through the PI3K-AKT and MEK1/2-ERK1/2 path...

  18. Fibroblastic rheumatism

    Directory of Open Access Journals (Sweden)

    Jyoti Ranjan Parida

    2017-01-01

    Full Text Available Fibroblastic rheumatism (FR is a rare dermoarthopathy reported from different parts of the world since 1980. Although the exact cause is unknown, few reports implicate infection may be a triggering event. Patients usually present with multiple skin nodules and polyarthropathy with progressive skin contractures. Laboratory parameters including acute phase reactants are usually normal. The confirmatory diagnosis is based on histopathologic study of skin nodules, which demonstrate fibroblastic proliferation, thickened collagen fibers, dermal fibrosis, and decreased number of elastic fibers. Immunoreactivity for b-catenin, smooth muscle actin, and the monoclonal antibody HHF35 show myofibroblastic differentiation. Treatments with oral prednisolone and other disease-modifying drugs such as methotrexate, infliximab, and interferon have been tried with variable success. In general, skin lesions respond more aptly than joint symptoms indicating that skin fibroblast is more amenable to treatment than synovial fibroblasts. Awareness regarding this orphan disease among clinicians and pathologists will help in more reporting of such cases and finding out optimal treatment regimen.

  19. Identification of lipid profile on γ-irradiated primary cultured human diploid fibroblasts by UPLC-ESI-QTOF-MS

    International Nuclear Information System (INIS)

    Lee, Eun Kyeong; Yun, Hyun Jin; Kim, Sun Young; Kim, So Ra; Kim, Eun Ju; Kang, Chang Mo; Hwang, Geum Sook

    2013-01-01

    The study of biomarkers in normal-tissue radiobiology is of great importance to people subjected to non-destructive testing, to nuclear power industry, and to radiological terrorism. Cytogenetics has remained the 'gold standard' for the detection of chromosomal changes, but this method is cumbersome and time consuming. Therefore, it is necessary to establish a rapid, high-throughput biological monitoring protocol to determine the exposure radiation dose in order to rapidly triage mass casualties. Metabolomics studies is an effective platform for identification and quantification of the complete set of metabolites in a biological system, and it has been successfully provides important insights into physiological and disease states and facilitate in depth understanding of underlying biochemical pathways. Radiation metabolomics has been used to identify radiation effects at the metabolite levels in cells and in urine or serum collected from laboratory animals such as mouse, rat and monkey. Recently, a few radiation metabolomics studies showed that multiple metabolites related to the purine, inositol or pyrimidine metabolism, fatty acid biosynthesis and glycolysis/gluconeogenesis could be potential minimal-invasive markers for radiation exposure. However, there is little consistency in these studies due to differences in analyzed sample (cells, urine, serum), animal models (mouse, rat and monkey). Furthermore, the biological effects of ionizing radiation (IR) were differently regulated in the different absorbed doses, dose-rates and type of IR. Thus, it is necessary to explore the entire biosphere profiling of metabolic markers to make reasonable biological explanation under radiation exposure. We hypothesized that the variations in lipid expression in IR-induced cells would result in a unique lipid profile in human diploid fibroblasts (HDF), which can be detected with the targeted and quantitative metabolomics approach. Irradiated HDF cells showed the dose

  20. Identification of lipid profile on γ-irradiated primary cultured human diploid fibroblasts by UPLC-ESI-QTOF-MS

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun Kyeong; Yun, Hyun Jin; Kim, Sun Young; Kim, So Ra; Kim, Eun Ju; Kang, Chang Mo [Division of Radiation Effect, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Hwang, Geum Sook [Integrated Metabolomics Research Group, Korea Basic Science Institute, Sejong (Korea, Republic of)

    2013-11-15

    The study of biomarkers in normal-tissue radiobiology is of great importance to people subjected to non-destructive testing, to nuclear power industry, and to radiological terrorism. Cytogenetics has remained the 'gold standard' for the detection of chromosomal changes, but this method is cumbersome and time consuming. Therefore, it is necessary to establish a rapid, high-throughput biological monitoring protocol to determine the exposure radiation dose in order to rapidly triage mass casualties. Metabolomics studies is an effective platform for identification and quantification of the complete set of metabolites in a biological system, and it has been successfully provides important insights into physiological and disease states and facilitate in depth understanding of underlying biochemical pathways. Radiation metabolomics has been used to identify radiation effects at the metabolite levels in cells and in urine or serum collected from laboratory animals such as mouse, rat and monkey. Recently, a few radiation metabolomics studies showed that multiple metabolites related to the purine, inositol or pyrimidine metabolism, fatty acid biosynthesis and glycolysis/gluconeogenesis could be potential minimal-invasive markers for radiation exposure. However, there is little consistency in these studies due to differences in analyzed sample (cells, urine, serum), animal models (mouse, rat and monkey). Furthermore, the biological effects of ionizing radiation (IR) were differently regulated in the different absorbed doses, dose-rates and type of IR. Thus, it is necessary to explore the entire biosphere profiling of metabolic markers to make reasonable biological explanation under radiation exposure. We hypothesized that the variations in lipid expression in IR-induced cells would result in a unique lipid profile in human diploid fibroblasts (HDF), which can be detected with the targeted and quantitative metabolomics approach. Irradiated HDF cells showed the dose

  1. Reconstitution of active telomerase in primary human foreskin fibroblasts : effects on proliferative characteristics and response to ionizing radiation

    NARCIS (Netherlands)

    Kampinga, H.H.; Waarde-Verhagen, M.A.W.H. van; Assen-Bolt, A.J. van; Rodemann, H.P.; Prowse, K.R.; Linskens, M.H.K.

    2004-01-01

    Purpose: Telomere shortening has been proposed to trigger senescence, and since most primary cells do not express active telomerase, reactivation of telomerase activity was proposed as a safe and non-transforming way of immortalizing cells. However, to study radiation responses, it is as yet unclear

  2. High expression of the taurine transporter TauT in primary cilia of NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Christensen, Søren Tvorup; Voss, Jesper W.; Teilmann, Stefan C.

    2005-01-01

    Taurine, present in high concentrations in various mammalian cells, is essential for regulation of cell volume, cellular oxidative status as well as the cellular Ca2+ homeostasis. Cellular taurine content is a balance between active uptake through the saturable, Na+-dependent taurine transporter...... TauT expression and (iii) long-term exposure to hypertonic taurine medium, i.e., growth medium supplemented with 100 mM taurine, reduces ciliary TauT expression. These results point to an important role of taurine in the regulation of physiological processes located to the primary cilium....

  3. Podoplanin-positive Cancer-associated Stromal Fibroblasts in Primary Tumor and Synchronous Lymph Node Metastases of HER2-overexpressing Breast Carcinomas.

    Science.gov (United States)

    Niemiec, Joanna; Adamczyk, Agnieszka; Harazin-Lechowska, Agnieszka; Ambicka, Aleksandra; Grela-Wojewoda, Aleksandra; Majchrzyk, Kaja; Kruczak, Anna; Sas-Korczyńska, Beata; Ryś, Janusz

    2018-04-01

    We compared the status of stromal podoplanin-positive cancer-associated fibroblasts (ppCAFs) between primary tumors and paired synchronous lymph node metastases (LNMs) and analyzed the prognostic significance of tumoral ppCAFs in 203 patients with human epidermal growth factor receptor 2-positive breast carcinoma. ppCAFs were found in 167/203 and in 35/87 tumors and LNM, respectively. ppCAFs were most frequently found in tumors and corresponding LNM (n=52, 59.8%; p=0.001). However, for all LNMs (n=12) without ppCAFs, their paired tumors also lacked ppCAFs. In both tumors and LNMs, ppCAFs were α-smooth muscle actin-positive and cluster of differentiation 21 protein-negative, suggesting them not to be resident lymph node cells. Moreover, in our series, the presence of ppCAFs in tumors was borderline related to poor disease-free survival (p=0.058). These results speak in favor of a hypothesis suggesting ppCAFs accompany metastatic cancer cells migrating from tumor to LNMs. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  4. The hallmarks of fibroblast ageing.

    Science.gov (United States)

    Tigges, Julia; Krutmann, Jean; Fritsche, Ellen; Haendeler, Judith; Schaal, Heiner; Fischer, Jens W; Kalfalah, Faiza; Reinke, Hans; Reifenberger, Guido; Stühler, Kai; Ventura, Natascia; Gundermann, Sabrina; Boukamp, Petra; Boege, Fritz

    2014-06-01

    Ageing is influenced by the intrinsic disposition delineating what is maximally possible and extrinsic factors determining how that frame is individually exploited. Intrinsic and extrinsic ageing processes act on the dermis, a post-mitotic skin compartment mainly consisting of extracellular matrix and fibroblasts. Dermal fibroblasts are long-lived cells constantly undergoing damage accumulation and (mal-)adaptation, thus constituting a powerful indicator system for human ageing. Here, we use the systematic of ubiquitous hallmarks of ageing (Lopez-Otin et al., 2013, Cell 153) to categorise the available knowledge regarding dermal fibroblast ageing. We discriminate processes inducible in culture from phenomena apparent in skin biopsies or primary cells from old donors, coming to the following conclusions: (i) Fibroblasts aged in culture exhibit most of the established, ubiquitous hallmarks of ageing. (ii) Not all of these hallmarks have been detected or investigated in fibroblasts aged in situ (in the skin). (iii) Dermal fibroblasts aged in vitro and in vivo exhibit additional features currently not considered ubiquitous hallmarks of ageing. (iv) The ageing process of dermal fibroblasts in their physiological tissue environment has only been partially elucidated, although these cells have been a preferred model of cell ageing in vitro for decades. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. The effect of injection speed and serial injection on propidium iodide entry into cultured HeLa and primary neonatal fibroblast cells using lance array nanoinjection.

    Science.gov (United States)

    Sessions, John W; Lewis, Tyler E; Skousen, Craig S; Hope, Sandra; Jensen, Brian D

    2016-01-01

    Although site-directed genetic engineering has greatly improved in recent years, particularly with the implementation of CRISPR-Cas9, the ability to deliver these molecular constructs to a wide variety of cell types without adverse reaction is still a challenge. One non-viral transfection method designed to address this challenge is a MEMS based biotechnology described previously as lance array nanoinjection (LAN). LAN delivery of molecular loads is based upon the combinational use of electrical manipulation of loads of interest and physical penetration of target cell membranes. This work explores an original procedural element to nanoinjection by investigating the effects of the speed of injection and also the ability to serially inject the same sample. Initial LAN experimentation demonstrated that injecting at speeds of 0.08 mm/s resulted in 99.3 % of cultured HeLa 229 cells remaining adherent to the glass slide substrate used to stage the injection process. These results were then utilized to examine whether or not target cells could be injected multiple times (1, 2, and 3 times) since the injection process was not pulling the cells off of the glass slide. Using two different current control settings (1.5 and 3.0 mA) and two different cell types (HeLa 229 cells and primary neonatal fibroblasts [BJ(ATCC(®) CRL-2522™)], treatment samples were injected with propidium iodide (PI), a cell membrane impermeable nucleic acid dye, to assess the degree of molecular load delivery. Results from the serial injection work indicate that HeLa cells treated with 3.0 mA and injected twice (×2) had the greatest mean PI uptake of 60.47 % and that neonatal fibroblasts treated with the same protocol reached mean PI uptake rates of 20.97 %. Both experimental findings are particularly useful because it shows that greater molecular modification rates can be achieved by multiple, serial injections via a slower injection process.

  6. Role of Circulating Fibroblast Growth Factor 21 Measurement in Primary Prevention of Coronary Heart Disease Among Chinese Patients With Type 2 Diabetes Mellitus.

    Science.gov (United States)

    Lee, Chi Ho; Woo, Yu Cho; Chow, Wing Sun; Cheung, Chloe Yu Yan; Fong, Carol Ho Yi; Yuen, Michele Mae Ann; Xu, Aimin; Tse, Hung Fat; Lam, Karen Siu Ling

    2017-06-06

    Fibroblast growth factor 21 (FGF21) has demonstrated beneficial effects on lipid and carbohydrate metabolism. In cross-sectional studies, an association of raised circulating FGF21 levels with coronary heart disease (CHD) was found in some but not all studies. Here we investigated prospectively whether baseline serum FGF21 levels could predict incident CHD in subjects with type 2 diabetes mellitus and no known cardiovascular diseases. Baseline serum FGF21 levels were measured in 3528 Chinese subjects with type 2 diabetes mellitus recruited from the Hong Kong West Diabetes Registry. The role of baseline serum FGF21 levels in predicting incident CHD over a median follow-up of 3.8 years was analyzed using Cox regression analysis. Among 3528 recruited subjects without known cardiovascular diseases, 147 (4.2%) developed CHD over a mean follow-up of 4 years. Baseline serum log-transformed FGF21 levels were significantly higher in those who had incident CHD than those who did not (222.7 pg/mL [92.8-438.4] versus 151.1 pg/mL [75.6-274.6]; P 1). On multivariable Cox regression analysis, baseline serum FGF21 levels, using an optimal cutoff of 206.22 pg/mL derived from our study, independently predicted incident CHD (hazard ratio, 1.55; 95% CI, 1.10-2.19; P =0.013) and significantly improved net reclassification index and integrated discrimination improvement after adjustment for conventional cardiovascular risk factors. We have demonstrated, for the first time, that serum FGF21 level is an independent predictor of incident CHD and might be usefully utilized as a biomarker for identifying type 2 diabetes mellitus subjects with raised CHD risk, for primary prevention. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  7. Generation of hematopoietic lineage cells from embryonic like cells

    Directory of Open Access Journals (Sweden)

    Gholam Reza Khamisipour

    2014-10-01

    Full Text Available Background: Epigenetic reprogramming of somatic cells into embryonic stem cells has attracted much attention, because of the potential for stem cell transplantation and compatibility with recipient. However, the therapeutic application of either nuclear transfer or nuclear fusion of somatic cell has been hindered by technical complications as well as ethical objections. Recently, a new method is reported whereby ectopic expression of embryonic specific transcription factors was shown to induce fibroblasts to become embryonic like SCs (induced pluripotent stem cells. A major limitation of this method is the use of potentially harmful genome integrating viruses such as reto- or lentivirus. The main aim of this investigation was generation of human hematopoietic stem cells from induced fibroblasts by safe adenovectors carrying embryonically active genes. Material and Methods: Isolated fibroblasts from foreskin were expanded and recombinant adenoviruses carrying human Sox2, Oct4, Klf4, cMyc genes were added to culture. After formation of embryonic like colonies and cell expansion, they were transferred to embryonic media without bFGF, and embryoid bodies were cultured on stromal and non-stromal differentiation media for 14 days. Results: Expression of CD34 gene and antigenic markers, CD34, CD38 & CD133 in stromal culture showed significant difference with non-differentiation and non-stromal media. Conclusion: These findings show high hematopoietic differentiation rate of Adeno-iPS cells in stromal culture and no need to use growth factors. While, there was no difference between non-differentiation and non-stromal media.

  8. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    International Nuclear Information System (INIS)

    Yue, Xiao-shan; Fujishiro, Masako; Toyoda, Masashi; Akaike, Toshihiro; Ito, Yoshihiro

    2010-01-01

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  9. Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

    Energy Technology Data Exchange (ETDEWEB)

    Yue, Xiao-shan [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Fujishiro, Masako [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Toyoda, Masashi [Department of Reproductive Biology, National Institute for Child Health and Development, 2-10-1, Okura, Setagaya-ku, Tokyo 157-8535 (Japan); Akaike, Toshihiro [Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan); Ito, Yoshihiro, E-mail: y-ito@riken.jp [Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Biomolecular Engineering, Graduate School of Bioscience and Technology, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501 (Japan)

    2010-04-16

    In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

  10. Inhibition of fibroblast growth by Notch1 signaling is mediated by induction of Wnt11-dependent WISP-1.

    Directory of Open Access Journals (Sweden)

    Zhao-Jun Liu

    Full Text Available Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM. They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1(Flox/Flox embryonic fibroblasts (MEFs. Notch1-deficient (Notch1(-/- MEFs displayed faster growth and motility rate compared to Notch1(Flox/Flox MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1 in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441, which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1. Functionally, "Notch-activated" stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4 in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression.

  11. Microarray mRNA expression analysis of Fanconi anemia fibroblasts.

    Science.gov (United States)

    Galetzka, D; Weis, E; Rittner, G; Schindler, D; Haaf, T

    2008-01-01

    Fanconi anemia (FA) cells are generally hypersensitive to DNA cross-linking agents, implying that mutations in the different FANC genes cause a similar DNA repair defect(s). By using a customized cDNA microarray chip for DNA repair- and cell cycle-associated genes, we identified three genes, cathepsin B (CTSB), glutaredoxin (GLRX), and polo-like kinase 2 (PLK2), that were misregulated in untreated primary fibroblasts from three unrelated FA-D2 patients, compared to six controls. Quantitative real-time RT PCR was used to validate these results and to study possible molecular links between FA-D2 and other FA subtypes. GLRX was misregulated to opposite directions in a variety of different FA subtypes. Increased CTSB and decreased PLK2 expression was found in all or almost all of the analyzed complementation groups and, therefore, may be related to the defective FA pathway. Transcriptional upregulation of the CTSB proteinase appears to be a secondary phenomenon due to proliferation differences between FA and normal fibroblast cultures. In contrast, PLK2 is known to play a pivotal role in processes that are linked to FA defects and may contribute in multiple ways to the FA phenotype: PLK2 is a target gene for TP53, is likely to function as a tumor suppressor gene in hematologic neoplasia, and Plk2(-/-) mice are small because of defective embryonal development. (c) 2008 S. Karger AG, Basel.

  12. Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

    Science.gov (United States)

    Epstein Shochet, Gali; Wollin, Lutz; Shitrit, David

    2018-03-12

    Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. Whether the ECM conditioned by IPF fibroblasts determines the phenotype of naïve fibroblasts is difficult to explore. IPF-derived primary fibroblasts were cultured on Matrigel and then cleared using ammonium hydroxide, creating an IPF-conditioned matrix (CM). Normal fibroblast CM served as control. Normal fibroblasts were cultured on both types of CM, and cell count, cell distribution and markers of myofibroblast differentiation; transforming growth factor beta (TGFβ) signalling; and ECM expression were assessed. The effects of the anti-fibrotic drugs nintedanib and pirfenidone at physiologically relevant concentrations were also explored. Normal fibroblasts cultured on IPF-CM arranged in large aggregates as a result of increased proliferation and migration. Moreover, increased levels of pSmad3, pSTAT3 (phospho signal transducer and activator of transcription 3), alpha smooth muscle actin (αSMA) and Collagen1a were found, suggesting a differentiation towards a myofibroblast-like phenotype. SB505124 (10 μmol/L) partially reversed these alterations, suggesting a TGFβ contribution. Furthermore, nintedanib at 100 nmol/L and, to a lesser extent, pirfenidone at 100 μmol/L prevented the IPF-CM-induced fibroblast phenotype alterations, suggesting an attenuation of the ECM-fibroblast interplay. IPF fibroblasts alter the ECM, thus creating a CM that further propagates an IPF-like phenotype in normal fibroblasts. This assay demonstrated differences in drug activities for approved IPF drugs at clinically relevant concentrations. Thus, the matrix-fibroblast phenotype interplay might be a relevant assay to explore drug candidates for IPF treatment. © 2018 Asian Pacific Society of Respirology.

  13. Markers of breast cancer stromal fibroblasts in the primary tumour site associated with lymph node metastasis : a systematic review including our case series

    NARCIS (Netherlands)

    Azevedo Koike Folgueira, Maria Aparecida; Maistro, Simone; Hirata Katayama, Maria Lucia; Roela, Rosimeire Aparecida; Lopes Mundim, Fiorita Gonzales; Nanogaki, Suely; de Bock, Geertruida H.; Brentani, M. Mitzi

    2013-01-01

    CAFs (cancer-associated fibroblasts), the most abundant cell type in breast cancer stroma, produce a plethora of chemokines, growth factors and ECM (extracellular matrix) proteins, that may contribute to dissemination and metastasis. Axillary nodes are the first metastatic site in breast cancer;

  14. Human embryonic stem cells handbook

    Directory of Open Access Journals (Sweden)

    Carlo Alberto Redi

    2013-03-01

    Full Text Available After the Nobel prize in physiology or medicine was awarded jointly to Sir John Gurdon and Shinya Yamanaka for the discovery that mature cells can be reprogrammed to become pluripotent it became imperative to write down the review for a book entirely devoted to human embryonic stem cells (hES, those cells that are a urgent need for researchers, those cells that rekindle the ethical debates and finally, last but not least, those cells whose study paved the way to obtain induced pluripotent stem cells by the OSKC’s Yamanaka method (the OSKC acronim refers, for those not familiar with the topic, to the four stemness genes used to transfect somatic fibroblasts: Oct4, Sox2, Klf4 and c-Myc....

  15. Primary Bovine Extra-Embryonic Cultured Cells: A New Resource for the Study of In Vivo Peri-Implanting Phenotypes and Mesoderm Formation.

    Directory of Open Access Journals (Sweden)

    Isabelle Hue

    Full Text Available In addition to nourishing the embryo, extra-embryonic tissues (EETs contribute to early embryonic patterning, primitive hematopoiesis, and fetal health. These tissues are of major importance for human medicine, as well as for efforts to improve livestock efficiency, but they remain incompletely understood. In bovines, EETs are accessible easily, in large amounts, and prior to implantation. We took advantage of this system to describe, in vitro and in vivo, the cell types present in bovine EETs at Day 18 of development. Specifically, we characterized the gene expression patterns and phenotypes of bovine extra-embryonic ectoderm (or trophoblast; bTC, endoderm (bXEC, and mesoderm (bXMC cells in culture and compared them to their respective in vivo micro-dissected cells. After a week of culture, certain characteristics (e.g., gene expression of the in vitro cells were altered with respect to the in vivo cells, but we were able to identify "cores" of cell-type-specific (and substrate-independent genes that were shared between in vitro and in vivo samples. In addition, many cellular phenotypes were cell-type-specific with regard to extracellular adhesion. We evaluated the ability of individual bXMCs to migrate and spread on micro-patterns, and observed that they easily adapted to diverse environments, similar to in vivo EE mesoderm cells, which encounter different EE epithelia to form chorion, yolk sac, and allantois. With these tissue interactions, different functions arose that were detected in silico and corroborated in vivo at D21-D25. Moreover, analysis of bXMCs allowed us to identify the EE cell ring surrounding the embryonic disc (ED at D14-15 as mesoderm cells, which had been hypothesized but not shown prior to this study. We envision these data will serve as a major resource for the future in the analysis of peri-implanting phenotypes in response to the maternal metabolism and contribute to subsequent studies of placental/fetal development in

  16. Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

    OpenAIRE

    Cong, Shan; Cao, Guifang; Liu, Dongjun

    2014-01-01

    To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic ...

  17. Impact of matrix stiffness on fibroblast function

    Energy Technology Data Exchange (ETDEWEB)

    El-Mohri, Hichem; Wu, Yang; Mohanty, Swetaparna; Ghosh, Gargi, E-mail: gargi@umich.edu

    2017-05-01

    Chronic non-healing wounds, caused by impaired production of growth factors and reduced vascularization, represent a significant burden to patients, health care professionals, and health care system. While several wound dressing biomaterials have been developed, the impact of the mechanical properties of the dressings on the residing cells and consequently on the healing of the wounds is largely overlooked. The primary focus of this study is to explore whether manipulation of the substrate mechanics can regulate the function of fibroblasts, particularly in the context of their angiogenic activity. A photocrosslinkable hydrogel platform with orthogonal control over gel modulus and cell adhesive sites was developed to explore the quantitative relationship between ECM compliance and fibroblast function. Increase in matrix stiffness resulted in enhanced fibroblast proliferation and stress fiber formation. However, the angiogenic activity of fibroblasts was found to be optimum when the cells were seeded on compliant matrices. Thus, the observations suggest that the stiffness of the wound dressing material may play an important role in the progression of wound healing. - Highlights: • Proliferation and stress fiber formation of fibroblasts increase with increasing matrix mechanics. • Cell area correlates with the growth of fibroblasts. • Angiogenic activity of fibroblasts optimum when cells seeded on compliant gels.

  18. Fibroblast growth factor receptors in human embryonic stem cells

    Czech Academy of Sciences Publication Activity Database

    Dvořáková, D.; Kosková, S.; Vodinská, M.; Hampl, Aleš; Dvořák, Petr

    2004-01-01

    Roč. 104, č. 11 (2004), s. 1 ISSN 1528-0020. [2004 Annual meeting of American Society of Hematology . 04.12.2004-07.12.2004, San Diego] Keywords : FRFRs Subject RIV: EB - Genetics ; Molecular Biology

  19. Fibroblast cultures in duchenne muscular dystrophy

    International Nuclear Information System (INIS)

    Ionasescu, V.; Lara-Braud, C.; Zellweger, H.; Ionasescu, R.; Burmeister, L.

    1977-01-01

    Primary skin fibroblast cultures were grown from forearm pinch skin biopsies obtained from 24 patients with Duchenne muscular dystrophy (DMD) and ten normal controls matched for sex and age. The first subcultures were grown for 7 days and incubated with L-( 3 H)-proline for 24 hours. Intracellular collagen incoption was significantly decreased (2.2 X) and extracellular collagen incorporation significantly increased (1.8 X) in fibroblast cultures from patients with DMD by both collagenase assay and polyacrylamide gel electrophoresis. The synthesis of noncollagen proteins showed low values from the DMD fibroblast cultures. The alterations in synthesis and secretion of collagen and noncollagen proteins were characteristic only for the log phase of DMD fibroblasts. (author)

  20. Myogenic conversion of bladder fibroblasts by construction and ...

    African Journals Online (AJOL)

    The cultured primary bladder fibroblasts were transfected by pEGFP-Myod1 with Lipofection 2000 reagent. The results showed that expression of Myod1 could cause myogenic differentiation of bladder fibroblasts. These findings support the possibility of an alternative approach to exploit the capacity of Myod1 to activate ...

  1. Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

    OpenAIRE

    Vanderperre, Beno?t; Herzig, S?bastien; Krznar, Petra; H?rl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

    2016-01-01

    Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic p...

  2. Heart Development, Diseases, and Regeneration - New Approaches From Innervation, Fibroblasts, and Reprogramming.

    Science.gov (United States)

    Ieda, Masaki

    2016-09-23

    It is well known that cardiac function is tightly controlled by neural activity; however, the molecular mechanism of cardiac innervation during development and the relationship with heart disease remain undetermined. My work has revealed the molecular networks that govern cardiac innervation and its critical roles in heart diseases such as silent myocardial ischemia and arrhythmias. Cardiomyocytes proliferate during embryonic development, but lose their proliferative capacity after birth. Cardiac fibroblasts are a major source of cells during fibrosis and induce cardiac hypertrophy after myocardial injury in the adult heart. Despite the importance of fibroblasts in the adult heart, the role of fibroblasts in embryonic heart development was previously not determined. I demonstrated that cardiac fibroblasts play important roles in myocardial growth and cardiomyocyte proliferation during embryonic development, and I identified key paracrine factors and signaling pathways. In contrast to embryonic cardiomyocytes, adult cardiomyocytes have little regenerative capacity, leading to heart failure and high mortality rates after myocardial infarction. Leveraging the knowledge of developmental biology, I identified cardiac reprogramming factors that can directly convert resident cardiac fibroblasts into cardiomyocytes for heart regeneration. These findings greatly improved our understanding of heart development and diseases, and provide a new strategy for heart regenerative therapy. (Circ J 2016; 80: 2081-2088).

  3. Establecimiento de una línea celular primaria a partir de huevos con embrión de Toxocara canis Establishing a primary cell line from Toxocara canis embryonated eggs

    Directory of Open Access Journals (Sweden)

    Fernando Iván Marín

    2011-09-01

    the basic aspects, as well as the development of diagnostic techniques. Objective: To obtain a primary cell line from embryonated eggs of T.canis. Methods: The parasites were extracted from the small intestines of dogs under one year old. Embryonic cells were obtained by embryogenesis of the eggs secreted by adult worms in four different media; two were rich in nutrients, one was 1% formaldehyde, and the other was distilled water. The cells were obtained by mechanical dissociation of embryonated eggs using 30G needles. Results: The estimated time for obtaining the cell line was fifteen days, from which seven were used for egg embryogenesis. The cells responded positively to the cryopreservation methods after two days or even two months, allowing a replication phase with four passes. Conclusions: We managed to obtain a cell line from T. canis embryonated eggs. This cell line will help the understanding of pathogenic relationships, potential therapeutic targets and for developing diagnostic methods.

  4. GROα regulates human embryonic stem cell self-renewal or adoption of a neuronal fate

    Science.gov (United States)

    Krtolica, Ana; Larocque, Nick; Genbacev, Olga; Ilic, Dusko; Coppe, Jean-Philippe; Patil, Christopher K.; Zdravkovic, Tamara; McMaster, Michael; Campisi, Judith; Fisher, Susan J.

    2012-01-01

    Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ~6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical–basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical–basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation—βIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell–cell contacts can trigger adoption of a neuronal fate. PMID:21396766

  5. Embryonic stem-like cells derived from in vitro produced bovine blastocysts

    Directory of Open Access Journals (Sweden)

    Erika Regina Leal de Freitas

    2011-06-01

    Full Text Available The aim of this work was to study the derivation of bovine embryonic stem-like (ES-like cells from the inner cell mass (ICM of in vitro produced blastocysts. The ICMs were mechanically isolated and six out of seventeen (35% ICMs could attach to a monolayer of murine embryonic fibroblasts (MEF. Ten days after, primary outgrowths were mechanically dissected into several small clumps and transferred to a new MEF layer. Cells were further propagated and passaged by physical dissociation over a 60 days period. The pluripotency of the bovine ES-like cells was confirmed by RT-PCR of Oct-4 and STAT-3 gene markers. The colonies were weakly stained for alkaline phosphatase and the mesoderm and endoderm differentiation gene markers such as GATA-4 and Flk-1, respectively, were not expressed. Embryoid bodies were spontaneously formed at the seventh passage. Results showed that bovine ES-like cells could be obtained and passaged by mechanical procedures from the fresh in vitro produced blastocysts.

  6. A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells.

    Science.gov (United States)

    Li, Bing; Su, Trent; Ferrari, Roberto; Li, Jing-Yu; Kurdistani, Siavash K

    2014-02-01

    The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.

  7. Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices.

    Science.gov (United States)

    Sharma, Manjinder; Dubey, Pawan K; Kumar, Rajesh; Nath, Amar; Kumar, G Sai; Sharma, G Taru

    2013-05-01

    Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA- 4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

  8. Dihydrotestosterone Potentiates EGF-Induced ERK Activation by Inducing SRC in Fetal Lung Fibroblasts

    Science.gov (United States)

    Smith, Susan M.; Murray, Sandy; Pham, Lucia D.; Minoo, Parviz; Nielsen, Heber C.

    2014-01-01

    Lung maturation is regulated by interactions between mesenchymal and epithelial cells, and is delayed by androgens. Fibroblast–Type II cell communications are dependent on extracellular signal-regulated kinases (ERK) 1/2 activation by the ErbB receptor ligands epidermal growth factor (EGF), transforming growth factor (TGF)-α, and neuregulin (Nrg). In other tissues, dihydrotestosterone (DHT) has been shown to activate SRC by a novel nontranscriptional mechanism, which phosphorylates EGF receptors to potentiate EGF-induced ERK1/2 activation. This study sought to determine if DHT potentiates EGFR signaling by a nontranscriptional mechanism. Embryonic day (E)17 fetal lung cells were isolated from dams treated with or without DHT since E12. Cells were exposed to 30 ng/ml DHT for periods of 30 minutes to 3 days before being stimulated with 100 ng/ml EGF, TGF-α, or Nrg for up to 30 minutes. Lysates were immunoblotted for ErbB and SRC pathway signaling intermediates. DHT increased ERK1/2 activation by EGF, TGF-α, and Nrg in fibroblasts and Type II cells. Characterization in fibroblasts showed that potentiation of the EGF pathway was significant after 60 minutes of DHT exposure and persisted in the presence of the translational inhibitor cycloheximide. SRC and EGF receptor phosphorylation was increased by DHT, as was EGF-induced SHC1 phosphorylation and subsequent association with GRB2. Finally, SRC silencing, SRC inhibition with PP2, and overexpression of a dominant-negative SRC each prevented DHT from increasing EGF-induced ERK1/2 phosphorylation. These results suggest that DHT activates SRC to potentiate the signaling pathway leading from the EGF receptor to ERK activation in primary fetal lung fibroblasts. PMID:24484548

  9. Differential programming of p53-deficient embryonic cells during rotenone block

    Science.gov (United States)

    Mitochondrial dysfunction has been implicated in chemical toxicities. The present study used an in vitro model to investigate the differential expression of metabolic pathways during cellular stress in p53- efficient embryonic fibroblasts compared to p53-deficient cells. These c...

  10. Adenoviral-mediated correction of methylmalonyl-CoA mutase deficiency in murine fibroblasts and human hepatocytes

    Directory of Open Access Journals (Sweden)

    Korson Mark

    2007-04-01

    Full Text Available Abstract Background Methylmalonic acidemia (MMA, a common organic aciduria, is caused by deficiency of the mitochondrial localized, 5'deoxyadenosylcobalamin dependent enzyme, methylmalonyl-CoA mutase (MUT. Liver transplantation in the absence of gross hepatic dysfunction provides supportive therapy and metabolic stability in severely affected patients, which invites the concept of using cell and gene delivery as future treatments for this condition. Methods To assess the effectiveness of gene delivery to restore the defective metabolism in this disorder, adenoviral correction experiments were performed using murine Mut embryonic fibroblasts and primary human methylmalonyl-CoA mutase deficient hepatocytes derived from a patient who harbored two early truncating mutations, E224X and R228X, in the MUT gene. Enzymatic and expression studies were used to assess the extent of functional correction. Results Primary hepatocytes, isolated from the native liver after removal subsequent to a combined liver-kidney transplantation procedure, or Mut murine fibroblasts were infected with a second generation recombinant adenoviral vector that expressed the murine methylmalonyl-CoA mutase as well as eGFP from distinct promoters. After transduction, [1-14C] propionate macromolecular incorporation studies and Western analysis demonstrated complete correction of the enzymatic defect in both cell types. Viral reconstitution of enzymatic expression in the human methylmalonyl-CoA mutase deficient hepatocytes exceeded that seen in fibroblasts or control hepatocytes. Conclusion These experiments provide proof of principle for viral correction in methylmalonic acidemia and suggest that hepatocyte-directed gene delivery will be an effective therapeutic treatment strategy in both murine models and in human patients. Primary hepatocytes from a liver that was unsuitable for transplantation provided an important resource for these studies.

  11. Porcine embryonic stem cells

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane

    2008-01-01

    The development of porcine embryonic stem cell lines (pESC) has received renewed interest given the advances being made in the production of immunocompatible transgenic pigs. However, difficulties are evident in the production of pESCs in-vitro. This may largely be attributable to differences...

  12. Effects of "second-hand" smoke on structure and function of fibroblasts, cells that are critical for tissue repair and remodeling

    Directory of Open Access Journals (Sweden)

    Yadav Madhav

    2004-04-01

    Full Text Available Abstract Background It is known that "second-hand" cigarette smoke leads to abnormal tissue repair and remodelling but the cellular mechanisms involved in these adverse effects are not well understood. Fibroblasts play a major role in repair and remodelling. They orchestrate these processes by proliferating, migrating, and secreting proteins such as, cytokines, growth factors and extracellular matrix molecules. Therefore, we focus our studies on the effects of "second-hand" cigarette smoke on the structure and function of these cells. Results We used sidestream whole (SSW smoke, a major component of "second-hand" smoke, primary embryonic fibroblasts, cells that behave very much like wound fibroblasts, and a variety of cellular and molecular approaches. We show that doses of smoke similar to those found in tissues cause cytoskeletal changes in the fibroblasts that may lead to a decrease in cell migration. In addition, we also show that these levels of cigarette smoke stimulate an increase in cell survival that is reflected in an increase and/or activation of stress/survival proteins such as cIL-8, grp78, PKB/Akt, p53, and p21. We further show that SSW affects the endomembrane system and that this effect is also accomplished by nicotine alone. Conclusions Taken together, our results suggest that: (i SSW may delay wound repair because of the inability of the fibroblasts to migrate into the wounded area, leading to an accumulation of these cells at the edge of the wound, thus preventing the formation of the healing tissue; (ii the increase in cell survival coupled to the decrease in cell migration can lead to a build-up of connective tissue, thereby causing fibrosis and excess scarring.

  13. Collagen expression in fibroblasts with a novel LMNA mutation

    International Nuclear Information System (INIS)

    Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko

    2007-01-01

    Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy

  14. Fibroblast growth factor 23

    African Journals Online (AJOL)

    Dr Olaleye

    Systemic phosphate homeostasis is maintained through several hormonal mechanisms which involve fibroblast growth factor 23 (FGF-23), α-klotho, vitamin D and parathyroid hormone. FGF-23 is known to be the major regulator of phosphate balance (Mirams et al., 2004). FGF-23 is a phosphaturic hormone, which is.

  15. Cytotoxicity of Betel leaf (Piper betel L. against primary culture of chicken embryo fibroblast and its effects on the production of proinflammatory cytokines by human peripheral blood mononuclear cells

    Directory of Open Access Journals (Sweden)

    Suprapto Ma’at

    2012-06-01

    Full Text Available Background: Betel leaf (Piper betel L. has been used in modern and traditional medicine as antiseptic, antibacterial, and also prevention of plaque accumulation, but it still can stimulate cancer in lime-piper betel quid. Betel leaf also has anti-inflammatory properties. Purpose: The purpose of this study was examine the cytotoxicity of Betel leaf extract (BLE against primary culture of chicken embryo fibroblast and its effects on the production of proinflammatory cytokines by peripheral blood mononuclear cells (PBMC stimulated with LPS. Methods: MTT assay was used to investigate the survival rate of the culture with the survival rate result of the given culture extract 4%, 2% and 1% about 82%, 83.4% and 85%. There was no significant difference between treatment with various concentrations of the extract and the control (p>0.05. To evaluate the effect of Betel leaf extracts on the production of cytokines, proinflammatory was conducted by incubating the extracts of betel leaf with peripheral blood mononuclear cells stimulated with lipopolysaccharide. Peripheral blood mononuclear cells were obtained from healthy volunteers isolated by density centrifugation method using Ficoll-Hypaque. Once coupled with various concentrations of betel leaf extract and lipopolysaccharide, and then incubated for 24 hours, the culture supernatant was used to determine the level of IFN-γ and TNF-α by ELISA method. Results: It is known that the survival rates of BLE 4%, 2% and 1% were 82%, 83.4% and 85%. There was no significant of difference between several concentrations of BLE and those in the control group (p>0.05. The production of IFN-γ and TNF-α stimulated with LPS was no significant difference between BLE 4%, 2% and 1% and that in the control group (p>0.05. Conclusion: It can be concluded that BLE is not toxic against primary culture of chicken embryo fibroblast, and the production of IFN-γ and TNF-α by PBMC was not affected by BLE.Latar belakang: Daun

  16. The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

    2017-04-06

    The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (Ep

  17. Promotion of hair follicle development and trichogenesis by Wnt-10b in cultured embryonic skin and in reconstituted skin

    International Nuclear Information System (INIS)

    Ouji, Yukiteru; Yoshikawa, Masahide; Shiroi, Akira; Ishizaka, Shigeaki

    2006-01-01

    We previously showed that Wnt-10b promoted the differentiation of primary skin epithelial cells (MPSEC) toward hair shaft and inner root sheath of the hair follicle (IRS) cells in vitro. In the present study, we found that Wnt-10b promotes the development of hair follicles using a culture of mouse embryonic skin tissue and trichogenesis using a reconstitution experiment with nude mice. Hair follicle development was observed in skin taken from mouse embryos on embryonic day 10.5 following a 2-day culture with recombinant Wnt-10b (rWnt-10b), however, not without rWnt-10b. Brown hair growth was observed at the site of reconstituted skin in Balb/c nude mice where dermal fibroblasts and keratinocytes, derived from C3H/HeN new born mice, were transplanted with Wnt-10b-producing COS cells (Wnt-COS). Without the co-transplantation of Wnt-COS, no hair growth was observed. Our results suggest an important role of Wnt-10b in the initiation of hair follicle development and following trichogenesis

  18. Establishment of rat embryonic stem-like cells from the morula using a combination of feeder layers.

    Science.gov (United States)

    Sano, Chiaki; Matsumoto, Asako; Sato, Eimei; Fukui, Emiko; Yoshizawa, Midori; Matsumoto, Hiromichi

    2009-08-01

    Embryonic stem (ES) cells are characterized by pluripotency, in particular the ability to form a germline on injection into blastocysts. Despite numerous attempts, ES cell lines derived from rat embryos have not yet been established. The reason for this is unclear, although certain intrinsic biological differences among species and/or strains have been reported. Herein, using Wistar-Imamichi rats, specific characteristics of preimplantation embryos are described. At the blastocyst stage, Oct4 (also called Pou5f1) was expressed in both the inner cell mass (ICM) and the trophectoderm (TE), whereas expression of Cdx2 was localized to the TE. In contrast, at an earlier stage, expression of Oct4 was detected in all the nuclei in the morula. These stages were examined using a combination of feeder layers (rat embryonic fibroblast [REF] for primary outgrowth and SIM mouse embryo-derived thioguanine- and ouabain-resistant [STO] cells for passaging) to establish rat ES-like cell lines. The rat ES-like cell lines obtained from the morula maintained expression of Oct4 over long-term culture, whereas cell lines derived from blastocysts lost pluripotency during early passage. The morula-derived ES-like cell lines showed Oct4 expression in a long-term culture, even after cryogenic preservation, thawing and EGFP transfection. These results indicate that rat ES-like cell lines with long-term Oct4 expression can be established from the morula of Wistar-Imamichi rats using a combination of feeder layers.

  19. Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

    Science.gov (United States)

    Hertsenberg, Andrew J; Funderburgh, James L

    2016-01-01

    Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

  20. Somatic donor cell type correlates with embryonic, but not extra-embryonic, gene expression in postimplantation cloned embryos.

    Directory of Open Access Journals (Sweden)

    Ryutaro Hirasawa

    Full Text Available The great majority of embryos generated by somatic cell nuclear transfer (SCNT display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts. The embryos retrieved from the uteri were separated into embryonic (epiblast and extraembryonic (extraembryonic ectoderm and ectoplacental cone tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs (>2-fold vs. controls than did the extraembryonic tissues (P<1.0 × 10(-26. In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1-5% per embryos transferred in our laboratory, because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

  1. Induction of pluripotent stem cells from fibroblast cultures.

    Science.gov (United States)

    Takahashi, Kazutoshi; Okita, Keisuke; Nakagawa, Masato; Yamanaka, Shinya

    2007-01-01

    Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

  2. Connective tissue fibroblasts and Tcf4 regulate myogenesis

    Science.gov (United States)

    Mathew, Sam J.; Hansen, Jody M.; Merrell, Allyson J.; Murphy, Malea M.; Lawson, Jennifer A.; Hutcheson, David A.; Hansen, Mark S.; Angus-Hill, Melinda; Kardon, Gabrielle

    2011-01-01

    Muscle and its connective tissue are intimately linked in the embryo and in the adult, suggesting that interactions between these tissues are crucial for their development. However, the study of muscle connective tissue has been hindered by the lack of molecular markers and genetic reagents to label connective tissue fibroblasts. Here, we show that the transcription factor Tcf4 (transcription factor 7-like 2; Tcf7l2) is strongly expressed in connective tissue fibroblasts and that Tcf4GFPCre mice allow genetic manipulation of these fibroblasts. Using this new reagent, we find that connective tissue fibroblasts critically regulate two aspects of myogenesis: muscle fiber type development and maturation. Fibroblasts promote (via Tcf4-dependent signals) slow myogenesis by stimulating the expression of slow myosin heavy chain. Also, fibroblasts promote the switch from fetal to adult muscle by repressing (via Tcf4-dependent signals) the expression of developmental embryonic myosin and promoting (via a Tcf4-independent mechanism) the formation of large multinucleate myofibers. In addition, our analysis of Tcf4 function unexpectedly reveals a novel mechanism of intrinsic regulation of muscle fiber type development. Unlike other intrinsic regulators of fiber type, low levels of Tcf4 in myogenic cells promote both slow and fast myogenesis, thereby promoting overall maturation of muscle fiber type. Thus, we have identified novel extrinsic and intrinsic mechanisms regulating myogenesis. Most significantly, our data demonstrate for the first time that connective tissue is important not only for adult muscle structure and function, but is a vital component of the niche within which muscle progenitors reside and is a critical regulator of myogenesis. PMID:21177349

  3. Carcino-Embryonic Antigen

    International Nuclear Information System (INIS)

    Akute, O.

    1999-02-01

    Tumour marker analysis has increased our understanding of the presence of tumours in the body. Carcino-embryonic antigen, CEA, is one of the best studied tumour markers and has proved an ideal diagnostic adjuvant. It has helped in quantifying the amount of disease present in a patient and thence to make accurate prognosis on the various diagnosed ailments. At UCH, it is observed that there is an increase in cancer related ailments and therefore the need for early diagnosis is more compelling in our environment to mitigate future cost of managing advanced manifestation

  4. Inositol-Requiring Enzyme 1-Mediated Downregulation of MicroRNA (miR)-146a and miR-155 in Primary Dermal Fibroblasts across Three TNFRSF1A Mutations Results in Hyperresponsiveness to Lipopolysaccharide.

    Science.gov (United States)

    Harrison, Stephanie R; Scambler, Thomas; Oubussad, Lylia; Wong, Chi; Wittmann, Miriam; McDermott, Michael F; Savic, Sinisa

    2018-01-01

    Tumor necrosis factor (TNF)-receptor-associated periodic fever syndrome (TRAPS) is a rare monogenic autoinflammatory disorder characterized by mutations in the TNFRSF1A gene, causing TNF-receptor 1 (TNFR1) misfolding, increased cellular stress, activation of the unfolded protein response (UPR), and hyperresponsiveness to lipopolysaccharide (LPS). Both microRNA (miR)-146a and miR-155 provide negative feedback for LPS-toll-like receptor 2/4 signaling and cytokine production, through regulation of nuclear factor kappa B (NF-κB). In this study, we hypothesized that proinflammatory cytokine signaling in TRAPS downregulates these two miRs, resulting in LPS-induced hyperresponsiveness in TRAPS dermal fibroblasts (DFs), irrespective of the underlying genetic mutation. Primary DF were isolated from skin biopsies of TRAPS patients and healthy controls (HC). TNFR1 cell surface expression was measured using immunofluorescence. DF were stimulated with LPS, interleukin (IL)-1β, thapsigargin, or TNF, with and without inositol-requiring enzyme 1 (IRE1) inhibitor (4u8C), following which miR-146a and miR-155 expression was measured by RT-qPCR. IL-1β, IL-6, and TNF secretion was measured by enzyme-linked immunosorbent assays, and baseline expression of 384 different miRs was assessed using microfluidics assays. TNFR1 was found to be expressed on the surface of HC DF but expression was deficient in all samples with TRAPS-associated mutations. HC DF showed significant dose-dependent increases in both miR-146a and miR-155 expression levels in response to LPS; however, TRAPS DF failed to upregulate either miR-146a or miR-155 under the same conditions. This lack of miR-146a and miR-155 upregulation was associated with increased proinflammatory cytokine production in TRAPS DF in response to LPS challenge, which was abrogated by 4u8C. Incubation of HC DF with IL-1β led to downregulation of miR-146a and miR-155 expression, which was dependent on IRE1 enzyme. We observed global

  5. LIF Mediates Proinvasive Activation of Stromal Fibroblasts in Cancer

    Directory of Open Access Journals (Sweden)

    Jean Albrengues

    2014-06-01

    Full Text Available Signaling crosstalk between tumor cells and fibroblasts confers proinvasive properties to the tumor microenvironment. Here, we identify leukemia inhibitory factor (LIF as a tumor promoter that mediates proinvasive activation of stromal fibroblasts independent of alpha-smooth muscle actin (α-SMA expression. We demonstrate that a pulse of transforming growth factor β (TGF-β establishes stable proinvasive fibroblast activation by inducing LIF production in both fibroblasts and tumor cells. In fibroblasts, LIF mediates TGF-β-dependent actomyosin contractility and extracellular matrix remodeling, which results in collective carcinoma cell invasion in vitro and in vivo. Accordingly, carcinomas from multiple origins and melanomas display strong LIF upregulation, which correlates with dense collagen fiber organization, cancer cell collective invasion, and poor clinical outcome. Blockade of JAK activity by Ruxolitinib (JAK inhibitor counteracts fibroblast-dependent carcinoma cell invasion in vitro and in vivo. These findings establish LIF as a proinvasive fibroblast producer independent of α-SMA and may open novel therapeutic perspectives for patients with aggressive primary tumors.

  6. Congestive heart failure effects on atrial fibroblast phenotype: differences between freshly-isolated and cultured cells.

    Directory of Open Access Journals (Sweden)

    Kristin Dawson

    Full Text Available Fibroblasts are important in the atrial fibrillation (AF substrate resulting from congestive heart failure (CHF. We previously noted changes in in vivo indices of fibroblast function in a CHF dog model, but could not detect changes in isolated cells. This study assessed CHF-induced changes in the phenotype of fibroblasts freshly isolated from control versus CHF dogs, and examined effects of cell culture on these differences.Left-atrial fibroblasts were isolated from control and CHF dogs (ventricular tachypacing 240 bpm × 2 weeks. Freshly-isolated fibroblasts were compared to fibroblasts in primary culture. Extracellular-matrix (ECM gene-expression was assessed by qPCR, protein by Western blot, fibroblast morphology with immunocytochemistry, and K(+-current with patch-clamp. Freshly-isolated CHF fibroblasts had increased expression-levels of collagen-1 (10-fold, collagen-3 (5-fold, and fibronectin-1 (3-fold vs. control, along with increased cell diameter (13.4 ± 0.4 µm vs control 8.4 ± 0.3 µm and cell spreading (shape factor 0.81 ± 0.02 vs. control 0.87 ± 0.02, consistent with an activated phenotype. Freshly-isolated control fibroblasts displayed robust tetraethylammonium (TEA-sensitive K(+-currents that were strongly downregulated in CHF. The TEA-sensitive K(+-current differences between control and CHF fibroblasts were attenuated after 2-day culture and eliminated after 7 days. Similarly, cell-culture eliminated the ECM protein-expression and shape differences between control and CHF fibroblasts.Freshly-isolated CHF and control atrial fibroblasts display distinct ECM-gene and morphological differences consistent with in vivo pathology. Culture for as little as 48 hours activates fibroblasts and obscures the effects of CHF. These results demonstrate potentially-important atrial-fibroblast phenotype changes in CHF and emphasize the need for caution in relating properties of cultured fibroblasts to in vivo systems.

  7. Gene targeting in adult rhesus macaque fibroblasts

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    Wolf Don P

    2008-03-01

    Full Text Available Abstract Background Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology. Results A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term in vitro manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer. Conclusion The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.

  8. High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

    Science.gov (United States)

    Chen, Fanfan; Zhang, Guoqiang; Yu, Ling; Feng, Yanye; Li, Xianghui; Zhang, Zhijun; Wang, Yongting; Sun, Dapeng; Pradhan, Sriharsa

    2016-07-30

    Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.

  9. Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    International Nuclear Information System (INIS)

    Selvey, Saxon; Haupt, Larisa M; Thompson, Erik W; Matthaei, Klaus I; Irving, Michael G; Griffiths, Lyn R

    2004-01-01

    Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms

  10. Stimulation of MMP-11 (stromelysin-3 expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Matthaei Klaus I

    2004-07-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14 and stromelysin-3 (MMP-11 are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs were: a treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b grown on collagens I, IV and V; c treated with fibronectin, con-A and matrigel; and d co-cultured with a range of HBC (human breast cancer cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

  11. Bacterial lipopolysaccharide promotes profibrotic activation of intestinal fibroblasts.

    LENUS (Irish Health Repository)

    Burke, J P

    2012-02-01

    BACKGROUND: Fibroblasts play a critical role in intestinal wound healing. Lipopolysaccharide (LPS) is a cell wall component of commensal gut bacteria. The effects of LPS on intestinal fibroblast activation were characterized. METHODS: Expression of the LPS receptor, toll-like receptor (TLR) 4, was assessed in cultured primary human intestinal fibroblasts using flow cytometry and confocal microscopy. Fibroblasts were treated with LPS and\\/or transforming growth factor (TGF) beta1. Nuclear factor kappaB (NFkappaB) pathway activation was assessed by inhibitory kappaBalpha (IkappaBalpha) degradation and NFkappaB promoter activity. Fibroblast contractility was measured using a fibroblast-populated collagen lattice. Smad-7, a negative regulator of TGF-beta1 signalling, and connective tissue growth factor (CTGF) expression were assessed using reverse transcriptase-polymerase chain reaction and western blot. The NFkappaB pathway was inhibited by IkappaBalpha transfection. RESULTS: TLR-4 was present on the surface of intestinal fibroblasts. LPS treatment of fibroblasts induced IkappaBalpha degradation, enhanced NFkappaB promoter activity and increased collagen contraction. Pretreatment with LPS (before TGF-beta1) significantly increased CTGF production relative to treatment with TGF-beta1 alone. LPS reduced whereas TGF-beta1 increased smad-7 expression. Transfection with an IkappaBalpha plasmid enhanced basal smad-7 expression. CONCLUSION: Intestinal fibroblasts express TLR-4 and respond to LPS by activating NFkappaB and inducing collagen contraction. LPS acts in concert with TGF-beta1 to induce CTGF. LPS reduces the expression of the TGF-beta1 inhibitor, smad-7.

  12. Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Kyle J Hewitt

    2011-02-01

    Full Text Available Human induced pluripotent stem (hiPS cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK and their hES-derived counterparts (EDK showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM-production (COL1A1 by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ, revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

  13. Expression and secretory profile of buffalo fetal fibroblasts and Wharton's jelly feeder layers.

    Science.gov (United States)

    Parmar, Mehtab S; Mishra, Smruti Ranjan; Somal, Anjali; Pandey, Sriti; Kumar, G Sai; Sarkar, Mihir; Chandra, Vikash; Sharma, G Taru

    2017-05-01

    The present study examined the comparative expression and secretory profile of vital signaling molecules in buffalo fetal fibroblasts (BFF) and Wharton's jelly (BWJ) feeder layers at different passages. Both feeder layers were expanded up to 8th passage. Signaling molecules viz. bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF) and transforming growth factor beta 1 (TGFB1) and pluripotency-associated transcriptional factors (POU5F1, SOX2, NANOG, KLF4, MYC and FOXD3) were immunolocalized in the both feeder types. A clear variation in the expression pattern of key signaling molecules with passaging was registered in both feeders compared to primary culture (0 passage). The conditioned media (CM) was collected from different passages (2, 4, 6, 8) of both the feeder layers and was quantified using enzyme-linked immunosorbent assay (ELISA). Concomitant to expression profile, protein quantification also revealed differences in the concentration of signaling molecules at different time points. Conjointly, expression and secretory profile revealed that 2nd passage of BFF and 6th passage of BWJ exhibit optimal levels of key signaling molecules thus may be selected as best passages for embryonic stem cells (ESCs) propagation. Further, the effect of mitomycin-C (MMC) treatment on the expression profile of signaling molecules in the selected passages of BFF and BWJ revealed that MMC modulates the expression profile of these molecules. In conclusion, the results indicate that feeder layers vary in expression and secretory pattern of vital signaling molecules with passaging. Based on these findings, the appropriate feeder passages may be selected for the quality propagation of buffalo ESCs. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Organelle-cytoskeleton relationships in fibroblasts: mitochondria, Golgi apparatus, and endoplasmic reticulum in phases of movement and growth

    DEFF Research Database (Denmark)

    Couchman, J R; Rees, D A

    1982-01-01

    When fibroblasts first emerge from explants of embryonic chick heart, most mitochondria are clustered tightly around the nucleus, with very few extending towards the leading lamella. Although cytoplasmic microtubules are well displayed, mitochondria do not obviously codistribute with them. As the...

  15. Mechanical signaling coordinates the embryonic heartbeat

    Science.gov (United States)

    Chiou, Kevin K.; Rocks, Jason W.; Chen, Christina Yingxian; Cho, Sangkyun; Merkus, Koen E.; Rajaratnam, Anjali; Robison, Patrick; Tewari, Manorama; Vogel, Kenneth; Majkut, Stephanie F.; Prosser, Benjamin L.; Discher, Dennis E.; Liu, Andrea J.

    2016-01-01

    In the beating heart, cardiac myocytes (CMs) contract in a coordinated fashion, generating contractile wave fronts that propagate through the heart with each beat. Coordinating this wave front requires fast and robust signaling mechanisms between CMs. The primary signaling mechanism has long been identified as electrical: gap junctions conduct ions between CMs, triggering membrane depolarization, intracellular calcium release, and actomyosin contraction. In contrast, we propose here that, in the early embryonic heart tube, the signaling mechanism coordinating beats is mechanical rather than electrical. We present a simple biophysical model in which CMs are mechanically excitable inclusions embedded within the extracellular matrix (ECM), modeled as an elastic-fluid biphasic material. Our model predicts strong stiffness dependence in both the heartbeat velocity and strain in isolated hearts, as well as the strain for a hydrogel-cultured CM, in quantitative agreement with recent experiments. We challenge our model with experiments disrupting electrical conduction by perfusing intact adult and embryonic hearts with a gap junction blocker, β-glycyrrhetinic acid (BGA). We find this treatment causes rapid failure in adult hearts but not embryonic hearts—consistent with our hypothesis. Last, our model predicts a minimum matrix stiffness necessary to propagate a mechanically coordinated wave front. The predicted value is in accord with our stiffness measurements at the onset of beating, suggesting that mechanical signaling may initiate the very first heartbeats. PMID:27457951

  16. YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers

    DEFF Research Database (Denmark)

    Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A

    2012-01-01

    oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted......The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high...... YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3ß, PDX1, CD34, p63, nestin, PAX6) markers. Double...

  17. Differences between Solution and Membrane Forms of Chitosan on the In Vitro Activity of Fibroblasts

    Directory of Open Access Journals (Sweden)

    Bahar Uslu

    2015-03-01

    Full Text Available Background: Chitosan, a linear polysaccharide, has been recently used in biomedical applications. In vitro studies have demonstrated its effect on cellular growth and its stimulatory action on cellular layer formation. Aims: The present study aims to compare the proliferative effects of chitosan in two forms, membranous and solution forms, on Swiss 3T3 mouse embryonic fibroblasts. Study Design: In vitro study. Methods: Three experimental groups were formed: cells were cultured in a normal medium without chitosan (Control Group; cells were cultured either in a medium containing 2.0% chitosan in membranous form (Membrane Group or chitosan solution at a concentration of 2.0% (Solution Group.Two different methods were used in the experiments: cells cultured on the medium containing chitosan in solution or membranous forms (method 1; and chitosan solution or membranous forms were added into the medium containing previously cultured cells (method 2. Results: Scanning electron microscopic investigations of the experimental groups revealed cells with well-defined cellular projections, intact cellular membranes and tight intercellular junctions. They were especially prominent in the membrane group of method 1 and in the membrane and solution groups of method 2. Mouse monoclonal anti-collagen 1 primary antibody was used to indicate collagen synthesis. Prominent collagen synthesis was detected in the membrane groups on the 10th day of culture for both methods. Bromodeoxyuridine (BrdU and MTT assays were performed in order to assess cellular proliferation and viability, respectively. BrdU labelling tests indicated a higher proliferation index in the membrane group of method 1 on the 5th and 10th days. For the second method, the membranous form on the 10th day and solution form on the 5th day were the most effective groups in terms of cellular proliferation. MTT results reflected a high cellular viability in method 1 on the 5th day of treatment with the

  18. Embryonic development rates of northern grasshoppers (Orthoptera: Acrididae): implications for climate change and habitat management

    Science.gov (United States)

    Temperature-dependent rates of embryonic development are a primary determinant of the life cycle of many species of grasshoppers which, in cold climates, spend two winters in the egg stage. Knowledge of embryonic developmental rates is important for an assessment of the effects of climate change and...

  19. Preclinical study of mouse pluripotent parthenogenetic embryonic stem cell derivatives for the construction of tissue-engineered skin equivalent.

    Science.gov (United States)

    Rao, Yang; Cui, Jihong; Yin, Lu; Liu, Wei; Liu, Wenguang; Sun, Mei; Yan, Xingrong; Wang, Ling; Chen, Fulin

    2016-10-22

    Embryonic stem cell (ESC) derivatives hold great promise for the construction of tissue-engineered skin equivalents (TESE). However, harvesting of ESCs destroys viable embryos and may lead to political and ethical concerns over their application. In the current study, we directed mouse parthenogenetic embryonic stem cells (pESCs) to differentiate into fibroblasts, constructed TESE, and evaluated its function in vivo. The stemness marker expression and the pluripotent differentiation ability of pESCs were tested. After embryoid body (EB) formation and adherence culture, mesenchymal stem cells (MSCs) were enriched and directed to differentiate into fibroblastic lineage. Characteristics of derived fibroblasts were assessed by quantitative real-time PCR and ELISA. Functional ability of the constructed TESE was tested by a mouse skin defects repair model. Mouse pESCs expressed stemness marker and could form teratoma containing three germ layers. MSCs could be enriched from outgrowths of EBs and directed to differentiate into fibroblastic lineage. These cells express a high level of growth factors including FGF, EGF, VEGF, TGF, PDGF, and IGF1, similar to those of ESC-derived fibroblasts and mouse fibroblasts. Seeded into collagen gels, the fibroblasts derived from pESCs could form TESE. Mouse skin defects could be successfully repaired 15 days after transplantation of TESE constructed by fibroblasts derived from pESCs. pESCs could be induced to differentiate into fibroblastic lineage, which could be applied to the construction of TESE and skin defect repair. Particularly, pESC derivatives avoid the limitations of political and ethical concerns, and provide a promising source for regenerative medicine.

  20. Growth properties and growth factor responsiveness in skin fibroblasts from centenarians.

    Science.gov (United States)

    Tesco, G; Vergelli, M; Grassilli, E; Salomoni, P; Bellesia, E; Sikora, E; Radziszewska, E; Barbieri, D; Latorraca, S; Fagiolo, U; Santacaterina, S; Amaducci, L; Tiozzo, R; Franceschi, C; Sorbi, S

    1998-03-27

    Human fibroblast cultures, which have a finite replicative lifespan in vitro, are the most widely used model for the study of senescence at the cellular level. An inverse relationship between replicative capability and donor age has been reported in human fibroblast strains. We studied the growth capacity of fibroblast primary cultures derived from people whose lifespan was as closer as possible to the expected maximum human lifespan, i.e. people over one hundred. Our data suggest that outgrowth of fibroblasts from biopsies, growth kinetics at different population doubling levels, capability to respond to a classical mitogenic stimulus (such as 20% serum) and a variety of growth factors, were remarkably similar in fibroblasts from centenarians and young controls. On the whole, our data challenge the tenet of a simple and strict relationship between in vivo aging and in vitro proliferative capability of human fibroblasts, at least at the individual level.

  1. Embryonic duplications in sheep.

    Science.gov (United States)

    Dennis, S M

    1975-02-01

    Twenty-seven embryonic duplications were examined during a 3-year investigation into the causes of perinatal lamb mortality. Twenty of the 27 were anomalous twins with 19 being conjoined (diplopagus 9 and heteropagus 10). The various duplications were: haloacardius acephalus 1, diprosopus 2, dicephalus 2, dipypus 3, diprosopus dipygus 1, syncephalus dipygus 1, pygopagus parasiticus 1, heteropagus dipygus 3, melodidymus 6, polyury 4, penile duplication 2, and bilateral otognathia 1. Four lambs were living and the time of death of the others was: parturient 8, and post-parturient 15. Average dry weight of the lambs was 3.35 kg (range 1.59 to 5.45 kg). Breed distribution was: Merino 77.8%, Crossbred 14.8%, Dorset Horn 3.7%, and Corriedale 3.7%. The caudal region was involved in 10 of the conjoined twins (52.6%), anterior region in 7 (36.9%), and both anterior and caudal regions in 2 (10.5%). Associated defects were present in 70.4% of the 27 lambs, the most common being atresia ani.

  2. Somatic Donor Cell Type Correlates with Embryonic, but Not Extra-Embryonic, Gene Expression in Postimplantation Cloned Embryos

    Science.gov (United States)

    Inoue, Kimiko; Ogura, Atsuo

    2013-01-01

    The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (Pcloning efficiency using SCNT. PMID:24146866

  3. Human skeletal muscle fibroblasts stimulate in vitro myogenesis and in vivo muscle regeneration.

    Science.gov (United States)

    Mackey, Abigail L; Magnan, Mélanie; Chazaud, Bénédicte; Kjaer, Michael

    2017-08-01

    Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. The extent of cross-talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell-culture studies. We observed a strong stimulation of myogenesis by human fibroblasts in cell culture. In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres. These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell-cell cross-talk during physiological and pathological muscle remodelling. Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross-sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co-cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable

  4. Chenodeoxycholic acid stimulated fibroblast growth factor 19 response

    DEFF Research Database (Denmark)

    Borup, C; Wildt, S; Rumessen, J J

    2017-01-01

    BACKGROUND: Bile acid diarrhoea is underdiagnosed and better diagnostic tests are needed. Fasting serum fibroblast growth factor-19 (FGF19) has insufficient diagnostic value, but this may be improved by stimulation. AIM: To explore if an impaired FGF19 response identifies primary bile acid...

  5. An atomic-force-microscopy study of the structure of surface layers of intact fibroblasts

    Science.gov (United States)

    Khalisov, M. M.; Ankudinov, A. V.; Penniyaynen, V. A.; Nyapshaev, I. A.; Kipenko, A. V.; Timoshchuk, K. I.; Podzorova, S. A.; Krylov, B. V.

    2017-02-01

    Intact embryonic fibroblasts on a collagen-treated substrate have been studied by atomic-force microscopy (AFM) using probes of two types: (i) standard probes with tip curvature radii of 2-10 nm and (ii) special probes with a calibrated 325-nm SiO2 ball radius at the tip apex. It is established that, irrespective of probe type, the average maximum fibroblast height is on a level of 1.7 μm and the average stiffness of the probe-cell contact amounts to 16.5 mN/m. The obtained AFM data reveal a peculiarity of the fibroblast structure, whereby its external layers move as a rigid shell relative to the interior and can be pressed inside to a depth dependent on the load only.

  6. Mechanobiology of embryonic limb development.

    Science.gov (United States)

    Nowlan, Niamh C; Murphy, Paula; Prendergast, Patrick J

    2007-04-01

    Considerable evidence exists to support the hypothesis that mechanical forces have an essential role in healthy embryonic skeletal development. Clinical observations and experimental data indicate the importance of muscle contractions for limb development. However, the influence of these forces is seldom referred to in biological descriptions of bone development, and perhaps this is due to the fact that the hypothesis that mechanical forces are essential for normal embryonic skeletal development is difficult to test and elaborate experimentally in vivo, particularly in humans. Computational modeling has the potential to address this issue by simulating embryonic growth under a range of loading conditions but the potential of such models has yet to be fully exploited. In this article, we review the literature on mechanobiology of limb development in three main sections: (a) experimental alteration of the mechanical environment, (b) mechanical properties of embryonic tissues, and (c) the use of computational models. Then we analyze the main issues, and suggest how experimental and computational fields could work closer together to enhance our understanding of mechanobiology of the embryonic skeleton.

  7. Pearl extract enhances the migratory ability of fibroblasts in a wound healing model.

    Science.gov (United States)

    Li, Yi-Chen; Chen, Chi-Ruei; Young, Tai-Horng

    2013-03-01

    For 2000 years, traditional Chinese medicine has been used as a remedy for general health improvement, including the fight against aging. Pearl powder has recently been used as a health food that has antioxidant, antiaging, antiradioactive, and tonic activities for cells; it is also applied to cure aphthous ulcer, gastric ulcer, and duodenal ulcer on clinical therapy. In addition, the mother of pearl, nacre, could enhance the cell adhesion and tissue regeneration of skin fibroblasts. Fibroblast is regarded as indispensable in the processes of wound healing. Therefore, the effect of pearl extract (PL) on fibroblasts is investigated in this study. PL is produced by a room temperature super extraction system (Taiwan patent no. I271 220). DMEM medium containing PL (300 μg/mL) was used to examine the effect of migration-promoting potential on human fibroblast cell line or human primary fibroblast cells in a wound healing model in vitro. Medium containing PL (300 μg/mL) demonstrated that the migratory cell numbers of fibroblasts were three times more than that without PL, and mRNA expression of collagen type III was higher than in collagen type I in fibroblasts. It revealed a migration-promoting potential of human fibroblasts in a wound healing model in vitro. The present study found that the migration-promoting effect in PL, which could be a supplement in cell culture. These data suggest PL could be useful for enhancing the wound healing of fibroblasts.

  8. Fibroblast growth factor receptors in breast cancer.

    Science.gov (United States)

    Wang, Shuwei; Ding, Zhongyang

    2017-05-01

    Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of breast cancer. Recent genetic studies have identified some subtypes of fibroblast growth factor receptors as strong genetic loci associated with breast cancer. In this article, we review the recent epidemiological findings and experiment results of fibroblast growth factor receptors in breast cancer. First, we summarized the structure and physiological function of fibroblast growth factor receptors in humans. Then, we discussed the common genetic variations in fibroblast growth factor receptors that affect breast cancer risk. In addition, we also introduced the potential roles of each fibroblast growth factor receptors isoform in breast cancer. Finally, we explored the potential therapeutics targeting fibroblast growth factor receptors for breast cancer. Based on the biological mechanisms of fibroblast growth factor receptors leading to the pathogenesis in breast cancer, targeting fibroblast growth factor receptors may provide new opportunities for breast cancer therapeutic strategies.

  9. Application of pulsed-magnetic field enhances non-viral gene delivery in primary cells from different origins

    Energy Technology Data Exchange (ETDEWEB)

    Kamau Chapman, Sarah W. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstr. 190, 8057 Zurich (Switzerland); Hassa, Paul O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstr. 190, 8057 Zurich (Switzerland); European Molecular Biology Laboratory (EMBL) Heidelberg, Meyerhofstrasse 1, 69117 Heidelberg (Germany); Koch-Schneidemann, Sabine; Rechenberg, Brigitte von [Musculoskeletal Research Unit, Equine Hospital, Vetsuisse Faculty Zurich, University of Zurich, Winterthurerstr. 260, 8057 Zurich (Switzerland); Hofmann-Amtenbrink, Margarethe [MatSearch, Chemin Jean Pavillard 14, 1009 Pully (Switzerland); Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich [Laboratory of Powder Technology, Ecole Polytechnique Federale de Lausanne (EPFL), Lausanne (Switzerland); Hottiger, Michael O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstr. 190, 8057 Zurich (Switzerland)], E-mail: hottiger@vetbio.uzh.ch

    2008-04-15

    Primary cell lines are more difficult to transfect when compared to immortalized/transformed cell lines, and hence new techniques are required to enhance the transfection efficiency in these cells. We isolated and established primary cultures of synoviocytes, chondrocytes, osteoblasts, melanocytes, macrophages, lung fibroblasts, and embryonic fibroblasts. These cells differed in several properties, and hence were a good representative sample of cells that would be targeted for expression and delivery of therapeutic genes in vivo. The efficiency of gene delivery in all these cells was enhanced using polyethylenimine-coated polyMAG magnetic nanoparticles, and the rates (17-84.2%) surpassed those previously achieved using other methods, especially in cells that are difficult to transfect. The application of permanent and pulsating magnetic fields significantly enhanced the transfection efficiencies in synoviocytes, chondrocytes, osteoblasts, melanocytes and lung fibroblasts, within 5 min of exposure to these magnetic fields. This is an added advantage for future in vivo applications, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.

  10. Cell proliferation in vitro modulates fibroblast collagenase activity

    International Nuclear Information System (INIS)

    Lindblad, W.J.; Flood, L.

    1986-01-01

    Collagenase enzyme activity is regulated by numerous control mechanisms which prevent excessive release and activation of this protease. A primary mechanism for regulating enzyme extracellular activity may be linked to cell division, therefore they have examined the release of collagenase by fibroblasts in vitro in response to cellular proliferation. Studies were performed using fibroblasts derived from adult rat dermis maintained in DMEM containing 10% newborn calf serum, 25 mM tricine buffer, and antibiotics. Cells between subculture 10 and 19 were used with enzyme activity determined with a 14 C-labelled soluble Type I collagen substrate with and without trypsin activation. Fibroblasts, trypsinized and plated at low density secreted 8.5 fold more enzyme than those cells at confluence (975 vs. 115 dpm/μg DNA). This diminution occurred gradually as the cells went from logrithmic growth towards confluence. Confluent fibroblast monolayers were scraped in a grid arrangement, stimulating the remaining cells to divide, without exposure to trypsin. Within 24-48 hr postscraping enzyme levels had increased 260-400%, accompanied by enhanced incorporation of 3 H-thymidine and 3 H-uridine into cell macromolecules. The burst of enzyme release began to subside 12 hr later. These results support a close relationship between fibroblast proliferation and collagenase secretion

  11. [Possibilities and limitations of fibroblast cultures in the study of animal aging].

    Science.gov (United States)

    Van Gansen, P; Van Lerberghe, N

    1987-01-01

    INTRODUCTION. Aging--the effect of time--occurs in every living organism. Senescence is the last period of the lifespan, leading to death. It happens in all animals, with the exception of a few didermic species (Hydras) having a stock of embryonic cells and being immortal. The causes of animal senescence are badly known. They depend both on genetic characters (maximal lifespan of a species) and on medium factors (mean expectation of life of the animals of a species). Animal senescence could depend on cell aging: 1) by senescence and death of the differentiated cells, 2) by modified proliferation and differentiation of the stem cells of differentiated tissues, 3) by alterations in the extracellular matrices, 4) by interactions between factors 1) 2) and 3) in each tissue, 5) by interactions between the several tissues of an organism. This complexity badly impedes the experimental study of animal senescence. Normal mammal cells are aging when they are cultivated (in vitro ageing): their phenotype varies and depends on the cell generation (in vitro differentiation); the last cell-generation doesn't divide anymore and declines until death of the culture (in vitro senescence). Analysis of these artificial but well controlled systems allows an experimental approach of the proliferation, differentiation, senescence and death of the cells and of the extracellular matrix functions. Present literature upon in vitro aging of cultivated human cells is essentially made of papers where proliferation and differentiation characteristics are compared between early ("young") and late ("old") cell-generations of the cultures. FIBROBLASTIC CELLS OF THE MOUSE SKIN. This cell type has been studied in our laboratory, using different systems: 1) Primary cultures isolated from peeled skins of 19 day old mouse embryos, 2) Mouse dermis analyzed in the animals, 3) Cultivated explants of skins, 4) Serial sub-cultures of fibroblasts isolated from these explants, 5) Cells cultivated comparably on

  12. Microprobe analysis of human fibroblasts

    International Nuclear Information System (INIS)

    Allan, G.L.; Zhu, J.; Legge, G.J.F.

    1985-01-01

    The Melbourne Proton Microprobe has been used to study the copper content in human skin fibroblast cells derived from patients with the genetic disease Menkes Syndrome. Both normal and diseased cells have been studied to investigate any elemental differences occurring between the two cell types. This paper details the preparatory techniques necessary for individual cell analysis and presents the elemental information with a new three dimensional contour mapping technique. These maps are used to highlight elemental differences between normal and mutant fibroblasts. The work also confirms the expected copper excess found in the Menkes cell and indicates that the microprobe can be used for rapid identification of a Menkes carrier

  13. Tubule-Derived Wnts Are Required for Fibroblast Activation and Kidney Fibrosis.

    Science.gov (United States)

    Zhou, Dong; Fu, Haiyan; Zhang, Lu; Zhang, Ke; Min, Yali; Xiao, Liangxiang; Lin, Lin; Bastacky, Sheldon I; Liu, Youhua

    2017-08-01

    Cell-cell communication via Wnt ligands is necessary in regulating embryonic development and has been implicated in CKD. Because Wnt ligands are ubiquitously expressed, the exact cellular source of the Wnts involved in CKD remains undefined. To address this issue, we generated two conditional knockout mouse lines in which Wntless (Wls), a dedicated cargo receptor that is obligatory for Wnt secretion, was selectively ablated in tubular epithelial cells or interstitial fibroblasts. Blockade of Wnt secretion by genetic deletion of Wls in renal tubules markedly inhibited myofibroblast activation and reduced renal fibrosis after unilateral ureteral obstruction. This effect associated with decreased activation of β -catenin and downstream gene expression and preserved tubular epithelial integrity. In contrast, fibroblast-specific deletion of Wls exhibited little effect on the severity of renal fibrosis after obstructive or ischemia-reperfusion injury. In vitro , incubation of normal rat kidney fibroblasts with tubule-derived Wnts promoted fibroblast proliferation and activation. Furthermore, compared with kidney specimens from patients without CKD, biopsy specimens from patients with CKD also displayed increased expression of multiple Wnt proteins, predominantly in renal tubular epithelium. These results illustrate that tubule-derived Wnts have an essential role in promoting fibroblast activation and kidney fibrosis via epithelial-mesenchymal communication. Copyright © 2017 by the American Society of Nephrology.

  14. [Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo].

    Science.gov (United States)

    Jiang, Hua; Feng, You-Ji; Xie, Yi; Han, Jin-Lan; Wang, Zack; Chen, Tong

    2008-10-14

    To establish a sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo. Human embryonic stem were (hESCs) were cultured on the mouse embryo fibroblasts and then were induced to differentiate to form three-dimensional EB. The hEBs were cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF)-4 of different concentrations for 3 days to observe the sprouting of the hEBs. 3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 h Immunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so as to determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturing condition was analyzed. The differentiated EBs formed sprouting structures in the collagen I matrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other and form vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from the EBs assimilated Dil-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. The concentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/ml of FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. The sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structures are indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonic vasculogenesis in humans.

  15. Induced Pluripotent Stem Cells: A novel frontier in the study of human primary immunodeficiencies

    Science.gov (United States)

    Pessach, Itai M.; Ordovas-Montanes, Jose; Zhang, Shen-Ying; Casanova, Jean-Laurent; Giliani, Silvia; Gennery, Andrew R.; Al-Herz, Waleed; Manos, Philip D.; Schlaeger, Thorsten M.; Park, In-Hyun; Rucci, Francesca; Agarwal, Suneet; Mostoslavsky, Gustavo; Daley, George Q.; Notarangelo, Luigi D.

    2010-01-01

    Background The novel ability to epigenetically reprogram somatic cells into induced pluripotent stem cells through the exogenous expression of transcription promises to revolutionize the study of human diseases. Objective Here we report on the generation of 25 induced pluripotent stem cell lines from 6 patients with various forms of Primary Immunodeficiencies, affecting adaptive and/or innate immunity. Methods Patients’ dermal fibroblasts were reprogrammed by expression of four transcription factors, OCT4, SOX2, KLF4, and c-MYC using a single excisable polycistronic lentiviral vector. Results Induced pluripotent stem cells derived from patients with primary immunodeficiencies show a stemness profile that is comparable to that observed in human embryonic stem cells. Following in vitro differentiation into embryoid bodies, pluripotency of the patient-derived indiced pluripotent stem cells lines was demonstrated by expression of genes characteristic of each of the three embryonic layers. We have confirmed the patient-specific origin of the induced pluripotent stem cell lines, and ascertained maintenance of karyotypic integrity. Conclusion By providing a limitless source of diseased stem cells that can be differentiated into various cell types in vitro, the repository of induced pluripotent stem cell lines from patients with primary immunodeficiencies represents a unique resource to investigate the pathophysiology of hematopoietic and extra-hematopoietic manifestations of these diseases, and may assist in the development of novel therapeutic approaches based on gene correction. PMID:21185069

  16. Expression of interleukin-17B in mouse embryonic limb buds and regulation by BMP-7 and bFGF

    International Nuclear Information System (INIS)

    You Zongbing; DuRaine, Grayson; Tien, Janet Y.L.; Lee, Corinne; Moseley, Timothy A.; Reddi, A. Hari

    2005-01-01

    Interleukin-17B (IL-17B) is a member of interleukin-17 family that displays a variety of proinflammatory and immune modulatory activities. In this study, we found that IL-17B mRNA was maximally expressed in the limb buds of 14.5 days post coitus (dpc) mouse embryo and declined to low level at 19.5 dpc. By immunohistochemical staining, the strongest IL-17B signals were observed in the cells of the bone collar in the primary ossification center. The chondrocytes in the resting and proliferative zones were stained moderately, while little staining was seen in the hypertrophic zone. Furthermore, in both C3H10T1/2 and MC3T3-E1 cells, the IL-17B mRNA was up-regulated by recombinant human bone morphogenetic protein-7, but down-regulated by basic fibroblast growth factor via the extracellular signal-regulated kinase pathway. This study provides the first evidence that IL-17B is expressed in the mouse embryonic limb buds and may play a role in chondrogenesis and osteogenesis

  17. [Effects of naloxone on the expression of stem cell factor and C-kit receptor in combined oxygen-glucose deprivation of primary cultured human embryonic neuron in vitro].

    Science.gov (United States)

    Zhu, Bo; Li, Lan-ying; Lü, Guo-yi; Xue, Yu-liang; Ye, Tie-hu

    2010-04-01

    To explore the effects of naloxone on the expression of c-kit receptor (c-kit R) and its ligand stem cell factor (SCF) in human embryo neuronal hypoxic injury. Serum-free cerebral cortical cultures prepared from embryonic human brains were deprived of both oxygen and glucose which would set up an environment more likely with that of in vivo ischemic injury. Neurons in 24-well culture plates were randomly divided into four groups: control group, hypoxia group, naloxone 0.5 microg/ml group and naloxone 10 microg/ml group. MTT assay and biological analysis were performed to study the cell death and the changes of extracellular concentrations of lactate dehydrogenase (LDH) after combined oxygen-glucose deprivation. Neurons in 25 ml culture flasks were also randomly allocated into four groups as previously described. Intracellular total RNA were extracted at different time points: pre-hypoxia, immediately after hypoxia, and 3, 6, 12, and 24 hours after reoxygenation. The changes of SCF/c-kit R mRNA expression in hypoxic neurons treated with different concentrations of naloxone pre and post oxygen-glucose deprivation were determined with RT-PCR. The cell vitality detected by MTT assay decreased significantly in hypoxia group and naloxone 0.5 microg/ml group when compared with control group (Pcontrol group. Extracellular concentration of LDH significantly increased in hypoxia group (Pcontrol group, between naloxone 0.5 microg/ml and hypoxia group, or between naloxone 10 microg/ml and control group (all P>0.05). Immediately after oxygen-glucose deprivation, the expression of SCF/c-kit R mRNA increased significantly (Pcontrol group (Pglucose deprivation. Naloxone 0.5 microg/ml can attenuate cell injuries and regulate the expression of SCF/c-kit R. Naloxone may protect neurons by modulating the expressions of some cytokines.

  18. Vascular Endothelial Growth Factor from Embryonic Status to Cardiovascular Pathology

    Directory of Open Access Journals (Sweden)

    Mohsen Azimi-Nezhad

    2014-05-01

    Full Text Available Vascular endothelial growth factor (VEGF is a multifunctional cytokine with distinct functions in angiogenesis, lymphangiogenesis, vascular permeability, and hematopoiesis. VEGF is a highly conserved, disulfide-bonded dimeric glycoprotein of 34 to 45 kDa produced by several cell types including fibroblasts, neutrophils, endothelial cells, and peripheral blood mononuclear cells, particularly T lymphocytes and macrophages. Six VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene, consisting of 121, 145, 165, 183, 189, or 206 amino acids. VEGF121, VEGF145, and VEGF165 are secreted whereas VEGF183, VEGF189, and VEGF206 are cell membrane-bound. VEGF145 has a key role during the vascularization of the human ovarian follicle and corpus luteum, in the placentation and embryonic periods, and in bone and wound healing, while VEGF165 is the most abundant and biologically active isoform. VEGF has been linked with a number of vascular pathologies including cardiovascular diseases such ischemic heart disease, heart failure, stroke, and diabetes and its related complications. In this review we aimed to present some important roles of VEGF in a number of clinical issues and indicate its involvement in several phenomena from the initial steps of the embryonic period to cardiovascular diseases.

  19. EDA-containing fibronectin increases proliferation of embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Noelia Losino

    Full Text Available Embryonic stem cells (ESC need a set of specific factors to be propagated. They can also grow in conditioned medium (CM derived from a bovine granulosa cell line BGC (BGC-CM, a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+. Here, we investigated if the FN EDA(+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-, and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy.

  20. Case report 511: Fibroblastic rheumatism

    International Nuclear Information System (INIS)

    Hernandez, R.J.; Martel, W.; Headington, J.T.; Kaufman, R.A.; Cincinnati Univ., OH

    1989-01-01

    We report a ten-year-old child with the newly described entity of fibroblastic rheumatism. This child developed rapid, progressive, symmetrical polyarthritis, similar to the radiographic appearance of juvenile rheumatoid arthritis, except for the rapidity of progression. The polyarthritis was preceded by the development of skin nodules with characteristic histological changes. (orig./GDG)

  1. Evaluation of biological effects of intermediate frequency magnetic field on differentiation of embryonic stem cell.

    Science.gov (United States)

    Yoshie, Sachiko; Ogasawara, Yuki; Ikehata, Masateru; Ishii, Kazuyuki; Suzuki, Yukihisa; Wada, Keiji; Wake, Kanako; Nakasono, Satoshi; Taki, Masao; Ohkubo, Chiyoji

    2016-01-01

    The embryotoxic effect of intermediate frequency (IF) magnetic field (MF) was evaluated using murine embryonic stem (ES) cells and fibroblast cells based on the embryonic stem cell test (EST). The cells were exposed to 21 kHz IF-MF up to magnetic flux density of 3.9 mT during the cell proliferation process (7 days) or the cell differentiation process (10 days) during which an embryonic body differentiated into myocardial cells. As a result, there was no significant difference in the cell proliferation between sham- and IF-MF-exposed cells for both ES and fibroblast cells. Similarly, the ratio of the number of ES-derived cell aggregates differentiated to myocardial cells to total number of cell aggregates was not changed by IF-MF exposure. In addition, the expressions of a cardiomyocytes-specific gene, Myl2 , and an early developmental gene, Hba-x , in the exposed cell aggregate were not altered. Since the magnetic flux density adopted in this study is much higher than that generated by an inverter of the electrical railway, an induction heating (IH) cooktop, etc . in our daily lives, these results suggested that IF-MF in which the public is exposed to in general living environment would not have embryotoxic effect.

  2. Directed Differentiation of Zebrafish Pluripotent Embryonic Cells to Functional Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Yao Xiao

    2016-09-01

    Full Text Available A cardiomyocyte differentiation in vitro system from zebrafish embryos remains to be established. Here, we have determined pluripotency window of zebrafish embryos by analyzing their gene-expression patterns of pluripotency factors together with markers of three germ layers, and have found that zebrafish undergoes a very narrow period of pluripotency maintenance from zygotic genome activation to a brief moment after oblong stage. Based on the pluripotency and a combination of appropriate conditions, we established a rapid and efficient method for cardiomyocyte generation in vitro from primary embryonic cells. The induced cardiomyocytes differentiated into functional and specific cardiomyocyte subtypes. Notably, these in vitro generated cardiomyocytes exhibited typical contractile kinetics and electrophysiological features. The system provides a new paradigm of cardiomyocyte differentiation from primary embryonic cells in zebrafish. The technology provides a new platform for the study of heart development and regeneration, in addition to drug discovery, disease modeling, and assessment of cardiotoxic agents.

  3. Influence of hyperthermia on the phosphorylation of ribosomal protein S6 from human skin fibroblasts and meningioma cells

    DEFF Research Database (Denmark)

    Richter, W W; Zang, K D; Issinger, O G

    1983-01-01

    Skin fibroblasts and meningioma cells, derived from primary cultures of the same patients have been used to study the influence of hyperthermia on (i) cell morphology and (ii) phosphorylation pattern of ribosomal and ribosome-associated proteins. Incubation of tumour cells and fibroblasts up to 7...

  4. Change of mitotic cycle and DNA repair in embryonic cells of rat, immortalized by E1 A oncogene and transformated by E1 A and c-Ha-Ras oncogenes under ionizing radiation action

    International Nuclear Information System (INIS)

    Kirillova, T.V.

    1997-01-01

    Comparison investigation into the repair of mitotic cycle and the reunion of DN single- and double-strand breaks in gamma-ray irradiated initial E1 A oncogene immortalized and E1 A and c-Ha-Ras oncogene transformed (mutant form) lines of rat embryonic fibroblasts was carried out. Possible involvement of Ras gene product in DNA repair speed governing and absence of tumor suppression function of p 53 protein in the embryonic and E1 A oncogene immortalized cells of rat fibroblast, as well as, presence of the mentioned function of p 53 protein in E1 A and c-Ha-Ras oncogene transformed cells were studied [ru

  5. Generation of KCL035 research grade human embryonic stem cell line carrying a mutation in HBB gene

    Directory of Open Access Journals (Sweden)

    Heema Hewitson

    2016-03-01

    Full Text Available The KCL035 human embryonic stem cell line was derived from an embryo donated for research that carried a mutation in the HBB gene, which is linked to the β-thalassemia syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  6. Effect of Fibroblast Co-culture on In Vitro Maturation and Fertilization of Mouse Preantral Follicles

    Directory of Open Access Journals (Sweden)

    Mahmoud Heidari

    2011-01-01

    Full Text Available Background: The aim of this study was to evaluate fibroblast co-culture on in vitro maturation andfertilization of prepubertal mouse preantral follicles.Materials and Methods: The ovaries of 12-14 day old mice were dissected and 120-150 μmintact preantral follicles with one or two layers of granulosa cells, and round oocytes were culturedindividually in α-minimal essential medium (α-MEM supplemented with 5% fetal bovine serum(FBS, 100 mIU/ml recombinant follicle stimulating hormone, 1% insulin, transferrin, seleniummix, 100 μg/ml penicillin and 50 μg/ml streptomycin as base medium for 12 days. A total number of226 follicules were cultured under two conditions: i base medium as control group (n=113; ii basemedium co-cultured with mouse embryonic fibroblast (MEF (n=113. Follicular diameters, alone,in addition to other factors were analyzed by student’s t-test and chi-square test, respectively.Results: The co-culture group showed significant differences (p<0.05 in growth rate (days 4, 6 and8 of the culture period and survival rate. However, there was no significant difference in antrumformation, ovulation rate and embryonic development of released oocytes. There were significantdifferences (p<0.05 in the estradiol and progesterone secretion at all days between the co-cultureand control groups.Conclusion: Fibroblast co-culture increased survival rate and steroid production of preantralfollicles by promoting granulosa cell proliferation.

  7. An Expandable, Inducible Hemangioblast State Regulated by Fibroblast Growth Factor

    Directory of Open Access Journals (Sweden)

    David T. Vereide

    2014-12-01

    Full Text Available During development, the hematopoietic and vascular lineages are thought to descend from common mesodermal progenitors called hemangioblasts. Here we identify six transcription factors, Gata2, Lmo2, Mycn, Pitx2, Sox17, and Tal1, that “trap” murine cells in a proliferative state and endow them with a hemangioblast potential. These “expandable” hemangioblasts (eHBs are capable, once released from the control of the ectopic factors, to give rise to functional endothelial cells, multilineage hematopoietic cells, and smooth muscle cells. The eHBs can be derived from embryonic stem cells, from fetal liver cells, or poorly from fibroblasts. The eHBs reveal a central role for fibroblast growth factor, which not only promotes their expansion, but also facilitates their ability to give rise to endothelial cells and leukocytes, but not erythrocytes. This study serves as a demonstration that ephemeral progenitor states can be harnessed in vitro, enabling the creation of tractable progenitor cell lines.

  8. L1TD1 Is a Marker for Undifferentiated Human Embryonic Stem Cells

    OpenAIRE

    Wong, Raymond Ching-Bong; Ibrahim, Abel; Fong, Helen; Thompson, Noelle; Lock, Leslie F.; Donovan, Peter J.

    2011-01-01

    Background Human embryonic stem cells (hESC) are stem cells capable of differentiating into cells representative of the three primary embryonic germ layers. There has been considerable interest in understanding the mechanisms regulating stem cell pluripotency, which will ultimately lead to development of more efficient methods to derive and culture hESC. In particular, Oct4, Sox2 and Nanog are transcription factors known to be important in maintenance of hESC. However, many of the downstream ...

  9. Generation of human induced pluripotent stem cell lines from human dermal fibroblasts using a non-integration system

    Directory of Open Access Journals (Sweden)

    Kyung-Ok Uhm

    2017-05-01

    Full Text Available We generated human induced pluripotent stem cells (hiPSCs from dermal fibroblasts using a Sendai virus (SeV-based gene delivery method. The generated hiPSC line, KSCBi002-A, has a normal karyotype (46,XY. The pluripotency and differentiation capacity were characterized by comparison with those of a human embryonic stem cell line. This cell line is registered and available from the National Stem Cell Bank, Korea National Institute of Health.

  10. Immunofluorescent histological studies of the role of fibronectin in the expression of the associative preferences of embryonic tissues.

    Science.gov (United States)

    Armstrong, P B; Armstrong, M T

    1981-08-01

    The identity of the chemical factors controlling the spreading behaviour of sheets of cells was examined in organ culture. When aggregates of two dissimilar tissues are apposed in organ culture, one tissue spreads reproducibly over the surface of the second. The present study employed indirect immunofluorescent localization techniques to evaluate the hypothesis that the spreading behaviour of chick embryonic heart tissue in culture is dominated by the presence or absence of the cell-surface and extracellular matrix protein fibronectin in the surface layers of the aggregates. Specifically, the hypothesis proposes that aggregates that display surface fibronectin earlier after culturing and/or in higher quantities segregate internally to aggregates that are slower to develop a surface layer of fibronectin or in which this layer contains reduced amounts of fibronectin. The hypothesis has been supported for 3 categories of behaviour of chick embryo heart tissue: (1) myocyte aggregates spread over myocyte aggregates containing a 20% admixture of heart fibroblasts, which in turn spread over heart fibroblast aggregates; (2) 5-day embryonic ventricle-tissue fragments maintained in culture for 0.5 days spread over ventricle fragments cultured for 2.5 days; and (3) 2-day embryonic ventricle spreads over 5-day ventricle. In all these situations, the aggregate type that segregates to an internal position displays more fibronectin at its surface than aggregate types that spread to occupy an external position. Evidence is presented that the fibronectin in heart tissue aggregates is elaborated by heart fibroblasts.

  11. Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

    International Nuclear Information System (INIS)

    Taru Sharma, G.; Dubey, Pawan K.; Verma, Om Prakash; Pratheesh, M.D.; Nath, Amar; Sai Kumar, G.

    2012-01-01

    Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: ► Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. ► Established 3D microenvironment for ES cells development and differentiation into three germ layers. ► Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to

  12. Collagen-IV supported embryoid bodies formation and differentiation from buffalo (Bubalus bubalis) embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Taru Sharma, G., E-mail: gts553@gmail.com [Reproductive Physiology Laboratory, Division of Physiology and Climatology, Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly, U.P. (India); Dubey, Pawan K.; Verma, Om Prakash; Pratheesh, M.D.; Nath, Amar; Sai Kumar, G. [Reproductive Physiology Laboratory, Division of Physiology and Climatology, Indian Veterinary Research Institute, Izatnagar-243 122, Bareilly, U.P. (India)

    2012-08-03

    Graphical abstract: EBs formation, characterization and expression of germinal layers marker genes of in vivo developed teratoma using four different types of extracellular matrices. Highlights: Black-Right-Pointing-Pointer Collagen-IV matrix is found cytocompatible for EBs formation and differentiation. Black-Right-Pointing-Pointer Established 3D microenvironment for ES cells development and differentiation into three germ layers. Black-Right-Pointing-Pointer Collagen-IV may be useful as promising candidate for ES cells based therapeutic applications. -- Abstract: Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5 days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it

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

    Energy Technology Data Exchange (ETDEWEB)

    Yamano, Noriko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Kimura, Tohru, E-mail: tkimura@patho.med.osaka-u.ac.jp [Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Watanabe-Kushima, Shoko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Shinohara, Takashi [Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501 (Japan); Nakano, Toru, E-mail: tnakano@patho.med.osaka-u.ac.jp [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)

    2010-02-12

    Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.

  14. Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes

    Science.gov (United States)

    Fang, Xiefan; Mei, Wenbin; Barbazuk, William B.; Rivkees, Scott A.

    2014-01-01

    Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20–60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3–65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes. PMID:25354728

  15. Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

    Science.gov (United States)

    Krznar, Petra; Hörl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

    2016-01-01

    Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic profiles, and both defects could be restored by reexpression of MPC1. Labeling experiments using 13C-labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose-derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains, together with major alterations of their metabolome including lactic acidosis, diminished TCA cycle intermediates, energy deficit and a perturbed balance of neurotransmitters. Strikingly, these changes were reversed when the pregnant dams were fed a ketogenic diet, which provides acetyl-CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups, even though they all died within a few minutes post-delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development, neurotransmitter balance and post-natal survival. PMID:27176894

  16. Mouse Rad9b is essential for embryonic development and promotes resistance to DNA damage

    Science.gov (United States)

    Leloup, Corinne; Hopkins, Kevin M.; Wang, Xiangyuan; Zhu, Aiping; Wolgemuth, Debra J.; Lieberman, Howard B.

    2010-01-01

    RAD9 participates in promoting resistance to DNA damage, cell cycle checkpoint control, DNA repair, apoptosis, embryogenesis, and regulation of transcription. A paralogue of RAD9 (named RAD9B) has been identified. To define the function of mouse Rad9b (Mrad9b), embryonic stem (ES) cells with a targeted gene deletion were constructed and used to generate Mrad9b mutant mice. Mrad9b−/− embryos are resorbed after E7.5 while some of the heterozygotes die between E12.5 and a few days after birth. Mrad9b is expressed in embryonic brain and Mrad9b+/− embryos exhibit abnormal neural tube closure. Mrad9b−/− mouse embryonic fibroblasts are not viable. Mrad9b−/− ES cells are more sensitive to gamma rays and mitomycin C than Mrad9b+/+ controls, but show normal gamma-ray-induced G2/M checkpoint control. There is no evidence of spontaneous genomic instability in Mrad9b−/− cells. Our findings thus indicate that Mrad9b is essential for embryonic development and mediates resistance to certain DNA damaging agents. PMID:20842695

  17. How the embryonic chick brain twists

    OpenAIRE

    Chen, Zi; Guo, Qiaohang; Dai, Eric; Forsch, Nickolas; Taber, Larry A.

    2016-01-01

    During early development, the tubular embryonic chick brain undergoes a combination of progressive ventral bending and rightward torsion, one of the earliest organ-level left–right asymmetry events in development. Existing evidence suggests that bending is caused by differential growth, but the mechanism for the predominantly rightward torsion of the embryonic brain tube remains poorly understood. Here, we show through a combination of in vitro experiments, a physical model of the embryonic m...

  18. Fibroblast growth factor-mediated proliferation of central nervous system precursors depends on endogenous production of insulin-like growth factor I

    International Nuclear Information System (INIS)

    Drago, J.; Murphy, M.; Carroll, S.M.; Harvey, R.P.; Bartlett, P.F.

    1991-01-01

    Fibroblast growth factor stimulates proliferation and subsequent differentiation of precursor cells isolated from the neuroepithelium of embryonic day 10 mice in vitro. Here we show that fibroblast growth factor-induced proliferation is dependent on the presence of insulin-like growth factors (IGFs) and that IGF-I is endogenously produced by the neuroepithelial cells. Blocking of endogenous IGF-I activity with anti-IGF-I antibodies results in complete inhibition of fibroblast growth factor-mediated proliferation and in cell death. IGF-I alone acts as a survival agent. These observations correlate with the detection of transcripts for IGF-I and basic fibroblast growth factor in freshly isolated neuroepithelium and are consistent with an autocrine action of these factors in early brain development in vivo

  19. The effects of acoustic vibration on fibroblast cell migration.

    Science.gov (United States)

    Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

    2016-12-01

    Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Anti-proliferative effect of olmesartan on Tenon's capsule fibroblasts

    Directory of Open Access Journals (Sweden)

    Xuan Wang

    2016-05-01

    Full Text Available AIM: To evaluate the inhibitive effect of olmesartan to fibroblast proliferation and the anti-scarring effect in Tenon’s capsule, both in vitro and in vivo. METHODS: Human primary Tenon’s capsule fibroblasts were cultured in vitro, treated with up titrating concentrations of olmesartan. The rate of inhibition was tested with methyl thiazol tetrazolium (MTT method. Real-time PCR was performed to analyze changes in mRNA expressions of the fibrosis-related factors: matrix metalloproteinase-2 (MMP-2, tissue inhibitor of metalloproteinase (TIMP-1,2 and proliferating cell nuclear antigen (PCNA. Thirty rabbits were divided into 5 groups (3, 7, 14, 21, and 28d. A rabbit conjunctiva flap model was created in each eye. Olmesartan solution was injected subconjunctivally and then evaluated its anti-proliferation and anti-fibrosis effects through the histological morphology and immunohistochemistry of MMP-2 and PCNA in each group. Only the 7d group was treated with Masson’s trichrome to compare the neovascularization in the subconjunctiva area. RESULTS: In vitro, cultured Tenon's capsule human fibroblasts showed a dose dependent inhibition by olmesartan in MTT. Olmesartan reduced mRNA expressions of MMP-2 and PCNA but increased mRNA expressions of TIMP-1 and TIMP-2. In vivo, the rabbit eyes treated with olmesartan at 3rd, 7th, 14th and 21st days demonstrated a significant reduced expressions of MMP-2 and PCNA compared with control eye, no significant difference observed in 28th day group. The cellular proliferation and neovascularization was suppressed by olmesartan in Masson’s trichrome observation. CONCLUSION: By inhibiting fibroblasts in vitro and in vivo, olmesartan prevents the proliferation and activity of fibroblasts in scar tissue formation, which might benefit glaucoma filtering surgery.

  1. Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach.

    Directory of Open Access Journals (Sweden)

    Jordan R Plews

    2010-12-01

    Full Text Available Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine.In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5'-aza-2'-deoxycytidine and cultured in human embryonic stem cell (ES medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days.Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.

  2. Glutamine deprivation induces interleukin-8 expression in ataxia telangiectasia fibroblasts.

    Science.gov (United States)

    Kim, Min-Hyun; Kim, Aryung; Yu, Ji Hoon; Lim, Joo Weon; Kim, Hyeyoung

    2014-05-01

    To investigate whether glutamine deprivation induces expression of inflammatory cytokine interleukin-8 (IL-8) by determining NF-κB activity and levels of oxidative indices (ROS, reactive oxygen species; hydrogen peroxide; GSH, glutathione) in fibroblasts isolated from patients with ataxia telangiectasia (A-T). We used A-T fibroblasts stably transfected with empty vector (Mock) or with human full-length ataxia telangiectasia mutated (ATM) cDNA (YZ5) and mouse embryonic fibroblasts (MEFs) transiently transfected with ATM small interfering RNA (siRNA) or with non-specific control siRNA. The cells were cultured with or without glutamine or GSH. ROS levels were determined using a fluorescence reader and confocal microscopy. IL-8 or murine IL-8 homolog, keratinocyte chemoattractant (KC), and hydrogen peroxide levels in the medium were determined by enzyme-linked immunosorbent assay and colorimetric assay. GSH level was assessed by enzymatic assay, while IL-8 (KC) mRNA level was measured by reverse transcription-polymerase chain reaction (RT-PCR) and/or quantitative real-time PCR. NF-κB DNA-binding activity was determined by electrophoretic mobility shift assay. Catalase activity and ATM protein levels were determined by O2 generation and Western blotting. While glutamine deprivation induced IL-8 expression and increased NF-κB DNA-binding activity in Mock cells, both processes were decreased by treatment of cells with glutamine or GSH or both glutamine and GSH. Glutamine deprivation had no effect on IL-8 expression or NF-κB DNA-binding activity in YZ5 cells. Glutamine-deprived Mock cells had higher oxidative stress indices (increases in ROS and hydrogen peroxide, reduction in GSH) than glutamine-deprived YZ5 cells. In Mock cells, glutamine deprivation-induced oxidative stress indices were suppressed by treatment with glutamine or GSH or both glutamine and GSH. GSH levels and catalase activity were lower in Mock cells than YZ5 cells. MEFs transfected with ATM siRNA and

  3. Coxsackievirus B3 induces the formation of autophagosomes in cardiac fibroblasts both in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Zhai, Xia, E-mail: zhai_xia_cool@126.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Qin, Ying, E-mail: qinyinggaofeng@163.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Chen, Yang, E-mail: cy_hmu@126.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Lin, Lexun, E-mail: linlexun@163.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Wang, Tianying, E-mail: wangty0929@163.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhong, Xiaoyan, E-mail: littlerock712@163.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Wu, Xiaoyu, E-mail: xiaoyu_wu2006@163.com [Department of Cardiology, The First Hospital of Harbin Medical University, 23 Youzheng Street, Harbin 150001 (China); Chen, Sijia, E-mail: chensj0802@163.com [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Li, Jing, E-mail: jing070822@163.com [Center of Electron Microscopy, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Wang, Yan, E-mail: wangyan@hrbmu.edu.cn [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhang, Fengmin, E-mail: fengminzhang@ems.hrbmu.edu.cn [Department of Microbiology and Wu Lien-Teh Institute, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhao, Wenran, E-mail: zhaowenran2002@aliyun.com [Department of Cell Biology, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); and others

    2016-12-10

    Coxsackievirus group B (CVB) is one of the common pathogens that cause myocarditis and cardiomyopathy. Evidence has shown that CVB replication in cardiomyocytes is responsible for the damage and loss of cardiac muscle and the dysfunction of the heart. However, it remains largely undefined how CVB would directly impact cardiac fibroblasts, the most abundant cells in human heart. In this study, cardiac fibroblasts were isolated from Balb/c mice and infected with CVB type 3 (CVB3). Increased double-membraned, autophagosome-like vesicles in the CVB3-infected cardiac fibroblasts were observed with electron microscope. Punctate distribution of LC3 and increased level of LC3-II were also detected in the infected cardiac fibroblasts. Furthermore, we observed that the expression of pro-inflammatory cytokines, IL-6 and TNF-α, was increased in the CVB3-infected cardiac fibroblasts, while suppressed autophagy by 3-MA and Atg7-siRNA inhibited cytokine expression. Consistent with the in vitro findings, increased formation of autophagosomes was observed in the cardiac fibroblasts of Balb/c mice infected with CVB3. In conclusion, our data demonstrated that cardiac fibroblasts respond to CVB3 infection with the formation of autophagosomes and the release of the pro-inflammatory cytokines. These results suggest that the autophagic response of cardiac fibroblasts may play a role in the pathogenesis of myocarditis caused by CVB3 infection. - Highlights: • CVB3 replication induced autophagosome assembly in primary cardiac fibroblasts. • Both IL-6 and TNF-α in cardiac fibroblasts infected by CVB3 were increased. • IL-6 and TNF-α were reduced in cardiac fibroblasts when autophagy was inhibited. • Autophagosome assembly in cardiac fibroblasts of CVB-infected mice was increased.

  4. Coxsackievirus B3 induces the formation of autophagosomes in cardiac fibroblasts both in vitro and in vivo

    International Nuclear Information System (INIS)

    Zhai, Xia; Qin, Ying; Chen, Yang; Lin, Lexun; Wang, Tianying; Zhong, Xiaoyan; Wu, Xiaoyu; Chen, Sijia; Li, Jing; Wang, Yan; Zhang, Fengmin; Zhao, Wenran

    2016-01-01

    Coxsackievirus group B (CVB) is one of the common pathogens that cause myocarditis and cardiomyopathy. Evidence has shown that CVB replication in cardiomyocytes is responsible for the damage and loss of cardiac muscle and the dysfunction of the heart. However, it remains largely undefined how CVB would directly impact cardiac fibroblasts, the most abundant cells in human heart. In this study, cardiac fibroblasts were isolated from Balb/c mice and infected with CVB type 3 (CVB3). Increased double-membraned, autophagosome-like vesicles in the CVB3-infected cardiac fibroblasts were observed with electron microscope. Punctate distribution of LC3 and increased level of LC3-II were also detected in the infected cardiac fibroblasts. Furthermore, we observed that the expression of pro-inflammatory cytokines, IL-6 and TNF-α, was increased in the CVB3-infected cardiac fibroblasts, while suppressed autophagy by 3-MA and Atg7-siRNA inhibited cytokine expression. Consistent with the in vitro findings, increased formation of autophagosomes was observed in the cardiac fibroblasts of Balb/c mice infected with CVB3. In conclusion, our data demonstrated that cardiac fibroblasts respond to CVB3 infection with the formation of autophagosomes and the release of the pro-inflammatory cytokines. These results suggest that the autophagic response of cardiac fibroblasts may play a role in the pathogenesis of myocarditis caused by CVB3 infection. - Highlights: • CVB3 replication induced autophagosome assembly in primary cardiac fibroblasts. • Both IL-6 and TNF-α in cardiac fibroblasts infected by CVB3 were increased. • IL-6 and TNF-α were reduced in cardiac fibroblasts when autophagy was inhibited. • Autophagosome assembly in cardiac fibroblasts of CVB-infected mice was increased.

  5. C-type natriuretic peptide ameliorates pulmonary fibrosis by acting on lung fibroblasts in mice.

    Science.gov (United States)

    Kimura, Toru; Nojiri, Takashi; Hino, Jun; Hosoda, Hiroshi; Miura, Koichi; Shintani, Yasushi; Inoue, Masayoshi; Zenitani, Masahiro; Takabatake, Hiroyuki; Miyazato, Mikiya; Okumura, Meinoshin; Kangawa, Kenji

    2016-02-19

    Pulmonary fibrosis has high rates of mortality and morbidity; however, no effective pharmacological therapy has been established. C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, selectively binds to the transmembrane guanylyl cyclase (GC)-B receptor and exerts anti-inflammatory and anti-fibrotic effects in various organs through vascular endothelial cells and fibroblasts that have a cell-surface GC-B receptor. Given the pathophysiological importance of fibroblast activation in pulmonary fibrosis, we hypothesized that the anti-fibrotic and anti-inflammatory effects of exogenous CNP against bleomycin (BLM)-induced pulmonary fibrosis were exerted in part by the effect of CNP on pulmonary fibroblasts. C57BL/6 mice were divided into two groups, CNP-treated (2.5 μg/kg/min) and vehicle, to evaluate BLM-induced (1 mg/kg) pulmonary fibrosis and inflammation. A periostin-CNP transgenic mouse model exhibiting CNP overexpression in fibroblasts was generated and examined for the anti-inflammatory and anti-fibrotic effects of CNP via fibroblasts in vivo. Additionally, we assessed CNP attenuation of TGF-β-induced differentiation into myofibroblasts by using immortalized human lung fibroblasts stably expressing GC-B receptors. Furthermore, to investigate whether CNP acts on human lung fibroblasts in a clinical setting, we obtained primary-cultured fibroblasts from surgically resected lungs of patients with lung cancer and analyzed levels of GC-B mRNA transcription. CNP reduced mRNA levels of the profibrotic cytokines interleukin (IL)-1β and IL-6, as well as collagen deposition and the fibrotic area in lungs of mice with bleomycin-induced pulmonary fibrosis. Furthermore, similar CNP effects were observed in transgenic mice exhibiting fibroblast-specific CNP overexpression. In cultured-lung fibroblasts, CNP treatment attenuated TGF-β-induced phosphorylation of Smad2 and increased mRNA and protein expression of α-smooth muscle actin and SM22

  6. Carcinoma-Associated Fibroblasts Are a Promising Therapeutic Target

    International Nuclear Information System (INIS)

    Togo, Shinsaku; Polanska, Urszula M.; Horimoto, Yoshiya; Orimo, Akira

    2013-01-01

    Human carcinomas frequently exhibit significant stromal reactions such as the so-called “desmoplastic stroma” or “reactive stroma”, which is characterised by the existence of large numbers of stromal cells and extracellular matrix proteins. Carcinoma-associated fibroblasts (CAFs), which are rich in activated fibroblast populations exemplified by myofibroblasts, are among the predominant cell types present within the tumour-associated stroma. Increased numbers of stromal myofibroblasts are often associated with high-grade malignancies with poor prognoses in humans. CAF myofibroblasts possess abilities to promote primary tumour development, growth and progression by stimulating the processes of neoangiogenesis as well as tumour cell proliferation, survival, migration and invasion. Moreover, it has been demonstrated that CAFs serve as a niche supporting the metastatic colonisation of disseminated carcinoma cells in distant organs. Their contribution to primary and secondary malignancies makes these fibroblasts a potential therapeutic target and they also appear to be relevant to the development of drug resistance and tumour recurrence. This review summarises our current knowledge of tumour-promoting CAFs and discusses the therapeutic feasibility of targeting these cells as well as disrupting heterotypic interactions with other cell types in tumours that may improve the efficacy of current anti-tumour therapies

  7. Vertebrate Embryonic Cleavage Pattern Determination.

    Science.gov (United States)

    Hasley, Andrew; Chavez, Shawn; Danilchik, Michael; Wühr, Martin; Pelegri, Francisco

    2017-01-01

    The pattern of the earliest cell divisions in a vertebrate embryo lays the groundwork for later developmental events such as gastrulation, organogenesis, and overall body plan establishment. Understanding these early cleavage patterns and the mechanisms that create them is thus crucial for the study of vertebrate development. This chapter describes the early cleavage stages for species representing ray-finned fish, amphibians, birds, reptiles, mammals, and proto-vertebrate ascidians and summarizes current understanding of the mechanisms that govern these patterns. The nearly universal influence of cell shape on orientation and positioning of spindles and cleavage furrows and the mechanisms that mediate this influence are discussed. We discuss in particular models of aster and spindle centering and orientation in large embryonic blastomeres that rely on asymmetric internal pulling forces generated by the cleavage furrow for the previous cell cycle. Also explored are mechanisms that integrate cell division given the limited supply of cellular building blocks in the egg and several-fold changes of cell size during early development, as well as cytoskeletal specializations specific to early blastomeres including processes leading to blastomere cohesion. Finally, we discuss evolutionary conclusions beginning to emerge from the contemporary analysis of the phylogenetic distributions of cleavage patterns. In sum, this chapter seeks to summarize our current understanding of vertebrate early embryonic cleavage patterns and their control and evolution.

  8. MicroRNA-33 promotes the replicative senescence of mouse embryonic fibroblasts by suppressing CDK6

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Shun; Huang, Haijiao; Li, Nanhong; Zhang, Bing; Jia, Yubin; Yang, Yukun; Yuan, Yuan; Xiong, Xing-dong; Wang, Dengchuan; Zheng, Hui-ling [Institute of Aging Research, Guangdong Medical University, Dongguan (China); Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical University, Zhanjiang (China); Liu, Xinguang, E-mail: xgliu64@126.com [Institute of Aging Research, Guangdong Medical University, Dongguan (China); Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical University, Zhanjiang (China)

    2016-05-13

    MicroRNAs are a large class of tiny noncoding RNAs, which have emerged as critical regulators of gene expression, and thus are involved in multiple cellular processes, including cellular senescence. MicroRNA-33 has previously been established to exert crucial effect on cell proliferation, lipid metabolism and cholesterol metabolism. Nonetheless, the association between microRNA-33 and cellular senescence and its underlying molecular mechanism are far to be elucidated. The present study has attempted to probe into the effect of microRNA-33 on MEFs senescence. Our data unveiled that microRNA-33 was dramatically down-regulated in senescent MEFs compared to the young MEFs, and ectopic expression of microRNA-33 promoted MEFs senescence, while knock-down of microRNA-33 exhibited a protective effect against senescence phenotype. Moreover, we verified CDK6 as a direct target of microRNA-33 in mouse. Silencing of CDK6 induced the premature senescence phenotype of MEFs similarly as microRNA-33, while enforced expression of CDK6 significantly reverse the senescence-induction effect of microRNA-33. Taken together, our results suggested that microRNA-33 enhanced the replicative senescence of MEFs potentially by suppressing CDK6 expression. -- Highlights: •MicroRNA-33 was dramatically down-regulated in senescent MEF cells. •Altered expression of microRNA-33 exerted a critical role in MEFs senescence. •MicroRNA-33 promoted the replicative senescence of MEFs via targeting of CDK6.

  9. MicroRNA-33 promotes the replicative senescence of mouse embryonic fibroblasts by suppressing CDK6

    International Nuclear Information System (INIS)

    Xu, Shun; Huang, Haijiao; Li, Nanhong; Zhang, Bing; Jia, Yubin; Yang, Yukun; Yuan, Yuan; Xiong, Xing-dong; Wang, Dengchuan; Zheng, Hui-ling; Liu, Xinguang

    2016-01-01

    MicroRNAs are a large class of tiny noncoding RNAs, which have emerged as critical regulators of gene expression, and thus are involved in multiple cellular processes, including cellular senescence. MicroRNA-33 has previously been established to exert crucial effect on cell proliferation, lipid metabolism and cholesterol metabolism. Nonetheless, the association between microRNA-33 and cellular senescence and its underlying molecular mechanism are far to be elucidated. The present study has attempted to probe into the effect of microRNA-33 on MEFs senescence. Our data unveiled that microRNA-33 was dramatically down-regulated in senescent MEFs compared to the young MEFs, and ectopic expression of microRNA-33 promoted MEFs senescence, while knock-down of microRNA-33 exhibited a protective effect against senescence phenotype. Moreover, we verified CDK6 as a direct target of microRNA-33 in mouse. Silencing of CDK6 induced the premature senescence phenotype of MEFs similarly as microRNA-33, while enforced expression of CDK6 significantly reverse the senescence-induction effect of microRNA-33. Taken together, our results suggested that microRNA-33 enhanced the replicative senescence of MEFs potentially by suppressing CDK6 expression. -- Highlights: •MicroRNA-33 was dramatically down-regulated in senescent MEF cells. •Altered expression of microRNA-33 exerted a critical role in MEFs senescence. •MicroRNA-33 promoted the replicative senescence of MEFs via targeting of CDK6.

  10. Nonhomologous DNA end joining and chromosome aberrations in human embryonic lung fibroblasts treated with environmental pollutants

    Czech Academy of Sciences Publication Activity Database

    Rössner ml., Pavel; Rössnerová, Andrea; Beskid, Olena; Tabashidze, Nana; Líbalová, Helena; Uhlířová, Kateřina; Topinka, Jan; Šrám, Radim

    763-764, MAY-JUN 2014 (2014), s. 28-38 ISSN 0027-5107 R&D Projects: GA ČR GAP503/11/0084 Institutional support: RVO:68378041 Keywords : benzo[a]pyrene * chromosome aberrations * double-strand DNA breaks Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.680, year: 2014

  11. Mouse embryonic fibroblasts derived from Odin deficient mice display a hyperproliiferative phenotype

    DEFF Research Database (Denmark)

    Kristiansen, Troels Zaccharias Glahn; Nielsen, Mogens Møller; Blagoev, Blagoy

    2004-01-01

    Odin is a recently identified cytosolic phosphotyrosine binding (PTB) domain containing negative regulatory protein, that was discovered on the basis of its ability to undergo tyrosine phosphorylation upon stimulation by epidermal growth factor in HeLa cells. The protein was originally obtained...

  12. Protein modification and replicative senescence of WI-38 human embryonic fibroblasts

    DEFF Research Database (Denmark)

    Ahmed, Emad K; Rogowska-Wrzesinska, Adelina; Roepstorff, Peter

    2010-01-01

    reflects a preferential accumulation of damaged proteins within the mitochondria during cellular senescence. Accumulation of AGE-modified proteins could be explained by the senescence-associated decreased activity of glyoxalase-I, the major enzyme involved in the detoxification of the glycating agents...... methylglyoxal and glyoxal, in both cytosol and mitochondria. This finding suggests a role of detoxification systems in the age-related build-up of damaged proteins. Moreover, the oxidized protein repair system methionine sulfoxide reductase was more affected in the mitochondria than in the cytosol during......Summary Oxidized proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and by glycation (AGE) have been shown to accumulate with aging in vivo and during replicative senescence in vitro. To better understand the mechanisms by which these damaged proteins...

  13. Nucleotide Excision Repair Is Not Induced in Human Embryonic Lung Fibroblasts Treated with Environmental Pollutants

    Czech Academy of Sciences Publication Activity Database

    Rössner Jr., P.; Mrhálková, A.; Uhlířová, Kateřina; Špátová, M.; Rössnerová, A.; Líbalová, H.; Schmuczerová, J.; Milcová, A.; Topinka, J.; Šrám, R.

    2013-01-01

    Roč. 8, č. 7 (2013), e69197 E-ISSN 1932-6203 Institutional support: RVO:60077344 Keywords : environmental pollutants * DNA * expression of DNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.534, year: 2013

  14. Nucleotide Excision Repair Is Not Induced in Human Embryonic Lung Fibroblasts Treated with Environmental Pollutants

    Czech Academy of Sciences Publication Activity Database

    Rössner ml., Pavel; Mrhálková, Andrea; Uhlířová, Kateřina; Špátová, Milada; Rössnerová, Andrea; Líbalová, Helena; Schmuczerová, Jana; Milcová, Alena; Topinka, Jan; Šrám, Radim

    2013-01-01

    Roč. 8, č. 7 (2013), e69197 E-ISSN 1932-6203 R&D Projects: GA ČR GAP503/11/0084 Institutional support: RVO:68378041 Keywords : environmental pollutants * DNA * expression of DNA Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.534, year: 2013

  15. Expression of TGF-β3 in Isolated Fibroblasts from Foreskin

    Directory of Open Access Journals (Sweden)

    Mahnaz Mahmoudi Rad

    2015-05-01

    Full Text Available Background: The multifunctional transforming growth factor beta (TGF-β is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3 in human foreskin fibroblasts (HFF’s. Methods: We obtained HFF’s from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA. Results: The highest TGF-β3 expression (8.3-fold greater than control was obtained by lipofection after 72 hours using 3 μl of transfection reagent. Expression was 1.4-fold greater than control by electroporation. Conclusions: In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring.

  16. An in vitro evaluation of cytotoxicity of curcumin against human dental pulp fibroblasts

    Directory of Open Access Journals (Sweden)

    Praveenkumar S Mandrol

    2016-01-01

    Full Text Available Objective: The objective of this study was to evaluate the cytotoxicity of curcumin to primary dental pulp fibroblasts in vitro. Materials and Methods: Dental pulp fibroblasts from primary maxillary central incisors were cultured and used for cytotoxicity tests after the fourth passage. Ninety-five percent curcumin was diluted with dimethylsulfoxide to prepare 100%, 50%, and 25% concentrations. Each concentration of curcumin was added in triplicate into 96-well microtiter plate containing the fibroblast culture at 104/well. Cells without treatment served as a control group. The number of viable cells after 48 hrs incubation at 37°C in a humidified atmosphere of 5 % CO2 and 95 % air was determined by the 3-(4, 5-dimethyl-thiazol-2-yl-2, 5-diphenyl-tetrazolium bromide (MTT assay. The relative viability of pulp cells was expressed as color intensity of the number in the experimental wells relative to that of the control group. Absorbances were read at 492 nm on a microplate reader with a background subtraction at 620 nm. Results: Cell viability of primary dental pulp fibroblasts to 25%, 50%, and 100% curcumin concentration was 174%, 310%, and 317%, respectively. Conclusions: Curcumin promotes cell viability and induces proliferation of primary dental pulp fibroblasts and has the potential to be developed into an economical and reliable medicament for vital pulp therapy.

  17. Usefulness of fibroblast culture for testing of cattle tissues polluted with heavy metals

    International Nuclear Information System (INIS)

    Weglarz, L.; Drozdz, M.Wa.; Wardas, M.; Kula, B.; Pawlaczyk-Szpilowa, M.

    1990-01-01

    Cattle tissues (liver, kidney, brain, and lung) that had been polluted with heavy metals were tested for their ability to alter fibroblast culture growth, cellular protein and DNA content, and fibroblast DNA synthesis. At 72 hr of incubation a significant increase in cellular DNA and [14C]thymidine incorporation was noted in the primary cultures as well as in the subcultures compared to controls. Fibroblast cultures also displayed growth inhibition and reduction in protein content. The measurement of basic biochemical parameters of the fibroblast culture may represent a sensitive means of assessing rapidly the activity of heavy metals deposited in the tissues of cattle as a result of their grazing on polluted soil

  18. Human fibroblasts display a differential focal adhesion phenotype relative to chimpanzee.

    Science.gov (United States)

    Advani, Alexander S; Chen, Annie Y; Babbitt, Courtney C

    2016-01-01

    There are a number of documented differences between humans and our closest relatives in responses to wound healing and in disease susceptibilities, suggesting a differential cellular response to certain environmental factors. In this study, we sought to look at a specific cell type, fibroblasts, to examine differences in cellular adhesion between humans and chimpanzees in visualized cells and in gene expression. We have found significant differences in the number of focal adhesions between primary human and chimpanzee fibroblasts. Additionally, we see that adhesion related gene ontology categories are some of the most differentially expressed between human and chimpanzee in normal fibroblast cells. These results suggest that human and chimpanzee fibroblasts may have somewhat different adhesive properties, which could play a role in differential disease phenotypes and responses to external factors. © The Author(s) 2016. Published by Oxford University Press on behalf of the Foundation for Evolution, Medicine, and Public Health.

  19. RNA-Guided Activation of Pluripotency Genes in Human Fibroblasts

    DEFF Research Database (Denmark)

    Xiong, Kai; Zhou, Yan; Blichfeld, Kristian Aabo

    2017-01-01

    -associated protein 9 (dCas9)-VP64 (CRISPRa) alone, or a combination of dCas9-VP64 and MS2-P65-HSF1 [synergistic activation mediator (SAM) system] mediated activation of five pluripotency genes: KLF4 (K), LIN28 (L), MYC (M), OCT4 (O), and SOX2 (S) in human cells (HEK293T, HeLa, HepG2, and primary fibroblasts...... could be obtained from these SAM fibroblasts. In conclusion, our study showed that CRISPR/Cas9-based ATFs are potent to activate and maintain transcription of endogenous human pluripotent genes. However, future improvements of the system are still required to improve activation efficiency and cellular...

  20. Bio-artificial pleura using an autologous dermal fibroblast sheet

    Science.gov (United States)

    Kanzaki, Masato; Takagi, Ryo; Washio, Kaoru; Kokubo, Mami; Yamato, Masayuki

    2017-10-01

    Air leaks (ALs) are observed after pulmonary resections, and without proper treatment, can produce severe complications. AL prevention is a critical objective for managing patients after pulmonary resection. This study applied autologous dermal fibroblast sheets (DFS) to close ALs. For sealing ALs in a 44-year-old male human patient with multiple bullae, a 5 × 15-mm section of skin was surgically excised. From this skin specimen, primary dermal fibroblasts were isolated and cultured for 4 weeks to produce DFSs that were harvested after a 10-day culture. ALs were completely sealed using surgical placement of these autologous DFSs. DFS were found to be a durable long-term AL sealant, exhibiting requisite flexibility, elasticity, durability, biocompatibility, and usability, resulting reliable AL closure. DFS should prove to be an extremely useful tissue-engineered pleura substitute.

  1. Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Karhemo, Piia-Riitta [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Räsänen, Kati [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Laakkonen, Pirjo [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Vaheri, Antti [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland)

    2016-07-01

    Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.

  2. Basal Cell Carcinoma in Gorlin's Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

    Science.gov (United States)

    Gache, Yannick; Brellier, Florence; Rouanet, Sophie; Al-Qaraghuli, Sahar; Goncalves-Maia, Maria; Burty-Valin, Elodie; Barnay, Stéphanie; Scarzello, Sabine; Ruat, Martial; Sevenet, Nicolas; Avril, Marie-Françoise; Magnaldo, Thierry

    2015-01-01

    Basal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings

  3. Neuronal protein gene product 9.5 (IEF SSP 6104) is expressed in cultured human MRC-5 fibroblasts of normal origin and is strongly down-regulated in their SV40 transformed counterparts

    DEFF Research Database (Denmark)

    Honoré, B; Rasmussen, H H; Vandekerckhove, J

    1991-01-01

    of proteins recovered from 2D gels we have identified PGP 9.5 UCH-L1 as polypeptide IEF SSP 6104 (Mr = 27,000, pI = 5.49) in the comprehensive 2D gel cellular protein database of human embryonal lung MRC-5 fibroblasts [(1989) Electrophoresis 10, 76 115; (1990) Electrophoresis 11, 1072 1113]. This protein...

  4. Radiosensitivity in cultured human fibroblasts

    International Nuclear Information System (INIS)

    Cox, R.; Masson, W.K.

    1980-01-01

    Caution is urged in the use of freshly isolated cultures of human diploid fibroblasts for quantitative studies of radiosensitivity. The distribution of x ray sensitivities of 'normal' human fibroblast cultures of foetal origin (10 subjects, skin or lung biopsy) and post-foetal origin (34 subjects, skin biopsy) are compared with the distribution in 12 patients with ataxia telangiectasia (probability of including any one of these in a normal post-foetal distribution is 0.01%). Cultures from nominally normal subjects showed a broad distribution of D 0 range of 98 +- 160 rad and assuming normal distribution, a mean +- one standard deviation of 122 +- 17 rad. Mean D 0 values for foetal origin cultures were 117 +- 12; values for post-foetal cultures D 0 were 124 +- 18. No systematic variation in D 0 was observed for age of donor, number of cell divisions in culture or for cloning efficiency. For ataxia telangiectasia D 0 values were 46 +- 7 rad. (U.K.)

  5. Protecting genomic integrity in somatic cells and embryonic stem cells

    International Nuclear Information System (INIS)

    Hong, Y.; Cervantes, R.B.; Tichy, E.; Tischfield, J.A.; Stambrook, P.J.

    2007-01-01

    Mutation frequencies at some loci in mammalian somatic cells in vivo approach 10 -4 . The majority of these events occur as a consequence of loss of heterozygosity (LOH) due to mitotic recombination. Such high levels of DNA damage in somatic cells, which can accumulate with age, will cause injury and, after a latency period, may lead to somatic disease and ultimately death. This high level of DNA damage is untenable for germ cells, and by extrapolation for embryonic stem (ES) cells, that must recreate the organism. ES cells cannot tolerate such a high frequency of damage since mutations will immediately impact the altered cell, and subsequently the entire organism. Most importantly, the mutations may be passed on to future generations. ES cells, therefore, must have robust mechanisms to protect the integrity of their genomes. We have examined two such mechanisms. Firstly, we have shown that mutation frequencies and frequencies of mitotic recombination in ES cells are about 100-fold lower than in adult somatic cells or in isogenic mouse embryonic fibroblasts (MEFs). A second complementary protective mechanism eliminates those ES cells that have acquired a mutational burden, thereby maintaining a pristine population. Consistent with this hypothesis, ES cells lack a G1 checkpoint, and the two known signaling pathways that mediate the checkpoint are compromised. The checkpoint kinase, Chk2, which participates in both pathways is sequestered at centrosomes in ES cells and does not phosphorylate its substrates (i.e. p53 and Cdc25A) that must be modified to produce a G1 arrest. Ectopic expression of Chk2 does not rescue the p53-mediated pathway, but does restore the pathway mediated by Cdc25A. Wild type ES cells exposed to ionizing radiation do not accumulate in G1 but do so in S-phase and in G2. ES cells that ectopically express Chk2 undergo cell cycle arrest in G1 as well as G2, and appear to be protected from apoptosis

  6. [Embryonic stem cells. Future perspectives].

    Science.gov (United States)

    Groebner, M; David, R; Franz, W M

    2006-05-01

    Embryonic stem cells (ES cells) are able to differentiate into any cell type, and therefore represent an excellent source for cellular replacement therapies in the case of widespread diseases, for example heart failure, diabetes, Parkinson's disease and spinal cord injury. A major prerequisite for their efficient and safe clinical application is the availability of pure populations for direct cell transplantation or tissue engineering as well as the immunological compatibility of the transplanted cells. The expression of human surface markers under the control of cell type specific promoters represents a promising approach for the selection of cardiomyocytes and other cell types for therapeutic applications. The first human clinical trial using ES cells will start in the United States this year.

  7. Vitamin K2 biosynthetic enzyme, UBIAD1 is essential for embryonic development of mice.

    Science.gov (United States)

    Nakagawa, Kimie; Sawada, Natsumi; Hirota, Yoshihisa; Uchino, Yuri; Suhara, Yoshitomo; Hasegawa, Tomoka; Amizuka, Norio; Okamoto, Tadashi; Tsugawa, Naoko; Kamao, Maya; Funahashi, Nobuaki; Okano, Toshio

    2014-01-01

    UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K2 biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1(-/-)) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1(-/-) embryonic stem (ES) cells failed to synthesize vitamin K2 but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1(+/-) mice developed normally, exhibiting normal growth and fertility. Vitamin K2 tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K2 synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1(+/-) E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1(-/-) mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1(+/-) mice. These results suggest that UBIAD1 is responsible for vitamin K2 synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K2, but may have additional functions beyond the biosynthesis of vitamin K2.

  8. Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance*

    Science.gov (United States)

    Soteriou, Despina; Iskender, Banu; Byron, Adam; Humphries, Jonathan D.; Borg-Bartolo, Simon; Haddock, Marie-Claire; Baxter, Melissa A.; Knight, David; Humphries, Martin J.; Kimber, Susan J.

    2013-01-01

    Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal. PMID:23658023

  9. Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

    International Nuclear Information System (INIS)

    Fuchigami, Takao; Kibe, Toshiro; Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio; Nishizawa, Yoshiaki; Hijioka, Hiroshi; Fujii, Tomomi; Ueda, Masahiro; Nakamura, Norifumi; Kiyono, Tohru; Kishida, Michiko

    2014-01-01

    Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

  10. Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Fuchigami, Takao [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kibe, Toshiro [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Nishizawa, Yoshiaki [Kagoshima University Faculty of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Hijioka, Hiroshi; Fujii, Tomomi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Ueda, Masahiro [Natural Science Centre for Research and Education, Kagoshima University, 1-21-24 Koorimoto, Kagoshima 890-8580 (Japan); Nakamura, Norifumi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kiyono, Tohru [Department of Virology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuouku, Tokyo 104-0045 (Japan); Kishida, Michiko, E-mail: kmichiko@m2.kufm.kagoshima-u.ac.jp [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

    2014-09-05

    Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

  11. GLUT3 gene expression is critical for embryonic growth, brain development and survival.

    Science.gov (United States)

    Carayannopoulos, Mary O; Xiong, Fuxia; Jensen, Penny; Rios-Galdamez, Yesenia; Huang, Haigen; Lin, Shuo; Devaskar, Sherin U

    2014-04-01

    Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Induction of Pluripotency in Adult Equine Fibroblasts without c-MYC

    Directory of Open Access Journals (Sweden)

    Khodadad Khodadadi

    2012-01-01

    Full Text Available Despite tremendous efforts on isolation of pluripotent equine embryonic stem (ES cells, to date there are few reports about successful isolation of ESCs and no report of in vivo differentiation of this important companion species. We report the induction of pluripotency in adult equine fibroblasts via retroviral transduction with three transcription factors using OCT4, SOX2, and KLF4 in the absence of c-MYC. The cell lines were maintained beyond 27 passages (more than 11 months and characterized. The equine iPS (EiPS cells stained positive for alkaline phosphatase by histochemical staining and expressed OCT4, NANOG, SSEA1, and SSEA4. Gene expression analysis of the cells showed the expression of OCT4, SOX2 NANOG, and STAT3. The cell lines retained a euploid chromosome count of 64 after long-term culture cryopreservation. The EiPS demonstrated differentiation capacity for the three embryonic germ layers both in vitro by embryoid bodies (EBs formation and in vivo by teratoma formation. In conclusion, we report the derivation of iPS cells from equine adult fibroblasts and long-term maintenance using either of the three reprogramming factors.

  13. Permissive cytomegalovirus infection of primary villous term and first trimester trophoblasts.

    Science.gov (United States)

    Hemmings, D G; Kilani, R; Nykiforuk, C; Preiksaitis, J; Guilbert, L J

    1998-06-01

    Forty percent of women with primary cytomegalovirus (CMV) infections during pregnancy infect their fetuses with complications for the baby varying from mild to severe. How CMV crosses the syncytiotrophoblast, the barrier between maternal blood and fetal tissue in the villous placenta, is unknown. Virus may cross by infection of maternal cells that pass through physical breaches in the syncytiotrophoblast or by direct infection of the syncytiotrophoblast, with subsequent transmission to underlying fetal placental cells. In this study, we show that pure (>99.99%), long-term and healthy (>3 weeks) cultures of syncytiotrophoblasts are permissively infected with CMV. Greater than 99% of infectious progeny virus remained cell associated throughout culture periods up to 3 weeks. Infection of term trophoblasts required a higher virus inoculum, was less efficient, and progressed more slowly than parallel infections of placental and human embryonic lung fibroblasts. Three laboratory strains (AD169, Towne, and Davis) and a clinical isolate from a congenitally infected infant all permissively infected trophoblasts, although infection efficiencies varied. The infection of first trimester syncytiotrophoblasts with strain AD169 occurred at higher frequency and progressed more rapidly than infection of term cells but less efficiently and rapidly than infection of fibroblasts. These results show that villous syncytiotrophoblasts can be permissively infected by CMV but that the infection requires high virus titers and proceeds slowly and that progeny virus remains predominantly cell associated.

  14. Fibroblastic osteosarcoma with epithelioid and squamous differentiation in a dog.

    Science.gov (United States)

    Jenkins, Tiffany L; Agnew, Dalen; Rissi, Daniel R

    2018-04-01

    A fibroblastic osteosarcoma with epithelioid and squamous differentiation in the distal femur of a 9-y-old spayed female Greyhound dog is described. Grossly, the tumor consisted of a pale-white, firm-to-hard mass that replaced the medullary and cortical areas of the distal end of the right femur. Histologically, the mass was composed predominantly of spindle cells admixed with areas of mineralized and non-mineralized osteoid matrix that were surrounded by stellate osteoblasts and scattered multinucleate giant cells, consistent with the diagnosis of a fibroblastic osteosarcoma. In addition, well-demarcated clusters of neoplastic epithelioid cells and foci of squamous differentiation with keratin pearls were present throughout the neoplasm. The spindle cells, epithelioid cells, and areas of squamous differentiation expressed cytoplasmic immunostaining for osteocalcin and osteonectin. The spindle cells and epithelioid cells were also immunopositive for vimentin. Epithelioid cells also expressed occasional cytoplasmic immunostaining for pancytokeratin (PCK) Lu-5, and areas of squamous differentiation were immunoreactive for PCK Lu-5 and high molecular weight CK; these areas were inconsistently immunoreactive for CK 5-6 and immunonegative for low molecular weight CK. Foci of squamous differentiation were not located within blood or lymphatic vessels, given that no immunoreactivity for factor VIII-related antigen was observed around these areas. A thorough autopsy and an evaluation of the medical history excluded a primary carcinoma or other neoplasm elsewhere in the dog. The findings were consistent with a diagnosis of fibroblastic osteosarcoma with epithelioid and squamous differentiation.

  15. Responses of fibroblasts and glial cells to nanostructured platinum surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Pennisi, C P; Sevcencu, C; Yoshida, K [Center for Sensory-Motor Interaction (SMI), Aalborg University, Aalborg (Denmark); Dolatshahi-Pirouz, A; Foss, M; Larsen, A Nylandsted; Besenbacher, F [Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus (Denmark); Hansen, J Lundsgaard [Department of Physics and Astronomy, Aarhus University, Aarhus (Denmark); Zachar, V, E-mail: cpennisi@hst.aau.d [Laboratory for Stem Cell Research, Aalborg University (Denmark)

    2009-09-23

    The chronic performance of implantable neural prostheses is affected by the growth of encapsulation tissue onto the stimulation electrodes. Encapsulation is associated with activation of connective tissue cells at the electrode's metallic contacts, usually made of platinum. Since surface nanotopography can modulate the cellular responses to materials, the aim of the present work was to evaluate the 'in vitro' responses of connective tissue cells to platinum strictly by modulating its surface nanoroughness. Using molecular beam epitaxy combined with sputtering, we produced platinum nanostructured substrates consisting of irregularly distributed nanopyramids and investigated their effect on the proliferation, cytoskeletal organization and cellular morphology of primary fibroblasts and transformed glial cells. Cells were cultured on these substrates and their responses to surface roughness were studied. After one day in culture, the fibroblasts were more elongated and their cytoskeleton less mature when cultured on rough substrates. This effect increased as the roughness of the surface increased and was associated with reduced cell proliferation throughout the observation period (4 days). Morphological changes also occurred in glial cells, but they were triggered by a different roughness scale and did not affect cellular proliferation. In conclusion, surface nanotopography modulates the responses of fibroblasts and glial cells to platinum, which may be an important factor in optimizing the tissue response to implanted neural electrodes.

  16. DIFFERENTIATION OF EMBRYONIC STEM CELLS: LESSONS FROM EMBRYONIC DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    EMOKE PALL

    2008-05-01

    Full Text Available Embryonic stem (ES cells, the undifferentiated cells of early embryos are established as permanent lines and are characterised by their self-renewal capacity and the ability to retain their developmental capacity in vivo and in vitro. The pluripotent properties of ES cells are the basis of gene targeting technologies used to create mutant mouse strains with inactivated genes by homologous recombination. There are several methods to induce the formation of EBs. One of them the formation by aggregating ES cells in hanging drops, using gravity as an aggregation force. This method presents the advantage of obtaining well-calibrated EBs almost identical in size. We used at our experiment the mouse ES cell line KA1/11/C3/C8 with a normal karyotype, at 14th passages. Immunohistochemical examination was aimed to identify tissue-restricted proteins for the two differentiated lineages: titin as a cell-specific antigen for cardiac and skeletal muscle, betaIII-tubulin for the neuronal differentiation, cytokeratin Endo-A (TROMA for the presence of mesenchymal progenitor cells, Oct-4 for the presence of the undifferentiated ES cells. The beating cardiac muscle clumps showed more synchronous rhythm than those seen in EBs obtained from suspension culture method, where the beating cardiac muscle clumps appeared later, had a lower frequency and were uneven. The synaptic networks of neuronal cells were best developed in EBs from suspension, compared to those observed in EBs from hanging-drop method.

  17. Altered Dermal Fibroblasts in Systemic Sclerosis Display Podoplanin and CD90.

    Science.gov (United States)

    Nazari, Banafsheh; Rice, Lisa M; Stifano, Giuseppina; Barron, Alexander M S; Wang, Yu Mei; Korndorf, Tess; Lee, Jungeun; Bhawan, Jag; Lafyatis, Robert; Browning, Jeffrey L

    2016-10-01

    Tissue injury triggers the activation and differentiation of multiple cell types to minimize damage and initiate repair processes. In systemic sclerosis, these repair processes appear to run unchecked, leading to aberrant remodeling and fibrosis of the skin and multiple internal organs, yet the fundamental pathological defect remains unknown. We describe herein a transition wherein the abundant CD34(+) dermal fibroblasts present in healthy human skin disappear in the skin of systemic sclerosis patients, and CD34(-), podoplanin(+), and CD90(+) fibroblasts appear. This transition is limited to the upper dermis in several inflammatory skin diseases, yet in systemic sclerosis, it can occur in all regions of the dermis. In vitro, primary dermal fibroblasts readily express podoplanin in response to the inflammatory stimuli tumor necrosis factor and IL-1β. Furthermore, we show that on acute skin injury in both human and murine settings, this transition occurs quickly, consistent with a response to inflammatory signaling. Transitioned fibroblasts partially resemble the cells that form the reticular networks in organized lymphoid tissues, potentially linking two areas of fibroblast research. These results allow for the visualization and quantification of a basic stage of fibroblast differentiation in inflammatory and fibrotic diseases in the skin. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  18. Blue light-irradiated human keloid fibroblasts: an in vitro study

    Science.gov (United States)

    Magni, Giada; Rossi, Francesca; Tatini, Francesca; Pini, Roberto; Coppi, Elisabetta; Cherchi, Federica; Fusco, Irene; Pugliese, Anna Maria; Pedata, Felicita; Fraccalvieri, Marco; Gasperini, Stefano; Pavone, Francesco S.; Tripodi, Cristina; Alfieri, Domenico; Targetti, Lorenzo

    2018-02-01

    Blue LED light irradiation is currently under investigation because of its effect in wound healing improvement. In this context, several mechanisms of action are likely to occur at the same time, consistently with the presence of different light absorbers within the skin. In our previous studies we observed the wound healing in superficial abrasions in an in vivo murine model. The results evidenced that both inflammatory infiltrate and myofibroblasts activity increase after irradiation. In this study we focused on evaluating the consequences of light absorption in fibroblasts from human cells culture: they play a key role in wound healing, both in physiological conditions and in pathological ones, such as keloid scarring. In particular we used keloids fibroblasts as a new target in order to investigate a possible metabolic or cellular mechanism correlation. Human keloid tissues were excised during standard surgery and immediately underwent primary cell culture extraction. Fibroblasts were allowed to grow in the appropriate conditions and then exposed to blue light. A metabolic colorimetric test (WST-8) was then performed. The tests evidenced an effect in mitochondrial activity, which could be modulated by the duration of the treatment. Electrophysiology pointed out a different behavior of irradiated fibroblasts. In conclusion, the Blue LED light affects the metabolic activity of fibroblasts and thus the cellular proliferation rate. No specific effect was found on keloid fibroblasts, thus indicating a very basic intracellular component, such as cytochromes, being the target of the treatment.

  19. Essential roles of BCCIP in mouse embryonic development and structural stability of chromosomes.

    Directory of Open Access Journals (Sweden)

    Huimei Lu

    2011-09-01

    Full Text Available BCCIP is a BRCA2- and CDKN1A(p21-interacting protein that has been implicated in the maintenance of genomic integrity. To understand the in vivo functions of BCCIP, we generated a conditional BCCIP knockdown transgenic mouse model using Cre-LoxP mediated RNA interference. The BCCIP knockdown embryos displayed impaired cellular proliferation and apoptosis at day E7.5. Consistent with these results, the in vitro proliferation of blastocysts and mouse embryonic fibroblasts (MEFs of BCCIP knockdown mice were impaired considerably. The BCCIP deficient mouse embryos die before E11.5 day. Deletion of the p53 gene could not rescue the embryonic lethality due to BCCIP deficiency, but partially rescues the growth delay of mouse embryonic fibroblasts in vitro. To further understand the cause of development and proliferation defects in BCCIP-deficient mice, MEFs were subjected to chromosome stability analysis. The BCCIP-deficient MEFs displayed significant spontaneous chromosome structural alterations associated with replication stress, including a 3.5-fold induction of chromatid breaks. Remarkably, the BCCIP-deficient MEFs had a ∼20-fold increase in sister chromatid union (SCU, yet the induction of sister chromatid exchanges (SCE was modestly at 1.5 fold. SCU is a unique type of chromatid aberration that may give rise to chromatin bridges between daughter nuclei in anaphase. In addition, the BCCIP-deficient MEFs have reduced repair of irradiation-induced DNA damage and reductions of Rad51 protein and nuclear foci. Our data suggest a unique function of BCCIP, not only in repair of DNA damage, but also in resolving stalled replication forks and prevention of replication stress. In addition, BCCIP deficiency causes excessive spontaneous chromatin bridges via the formation of SCU, which can subsequently impair chromosome segregations in mitosis and cell division.

  20. The radiation response of human dermal fibroblasts

    Science.gov (United States)

    Mitchell, Stephen Andrew

    A clinically reliable predictive assay based on normal-tissue radiosensitivity may lead to improved tumour control through individualised dose prescriptions. In-vitro fibroblast radiosensitivity has been shown, in several studies, to correlate with late radiation morbidity. The aim of this study was to investigate some of the cellular mechanisms underlying the normal-tissue response. In this study, seventeen primary fibroblast strains were established by enzymatic disaggregation of skin biopsies obtained from patients. These comprised seven who experienced acute tissue reactions to radiotherapy, four patients with a normal response and six non-cancer volunteers. An AT cell line was included as a positive control for radiosensitivity. In-vitro radiosensitivity was measured using a clonogenic assay at both high (HDR: 1.6 Gymin-1) and low dose rate (LDR: 0.01 Gymin-1). The radiation parameter HDR SF2 was the most sensitive in discriminating the seven sensitive patients from the remaining ten normal patients (range 0.11-0.19 sensitive patients compared with 0.17-0.34 control patients: puse of an internal control or LDR radiation protocol increased this discrimination. Pulsed-field gel electrophoresis (PFGE) was used to measure the level of initial and residual double-strand breaks following irradiation. No correlation was found between HDR SF2 and initial DNA damage. However, a strong correlation was found between clonogenic survival and both residual DNA damage (measured over 10-70 Gy, allowing 4 h repair, correlation coefficient: 0.90, <0.0001) and the ratio of residual/initial DNA damage, with the sensitive cell lines generally showing a higher level of residual DNA damage. Cell-cycle delays were found in all 18 cell strains in response to 2 Gy irradiation, but were not found to discriminate between sensitive and normal patients. Associated studies found no mutations of the ATM gene in the five radiosensitive patients studied. However, a coding sequence alteration

  1. Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yanfu [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Chai, Jiake, E-mail: cjk304@126.com [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Sun, Tianjun; Li, Dongjie; Tao, Ran [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China)

    2011-10-07

    Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue

  2. Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

    International Nuclear Information System (INIS)

    Han, Yanfu; Chai, Jiake; Sun, Tianjun; Li, Dongjie; Tao, Ran

    2011-01-01

    Highlights: → Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. → Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. → We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. → Collagen type I and collagen type III mRNA level was higher in differentiated cells. → UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue-engineered dermis.

  3. Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria

    DEFF Research Database (Denmark)

    Christensen, Rikke; Güttler, Flemming; Jensen, Thomas G

    2002-01-01

    gene therapy. We have previously shown that overexpression of PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH(4) supplementation [Gene Ther. 7 (2000) 1971]. Here, we investigate the capacity of fibroblasts, another cell type from the skin......, to metabolize phenylalanine. After retroviral gene transfer of PAH and GTP-CH both normal and PKU patient fibroblasts were able to metabolize phenylalanine, however, in lower amounts compared to genetically modified keratinocytes. Further comparative analyses between keratinocytes and fibroblasts revealed...

  4. Guidelines for human embryonic stem cell research

    National Research Council Canada - National Science Library

    Committee on Guidelines for Human Embryonic Stem Cell Research, National Research Council

    2005-01-01

    Since 1998, the volume of research being conducted using human embryonic stem (hES) cells has expanded primarily using private funds because of restrictions on the use of federal funds for such research...

  5. Effect of chloramphenicol on sister chromatid exchange in bovine fibroblasts.

    Science.gov (United States)

    Arruga, M V; Catalan, J; Moreno, C

    1992-03-01

    The genotoxic potential of different chloramphenicol concentrations (5, 20, 40 and 60 micrograms ml-1) was investigated in bovine fibroblast primary lines by sister chromatid exchange assay. Chloramphenicol acted for long enough to ensure similar effects to persistent storage in the kidney. In this experiment 10 micrograms ml-1 of 5-bromodeoxyuridine was added for 60 hours for all doses of chloramphenicol and to the control. When the tissue culture cells were exposed to increasing doses, increased numbers of sister chromatid exchanges developed. Differences were significantly different to the control.

  6. Effect of saccharin on metabolic cooperation between human fibroblasts

    International Nuclear Information System (INIS)

    Mosser, D.D.; Bols, N.C.

    1983-01-01

    Autoradiography was used to study the effect of saccharin on metabolic cooperation between human diploid fibroblasts. When the donors, HGPRT+ cells, and recipients, HGPRT- cells, were plated together in the presence of saccharin, all the interactions that developed in 4 and 24 h were positive for metabolic cooperation. When saccharin was added after donor cells and recipient cells had made contact, the proportion of interactions that were positive for metabolic cooperation was unchanged but the number of grains over primary recipients was reduced. However, in donor cells saccharin caused a reduction in [ 3 H]hypoxanthine incorporation into both acid-soluble and acid-insoluble fractions, although the relative distribution of radioactivity between these two fractions and between the phosphorylated and non-phosphorylated derivatives of [ 3 H]hypoxanthine was unchanged. Metabolic cooperation was studied under conditions in which the number of grains over the nuclei of both the primary recipient and the primary recipient's donor could be counted. The change in the number of grains over these two cell types in response to saccharin was compared and found to be the same. Thus in normal human fibroblasts saccharin does not appear to affect metabolic cooperation, which is a measure of cell-to-cell communication

  7. Comparison of the early response of human embryonic stem cells and human induced pluripotent stem cells to ionizing radiation.

    Science.gov (United States)

    Suchorska, Wiktoria Maria; Augustyniak, Ewelina; Łukjanow, Magdalena

    2017-04-01

    Despite the well-demonstrated efficacy of stem cell (SC) therapy, this approach has a number of key drawbacks. One important concern is the response of pluripotent SCs to treatment with ionizing radiation (IR), given that SCs used in regenerative medicine will eventually be exposed to IR for diagnostic or treatment‑associated purposes. Therefore, the aim of the present study was to examine and compare early IR‑induced responses of pluripotent SCs to assess their radioresistance and radiosensitivity. In the present study, 3 cell lines; human embryonic SCs (hESCs), human induced pluripotent SCs (hiPSCs) and primary human dermal fibroblasts (PHDFs); were exposed to IR at doses ranging from 0 to 15 gray (Gy). Double strand breaks (DSBs), and the gene expression of the following DNA repair genes were analyzed: P53; RAD51; BRCA2; PRKDC; and XRCC4. hiPSCs demonstrated greater radioresistance, as fewer DSBs were identified, compared with hESCs. Both pluripotent SC lines exhibited distinct gene expression profiles in the most common DNA repair genes that are involved in homologous recombination, non‑homologous end‑joining and enhanced DNA damage response following IR exposure. Although hESCs and hiPSCs are equivalent in terms of capacity for pluripotency and differentiation into 3 germ layers, the results of the present study indicate that these 2 types of SCs differ in gene expression following exposure to IR. Consequently, further research is required to determine whether hiPSCs and hESCs are equally safe for application in clinical practice. The present study contributes to a greater understanding of DNA damage response (DDR) mechanisms activated in pluripotent SCs and may aid in the future development of safe SC‑based clinical protocols.

  8. Biomechanical forces promote embryonic haematopoiesis

    Science.gov (United States)

    Adamo, Luigi; Naveiras, Olaia; Wenzel, Pamela L.; McKinney-Freeman, Shannon; Mack, Peter J.; Gracia-Sancho, Jorge; Suchy-Dicey, Astrid; Yoshimoto, Momoko; Lensch, M. William; Yoder, Mervin C.; García-Cardeña, Guillermo; Daley, George Q.

    2009-01-01

    Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system1,2. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3–5), a master regulator of haematopoiesis, and give rise to haematopoietic cells4. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential6. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41+c-Kit+ haematopoietic progenitor cells7,concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the paraaortic splanchnopleura/aorta–gonads–mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling8, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development. PMID:19440194

  9. Human embryonic stem cells have enhanced repair of multiple forms of DNA damage

    DEFF Research Database (Denmark)

    Maynard, Scott; Swistowska, Anna Maria; Lee, Jae Wan

    2008-01-01

    cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary...

  10. Meningeal norepinephrine produces headache behaviors in rats via actions both on dural afferents and fibroblasts.

    Science.gov (United States)

    Wei, Xiaomei; Yan, Jin; Tillu, Dipti; Asiedu, Marina; Weinstein, Nicole; Melemedjian, Ohannes; Price, Theodore; Dussor, Gregory

    2015-10-01

    Stress is commonly reported to contribute to migraine although mechanisms by which this may occur are not fully known. The purpose of these studies was to examine whether norepinephrine (NE), the primary sympathetic efferent transmitter, acts on processes in the meninges that may contribute to the pain of migraine. NE was applied to rat dura using a behavioral model of headache. Primary cultures of rat trigeminal ganglia retrogradely labeled from the dura mater and of rat dural fibroblasts were prepared. Patch-clamp electrophysiology, Western blot, and ELISA were performed to examine the effects of NE. Conditioned media from NE-treated fibroblast cultures was applied to the dura using the behavioral headache model. Dural injection both of NE and media from NE-stimulated fibroblasts caused cutaneous facial and hindpaw allodynia in awake rats. NE application to cultured dural afferents increased action potential firing in response to current injections. Application of NE to dural fibroblasts increased phosphorylation of ERK and caused the release of interleukin-6 (IL-6). These data demonstrate that NE can contribute to pro-nociceptive signaling from the meninges via actions on dural afferents and dural fibroblasts. Together, these actions of NE may contribute to the headache phase of migraine. © International Headache Society 2015.

  11. DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

    Energy Technology Data Exchange (ETDEWEB)

    Cogan, Nicola [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Baird, Duncan M. [Department of Pathology School of Medicine, Cardiff University, Henry Wellcome Building for Biomedical Research in Wales, Heath Park, Cardiff, CF14 4XN (United Kingdom); Phillips, Ryan [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Crompton, Lucy A.; Caldwell, Maeve A. [Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, BS1 3NY (United Kingdom); Rubio, Miguel A. [Center of Regenerative Medicine in Barcelona, CMRB Dr. Aiguader, 88, 7th Floor, 08003 Barcelona (Spain); Newson, Roger [Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Dublin 2 (Ireland); Lyng, Fiona [National Heart and Lung Institute, Imperial College London, London, SW7 2AZ (United Kingdom); Case, C. Patrick, E-mail: c.p.case@bristol.ac.uk [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom)

    2010-01-05

    The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

  12. DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

    International Nuclear Information System (INIS)

    Cogan, Nicola; Baird, Duncan M.; Phillips, Ryan; Crompton, Lucy A.; Caldwell, Maeve A.; Rubio, Miguel A.; Newson, Roger; Lyng, Fiona; Case, C. Patrick

    2010-01-01

    The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

  13. The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts.

    Science.gov (United States)

    Yhee, Ji Young; Yoon, Hong Yeol; Kim, Hyunjoon; Jeon, Sangmin; Hergert, Polla; Im, Jintaek; Panyam, Jayanth; Kim, Kwangmeyung; Nho, Richard Seonghun

    2017-01-01

    Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6-78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts.

  14. Deregulated MAPK activity prevents adipocyte differentiation of fibroblasts lacking the retinoblastoma protein

    DEFF Research Database (Denmark)

    Hansen, Jacob B; Petersen, Rasmus K; Jørgensen, Claus

    2002-01-01

    A functional retinoblastoma protein (pRB) is required for adipose conversion of preadipocyte cell lines and primary mouse embryo fibroblasts (MEFs) in response to treatment with standard adipogenic inducers. Interestingly, lack of functional pRB in MEFs was recently linked to elevated Ras activity...

  15. Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells

    DEFF Research Database (Denmark)

    Hattori, Naoko; Niwa, Tohru; Kimura, Kana

    2013-01-01

    . Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell......Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which...... population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination...

  16. Fibroblasts in fibrosis: novel roles and mediators

    Directory of Open Access Journals (Sweden)

    Ryan Thomas Kendall

    2014-05-01

    Full Text Available Fibroblasts are the most common cell type of the connective tissues found throughout the body and the principal source of the extensive extracellular matrix (ECM characteristic of these tissues. They are also the central mediators of the pathological fibrotic accumulation of ECM and the cellular proliferation and differentiation that occurs in response to prolonged tissue injury and chronic inflammation. The transformation of the fibroblast cell lineage involves classical developmental signaling programs and includes a surprisingly diverse range of precursor cell types—most notably, myofibroblasts that are the apex of the fibrotic phenotype. Myofibroblasts display exaggerated ECM production; constitutively secrete and are hypersensitive to chemical signals such as cytokines, chemokines, and growth factors; and are endowed with a contractile apparatus allowing them to manipulate the ECM fibers physically to close open wounds. In addition to ECM production, fibroblasts have multiple concomitant biological roles, such as in wound healing, inflammation, and angiogenesis, which are each interwoven with the process of fibrosis. We now recognize many common fibroblast-related features across various physiological and pathological protracted processes. Indeed, a new appreciation has emerged for the role of noncancerous fibroblast interactions with tumors in cancer progression. Although the predominant current clinical treatments of fibrosis involve nonspecific immunosuppressive and anti-proliferative drugs, a variety of potential therapies under investigation specifically target fibroblast biology.

  17. Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Braam, S.R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2008-01-01

    Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired

  18. Generation of KCL025 research grade human embryonic stem cell line carrying a mutation in NF1 gene

    Directory of Open Access Journals (Sweden)

    Heema Hewitson

    2016-03-01

    Full Text Available The KCL025 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neurofibromin (c.3739–3742 ΔTTTG. Mutations in this gene have been linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  19. Generation of KCL018 research grade human embryonic stem cell line carrying a mutation in the DMPK gene

    Directory of Open Access Journals (Sweden)

    Cristian Miere

    2016-03-01

    Full Text Available The KCL018 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the DMPK gene encoding the dystrophia myotonica protein kinase (2200 trinucleotide repeats; 14 for the normal allele. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  20. Generation of KCL028 research grade human embryonic stem cell line carrying a mutation in the HTT gene

    Directory of Open Access Journals (Sweden)

    Laureen Jacquet

    2016-03-01

    Full Text Available The KCL028 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting one allele of the HTT gene encoding huntingtin (43 trinucleotide repeats; 21 for the normal allele. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays.

  1. Relationship between delayed embryonic development and metabolic factors and fat deposition in fruit bat Cynopterus sphinx.

    Science.gov (United States)

    Banerjee, Arnab; Meenakumari, K J; Krishna, Amitabh

    2007-01-01

    The present study was undertaken in the fruit bat Cynopterus sphinx, which breeds twice in quick succession at Varanasi, India. Its gestation period varies significantly in the two successive pregnancies of the year owing to delayed embryonic development during the first (winter) pregnancy. The primary aim of the present study was to determine the role of metabolic factors in delayed embryonic development in the fruit bat C. sphinx. Variation in bodyweight, fat deposition, oxygen (O(2)) consumption rate, basal metabolic rate (BMR), body temperature (Tb) and hepatic succinate dehydrogenase (SDH) activity, along with circulating levels of thyroid hormones (tri-iodothyronine and thyroxine), were examined as metabolic factors during the two successive pregnancies in C. sphinx. The increase in bodyweight observed in November was due to accumulation of white adipose tissue in the posterior abdominal region. A significant decline in O(2) consumption rate, BMR, Tb and SDH activity was found in early winter in November-December, which coincides closely with the period of fat accumulation and with the period of delayed embryonic development in C. sphinx. A significantly higher O(2) consumption rate, BMR, Tb and SDH activity was noted during the second pregnancy in, when embryonic development was relatively faster. Thyroid hormone levels were high during the period of embryonic delay compared with levels during the remaining months. The results of the present study suggest that the delayed embryonic development in C. sphinx during early winter may be due to a low O(2) consumption rate, BMR, Tb and SDH activity in November-December. The energy saved by suppressing embryonic development in this species may be advantageous for fat accumulation. Increased thyroid hormone levels during the early winter period might facilitate fat accumulation in C. sphinx.

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

    Science.gov (United States)

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

    2010-11-01

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

  3. Colony size distributions according to in vitro aging in human skin fibroblasts

    International Nuclear Information System (INIS)

    Kim, Jun Sang; Kim, Jae Sung; Cho, Moon June; Park, Jeong Kyu; Paik, Tae Hyun

    1999-01-01

    To investigate the percentage of colonies with 16 or more cells distribution of human skin fibroblast according to in vitro aging, and to evaluate the relationship between percentage of colonies with 16 or more cells and in vivo donor age in human skin fibroblast culture. C1, C2, C3a, and C3b human skin fibroblast samples from three breast cancer patients were used as subjects. The C1, C2, and C3a donor were 44, 54, and 55 years old, respectively. C3a and C3b cells were isolated from the same person. Single cell suspension of skin fibroblasts was prepared with primary explant technique. One hundred cells are plated into 100ml tissue culture flask and cultured for two weeks. The colony size was defined as colonies with 16 or more cells. The cultured cell was stained with crystal violet, and number of cells in each colony was determined with stereo microscope at x 10 magnification. Passage number of C1, C2, C3a and C3b skin fibroblast were 12th, 17th, and 14th, respectively. Percentage of colonies with 16 or more cells of skin fibroblast samples decreased with increasing in vitro passage number. In contrast, cumulative population doublings of skin fibroblast sample increased with increasing in vitro passage number. Percentage of colonies with 16 or more cells also decreased with increasing population doublings in human skin fibroblast culture. There was strong correlation with percentage of colonised with 16 or more cells and population doublings in C3a skin fibroblast sample. At the same point of population doublings, the percentage of colonies with 16 or more cells of the young C1 donor was higher level than the old C3a donor. The population doublings increased with increasing in vitro passage number but percentage of colonies with 16 or more cells decreased. The results of this study imply that percentage of colonies with 16 or more cells is useful as a indicator of in vitro human skin fibroblast aging and may estimate the in vivo donor age

  4. Identification of colonic fibroblast secretomes reveals secretory factors regulating colon cancer cell proliferation.

    Science.gov (United States)

    Chen, Sun-Xia; Xu, Xiao-En; Wang, Xiao-Qing; Cui, Shu-Jian; Xu, Lei-Lei; Jiang, Ying-Hua; Zhang, Yang; Yan, Hai-Bo; Zhang, Qian; Qiao, Jie; Yang, Peng-Yuan; Liu, Feng

    2014-10-14

    Stromal microenvironment influences tumor cell proliferation and migration. Fibroblasts represent the most abundant stromal constituents. Here, we established two pairs of normal fibroblast (NF) and cancer-associated fibroblast (CAF) cultures from colorectal adenocarcinoma tissues and the normal counterparts. The NFs and CAFs were stained positive for typical fibroblast markers and inhibited colon cancer (CC) cell proliferation in in vitro cocultures and in xenograft mouse models. The fibroblast conditioned media were analyzed using LC-MS and 227 proteins were identified at a false discovery rate of 1.3%, including 131 putative secretory and 20 plasma membrane proteins. These proteins were enriched for functional categories of extracellular matrix, adhesion, cell motion, inflammatory response, redox homeostasis and peptidase inhibitor. Secreted protein acidic and rich in cysteine, transgelin, follistatin-related protein 1 (FSTL1) and decorin was abundant in the fibroblast secretome as confirmed by Western blot. Silencing of FSTL1 and transgelin in colonic fibroblast cell line CCD-18Co induced an accelerated proliferation of CC cells in cocultures. Exogenous FSTL1 attenuates CC cell proliferation in a negative fashion. FSTL1 was upregulated in CC patient plasma and cancerous tissues but had no implication in prognosis. Our results provided novel insights into the molecular signatures and modulatory role of CC associated fibroblasts. In this study, a label-free LC-MS was performed to analyze the secretomes of two paired primary fibroblasts, which were isolated from fresh surgical specimen of colorectal adenocarcinoma and adjacent normal colonic tissues and exhibited negative modulatory activity for colon cancer cell growth in in vitro cocultures and in vivo xenograph mouse models. Follistatin-related protein 1 was further revealed to be one of the stroma-derived factors of potential suppression role for colon cancer cell proliferation. Our results provide novel

  5. Bryostatin and its synthetic analog, picolog rescue dermal fibroblasts from prolonged stress and contribute to survival and rejuvenation of human skin equivalents.

    Science.gov (United States)

    Khan, Tapan K; Wender, Paul A; Alkon, Daniel L

    2018-02-01

    Skin health is associated with the day-to-day activity of fibroblasts. The primary function of fibroblasts is to synthesize structural proteins, such as collagen, extracellular matrix proteins, and other proteins that support the structural integrity of the skin and are associated with younger, firmer, and more elastic skin that is better able to resist and recover from injury. At sub-nanomolar concentrations (0.03-0.3 nM), bryostatin-1 and its synthetic analog, picolog (0.1-10 nM) sustained the survival and activation of human dermal fibroblasts cultured under the stressful condition of prolonged serum deprivation. Bryostatin-1 treatment stabilized human skin equivalents (HSEs), a bioengineered combination of primary human skin cells (keratinocytes and dermal fibroblasts) on an extracellular matrix composed of mainly collagen. Fibroblasts activated by bryostatin-1 protected the structural integrity of HSEs. Bryostatin-1 and picolog prolonged activation of Erk in fibroblasts to promote cell survival. Chronic stress promotes the progression of apoptosis. Dermal fibroblasts constitutively express all components of Fas associated apoptosis, including caspase-8, an initiator enzyme of apoptosis. Prolong bryostatin-1 treatment reduced apoptosis by decreasing caspase-8 and protected dermal fibroblasts. Our data suggest that bryostatin-1 and picolog could be useful in anti-aging skincare, and could have applications in tissue engineering and regenerative medicine. © 2017 Wiley Periodicals, Inc.

  6. CNS embryonal tumours: WHO 2016 and beyond.

    Science.gov (United States)

    Pickles, J C; Hawkins, C; Pietsch, T; Jacques, T S

    2018-02-01

    Embryonal tumours of the central nervous system (CNS) present a significant clinical challenge. Many of these neoplasms affect young children, have a very high mortality and therapeutic strategies are often aggressive with poor long-term outcomes. There is a great need to accurately diagnose embryonal tumours, predict their outcome and adapt therapy to the individual patient's risk. For the first time in 2016, the WHO classification took into account molecular characteristics for the diagnosis of CNS tumours. This integration of histological features with genetic information has significantly changed the diagnostic work-up and reporting of tumours of the CNS. However, this remains challenging in embryonal tumours due to their previously unaccounted tumour heterogeneity. We describe the recent revisions made to the 4th edition of the WHO classification of CNS tumours and review the main changes, while highlighting some of the more common diagnostic testing strategies. © 2017 British Neuropathological Society.

  7. N-cadherin is overexpressed in Crohn's stricture fibroblasts and promotes intestinal fibroblast migration.

    LENUS (Irish Health Repository)

    Burke, John P

    2012-02-01

    BACKGROUND: Intestinal fibroblasts mediate stricture formation in Crohn\\'s disease (CD). Transforming growth factor-beta (TGF-beta) is important in fibroblast activation, while cell attachment and migration is regulated by the adhesion molecule N-cadherin. The aim of this study was to investigate the expression and function of N-cadherin in intestinal fibroblasts in patients with fibrostenosing CD. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies from patients undergoing resection for terminal ileal fibrostenosing CD (n = 14) or controls patients (n = 8). N-cadherin expression was assessed using Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Fibroblasts were stimulated with TGF-beta and selective pathway inhibitors Y27632, PD98050, and LY294002 were used to examine the Rho\\/ROCK, ERK-1\\/2, and Akt signaling pathways, respectively. Cell migration was assessed using a scratch wound assay. N-cadherin was selectively overexpressed using a plasmid. RESULTS: Fibroblasts from fibrostenosing CD express increased constitutive N-cadherin mRNA and protein and exhibit enhanced basal cell migration relative to those from directly adjacent normal bowel. Control fibroblasts treated with TGF-beta induced N-cadherin in a dose-dependent manner which was inhibited by Rho\\/ROCK and Akt pathway modulation. Control fibroblasts exhibited enhanced cell migration in response to treatment with TGF-beta or transfection with an N-cadherin plasmid. CONCLUSIONS: Fibroblasts from strictures in CD express increased constitutive N-cadherin and exhibit enhanced basal cell migration. TGF-beta is a potent inducer of N-cadherin in intestinal fibroblasts resulting in enhanced cell migration. The TGF-beta-mediated induction of N-cadherin may potentiate Crohn\\'s stricture formation.

  8. p53/PUMA expression in human pulmonary fibroblasts mediates cell activation and migration in silicosis.

    Science.gov (United States)

    Wang, Wei; Liu, Haijun; Dai, Xiaoniu; Fang, Shencun; Wang, Xingang; Zhang, Yingming; Yao, Honghong; Zhang, Xilong; Chao, Jie

    2015-11-18

    Phagocytosis of SiO2 into the lung causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Clinical evidence has indicated that the activation of alveolar macrophages by SiO2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1, which, in turn, induces fibrosis. However, the details of events downstream of monocyte chemotactic protein 1 activity in pulmonary fibroblasts remain unclear. Here, to elucidate the role of p53 in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Experiments using primary cultured adult human pulmonary fibroblasts led to the following results: 1) SiO2 treatment resulted in a rapid and sustained increase in p53 and PUMA protein levels; 2) the MAPK and PI3K pathways were involved in the SiO2-induced alteration of p53 and PUMA expression; and 3) RNA interference targeting p53 and PUMA prevented the SiO2-induced increases in fibroblast activation and migration. Our study elucidated a link between SiO2-induced p53/PUMA expression in fibroblasts and cell migration, thereby providing novel insight into the potential use of p53/PUMA in the development of novel therapeutic strategies for silicosis treatment.

  9. Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Jason A Neidleman

    2017-02-01

    Full Text Available Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells-by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV-without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy.

  10. Cell Surface Glycoprotein of Reactive Stromal Fibroblasts as a Potential Antibody Target in Human Epithelial Cancers

    Science.gov (United States)

    Garin-Chesa, Pilar; Old, Lloyd J.; Rettig, Wolfgang J.

    1990-09-01

    The F19 antigen is a cell surface glycoprotein (M_r, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19^-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19^+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19^+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19^- normal tissues and benign tumors. Our results suggest that the F19^+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.

  11. Inhibiting aerobic glycolysis suppresses renal interstitial fibroblast activation and renal fibrosis.

    Science.gov (United States)

    Ding, Hao; Jiang, Lei; Xu, Jing; Bai, Feng; Zhou, Yang; Yuan, Qi; Luo, Jing; Zen, Ke; Yang, Junwei

    2017-09-01

    Chronic kidney diseases generally lead to renal fibrosis. Despite great progress having been made in identifying molecular mediators of fibrosis, the mechanism that governs renal fibrosis remains unclear, and so far no effective therapeutic antifibrosis strategy is available. Here we demonstrated that a switch of metabolism from oxidative phosphorylation to aerobic glycolysis (Warburg effect) in renal fibroblasts was the primary feature of fibroblast activation during renal fibrosis and that suppressing renal fibroblast aerobic glycolysis could significantly reduce renal fibrosis. Both gene and protein assay showed that the expression of glycolysis enzymes was upregulated in mouse kidneys with unilateral ureter obstruction (UUO) surgery or in transforming growth factor-β1 (TGF-β1)-treated renal interstitial fibroblasts. Aerobic glycolysis flux, indicated by glucose uptake and lactate production, was increased in mouse kidney with UUO nephropathy or TGF-β1-treated renal interstitial fibroblasts and positively correlated with fibrosis process. In line with this, we found that increasing aerobic glycolysis can remarkably induce myofibroblast activation while aerobic glycolysis inhibitors shikonin and 2-deoxyglucose attenuate UUO-induced mouse renal fibrosis and TGF-β1-stimulated myofibroblast activation. Furthermore, mechanistic study indicated that shikonin inhibits renal aerobic glycolysis via reducing phosphorylation of pyruvate kinase type M2, a rate-limiting glycolytic enzyme associated with cell reliance on aerobic glycolysis. In conclusion, our findings demonstrate the critical role of aerobic glycolysis in renal fibrosis and support treatment with aerobic glycolysis inhibitors as a potential antifibrotic strategy. Copyright © 2017 the American Physiological Society.

  12. Effects of titanium surface topography on morphology and in vitro activity of human gingival fibroblasts.

    Science.gov (United States)

    Ramaglia, L; Capece, G; Di Spigna, G; Bruno, M P; Buonocore, N; Postiglione, L

    2013-01-01

    The aim of the present study was to evaluate in vitro the biological behavior of human gingival fibroblasts cultured on two different titanium surfaces. Titanium test disks were prepared with a machined, relatively smooth (S) surface or a rough surface (O) obtained by a double acid etching procedure. Primary cultures of human gingival fibroblasts were plated on the experimental titanium disks and cultured up to 14 days. Titanium disk surfaces were analysed by scanning electron microscopy (SEM). Cell proliferation and a quantitative analysis by ELISA in situ of ECM components as CoI, FN and TN were performed. Results have shown different effects of titanium surface microtopography on cell expression and differentiation. At 96 hours of culture on experimental surfaces human gingival fibroblasts displayed a favourable cell attachment and proliferation on both surfaces although showing some differences. Both the relatively smooth and the etched surfaces interacted actively with in vitro cultures of human gingival fibroblasts, promoting cell proliferation and differentiation. Results suggested that the microtopography of a double acid-etched rough surface may induce a greater Co I and FN production, thus conditioning in vivo the biological behaviour of human gingival fibroblasts during the process of peri-implant soft tissue healing.

  13. Inability of Kaplan radiation leukemia virus to replicate on mouse fibroblasts is conferred by its long terminal repeat

    International Nuclear Information System (INIS)

    Rassart, E.; Paquette, Y.; Jolicoeur, P.

    1988-01-01

    The molecularly cloned infectious Kaplan radiation leukemia virus has previously been shown to be unable to replicate on mouse fibroblasts. To map the viral sequences responsible for this, we constructed chimeric viral DNA genomes in vitro with parental cloned infectious viral DNAs from the nonfibrotropic (F-) BL/VL3 V-13 radiation leukemia virus and the fibrotropic (F+) endogenous BALB/c or Moloney murine leukemia viruses (MuLV). Infectious chimeric MuLVs, recovered after transfection of Ti-6 lymphocytes with these recombinant DNAs, were tested for capacity to replicate on mouse fibroblasts in vitro. We found that chimeric MuLVs harboring the long terminal repeat (LTR) of a fibrotropic MuLV replicated well on mouse fibroblasts. Conversely, chimeric MuLVs harboring the LTR of a nonfibrotropic MuLV were restricted on mouse fibroblasts. These results indicate that the LTR of BL/VL3 radiation leukemia virus harbors the primary determinant responsible for its inability to replicate on mouse fibroblasts in vitro. Our results also show that the primary determinant allowing F+ MuLVs (endogenous BALB/c and Moloney MuLVs) to replicate on mouse fibroblasts in vitro resides within the LTR

  14. Adventitial fibroblasts induce a distinct proinflammatory/profibrotic macrophage phenotype in pulmonary hypertension.

    Science.gov (United States)

    El Kasmi, Karim C; Pugliese, Steven C; Riddle, Suzette R; Poth, Jens M; Anderson, Aimee L; Frid, Maria G; Li, Min; Pullamsetti, Soni S; Savai, Rajkumar; Nagel, Maria A; Fini, Mehdi A; Graham, Brian B; Tuder, Rubin M; Friedman, Jacob E; Eltzschig, Holger K; Sokol, Ronald J; Stenmark, Kurt R

    2014-07-15

    Macrophage accumulation is not only a characteristic hallmark but is also a critical component of pulmonary artery remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Using multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, and primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive pulmonary arteries (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL-6 and STAT3, HIF1, and C/EBPβ signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL-4/IL-13-STAT6 and TLR-MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation, complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, and deficiency in C/EBPβ or HIF1 attenuated fibroblast-driven macrophage activation. These findings challenge the current paradigm of IL-4/IL-13-STAT6-mediated alternative macrophage activation as the sole driver of vascular remodeling in PH, and uncover a cross-talk between adventitial fibroblasts and macrophages in which paracrine IL-6-activated STAT3, HIF1α, and C/EBPβ signaling are critical for macrophage activation and polarization. Thus, targeting IL-6 signaling in macrophages by completely inhibiting C/EBPβ or HIF1α or by partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL-6 and absent IL-4/IL-13 signaling. Copyright © 2014 by The American Association of Immunologists

  15. Adventitial Fibroblasts induce a distinct Pro-inflammatory/Pro-fibrotic Macrophage Phenotype in Pulmonary Hypertension

    Science.gov (United States)

    El Kasmi, Karim C.; Pugliese, Steven C.; Riddle, Suzette R.; Poth, Jens M.; Anderson, Aimee L.; Frid, Maria G.; Li, Min; Pullamsetti, Soni S.; Savai, Rajkumar; Nagel, Maria A.; Fini, Mehdi A.; Graham, Brian B.; Tuder, Rubin M.; Friedman, Jacob E.; Eltzschig, Holger K.; Sokol, Ronald J.; Stenmark, Kurt R.

    2014-01-01

    Macrophage accumulation is not only a characteristic hallmark but also a critical component of pulmonary artery (PA) remodeling associated with pulmonary hypertension (PH). However, the cellular and molecular mechanisms that drive vascular macrophage activation and their functional phenotype remain poorly defined. Utilizing multiple levels of in vivo (bovine and rat models of hypoxia-induced PH, together with human tissue samples) and in vitro (primary mouse, rat, and bovine macrophages, human monocytes, as well as primary human and bovine fibroblasts) approaches, we observed that adventitial fibroblasts derived from hypertensive Pas (bovine and human) regulate macrophage activation. These fibroblasts activate macrophages through paracrine IL6 and STAT3, HIF1, and C/EBPβ signaling to drive expression of genes previously implicated in chronic inflammation, tissue remodeling, and PH. This distinct fibroblast-activated macrophage phenotype was independent of IL4/IL13-STAT6 and TLR-MyD88 signaling. We found that genetic STAT3 haplodeficiency in macrophages attenuated macrophage activation while complete STAT3 deficiency increased macrophage activation through compensatory upregulation of STAT1 signaling, while deficiency in C/EBPβ or HIF1 attenuated fibroblast driven macrophage activation. These findings challenge the current paradigm of IL4/IL13-STAT6 mediated alternative macrophage activation as the sole driver of vascular remodeling in PH and uncover a crosstalk between adventitial fibroblasts and macrophages in which paracrine IL6 activated STAT3, HIF1, and C/EBPβ signaling is critical for macrophage activation and polarization. Thus, targeting IL6 signaling in macrophages by completely inhibiting C/EBPβ, HIF1a or partially inhibiting STAT3 may hold therapeutic value for treatment of PH and other inflammatory conditions characterized by increased IL6 and absent IL4/IL13 signaling. PMID:24928992

  16. Primary acalvaria: a case report

    International Nuclear Information System (INIS)

    Rios, Livia Teresa Moreira; Martins, Marilia da Gloria; Simoes, Vanda Maria Ferreira; Nunes, Marynea do Vale; Marques, Patricia Franco; Godoy, Silvia Helena Cavalcante de Souza

    2010-01-01

    Acalvaria is a rare congenital malformation of unknown pathogenesis characterized by the absence of the flat bones of the cranial vault, dura mater and associated muscles, while the central nervous system is usually preserved. The most accepted physiopathogenic theory suggests the presence of a post neurulation defect with normal placement the embryonic ectoderm. The present report describes neonatal imaging findings of primary acalvaria. (author)

  17. Primary acalvaria: a case report

    Energy Technology Data Exchange (ETDEWEB)

    Rios, Livia Teresa Moreira, E-mail: ltlrios@terra.com.b [Universidade Federal do Maranhao (UFMA), Sao Luis, MA (Brazil). Hospital Universitario. Unidade de Diagnostico por Imagem; Martins, Marilia da Gloria [Universidade Federal do Maranhao (UFMA), Sao Luis, MA (Brazil). Hospital Universitario. Servico de Ginecologia e Obstetricia; Simoes, Vanda Maria Ferreira; Nunes, Marynea do Vale; Marques, Patricia Franco; Godoy, Silvia Helena Cavalcante de Souza [Universidade Federal do Maranhao (UFMA), Sao Luis, MA (Brazil). Hospital Universitario. Servico de Neonatologia

    2010-07-15

    Acalvaria is a rare congenital malformation of unknown pathogenesis characterized by the absence of the flat bones of the cranial vault, dura mater and associated muscles, while the central nervous system is usually preserved. The most accepted physiopathogenic theory suggests the presence of a post neurulation defect with normal placement the embryonic ectoderm. The present report describes neonatal imaging findings of primary acalvaria. (author)

  18. Chromatin in embryonic stem cell neuronal differentiation.

    Science.gov (United States)

    Meshorer, E

    2007-03-01

    Chromatin, the basic regulatory unit of the eukaryotic genetic material, is controlled by epigenetic mechanisms including histone modifications, histone variants, DNA methylation and chromatin remodeling. Cellular differentiation involves large changes in gene expression concomitant with alterations in genome organization and chromatin structure. Such changes are particularly evident in self-renewing pluripotent embryonic stem cells, which begin, in terms of cell fate, as a tabula rasa, and through the process of differentiation, acquire distinct identities. Here I describe the changes in chromatin that accompany neuronal differentiation, particularly of embryonic stem cells, and discuss how chromatin serves as the master regulator of cellular destiny.

  19. Serum-free keloid fibroblast cell culture: an in vitro model for the study of aberrant wound healing.

    Science.gov (United States)

    Koch, R J; Goode, R L; Simpson, G T

    1997-04-01

    The purpose of this study was to develop an in vitro serum-free keloid fibroblast model. Keloid formation remains a problem for every surgeon. Prior evaluations of fibroblast characteristics in vitro, especially those of growth factor measurement, have been confounded by the presence of serum-containing tissue culture media. The serum itself contains growth factors, yet has been a "necessary evil" to sustain cell growth. The design of this study is laboratory-based and uses keloid fibroblasts obtained from five patients undergoing facial (ear lobule) keloid removal in a university-affiliated clinic. Keloid fibroblasts were established in primary cell culture and then propagated in a serum-free environment. The main outcome measures included sustained keloid fibroblast growth and viability, which was comparable to serum-based models. The keloid fibroblast cell cultures exhibited logarithmic growth, sustained a high cellular viability, maintained a monolayer, and displayed contact inhibition. Demonstrating model consistency, there was no statistically significant difference between the mean cell counts of the five keloid fibroblast cell lines at each experimental time point. The in vitro growth of keloid fibroblasts in a serum-free model has not been done previous to this study. The results of this study indicate that the proliferative characteristics described are comparable to those of serum-based models. The described model will facilitate the evaluation of potential wound healing modulators, and cellular effects and collagen modifications of laser resurfacing techniques, and may serve as a harvest source for contaminant-free fibroblast autoimplants. Perhaps its greatest utility will be in the evaluation of endogenous and exogenous growth factors.

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

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... hepatocyte transplantation therapy and toxicity screening in drug discovery. Key words: Embryonic stem cells, hepatic-like cells, in vitro differentiation, sodium butyrate, ... from embryonic stem (ES) cell or induced pluripotent.

  1. Propagation of senescent mice using nuclear transfer embryonic stem cell lines.

    Science.gov (United States)

    Mizutani, Eiji; Ono, Tetsuo; Li, Chong; Maki-Suetsugu, Rinako; Wakayama, Teruhiko

    2008-09-01

    Senescent mice are often infertile, and the cloning success rate decreases with age, making it almost impossible to produce cloned progeny directly from such animals. In this study, we tried to produce offspring from such "unclonable" senescent mice using nuclear transfer techniques. Donor fibroblasts were obtained from the tail tips of mice aged up to 2 years and 9 months. Although most attempts failed to produce cloned mice by direct somatic cell nuclear transfer, we managed to establish nuclear transfer embryonic stem (ntES) cell lines from all aged mice with an establishment rate of 10-25%, irrespective of sex or strain. Finally, cloned mice were obtained from these ntES cells by a second round of nuclear transfer. In addition, healthy offspring was obtained from all aged donors via germline transmission of ntES cells in chimeric mice. This technique is thus applicable to the propagation of a variety of animals, irrespective of age or fertile potential.

  2. Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes

    DEFF Research Database (Denmark)

    Menzorov, Aleksei G; Matveeva, Natalia M.; Markakis, Marios Nektarios

    2015-01-01

    BACKGROUND: Recently fibroblasts of many mammalian species have been reprogrammed to pluripotent state using overexpression of several transcription factors. This technology allows production of induced pluripotent stem (iPS) cells with properties similar to embryonic stem (ES) cells....... The completeness of reprogramming process is well studied in such species as mouse and human but there is not enough data on other species. We produced American mink (Neovison vison) ES and iPS cells and compared these cells using transcriptome analysis. RESULTS: We report the generation of 10 mink ES and 22 i......PS cell lines. The majority of the analyzed cell lines had normal diploid chromosome number. The only ES cell line with XX chromosome set had both X-chromosomes in active state that is characteristic of pluripotent cells. The pluripotency of ES and iPS cell lines was confirmed by formation of teratomas...

  3. Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

    International Nuclear Information System (INIS)

    Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

    1982-01-01

    The metabolism of [1,2,6,7-3H]testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC

  4. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Zhong Xin [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Sun, Cong Cong [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Zheng, Jia Yong [Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Zhou, Xuan [Ningbo First Hospital, Ningbo, Zhejiang (China); Cong, Wei Tao [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Li, Xiao Kun, E-mail: proflxk@163.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Jin, Li Tai, E-mail: jin_litai@126.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China)

    2017-06-15

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

  5. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

    International Nuclear Information System (INIS)

    Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

    2017-01-01

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.

  6. The chalcone butein from Rhus verniciflua Stokes inhibits clonogenic growth of human breast cancer cells co-cultured with fibroblasts

    Directory of Open Access Journals (Sweden)

    Tan Jenny

    2005-03-01

    Full Text Available Abstract Background Butein (3,4,2',4'-tetrahydroxychalone, a plant polyphenol, is a major biologically active component of the stems of Rhus verniciflua Stokes. It has long been used as a food additive in Korea and as an herbal medicine throughout Asia. Recently, butein has been shown to suppress the functions of fibroblasts. Because fibroblasts are believed to play an important role in promoting the growth of breast cancer cells, we investigated the ability of butein to inhibit the clonogenic growth of small numbers of breast cancer cells co-cultured with fibroblasts in vitro. Methods We first measured the clonogenic growth of small numbers of the UACC-812 human breast cancer cell line co-cultured on monolayers of serum-activated, human fibroblasts in the presence of butein (2 μg/mL or various other modulators of fibroblast function (troglitazone-1 μg/mL; GW9662-1 μM; meloxican-1 μM; and 3,4 dehydroproline-10 μg/mL. In a subsequent experiment, we measured the dose-response effect on the clonogenic growth of UACC-812 breast cancer cells by pre-incubating the fibroblasts with varying concentrations of butein (10 μg/ml-1.25 μg/mL. Finally, we measured the clonogenic growth of primary breast cancer cells obtained from 5 clinical specimens with normal fibroblasts and with fibroblasts that had been pre-treated with a fixed dose of butein (2.5 μg/mL. Results Of the five modulators of fibroblast function that we tested, butein was by far the most potent inhibitor of clonogenic growth of UACC-812 breast cancer cells co-cultured with fibroblasts. Pre-treatment of fibroblasts with concentrations of butein as low as 2.5 μg/mL nearly abolished subsequent clonogenic growth of UACC-812 breast cancer cells co-cultured with the fibroblasts. A similar dose of butein had no effect on the clonogenic growth of breast cancer cells cultured in the absence of fibroblasts. Significantly, clonogenic growth of the primary breast cancer cells was also

  7. the production of mouse embryonic stem cells

    Indian Academy of Sciences (India)

    MADU

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

  8. Testicular Embryonic Rhabdomyosarcoma, Case report with brief ...

    African Journals Online (AJOL)

    Testicular Embryonic Rhabdomyosarcoma, Case report with brief literature review. AM Adam, MMAM Ibnouf, IAF Allah. Abstract. Background: Rhabdomyosarcoma (RMS) is a malignant solid tumour arising from mesenchymal tissues which normally differentiate to form striated muscle. It can occur in a wide variety of sites.

  9. How the embryonic chick brain twists.

    Science.gov (United States)

    Chen, Zi; Guo, Qiaohang; Dai, Eric; Forsch, Nickolas; Taber, Larry A

    2016-11-01

    During early development, the tubular embryonic chick brain undergoes a combination of progressive ventral bending and rightward torsion, one of the earliest organ-level left-right asymmetry events in development. Existing evidence suggests that bending is caused by differential growth, but the mechanism for the predominantly rightward torsion of the embryonic brain tube remains poorly understood. Here, we show through a combination of in vitro experiments, a physical model of the embryonic morphology and mechanics analysis that the vitelline membrane (VM) exerts an external load on the brain that drives torsion. Our theoretical analysis showed that the force is of the order of 10 micronewtons. We also designed an experiment to use fluid surface tension to replace the mechanical role of the VM, and the estimated magnitude of the force owing to surface tension was shown to be consistent with the above theoretical analysis. We further discovered that the asymmetry of the looping heart determines the chirality of the twisted brain via physical mechanisms, demonstrating the mechanical transfer of left-right asymmetry between organs. Our experiments also implied that brain flexure is a necessary condition for torsion. Our work clarifies the mechanical origin of torsion and the development of left-right asymmetry in the early embryonic brain. © 2016 The Author(s).

  10. Embryonal rhabdomyosarcoma of the cervix | Ocheke | African ...

    African Journals Online (AJOL)

    Embryonal rhabdomyosarcoma (sarcoma botyroides) of the cervix, which is rare, is described in a 16-yearold. The combined use of chemotherapy, radiotherapy and surgery has markedly improved survival in those with this condition. However, our patient did not benefit from this treatment modality due to late presentation ...

  11. Embryonic Stem Cells and their Genetic Modification

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 13; Issue 2. Embryonic Stem Cells and their Genetic Modification - The Nobel Prize in Physiology or Medicine 2007. Mitradas M Panicker. General Article Volume 13 Issue 2 February 2008 pp 172-180 ...

  12. Transcriptome Landscapes of Mammalian Embryonic Cells

    NARCIS (Netherlands)

    Brinkhof, B.

    2015-01-01

    This thesis describes research on gene expression profiles from different embryonic stages and cell types to identify genes involved in pluripotency or differentiation in bovine and porcine cells. The results are compared with data from other mammals. RNA expression profiles of morula and blastocyst

  13. Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Zhenqiang Zhao

    2016-12-01

    Full Text Available Mouse embryonic fibroblasts (MEFs and human foreskin fibroblasts (HFFs are used for the culture of human embryonic stem cells (hESCs. MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs =1:1 and HFFs feeder respectively, and then were differentiated into DA neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR (qRT-PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of TH positive cells and expressed higher levels of FOXA2, PITX3, NURR1 and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons.

  14. A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

    1992-01-01

    major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10......Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...

  15. PLZF regulates fibroblast growth factor responsiveness and maintenance of neural progenitors.

    Science.gov (United States)

    Gaber, Zachary B; Butler, Samantha J; Novitch, Bennett G

    2013-10-01

    Distinct classes of neurons and glial cells in the developing spinal cord arise at specific times and in specific quantities from spatially discrete neural progenitor domains. Thus, adjacent domains can exhibit marked differences in their proliferative potential and timing of differentiation. However, remarkably little is known about the mechanisms that account for this regional control. Here, we show that the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF) plays a critical role shaping patterns of neuronal differentiation by gating the expression of Fibroblast Growth Factor (FGF) Receptor 3 and responsiveness of progenitors to FGFs. PLZF elevation increases FGFR3 expression and STAT3 pathway activity, suppresses neurogenesis, and biases progenitors towards glial cell production. In contrast, PLZF loss reduces FGFR3 levels, leading to premature neuronal differentiation. Together, these findings reveal a novel transcriptional strategy for spatially tuning the responsiveness of distinct neural progenitor groups to broadly distributed mitogenic signals in the embryonic environment.

  16. Use of fibroblast growth factor 2 for expansion of chondrocytes and tissue engineering

    Science.gov (United States)

    Vunjak-Novakovic, Gordana (Inventor); Martin, Ivan (Inventor); Freed, Lisa E. (Inventor); Langer, Robert (Inventor)

    2003-01-01

    The present invention provides an improved method for expanding cells for use in tissue engineering. In particular the method provides specific biochemical factors to supplement cell culture medium during the expansion process in order to reproduce events occurring during embryonic development with the goal of regenerating tissue equivalents that resemble natural tissues both structurally and functionally. These specific biochemical factors improve proliferation of the cells and are capable of de-differentiation mature cells isolated from tissue so that the differentiation potential of the cells is preserved. The bioactive molecules also maintain the responsiveness of the cells to other bioactive molecules. Specifically, the invention provides methods for expanding chondrocytes in the presence of fibroblast growth factor 2 for use in regeneration of cartilage tissue.

  17. Random integration of SV40 in SV40-transformed, immortalized human fibroblasts.

    Science.gov (United States)

    Hara, H; Kaji, H

    1987-02-01

    We have studied the relationship between immortalization of SV40-transformed human embryonic fibroblasts and their SV40 integration sites. From several independently transformed cell pools, we have isolated clones which do not harbor unintegrated SV40 DNA. We have analysed whole-cell DNA from these clones, using the Southern blot method. Our results suggest that no specific integration sites in the cellular genome exist which are a prerequisite for the immortalization process. Although some integration sites were found to be predominant in pre-crisis clones, they could not be detected in the post-crisis clones. This suggests that none of these predominating sites is selected for during the crisis period.

  18. Role of periodontal ligament fibroblasts in osteoclastogenesis: a review

    NARCIS (Netherlands)

    Sokos, D.; Everts, V.; de Vries, T.J.

    2015-01-01

    During the last decade it has become clear that periodontal ligament fibroblasts may contribute to the in vitro differentiation of osteoclasts. We surveyed the current findings regarding their osteoclastogenesis potential. Periodontal ligament fibroblasts have the capacity to select and attract

  19. Derivation of keratinocytes from chicken embryonic stem cells: Establishment and characterization of differentiated proliferative cell populations

    Directory of Open Access Journals (Sweden)

    Mathilde Couteaudier

    2015-03-01

    Full Text Available A common challenge in avian cell biology is the generation of differentiated cell-lines, especially in the keratinocyte lineage. Only a few avian cell-lines are available and very few of them show an interesting differentiation profile. During the last decade, mammalian embryonic stem cell-lines were shown to differentiate into almost all lineages, including keratinocytes. Although chicken embryonic stem cells had been obtained in the 1990s, few differentiation studies toward the ectodermal lineage were reported. Consequently, we explored the differentiation of chicken embryonic stem cells toward the keratinocyte lineage by using a combination of stromal induction, ascorbic acid, BMP4 and chicken serum. During the induction period, we observed a downregulation of pluripotency markers and an upregulation of epidermal markers. Three homogenous cell populations were derived, which were morphologically similar to chicken primary keratinocytes, displaying intracellular lipid droplets in almost every pavimentous cell. These cells could be serially passaged without alteration of their morphology and showed gene and protein expression profiles of epidermal markers similar to chicken primary keratinocytes. These cells represent an alternative to the isolation of chicken primary keratinocytes, being less cumbersome to handle and reducing the number of experimental animals used for the preparation of primary cells.

  20. RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

    Science.gov (United States)

    Pandolfini, Luca; Luzi, Ettore; Bressan, Dario; Ucciferri, Nadia; Bertacchi, Michele; Brandi, Rossella; Rocchiccioli, Silvia; D'Onofrio, Mara; Cremisi, Federico

    2016-05-06

    Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.

  1. Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts.

    Science.gov (United States)

    Streckfuss-Bömeke, Katrin; Wolf, Frieder; Azizian, Azadeh; Stauske, Michael; Tiburcy, Malte; Wagner, Stefan; Hübscher, Daniela; Dressel, Ralf; Chen, Simin; Jende, Jörg; Wulf, Gerald; Lorenz, Verena; Schön, Michael P; Maier, Lars S; Zimmermann, Wolfram H; Hasenfuss, Gerd; Guan, Kaomei

    2013-09-01

    Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes. We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated. Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell

  2. Fibroblast growth factor receptor 4 (FGFR4) and fibroblast growth factor 19 (FGF19) autocrine enhance breast cancer cells survival.

    Science.gov (United States)

    Tiong, Kai Hung; Tan, Boon Shing; Choo, Heng Lungh; Chung, Felicia Fei-Lei; Hii, Ling-Wei; Tan, Si Hoey; Khor, Nelson Tze Woei; Wong, Shew Fung; See, Sze-Jia; Tan, Yuen-Fen; Rosli, Rozita; Cheong, Soon-Keng; Leong, Chee-Onn

    2016-09-06

    Basal-like breast cancer is an aggressive tumor subtype with poor prognosis. The discovery of underlying mechanisms mediating tumor cell survival, and the development of novel agents to target these pathways, is a priority for patients with basal-like breast cancer. From a functional screen to identify key drivers of basal-like breast cancer cell growth, we identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of cell survival. We found that FGFR4 mediates cancer cell survival predominantly via activation of PI3K/AKT. Importantly, a subset of basal-like breast cancer cells also secrete fibroblast growth factor 19 (FGF19), a canonical ligand specific for FGFR4. siRNA-mediated silencing of FGF19 or neutralization of extracellular FGF19 by anti-FGF19 antibody (1A6) decreases AKT phosphorylation, suppresses cancer cell growth and enhances doxorubicin sensitivity only in the FGFR4+/FGF19+ breast cancer cells. Consistently, FGFR4/FGF19 co-expression was also observed in 82 out of 287 (28.6%) primary breast tumors, and their expression is strongly associated with AKT phosphorylation, Ki-67 staining, higher tumor stage and basal-like phenotype. In summary, our results demonstrated the presence of an FGFR4/FGF19 autocrine signaling that mediates the survival of a subset of basal-like breast cancer cells and suggest that inactivation of this autocrine loop may potentially serve as a novel therapeutic intervention for future treatment of breast cancers.

  3. Fibroblast growth factor 23 - et fosfatregulerende hormon

    DEFF Research Database (Denmark)

    Beck-Nielsen, Signe; Pedersen, Susanne Møller; Kassem, Moustapha

    2010-01-01

    Fibroblast growth factor 23 (FGF23) er et nyligt identificeret fosfatonin. FGF23's fysiologiske hovedfunktion er at opretholde normalt serumfosfat og at virke som et D-vitaminmodregulatorisk hormon. Sygdomme, der er koblet til forhøjet serum FGF23, er hypofosfatæmisk rakitis, fibrøs dysplasi og t...

  4. Cryopreservation of canine ovarian and testicular fibroblasts.

    Science.gov (United States)

    Yu, Il-Jeoung; Leibo, S P; Songsasen, Nucharin; Dresser, Betsy L; Kim, In-Shik

    2009-01-01

    To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5 degree C in a humidified atmosphere of 5 percent CO2 95 percent air, and then their membrane integrity was assayed with a double fluorescent stain, Fertilight. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70 percent and functional survival ranged from 20-40 percent. With frozen-thawed ovarian cells, the average membrane integrity was 55-75 percent and the average functional survival was 35-40 percent. When frozen in ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P less than 0.05). These methods should prove useful to preserve cells collected from canids in the wild.

  5. Ca2+ influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    International Nuclear Information System (INIS)

    Murata, Naohiko; Ito, Satoru; Furuya, Kishio; Takahara, Norihiro; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-01-01

    Highlights: • Uniaxial stretching activates Ca 2+ signaling in human lung fibroblasts. • Stretch-induced intracellular Ca 2+ elevation is mainly via Ca 2+ influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca 2+ influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca 2+ concentration ([Ca 2+ ] i ) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca 2+ ] i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca 2+ ] i . The stretch-induced [Ca 2+ ] i elevation was attenuated in Ca 2+ -free solution. In contrast, the increase of [Ca 2+ ] i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd 3+ , ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca 2+ ] i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca 2+ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP

  6. Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta.

    Science.gov (United States)

    McCoy, Sara S; Reed, Tamra J; Berthier, Celine C; Tsou, Pei-Suen; Liu, Jianhua; Gudjonsson, Johann E; Khanna, Dinesh; Kahlenberg, J Michelle

    2017-11-01

    SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect. Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-β. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (α-SMA), collagen type 1 (COL1A1) and CCL5 expression. SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-β. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes. Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-β-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  7. Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Murata, Naohiko [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Furuya, Kishio [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Takahara, Norihiro [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Naruse, Keiji [Department of Cardiovascular Physiology, Okayama University Graduate School of Medicine, Okayama 700-8558 (Japan); Aso, Hiromichi; Kondo, Masashi [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Sokabe, Masahiro [Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan); Hasegawa, Yoshinori [Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya 466-8550 (Japan)

    2014-10-10

    Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

  8. Early embryonic chromosome instability results in stable mosaic pattern in human tissues.

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    Hasmik Mkrtchyan

    Full Text Available The discovery of copy number variations (CNV in the human genome opened new perspectives on the study of the genetic causes of inherited disorders and the aetiology of common diseases. Here, a single-cell-level investigation of CNV in different human tissues led us to uncover the phenomenon of mitotically derived genomic mosaicism, which is stable in different cell types of one individual. The CNV mosaic ratios were different between the 10 individuals studied. However, they were stable in the T lymphocytes, immortalized B lymphoblastoid cells, and skin fibroblasts analyzed in each individual. Because these cell types have a common origin in the connective tissues, we suggest that mitotic changes in CNV regions may happen early during embryonic development and occur only once, after which the stable mosaic ratio is maintained throughout the differentiated tissues. This concept is further supported by a unique study of immortalized B lymphoblastoid cell lines obtained with 20 year difference from two subjects. We provide the first evidence of somatic mosaicism for CNV, with stable variation ratios in different cell types of one individual leading to the hypothesis of early embryonic chromosome instability resulting in stable mosaic pattern in human tissues. This concept has the potential to open new perspectives in personalized genetic diagnostics and can explain genetic phenomena like diminished penetrance in autosomal dominant diseases. We propose that further genomic studies should focus on the single-cell level, to better understand the aetiology of aging and diseases mediated by somatic mutations.

  9. Generation of induced pluripotent stem cells with high efficiency from human embryonic renal cortical cells.

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    Yao, Ling; Chen, Ruifang; Wang, Pu; Zhang, Qi; Tang, Hailiang; Sun, Huaping

    2016-01-01

    Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) emerges as a prospective therapeutic angle in regenerative medicine and a tool for drug screening. Although increasing numbers of iPSCs from different sources have been generated, there has been limited progress in yield of iPSC. Here, we show that four Yamanaka factors Oct4, Sox2, Klf4 and c-Myc can convert human embryonic renal cortical cells (hERCCs) to pluripotent stem cells with a roughly 40-fold higher reprogramming efficiency compared with that of adult human dermal fibroblasts. These iPSCs show pluripotency in vitro and in vivo, as evidenced by expression of pluripotency associated genes, differentiation into three embryonic germ layers by teratoma tests, as well as neuronal fate specification by embryoid body formation. Moreover, the four exogenous genes are effectively silenced in these iPSCs. This study highlights the use of hERCCs to generate highly functional human iPSCs which may aid the study of genetic kidney diseases and accelerate the development of cell-based regenerative therapy.

  10. Impact of transient down-regulation of DREAM in human embryonic stem cell pluripotency

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    A. Fontán-Lozano

    2016-05-01

    Full Text Available Little is known about the functions of downstream regulatory element antagonist modulator (DREAM in embryonic stem cells (ESCs. However, DREAM interacts with cAMP response element-binding protein (CREB in a Ca2+-dependent manner, preventing CREB binding protein (CBP recruitment. Furthermore, CREB and CBP are involved in maintaining ESC self-renewal and pluripotency. However, a previous knockout study revealed the protective function of DREAM depletion in brain aging degeneration and that aging is accompanied by a progressive decline in stem cells (SCs function. Interestingly, we found that DREAM is expressed in different cell types, including human ESCs (hESCs, human adipose-derived stromal cells (hASCs, human bone marrow-derived stromal cells (hBMSCs, and human newborn foreskin fibroblasts (hFFs, and that transitory inhibition of DREAM in hESCs reduces their pluripotency, increasing differentiation. We stipulate that these changes are partly mediated by increased CREB transcriptional activity. Overall, our data indicates that DREAM acts in the regulation of hESC pluripotency and could be a target to promote or prevent differentiation in embryonic cells.

  11. Three-dimensional epithelial tissues generated from human embryonic stem cells.

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    Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

    2009-11-01

    The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

  12. Effects of recombinant human epidermal growth factor on the proliferation and radiation survival of human fibroblast cell lines in vitro

    International Nuclear Information System (INIS)

    Kim, Hyun Sook; Kang, Ki Mun; Na, Jae Boem; Chai, Gyu Young; Lee, Sang Wook

    2006-01-01

    To explore the effect of recombinant human EGF on the proliferation and survival of human fibroblast cell lines following irradiation. Fibroblast was originated human skin and primary cultured. The trypan blue stain assay and MTT assay were used to study the proliferative effects of EGF on human fibroblast cell lines in vitro. An incubation of fibroblasts with rhEGF for 24 hours immediately after irradiation was counted everyday. Cell cycle distributions were analyzed by FACS analysis. Number of fibroblast was significant more increased rhEGF (1.0 nM, 10 nM, 100 nM, 1,000 nM) treated cell than control after 8 Gy irradiation. Most effective dose of rhEGF was at 160 nM. These survival differences were maintained at 1 week later. Proportion of S phase was significantly increased on rhEGF treated cells. rhEGF cause increased fibroblast proliferation following irradiation. We expect that rhEGF was effective for radiation induced wound healing

  13. The physiological period length of the human circadian clock in vivo is directly proportional to period in human fibroblasts.

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    Lucia Pagani

    Full Text Available BACKGROUND: Diurnal behavior in humans is governed by the period length of a circadian clock in the suprachiasmatic nuclei of the brain hypothalamus. Nevertheless, the cell-intrinsic mechanism of this clock is present in most cells of the body. We have shown previously that for individuals of extreme chronotype ("larks" and "owls", clock properties measured in human fibroblasts correlated with extreme diurnal behavior. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have measured circadian period in human primary fibroblasts taken from normal individuals and, for the first time, compared it directly with physiological period measured in vivo in the same subjects. Human physiological period length was estimated via the secretion pattern of the hormone melatonin in two different groups of sighted subjects and one group of totally blind subjects, each using different methods. Fibroblast period length was measured via cyclical expression of a lentivirally delivered circadian reporter. Within each group, a positive linear correlation was observed between circadian period length in physiology and in fibroblast gene expression. Interestingly, although blind individuals showed on average the same fibroblast clock properties as sighted ones, their physiological periods were significantly longer. CONCLUSIONS/SIGNIFICANCE: We conclude that the period of human circadian behaviour is mostly driven by cellular clock properties in normal individuals and can be approximated by measurement in peripheral cells such as fibroblasts. Based upon differences among sighted and blind subjects, we also speculate that period can be modified by prolonged unusual conditions such as the total light deprivation of blindness.

  14. Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes.

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    Ou, Dong-Bo; He, Yong; Chen, Rui; Teng, Ji-Wei; Wang, Hong-Tao; Zeng, Di; Liu, Xiong-Tao; Ding, Lu; Huang, Jin-Yan; Zheng, Qiang-Sun

    2011-12-01

    The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 µm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells. Copyright © 2011 Wiley Periodicals, Inc.

  15. Acetylome in Human Fibroblasts From Parkinson's Disease Patients

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    Sokhna M. S. Yakhine-Diop

    2018-04-01

    Full Text Available Parkinson's disease (PD is a multifactorial neurodegenerative disorder. The pathogenesis of this disease is associated with gene and environmental factors. Mutations in leucine-rich repeat kinase 2 (LRRK2 are the most frequent genetic cause of familial and sporadic PD. Moreover, posttranslational modifications, including protein acetylation, are involved in the molecular mechanism of PD. Acetylation of lysine proteins is a dynamic process that is modulated in PD. In this descriptive study, we characterized the acetylated proteins and peptides in primary fibroblasts from idiopathic PD (IPD and genetic PD harboring G2019S or R1441G LRRK2 mutations. Identified acetylated peptides are modulated between individuals' groups. Although acetylated nuclear proteins are the most represented in cells, they are hypoacetylated in IPD. Results display that the level of hyperacetylated and hypoacetylated peptides are, respectively, enhanced in genetic PD and in IPD cells.

  16. Eighteen-Year Cryopreservation Does Not Negatively Affect the Pluripotency of Human Embryos: Evidence from Embryonic Stem Cell Derivation

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    Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Isarasena, Nipan; Virutamasen, Pramuan

    2012-01-01

    Abstract Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research. PMID:23514952

  17. Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

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    Jiafei Xi

    Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

  18. Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

    Science.gov (United States)

    Shuda, Masahiro; Guastafierro, Anna; Geng, Xuehui; Shuda, Yoko; Ostrowski, Stephen M; Lukianov, Stefan; Jenkins, Frank J; Honda, Kord; Maricich, Stephen M; Moore, Patrick S; Chang, Yuan

    2015-01-01

    Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

  19. RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

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    Hoban PR

    2006-06-01

    Full Text Available Abstract Background Idiopathic Pulmonary Fibrosis (IPF is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression. Methods Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies. Results Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p Conclusion These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.

  20. YKL-40 is differentially expressed in human embryonic stem cells and in cell progeny of the three germ layers.

    Science.gov (United States)

    Brøchner, Christian B; Johansen, Julia S; Larsen, Lars A; Bak, Mads; Mikkelsen, Hanne B; Byskov, Anne Grete; Andersen, Claus Yding; Møllgård, Kjeld

    2012-03-01

    The secreted glycoprotein YKL-40 participates in cell differentiation, inflammation, and cancer progression. High YKL-40 expression is reported during early human development, but its functions are unknown. Six human embryonic stem cell (hESC) lines were cultured in an atmosphere of low or high oxygen tension, in culture medium with or without basic fibroblast growth factor, and on feeder layers comprising mouse embryonic fibroblasts or human foreskin fibroblasts to evaluate whether hESCs and their progeny produced YKL-40 and to characterize YKL-40 expression during differentiation. Secreted YKL-40 protein and YKL-40 mRNA expression were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative RT-PCR. Serial-sectioned colonies were stained for YKL-40 protein and for pluripotent hESC (OCT4, NANOG) and germ layer (HNF-3β, PDX1, CD34, p63, nestin, PAX6) markers. Double-labeling showed YKL-40 expression in OCT4-positive hESCs, PAX6-positive neuroectodermal cells, and HNF-3β-positive endodermal cells. The differentiating progeny showed strong YKL-40 expression. Abrupt transition between YKL-40 and OCT4-positive hESCs and YKL-40-positive ecto- and neuroectodermal lineages was observed within the same epithelial-like layer. YKL-40-positive cells within deeper layers lacked contact with OCT4-positive cells. YKL-40 may be important in initial cell differentiation from hESCs toward ectoderm and neuroectoderm, with retained epithelial morphology, whereas later differentiation into endoderm and mesoderm involves a transition into the deeper layers of the colony.

  1. Generation of human-induced pluripotent stem cells from burn patient-derived skin fibroblasts using a non-integrative method.

    Science.gov (United States)

    Fu, Shangfeng; Ding, Jianwu; Liu, Dewu; Huang, Heping; Li, Min; Liu, Yang; Tu, Longxiang; Liu, Deming

    2018-01-01

    Patient specific induced pluripotent stem cells (iPSCs) have been recognized as a possible source of cells for skin tissue engineering. They have the potential to greatly benefit patients with large areas of burned skin or skin defects. However, the integration virus-based reprogramming method is associated with a high risk of genetic mutation and mouse embryonic fibroblast feeder-cells may be a pollutant. In the present study, human skin fibroblasts (HSFs) were successfully harvested from patients with burns and patient-specific iPSCs were generated using a non-integration method with a feeder-free approach. The octamer-binding transcription factor 4 (OCT4), sex-determining region Y box 2 (SOX2) and NANOG transcription factors were delivered using Sendai virus vectors. iPSCs exhibited representative human embryonic stem cell-like morphology and proliferation characteristics. They also expressed pluripotent markers, including OCT4, NANOG, SOX2, TRA181, stage-specific embryonic antigen 4 and TRA-160, and exhibited a normal karyotype. Teratoma and embryoid body formation revealed that iPSCs were able to differentiate into cells of all three germ layers in vitro and in vivo. The results of the present study demonstrate that HSFs derived from patients with burns, may be reprogrammed into stem cells with pluripotency, which provides a basis for cell‑based skin tissue engineering in the future.

  2. Neural stem cell regulation, fibroblast growth factors, and the developmental origins of neuropsychiatric disorders

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    Hanna E Stevens

    2010-09-01

    Full Text Available There is increasing appreciation for the neurodevelopmental underpinnings of many psychiatric disorders. Disorders that begin in childhood such as autism, language disorders or mental retardation as well as adult-onset mental disorders may have origins early in neurodevelopment. Neural stem cells (NSCs can be defined as self-renewing, multipotent cells that are present in both the embryonic and adult brain. Several recent research findings demonstrate that psychiatric illness may begin with abnormal specification, growth, expansion and differentiation of embryonic NSCs. For example, candidate susceptibility genes for schizophrenia, autism and major depression include the signaling molecule Disrupted In Schizophrenia-1 (DISC-1, the homeodomain gene engrailed-2 (EN-2, and several receptor tyrosine kinases (RTKs, including MET, brain-derived growth factor (BDNF and fibroblast growth factors (FGF, all of which have been shown to play important roles in NSCs or neuronal precursors. We will discuss here stem cell biology, signaling factors that affect these cells, and the potential contribution of these processes to the etiology of neuropsychiatric disorders. Hypotheses about how some of these factors relate to psychiatric disorders will be reviewed.

  3. Ontogeny of basic fibroblast growth factor binding sites in mouse ocular tissues

    International Nuclear Information System (INIS)

    Fayein, N.A.; Courtois, Y.; Jeanny, J.C.

    1990-01-01

    Basic fibroblast growth factor (bFGF) binding to ocular tissues has been studied by autoradiographical and biochemical approaches directly performed on sections during mouse embryonic and postnatal development. Frozen sections of embryos (9 to 18 days), newborns, and adults (1 day to 6 months) were incubated with iodinated bFGF. One specific FGF binding site (KD = 2.5 nM) is colocalized with heparan sulfate proteoglycans of the basement membranes and is heparitinase sensitive. It first appears at Day 9 around the neural tube, the optic vesicles, and below the head ectoderm and by Day 14 of embryonic development is found in all basement membranes of the eye. At Day 16, very intensely labeled patches appear, corresponding to mast cells which have been characterized by metachromatic staining of their heparin-rich granulations with toluidine blue. In addition to the latter binding, we have also observed a general diffuse distribution of silver grains on all tissues and preferentially in the ecto- and neuroectodermic tissues. From Days 17-18, there is heterogeneous labeling inside the retina, localized in the pigmented epithelium and in three different layers colocalized with the inner and outer plexiform layers and with the inner segments of the photoreceptors. This binding is heparitinase resistant but N-glycanase sensitive and may represent a second specific binding site corresponding to cellular FGF receptors (KD = 280 pM). Both types of binding patterns observed suggest a significant role for bFGF in eye development and physiology

  4. Interplay between Selenium Levels and Replicative Senescence in WI-38 Human Fibroblasts: A Proteomic Approach.

    Science.gov (United States)

    Hammad, Ghania; Legrain, Yona; Touat-Hamici, Zahia; Duhieu, Stéphane; Cornu, David; Bulteau, Anne-Laure; Chavatte, Laurent

    2018-01-20

    Selenoproteins are essential components of antioxidant defense, redox homeostasis, and cell signaling in mammals, where selenium is found in the form of a rare amino acid, selenocysteine. Selenium, which is often limited both in food intake and cell culture media, is a strong regulator of selenoprotein expression and selenoenzyme activity. Aging is a slow, complex, and multifactorial process, resulting in a gradual and irreversible decline of various functions of the body. Several cellular aspects of organismal aging are recapitulated in the replicative senescence of cultured human diploid fibroblasts, such as embryonic lung fibroblast WI-38 cells. We previously reported that the long-term growth of young WI-38 cells with high (supplemented), moderate (control), or low (depleted) concentrations of selenium in the culture medium impacts their replicative lifespan, due to rapid changes in replicative senescence-associated markers and signaling pathways. In order to gain insight into the molecular link between selenium levels and replicative senescence, in the present work, we have applied a quantitative proteomic approach based on 2-Dimensional Differential in-Gel Electrophoresis (2D-DIGE) to the study of young and presenescent cells grown in selenium-supplemented, control, or depleted media. Applying a restrictive cut-off (spot intensity ±50% and a p value iii) spots varying in response to selenium concentration in presenescent cells. Interestingly, a 72% overlap between the impact of senescence and selenium was observed in our proteomic results, demonstrating a strong interplay between selenium, selenoproteins, and replicative senescence.

  5. [Comparison of fibroblastic cell compatibility of type I collagen-immobilized titanium between electrodeposition and immersion].

    Science.gov (United States)

    Kyuragi, Takeru

    2014-03-01

    Titanium is widely used for medical implants. While many techniques for surface modification have been studied for optimizing its biocompatibility with hard tissues, little work has been undertaken to explore ways of maximizing its biocompatibility with soft tissues. We investigated cell attachment to titanium surfaces modified with bovine Type I collagen immobilized by either electrodeposition or a conventional immersion technique. The apparent thickness and durability of the immobilized collagen layer were evaluated prior to incubation of the collagen-immobilized titanium surfaces with NIH/3T3 mouse embryonic fibroblasts. The initial cell attachment and expression of actin and vinculin were evaluated. We determined that the immobilized collagen layer was much thicker and more durable when placed using the electrodeposition technique than the immersion technique. Both protocols produced materials that promoted better cell attachment, growth and structural protein expression than titanium alone. However, electrodeposition was ultimately superior to immersion because it is quicker to perform and produces a more durable collagen coating. We conclude that electrodeposition is an effective technique for immobilizing type I collagen on titanium surfaces, thus improving their cytocompatibility with fibroblasts.

  6. Embryonic vaccines against cancer: an early history.

    Science.gov (United States)

    Brewer, Bradley G; Mitchell, Robert A; Harandi, Amir; Eaton, John W

    2009-06-01

    Almost 100 years have passed since the seminal observations of Schöne showing that vaccination of animals with fetal tissue would prevent the growth of transplantable tumors. Many subsequent reports have affirmed the general idea that immunologic rejection of transplantable tumors, as well as prevention of carcinogenesis, may be affected by vaccination with embryonic/fetal material. Following a decade of intense research on this phenomenon during approximately 1964-1974, interest appears to have waned. This earlier experimental work may be particularly pertinent in view of the rising interest in so-called cancer stem cells. We believe that further work - perhaps involving the use of embryonic stem cells as immunogens - is warranted and that the results reviewed herein support the concept that vaccination against the appearance of cancers of all kinds is a real possibility.

  7. Cytokine signalling in embryonic stem cells

    DEFF Research Database (Denmark)

    Kristensen, David Møbjerg; Kalisz, Mark; Nielsen, Jens Høiriis

    2006-01-01

    Cytokines play a central role in maintaining self-renewal in mouse embryonic stem (ES) cells through a member of the interleukin-6 type cytokine family termed leukemia inhibitory factor (LIF). LIF activates the JAK-STAT3 pathway through the class I cytokine receptor gp130, which forms a trimeric...... pathways seem to converge on c-myc as a common target to promote self-renewal. Whereas LIF does not seem to stimulate self-renewal in human embryonic stem cells it cannot be excluded that other cytokines are involved. The pleiotropic actions of the increasing number of cytokines and receptors signalling...... via JAKs, STATs and SOCS exhibit considerable redundancy, compensation and plasticity in stem cells in accordance with the view that stem cells are governed by quantitative variations in strength and duration of signalling events known from other cell types rather than qualitatively different stem...

  8. Molecular fingerprinting of TGFbeta-treated embryonic maxillary mesenchymal cells.

    Science.gov (United States)

    Pisano, M M; Mukhopadhyay, P; Greene, R M

    2003-11-01

    The transforming growth factor-beta (TGF(beta)) family represents a class of signaling molecules that plays a central role in normal embryonic development, specifically in development of the craniofacial region. Members of this family are vital to development of the secondary palate where they regulate maxillary and palate mesenchymal cell proliferation and extracellular matrix synthesis. The function of this growth factor family is particularly critical in that perturbation of either process results in a cleft of the palate. While the cellular and phenotypic effects of TGF(beta) on embryonic craniofacial tissue have been extensively cataloged, the specific genes that function as downstream mediators of TGF(beta) in maxillary/palatal development are poorly defined. Gene expression arrays offer the ability to conduct a rapid, simultaneous assessment of hundreds to thousands of differentially expressed genes in a single study. Inasmuch as the downstream sequelae of TGF(beta) action are only partially defined, a complementary DNA (cDNA) expression array technology (Clontech's Atlas Mouse cDNA Expression Arrays), was utilized to delineate a profile of differentially expressed genes from TGF(beta)-treated primary cultures of murine embryonic maxillary mesenchymal cells. Hybridization of a membrane-based cDNA array (1178 genes) was performed with 32P-labeled cDNA probes synthesized from RNA isolated from either TGF(beta)-treated or vehicle-treated embryonic maxillary mesenchymal cells. Resultant phosphorimages were subject to AtlasImage analysis in order to determine differences in gene expression between control and TGF(beta)-treated maxillary mesenchymal cells. Of the 1178 arrayed genes, 552 (47%) demonstrated detectable levels of expression. Steady state levels of 22 genes were up-regulated, while those of 8 other genes were down-regulated, by a factor of twofold or greater in response to TGF(beta). Affected genes could be grouped into three general functional

  9. Establishment of human induced pluripotent stem cell lines from normal fibroblast TIG-1.

    Science.gov (United States)

    Kumazaki, Tsutomu; Kurata, Sayaka; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

    2011-06-01

    Normal human cells have a replicative life span and therefore senesce. Usually, normal human cell strains are differentiated cells and reach a terminally differentiated state after a number of cell divisions. At present, definitive differences are not known between replicative senescence and terminal differentiation. TIG-1 is a human fibroblast strain established from fetal lung and has been used extensively in studies of cellular senescence, and numerous data were accumulated at the molecular level. Recently, a method for generating induced pluripotent stem cells (iPSCs) was developed. Using the method, we introduced four reprogramming genes to TIG-1 fibroblasts and succeeded in isolating colonies that had embryonic stem cell (ESC)-like morphologies. They showed alkaline phosphatase activity and expressed ESC markers, as shown by immunostaining of OCT4, SOX2, SSEA4, and TRA-1-81 as well as reverse-transcription polymerase chain reaction (RT-PCR) for OCT4 and NANOG transcripts. Thus, we succeeded in establishing iPSC clones from TIG-1. The iPSC clones could differentiate to cells originated from all three germ-cell layers, as shown by RT-PCR, for messenger RNA (mRNA) expression of α-fetoprotein (endoderm), MSX1 (mesoderm) and microtubule-associated protein 2 (ectoderm), and by immunostaining for α-fetoprotein (endoderm), α-smooth muscle actin (mesoderm), and β-III-tubulin (ectoderm). The iPSCs formed teratoma containing the structures developed from all three germ-cell layers in severe combined immune-deficiency mice. Thus, by comparing the aging process of parental TIG-1 cells and the differentiation process of iPSC-derived fibrocytes to fibroblasts, we can reveal the exact differences in processes between senescence and terminal differentiation.

  10. Optimization of culture conditions to support long-term self-renewal of buffalo (Bubalus bubalis) embryonic stem cell-like cells.

    Science.gov (United States)

    Sharma, Ruchi; George, Aman; Kamble, Nitin Manchindra; Singh, Karn Pratap; Chauhan, Manmohan Singh; Singla, Suresh Kumar; Manik, Radhey Sham; Palta, Prabhat

    2011-12-01

    A culture system capable of sustaining self-renewal of buffalo embryonic stem (ES) cell-like cells in an undifferentiated state over a long period of time was developed. Inner cell masses were seeded on KO-DMEM+15% KO-serum replacer on buffalo fetal fibroblast feeder layer. Supplementation of culture medium with 5 ng/mL FGF-2 and 1000 IU/mL mLIF gave the highest (p<0.05) rate of primary colony formation. The ES cell-like cells' colony survival rate and increase in colony size were highest (p<0.05) following supplementation with FGF-2 and LIF compared to other groups examined. FGF-2 supplementation affected the quantitative expression of NANOG, SOX-2, ACTIVIN A, BMP 4, and TGFβ1, but not OCT4 and GREMLIN. Supplementation with SU5402, an FGFR inhibitor (≥20 μM) increased (p<0.05) the percentage of colonies that differentiated. FGFR1-3 and ERK1, K-RAS, E-RAS, and SHP-2, key signaling intermediates of FGF signaling, were detected in ES cell-like cells. Under culture conditions described, three ES cell lines were derived that, to date, have been maintained for 135, 95, and 85 passages for over 27, 19, and 17 months, respectively, whereas under other conditions examined, ES cell-like cells did not survive beyond passage 10. The ES cell-like cells were regularly monitored for expression of pluripotency markers and their potency to form embryoid bodies.

  11. Hedgehog Signalling in the Embryonic Mouse Thymus

    Directory of Open Access Journals (Sweden)

    Alessandro Barbarulo

    2016-07-01

    Full Text Available T cells develop in the thymus, which provides an essential environment for T cell fate specification, and for the differentiation of multipotent progenitor cells into major histocompatibility complex (MHC-restricted, non-autoreactive T cells. Here we review the role of the Hedgehog signalling pathway in T cell development, thymic epithelial cell (TEC development, and thymocyte–TEC cross-talk in the embryonic mouse thymus during the last week of gestation.

  12. Ear embryonic rabdomiosarcoma. A case report

    International Nuclear Information System (INIS)

    Cueto, L.; Canabal, A.; Blanco, A.; Sabate, J.

    2002-01-01

    A case of embryonic rabdomiosarcoma in the ear of a 5-year-old girl who initially shows clinical symptoms of otitis media. The CT reveals a dense lesion of soft tissue which shows up slightly in the right external auditory channel. Also of interest were osteolytic areas in the petrous, clivus and zygomatic arch. A hypointensive lesion with marked enhancement after Gd-DPTA injection is observed. Discussed are the imaging methods used in the diagnosis of this tumor. (Author) 10 refs

  13. The Use of Embryonic Stem Cells

    OpenAIRE

    Corkery, Padraig

    2002-01-01

    Over the past year there has been great interest, optimism and anxiety in many societies about developments in the use of embryonic stem cells (ES cells). Within the scientific community there has been debate for some time on the merits and ethical implications of using ES cells. The discussion entered the public domain inthe decisive way during the past year when there were significant changes in legislation governing the use of such cells in Britain and the United States. These changes c...

  14. The effect of tranilast on fibroblast activation protein α (FAP-α expression in normal and keloid fibroblasts in vitro

    Directory of Open Access Journals (Sweden)

    Paweł P. Antończak

    2017-07-01

    Full Text Available Introduction . Tranilast (N-(3’,4’-demethoxycinnamoyl-anthranilic acid is an anti-allergic drug. Its mechanism of action is based on the inhibition of antigen-induced release of chemical mediators from mast cells and basophils. It also reveals antifibroproliferative activities. These properties of tranilast are used in the treatment of hypertrophic scars and keloids. Keloids are characterized by incorrect extracellular matrix components turnover. Fibroblasts derived from keloids reveal overproduction of collagen type I and decreased degradation of extracellular matrix in comparison with normal fibroblasts. Fibroblast activation protein α (FAP-α may play an important role in remodeling of extracellular matrix and the invasive properties of keloids. Objective . In the present study, the effect of tranilast on expression of FAP-α gene and its protein was evaluated in normal human dermal fibroblasts and fibroblasts derived from keloids cultured in vitro . Materials and methods. In the first stage of the study, the influence of tranilast on cell viability was estimated. The second stage of the study included the quantitative evaluation of FAP-α mRNA expression in normal and keloid fibroblasts treated with tranilast. The third stage of the study comprised fibroblast activation protein α expression analysis in the examined cells treated with tranilast. Results and conclusions . The expression of FAP-α gene and fibroblast activation protein α is higher in keloid fibroblasts. Tranilast at concentrations of 3 μM and 30 μM up-regulated mRNA FAP-α expression in normal fibroblasts but did not influence keloid fibroblasts. The drug, at concentrations of 30 μM and 300 μM up-regulated fibroblast activation protein α expression in normal fibroblasts and did not influence keloid fibroblasts. Tranilast antiproliferative effect is not associated with FAP-α expression in keloid fibroblasts.

  15. /sup 125/I-labelled tetanus toxin as a neuronal marker in tissue cultures derived from embryonic CNS

    Energy Technology Data Exchange (ETDEWEB)

    Dimpfel, W; Neale, J H; Habermann, E [Giessen Univ. (F.R. Germany). Pharmakologisches Inst.; National Inst. of Child Health and Human Development, Bethesda, Md. (USA). Behavioural Biology Branch)

    1975-01-01

    Primary cultures derived from embryonic mouse brain and spinal cord were exposed to /sup 125/I-labelled tetanus toxin and subjected to autoradioraphy. Cells with neuronal, but not glial, morphology selectively accumulated the toxin. The distribution of the grains over these cells and their processes was not uniform, discrete processes showing heavier labelling.

  16. Do embryonic polar bodies commit suicide?

    Science.gov (United States)

    Fabian, Dušan; Čikoš, Štefan; Rehák, Pavol; Koppel, Juraj

    2014-02-01

    The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

  17. Metabolomic Elucidation of the Effects of Curcumin on Fibroblast-Like Synoviocytes in Rheumatoid Arthritis

    OpenAIRE

    Ahn, Joong Kyong; Kim, Sooah; Hwang, Jiwon; Kim, Jungyeon; Lee, You Sun; Koh, Eun-Mi; Kim, Kyoung Heon; Cha, Hoon-Suk

    2015-01-01

    Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterized by synovial inflammation and joint disability. Curcumin is known to be effective in ameliorating joint inflammation in RA. To obtain new insights into the effect of curcumin on primary fibroblast-like synoviocytes (FLS, N = 3), which are key effector cells in RA, we employed gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS)-based metabolomics. Metabolomic profiling of tumor necrosis factor (TNF)-α...

  18. CDCP1 identifies a CD146 negative subset of marrow fibroblasts involved with cytokine production.

    Directory of Open Access Journals (Sweden)

    Mineo Iwata

    Full Text Available In vitro expanded bone marrow stromal cells contain at least two populations of fibroblasts, a CD146/MCAM positive population, previously reported to be critical for establishing the stem cell niche and a CD146-negative population that expresses CUB domain-containing protein 1 (CDCP1/CD318. Immunohistochemistry of marrow biopsies shows that clusters of CDCP1+ cells are present in discrete areas distinct from areas of fibroblasts expressing CD146. Using a stromal cell line, HS5, which approximates primary CDCP1+ stromal cells, we show that binding of an activating antibody against CDCP1 results in tyrosine-phosphorylation of CDCP1, paralleled by phosphorylation of Src Family Kinases (SFKs Protein Kinase C delta (PKC-δ. When CDCP1 expression is knocked-down by siRNA, the expression and secretion of myelopoietic cytokines is increased. These data suggest CDCP1 expression can be used to identify a subset of marrow fibroblasts functionally distinct from CD146+ fibroblasts. Furthermore the CDCP1 protein may contribute to the defining function of these cells by regulating cytokine expression.

  19. Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

    DEFF Research Database (Denmark)

    Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders

    2002-01-01

    The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations...... days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When...... cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether...

  20. Lung Fibroblasts, Aging, and Idiopathic Pulmonary Fibrosis.

    Science.gov (United States)

    Pardo, Annie; Selman, Moisés

    2016-12-01

    Idiopathic pulmonary fibrosis (IPF) is an aging-associated, progressive, and irreversible lung disease of unknown etiology, elusive pathogenesis, and very limited therapeutic options. The hallmarks of IPF are aberrant activation of alveolar epithelial cells and accumulation of fibroblasts and myofibroblasts along with excessive production of extracellular matrix. The linkage of aging with this disorder is uncertain, but a number of changes associated with aging, including telomere attrition, cell senescence, and mitochondrial dysfunction, have been revealed in IPF lungs. Also, aging seems to confer a profibrotic phenotype upon fibroblasts and to increase the severity of the fibrogenic response in non-IPF fibrotic lung disorders. Better knowledge of the pathophysiological mechanisms linking aging to IPF will advance understanding of its pathogenesis and may provide new therapeutic windows to treatment of this devastating disease.

  1. Nodal signals mediate interactions between the extra-embryonic and embryonic tissues in zebrafish

    OpenAIRE

    Xiang, Fan; Hagos, Engda G.; Xu, Bo; Sias, Christina; Kawakami, Koichi; Burdine, Rebecca D.; Dougan, Scott T.

    2007-01-01

    In many vertebrates, extra-embryonic tissues are important signaling centers that induce and pattern the germ layers. In teleosts, the mechanism by which the extra-embryonic yolk syncytial layer (YSL) patterns the embryo is not understood. Although the Nodal-related protein Squint is expressed in the YSL, its role in this tissue is not known. We generated a series of stable transgenic lines with GFP under the control of squint genomic sequences. In all species, nodal-related genes induce thei...

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

    Science.gov (United States)

    Rajanahalli Krishnamurthy, Pavan

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

  3. Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

    Science.gov (United States)

    Chang, Tien-Cheng; Liu, Ya-Guang; Eddy, Carlton A; Jacoby, Ethan S; Binkley, Peter A; Brzyski, Robert G; Schenken, Robert S

    2011-06-01

    The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

  4. Isolation of a primate embryonic stem cell line.

    OpenAIRE

    Thomson, J A; Kalishman, J; Golos, T G; Durning, M; Harris, C P; Becker, R A; Hearn, J P

    1995-01-01

    Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, st...

  5. Increased superoxide accumulation in pyruvate dehydrogenase complex deficient fibroblasts.

    Science.gov (United States)

    Glushakova, Lyudmyla G; Judge, Sharon; Cruz, Alex; Pourang, Deena; Mathews, Clayton E; Stacpoole, Peter W

    2011-11-01

    The pyruvate dehydrogenase complex (PDC) oxidizes pyruvate to acetyl CoA and is critically important in maintaining normal cellular energy homeostasis. Loss-of-function mutations in PDC give rise to congenital lactic acidosis and to progressive cellular energy failure. However, the subsequent biochemical consequences of PDC deficiency that may contribute to the clinical manifestations of the disorder are poorly understood. We postulated that altered flux through PDC would disrupt mitochondrial electron transport, resulting in oxidative stress. Compared to cells from 4 healthy subjects, primary cultures of skin fibroblasts from 9 patients with variable mutations in the gene encoding the alpha subunit (E1α) of pyruvate dehydrogenase (PDA1) demonstrated reduced growth and viability. Superoxide (O(2)(.-)) from the Qo site of complex III of the electron transport chain accumulated in these cells and was associated with decreased activity of manganese superoxide dismutase. The expression of uncoupling protein 2 was also decreased in patient cells, but there were no significant changes in the expression of cellular markers of protein or DNA oxidative damage. The expression of hypoxia transcription factor 1 alpha (HIF1α) also increased in PDC deficient fibroblasts. We conclude that PDC deficiency is associated with an increase in O(2)(.-) accumulation coupled to a decrease in mechanisms responsible for its removal. Increased HIF1α expression may contribute to the increase in glycolytic flux and lactate production in PDC deficiency and, by trans-activating pyruvate dehydrogenase kinase, may further suppress residual PDC activity through phosphorylation of the E1α subunit. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Role of human pulmonary fibroblast-derived MCP-1 in cell activation and migration in experimental silicosis

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xueting [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Fang, Shencun [Nine Department of Respiratory Medicine, Nanjing Chest Hospital, Nanjing, Jiangsu 210029 (China); Liu, Haijun [Neurobiology Laboratory, New Drug Screening Centre, China Pharmaceutical University, Nanjing, Jiangsu 210009 (China); Wang, Xingang; Dai, Xiaoniu; Yin, Qing; Yun, Tianwei [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Wang, Wei; Zhang, Yingming [Nine Department of Respiratory Medicine, Nanjing Chest Hospital, Nanjing, Jiangsu 210029 (China); Liao, Hong [Neurobiology Laboratory, New Drug Screening Centre, China Pharmaceutical University, Nanjing, Jiangsu 210009 (China); Zhang, Wei [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Yao, Honghong [Department of Pharmacology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Chao, Jie, E-mail: chaojie@seu.edu.cn [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China)

    2015-10-15

    Background: Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO{sub 2}). Phagocytosis of SiO{sub 2} in the lung initiates an inflammatory cascade that results in fibroblast proliferation and migration and subsequent fibrosis. Clinical evidence indicates that the activation of alveolar macrophages by SiO{sub 2} produces rapid and sustained inflammation that is characterized by the generation of monocyte chemotactic protein 1 (MCP-1), which induces fibrosis. Pulmonary fibroblast-derived MCP-1 may play a critical role in fibroblast proliferation and migration. Methods and results: Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following results: 1) SiO{sub 2} treatment resulted in the rapid and sustained induction of MCP-1 as well as the elevation of the CC chemokine receptor type 2 (CCR2) protein levels; 2) pretreatment of HPF-a with RS-102895, a specific CCR2 inhibitor, abolished the SiO{sub 2}-induced increase in cell activation and migration in both 2D and 3D culture systems; and 3) RNA interference targeting CCR2 prevented the SiO{sub 2}-induced increase in cell migration. Conclusion: These data demonstrated that the up-regulation of pulmonary fibroblast-derived MCP-1 is involved in pulmonary fibroblast migration induced by SiO{sub 2}. CCR2 was also up-regulated in response to SiO{sub 2}, and this up-regulation facilitated the effect of MCP-1 on fibroblasts. Our study deciphered the link between fibroblast-derived MCP-1 and SiO{sub 2}-induced cell migration. This finding provides novel insight into the potential of MCP-1 in the development of novel therapeutic strategies for silicosis. - Highlights: • Role of pulmonary fibroblast-derived MCP-1 in experimental silicosis was studied. • SiO{sub 2} induced MCP-1 release from cultured human pulmonary fibroblast (HPF-a). • SiO{sub 2} directly activated HPF-a via the MCP-1/CCR2 pathway. • SiO{sub 2} increased HPF-a migration in both 2D and 3D

  7. Role of human pulmonary fibroblast-derived MCP-1 in cell activation and migration in experimental silicosis

    International Nuclear Information System (INIS)

    Liu, Xueting; Fang, Shencun; Liu, Haijun; Wang, Xingang; Dai, Xiaoniu; Yin, Qing; Yun, Tianwei; Wang, Wei; Zhang, Yingming; Liao, Hong; Zhang, Wei; Yao, Honghong; Chao, Jie

    2015-01-01

    Background: Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO 2 ). Phagocytosis of SiO 2 in the lung initiates an inflammatory cascade that results in fibroblast proliferation and migration and subsequent fibrosis. Clinical evidence indicates that the activation of alveolar macrophages by SiO 2 produces rapid and sustained inflammation that is characterized by the generation of monocyte chemotactic protein 1 (MCP-1), which induces fibrosis. Pulmonary fibroblast-derived MCP-1 may play a critical role in fibroblast proliferation and migration. Methods and results: Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following results: 1) SiO 2 treatment resulted in the rapid and sustained induction of MCP-1 as well as the elevation of the CC chemokine receptor type 2 (CCR2) protein levels; 2) pretreatment of HPF-a with RS-102895, a specific CCR2 inhibitor, abolished the SiO 2 -induced increase in cell activation and migration in both 2D and 3D culture systems; and 3) RNA interference targeting CCR2 prevented the SiO 2 -induced increase in cell migration. Conclusion: These data demonstrated that the up-regulation of pulmonary fibroblast-derived MCP-1 is involved in pulmonary fibroblast migration induced by SiO 2 . CCR2 was also up-regulated in response to SiO 2 , and this up-regulation facilitated the effect of MCP-1 on fibroblasts. Our study deciphered the link between fibroblast-derived MCP-1 and SiO 2 -induced cell migration. This finding provides novel insight into the potential of MCP-1 in the development of novel therapeutic strategies for silicosis. - Highlights: • Role of pulmonary fibroblast-derived MCP-1 in experimental silicosis was studied. • SiO 2 induced MCP-1 release from cultured human pulmonary fibroblast (HPF-a). • SiO 2 directly activated HPF-a via the MCP-1/CCR2 pathway. • SiO 2 increased HPF-a migration in both 2D and 3D model via the MCP-1/CCR2 pathway. • RNA-i of MCP-1/CCR2

  8. Generation of a human induced pluripotent stem cell line (MUSIi001-A from caesarean section scar fibroblasts using Sendai viral vectors

    Directory of Open Access Journals (Sweden)

    Methichit Wattanapanitch

    2018-03-01

    Full Text Available We generated a human induced pluripotent stem cell (iPSC line from caesarean section scar fibroblasts of a 33-year-old healthy woman using transgene-free Sendai viral vectors under feeder-free condition. The established iPSC line, designated as MUSIi001-A, exhibited a normal karyotype, expressed pluripotent markers, differentiated into cells of three embryonic germ layers. Further analyses showed that the Sendai viral genome was absent at passage 25. The MUSIi001-A line can serve as a control for studying developmental biology and phenotypic comparison with disease-specific iPSCs.

  9. Sirtuin 7 is decreased in pulmonary fibrosis and regulates the fibrotic phenotype of lung fibroblasts.

    Science.gov (United States)

    Wyman, Anne E; Noor, Zahid; Fishelevich, Rita; Lockatell, Virginia; Shah, Nirav G; Todd, Nevins W; Atamas, Sergei P

    2017-06-01

    Pulmonary fibrosis is a severe condition with no cure and limited therapeutic options. A better understanding of its pathophysiology is needed. Recent studies have suggested that pulmonary fibrosis may be driven by accelerated aging-related mechanisms. Sirtuins (SIRTs), particularly SIRT1, SIRT3, and SIRT6, are well-known mediators of aging; however, limited data exist on the contribution of sirtuins to lung fibrosis. We assessed the mRNA and protein levels of all seven known sirtuins in primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) in comparison with lung fibroblasts from healthy controls. These unbiased tests revealed a tendency for all sirtuins to be expressed at lower levels in fibroblasts from patients compared with controls, but the greatest decrease was observed with SIRT7. Similarly, SIRT7 was decreased in lung tissues of bleomycin-challenged mice. Inhibition of SIRT7 with siRNA in cultured lung fibroblasts resulted in an increase in collagen and α-smooth muscle actin (α-SMA). Reciprocally, overexpression of SIRT7 resulted in lower basal and TGF-β-induced levels of COL1A1, COL1A2, COL3A1, and α-SMA mRNAs, as well as collagen and α-SMA proteins. Induced changes in SIRT7 had no effect on endogenous TGF-β mRNA levels or latent TGF-β activation, but overexpression of SIRT7 reduced the levels of Smad3 mRNA and protein. In conclusion, the decline in SIRT7 in lung fibroblasts has a profibrotic effect, which is mediated by changes in Smad3 levels.

  10. Diffuse colonies of human skin fibroblasts in relation to cellular senescence and proliferation.

    Science.gov (United States)

    Zorin, Vadim; Zorina, Alla; Smetanina, Nadezhda; Kopnin, Pavel; Ozerov, Ivan V; Leonov, Sergey; Isaev, Artur; Klokov, Dmitry; Osipov, Andreyan N

    2017-05-16

    Development of personalized skin treatment in medicine and skin care may benefit from simple and accurate evaluation of the fraction of senescent skin fibroblasts that lost their proliferative capacity. We examined whether enriched analysis of colonies formed by primary human skin fibroblasts, a simple and widely available cellular assay, could reveal correlations with the fraction of senescent cells in heterogenic cell population. We measured fractions of senescence associated β-galactosidase (SA-βgal) positive cells in either mass cultures or colonies of various morphological types (dense, mixed and diffuse) formed by skin fibroblasts from 10 human donors. Although the donors were chosen to be within the same age group (33-54 years), the colony forming efficiency of their fibroblasts (ECO-f) and the percentage of dense, mixed and diffuse colonies varied greatly among the donors. We showed, for the first time, that the SA-βgal positive fraction was the largest in diffuse colonies, confirming that they originated from cells with the least proliferative capacity. The percentage of diffuse colonies was also found to correlate with the SA-βgal positive cells in mass culture. Using Ki67 as a cell proliferation marker, we further demonstrated a strong inverse correlation (r=-0.85, p=0.02) between the percentage of diffuse colonies and the fraction of Ki67+ cells. Moreover, a significant inverse correlation (r=-0.94, p=0.0001) between the percentage of diffuse colonies and ECO-f was found. Our data indicate that quantification of a fraction of diffuse colonies may provide a simple and useful method to evaluate the extent of cellular senescence in human skin fibroblasts.

  11. Evaluation of circadian phenotypes utilizing fibroblasts from patients with circadian rhythm sleep disorders.

    Science.gov (United States)

    Hida, A; Ohsawa, Y; Kitamura, S; Nakazaki, K; Ayabe, N; Motomura, Y; Matsui, K; Kobayashi, M; Usui, A; Inoue, Y; Kusanagi, H; Kamei, Y; Mishima, K

    2017-04-25

    We evaluated the circadian phenotypes of patients with delayed sleep-wake phase disorder (DSWPD) and non-24-hour sleep-wake rhythm disorder (N24SWD), two different circadian rhythm sleep disorders (CRSDs) by measuring clock gene expression rhythms in fibroblast cells derived from individual patients. Bmal1-luciferase (Bmal1-luc) expression rhythms were measured in the primary fibroblast cells derived from skin biopsy samples of patients with DSWPD and N24SWD, as well as control subjects. The period length of the Bmal1-luc rhythm (in vitro period) was distributed normally and was 22.80±0.47 (mean±s.d.) h in control-derived fibroblasts. The in vitro periods in DSWPD-derived fibroblasts and N24SWD-derived fibroblasts were 22.67±0.67 h and 23.18±0.70 h, respectively. The N24SWD group showed a significantly longer in vitro period than did the control or DSWPD group. Furthermore, in vitro period was associated with response to chronotherapy in the N24SWD group. Longer in vitro periods were observed in the non-responders (mean±s.d.: 23.59±0.89 h) compared with the responders (mean±s.d.: 22.97±0.47 h) in the N24SWD group. Our results indicate that prolonged circadian periods contribute to the onset and poor treatment outcome of N24SWD. In vitro rhythm assays could be useful for predicting circadian phenotypes and clinical prognosis in patients with CRSDs.

  12. Exploring the elasticity and adhesion behavior of cardiac fibroblasts by atomic force microscopy indentation

    Energy Technology Data Exchange (ETDEWEB)

    Codan, B.; Del Favero, G. [Department of Engineering and Architecture, University of Trieste (Italy); Martinelli, V. [Department of Engineering and Architecture, University of Trieste (Italy); International Center for Genetic Engineering and Biotechnology, Trieste (Italy); Long, C.S.; Mestroni, L. [University of Colorado Cardiovascular Institute, University of Colorado Denver, Aurora, CO (United States); Sbaizero, O., E-mail: sbaizero@units.it [Department of Engineering and Architecture, University of Trieste (Italy)

    2014-07-01

    AFM was used to collect the whole force–deformation cell curves. They provide both the elasticity and adhesion behavior of mouse primary cardiac fibroblasts. To confirm the hypothesis that a link exists between the membrane receptors and the cytoskeletal filaments causing therefore changing in both elasticity and adhesion behavior, actin-destabilizing Cytochalsin D was administrated to the fibroblasts. From immunofluorescence observation and AFM loading/unloading curves, cytoskeletal reorganization as well as a change in the elasticity and adhesion was indeed observed. Elasticity of control fibroblasts is three times higher than that for fibroblasts treated with 0.5 μM Cytochalasin. Moreover, AFM loading–unloading curves clearly show the different mechanical behavior of the two different cells analyzed: (i) for control cells the AFM cantilever rises during the dwell time while cells with Cytochalasin fail to show such an active resistance; (ii) the maximum force to deform control cells is quite higher and as far as adhesion is concern (iii) the maximum separation force, detachment area and the detachment process time are much larger for control compared to the Cytochalasin treated cells. Therefore, alterations in the cytoskeleton suggest that a link must exist between the membrane receptors and the cytoskeletal filaments beneath the cellular surface and inhibition of actin polymerization has effects on the whole cell mechanical behavior as well as adhesion. - Highlights: • The whole AFM force–deformation cell curves were analyzed. • They provide information on both the elasticity and adhesion behavior. • Actin-destabilizing Cytochalasin D was administrated to the fibroblasts. • Change in elasticity and adhesion was ascribed to cytoskeletal reorganization. • A link exists between the membrane receptors and the cytoskeletal filaments.

  13. Exploring the elasticity and adhesion behavior of cardiac fibroblasts by atomic force microscopy indentation

    International Nuclear Information System (INIS)

    Codan, B.; Del Favero, G.; Martinelli, V.; Long, C.S.; Mestroni, L.; Sbaizero, O.

    2014-01-01

    AFM was used to collect the whole force–deformation cell curves. They provide both the elasticity and adhesion behavior of mouse primary cardiac fibroblasts. To confirm the hypothesis that a link exists between the membrane receptors and the cytoskeletal filaments causing therefore changing in both elasticity and adhesion behavior, actin-destabilizing Cytochalsin D was administrated to the fibroblasts. From immunofluorescence observation and AFM loading/unloading curves, cytoskeletal reorganization as well as a change in the elasticity and adhesion was indeed observed. Elasticity of control fibroblasts is three times higher than that for fibroblasts treated with 0.5 μM Cytochalasin. Moreover, AFM loading–unloading curves clearly show the different mechanical behavior of the two different cells analyzed: (i) for control cells the AFM cantilever rises during the dwell time while cells with Cytochalasin fail to show such an active resistance; (ii) the maximum force to deform control cells is quite higher and as far as adhesion is concern (iii) the maximum separation force, detachment area and the detachment process time are much larger for control compared to the Cytochalasin treated cells. Therefore, alterations in the cytoskeleton suggest that a link must exist between the membrane receptors and the cytoskeletal filaments beneath the cellular surface and inhibition of actin polymerization has effects on the whole cell mechanical behavior as well as adhesion. - Highlights: • The whole AFM force–deformation cell curves were analyzed. • They provide information on both the elasticity and adhesion behavior. • Actin-destabilizing Cytochalasin D was administrated to the fibroblasts. • Change in elasticity and adhesion was ascribed to cytoskeletal reorganization. • A link exists between the membrane receptors and the cytoskeletal filaments

  14. Luteal cell steroidogenesis in relation to delayed embryonic development in the Indian short-nosed fruit bat, Cynopterus sphinx.

    Science.gov (United States)

    Meenakumari, Karukayil J; Banerjee, Arnab; Krishna, Amitabh

    2009-01-01

    The primary aim of this study was to determine the possible cause of slow or delayed embryonic development in Cynopterus sphinx by investigating morphological and steroidogenic changes in the corpus luteum (CL) and circulating hormone concentrations during two pregnancies of a year. This species showed delayed post-implantational embryonic development during gastrulation of the first pregnancy. Morphological features of the CL showed normal luteinization during both pregnancies. The CL did not change significantly in luteal cell size during the delay period of the first pregnancy as compared with the second pregnancy. The circulating progesterone and 17beta-estradiol concentrations were significantly lower during the period of delayed embryonic development as compared with the same stage of embryonic development during the second pregnancy. We also showed a marked decline in the activity of 3beta-hydroxysteroid dehydrogenase, P450 side chain cleavage enzyme, and steroidogenic acute regulatory peptide in the CL during the delay period. This may cause low circulating progesterone and estradiol synthesis and consequently delay embryonic development. What causes the decrease in steroidogenic factors in the CL during the period of delayed development in C. sphinx is under investigation.

  15. Cardiomyocytes derived from embryonic stem cells resemble cardiomyocytes of the embryonic heart tube

    NARCIS (Netherlands)

    Fijnvandraat, Arnoud C.; van Ginneken, Antoni C. G.; de Boer, Piet A. J.; Ruijter, Jan M.; Christoffels, Vincent M.; Moorman, Antoon F. M.; Lekanne Deprez, Ronald H.

    2003-01-01

    OBJECTIVE: After formation of the linear heart tube a chamber-specific program of gene expression becomes active that underlies the formation of the chamber myocardium. To assess whether this program is recapitulated in in vitro differentiated embryonic stem cells, we performed qualitative and

  16. Chromosome aberration induction in human diploid fibroblast and epithelial cells

    International Nuclear Information System (INIS)

    Scott, D.

    1986-01-01

    The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

  17. Replication of Brucella abortus and Brucella melitensis in fibroblasts does not require Atg5-dependent macroautophagy.

    Science.gov (United States)

    Hamer, Isabelle; Goffin, Emeline; De Bolle, Xavier; Letesson, Jean-Jacques; Jadot, Michel

    2014-09-02

    Several intracellular bacterial pathogens have evolved subtle strategies to subvert vesicular trafficking pathways of their host cells to avoid killing and to replicate inside the cells. Brucellae are Gram-negative facultative intracellular bacteria that are responsible for brucellosis, a worldwide extended chronic zoonosis. Following invasion, Brucella abortus is found in a vacuole that interacts first with various endosomal compartments and then with endoplasmic reticulum sub-compartments. Brucella establishes its replication niche in ER-derived vesicles. In the past, it has been proposed that B. abortus passed through the macroautophagy pathway before reaching its niche of replication. However, recent experiments provided evidence that the classical macroautophagy pathway was not involved in the intracellular trafficking and the replication of B. abortus in bone marrow-derived macrophages and in HeLa cells. In contrast, another study showed that macroautophagy favoured the survival and the replication of Brucella melitensis in infected RAW264.7 macrophages. This raises the possibility that B. abortus and B. melitensis followed different intracellular pathways before replicating. In the present work, we have addressed this issue by comparing the replication rate of B. abortus and B. melitensis in embryonic fibroblasts derived from wild-type and Atg5-/- mice, Atg5 being a core component of the canonical macroautophagic pathway. Our results indicate that both B. abortus S2308 and B. melitensis 16M strains are able to invade and replicate in Atg5-deficient fibroblasts, suggesting that the canonical Atg5-dependent macroautophagic pathway is dispensable for Brucella replication. The number of viable bacteria was even slightly higher in Atg5-/- fibroblasts than in wild-type fibroblasts. This increase could be due to a more efficient uptake or to a better survival rate of bacteria before the beginning of the replication in Atg5-deficient cells as compared to wild

  18. Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

    OpenAIRE

    Wei, Hongbo; Zhao, Lingzhou; Chen, Bangdao; Bai, Shizhu; Zhao, Yimin

    2014-01-01

    Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; ...

  19. The microRNA-132/212 family fine-tunes multiple targets in Angiotensin II signalling in cardiac fibroblasts

    DEFF Research Database (Denmark)

    Eskildsen, Tilde V; Schneider, Mikael; Sandberg, Maria B

    2015-01-01

    INTRODUCTION: MicroRNAs (miRNAs) are emerging as key regulators of cardiovascular development and disease; however, the cardiac miRNA target molecules are not well understood. We and others have described the Angiotensin II (AngII)-induced miR-132/212 family as novel regulators of cardiovascular...... in silico and in vitro experiments to identify miR-132/212 molecular targets in primary rat cardiac fibroblasts. RESULTS: MiR-132/212 overexpression increased fibroblast cell size and mRNA arrays detected several hundred genes that were differentially expressed, including a wide panel of receptors...... pathways that fine-tuned by miR-132/212, suggesting a role for this miRNA family as master signalling switches in cardiac fibroblasts. Our data underscore the potential for miRNA tools to manipulate a large array of molecules and thereby control biological function....

  20. Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: implications for breast cancer and DCIS therapy with autophagy inhibitors.

    Science.gov (United States)

    Martinez-Outschoorn, Ubaldo E; Pavlides, Stephanos; Whitaker-Menezes, Diana; Daumer, Kristin M; Milliman, Janet N; Chiavarina, Barbara; Migneco, Gemma; Witkiewicz, Agnieszka K; Martinez-Cantarin, Maria P; Flomenberg, Neal; Howell, Anthony; Pestell, Richard G; Lisanti, Michael P; Sotgia, Federica

    2010-06-15

    Loss of stromal caveolin 1 (Cav-1) is a novel biomarker for cancer-associated fibroblasts that predicts poor clinical outcome in breast cancer and DCIS patients. We hypothesized that epithelial cancer cells may have the ability to drive Cav-1 downregulation in adjacent normal fibroblasts, thereby promoting the cancer associated fibroblast phenotype. To test this hypothesis directly, here we developed a novel co-culture model employing (i) human breast cancer cells (MCF7), and (ii) immortalized fibroblasts (hTERT-BJ1), which are grown under defined experimental conditions. Importantly, we show that co-culture of immortalized human fibroblasts with MCF7 breast cancer cells leads to Cav-1 downregulation in fibroblasts. These results were also validated using primary cultures of normal human mammary fibroblasts co-cultured with MCF7 cells. In this system, we show that Cav-1 downregulation is mediated by autophagic/lysosomal degradation, as pre-treatment with lysosome-specific inhibitors rescues Cav-1 expression. Functionally, we demonstrate that fibroblasts co-cultured with MCF7 breast cancer cells acquire a cancer associated fibroblast phenotype, characterized by Cav-1 downregulation, increased expression of myofibroblast markers and extracellular matrix proteins, and constitutive activation of TGFβ/Smad2 signaling. siRNA-mediated Cav-1 downregulation mimics several key changes that occur in co-cultured fibroblasts, clearly indicating that a loss of Cav-1 is a critical initiating factor, driving stromal fibroblast activation during tumorigenesis. As such, this co-culture system can now be used as an experimental model for generating "synthetic" cancer associated fibroblasts (CAFs). More specifically, these "synthetic" CAFs could be used for drug screening to identify novel therapeutics that selectively target the Cav-1-negative tumor micro-environment. Our findings also suggest that chloroquine, or other autophagy/lysosome inhibitors, may be useful as anti

  1. The effect of phorbols on metabolic cooperation between human fibroblasts

    International Nuclear Information System (INIS)

    Mosser, D.D.; Bols, N.C.

    1982-01-01

    Autoradiography has been used to study the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), 4-O-methyl TPA, and phorbol on metabolic cooperation between human diploid fibroblasts. When the donors, hypoxanthine-guanine phosphoribosyl transferase+ (HGPRT+) cells, and recipients, HGPRT- cells, were plated together in the presence of [ 3 H]hypoxanthine and either 4-O-methyl TPA or phorbol, nearly all interactions that developed in 4 h were positive for metabolic cooperation whereas when high concentrations of TPA were used, the number of positive interactions was significantly less than the control. If the phorbol analogs were added after the donors and recipients had made contact, the number of positive interactions was the same as the control in all cases. However, although primary recipients in the cultures that had been treated with phorbol had the same number of grains as those in the control, primary recipients in cultures that had been treated with TPA or high concentrations of 4-O-methyl TPA had significantly fewer grains than those in the control. TPA treatment for 4 h had no effect on total [ 3 H]hypoxanthine incorporation or incorporation into acid-soluble and acid-insoluble fractions. Thus, the effect of TPA on metabolic cooperation is interpreted as a reduction in the transfer of [ 3 H]nucleotides and is an indication of an interference with intercellular communication

  2. Tumor-secreted LOXL2 activates fibroblasts through FAK signaling

    DEFF Research Database (Denmark)

    Barker, Holly E; Bird, Demelza; Lang, Georgina

    2013-01-01

    models. Here, we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of α-smooth muscle actin (α-SMA). Using a marker for reticular....... Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix. Moreover, LOXL2 induced the expression of α...

  3. Effect of SMAD7 gene overexpression on TGF-β1-induced profibrotic responses in fibroblasts derived from Peyronie's plaque

    Directory of Open Access Journals (Sweden)

    Min Ji Choi

    2015-06-01

    Full Text Available Transforming growth factor-β1 (TGF-β1 has been identified as one of the most important fibrogenic cytokines associated with Peyronie's disease (PD. The mothers against decapentaplegic homolog 7 (SMAD7 is an inhibitory Smad protein that blocks TGF-β signaling pathway. The aim of this study was to examine the anti-fibrotic effect of the SMAD7 gene in primary fibroblasts derived from human PD plaques. PD fibroblasts were pretreated with the SMAD7 gene and then stimulated with TGF-β1. Treated fibroblasts were used for Western blotting, fluorescent immunocytochemistry, hydroxyproline determination, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. Overexpression of the SMAD7 gene inhibited TGF-β1-induced phosphorylation and nuclear translocation of SMAD2 and SMAD3, transdifferentiation of fibroblasts into myofibroblasts, and quashed TGF-β1-induced production of extracellular matrix protein and hydroxyproline. Overexpression of the SMAD7 gene decreased the expression of cyclin D1 (a positive cell cycle regulator and induced the expression of poly (ADP-ribose polymerase 1, which is known to terminate Smad-mediated transcription, in PD fibroblasts. These findings suggest that the blocking of the TGF-β pathway by use of SMAD7 may be a promising therapeutic strategy for the treatment of PD.

  4. Wnt-expressing rat embryonic fibroblasts suppress Apo2L/TRAIL-induced apoptosis of human leukemia cells

    Czech Academy of Sciences Publication Activity Database

    Doubravská, Lenka; Šímová, Šárka; Čermák, Lukáš; Valenta, Tomáš; Kořínek, Vladimír; Anděra, Ladislav

    2008-01-01

    Roč. 13, č. 4 (2008), s. 573-587 ISSN 1360-8185 Institutional research plan: CEZ:AV0Z50520514 Keywords : Wnt * Apo2L/TRAIL * pre-B lymphocyte Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.971, year: 2008

  5. Elevated nuclear sphingoid base-1-phosphates and decreased histone deacetylase activity after Fumonisin B1 treatment in mouse embryonic fibroblasts

    Science.gov (United States)

    Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. ...

  6. Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles

    Czech Academy of Sciences Publication Activity Database

    Líbalová, Helena; Uhlířová, Kateřina; Kléma, J.; Machala, M.; Šrám, Radim; Ciganek, M.; Topinka, Jan

    2012-01-01

    Roč. 9, č. 1 (2012), s. 1-16 ISSN 1743-8977 R&D Projects: GA MŠk 2B08005; GA ČR GAP503/11/0142 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50390703 Institutional support: RVO:68378041 Keywords : air pollution * complex mixtures * HEL cells Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 9.178, year: 2012

  7. ER stress responses in the absence of apoptosome: a comparative study in CASP9 proficient vs deficient mouse embryonic fibroblasts.

    Science.gov (United States)

    Deegan, Shane; Saveljeva, Svetlana; Gupta, Sanjeev; MacDonald, David C; Samali, Afshin

    2014-08-29

    Cells respond to endoplasmic reticulum (ER) stress through the unfolded protein response (UPR), autophagy and cell death. In this study we utilized casp9(+/+) and casp9(-/-) MEFs to determine the effect of inhibition of mitochondrial apoptosis pathway on ER stress-induced-cell death, UPR and autophagy. We observed prolonged activation of UPR and autophagy in casp9(-/-) cells as compared with casp9(+/+) MEFs, which displayed transient activation of both pathways. Furthermore we showed that while casp9(-/-) MEFs were resistant to ER stress, prolonged exposure led to the activation of a non-canonical, caspase-mediated mode of cell death. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Establishment, characterization and immortalization of a fibroblast cell line from the Chinese red belly toad Bombina maxima skin.

    Science.gov (United States)

    Xiang, Yang; Gao, Qian; Su, Weiting; Zeng, Lin; Wang, Jinhuan; Hu, Yi; Nie, Wenhui; Ma, Xutong; Zhang, Yong; Lee, Wenhui; Zhang, Yun

    2012-01-01

    The skin of the amphibian Bombina maxima is rich in biologically active proteins and peptides, most of which have mammalian analogues. The physiological functions of most of the mammalian analogues are still unknown. Thus, Bombina maxima skin may be a promising model to reveal the physiological role of these proteins and peptides because of their large capacity for secretion. To investigate the physiological role of these proteins and peptides in vitro, a fibroblast cell line was successfully established from Bombina maxima tadpole skin. The cell line grew to form a monolayer with cells of a uniform shape and abundant rough endoplasmic reticulum, which are typical characteristics of fibroblasts. Further identification at a molecular level revealed that they strongly expressed the fibroblast marker protein vimentin. The chromosome number of these cells is 2n = 28, and most of them were diploid. Growth property analysis showed that they grew well for 14 passages. However, cells showed decreased proliferative ability after passage 15. Thus, we tried to immortalize the cells through the overexpression of SV40 T antigen. After selecting by G418, cells stably expressed SV40 large T antigen and showed enhanced proliferative ability and increased telomerase activity. Signal transduction analysis revealed functional p42 mitogen-activated protein (MAP) kinase in immortalized Bombina maxima dermal fibroblasts. Primary fibroblast cells and the immortalized fibroblast cells from Bombina maxima cultured in the present study can be used to investigate the physiological role of Bombina maxima skin-secreted proteins and peptides. In addition, the methods for primary cell culturing and cell immortalization will be useful for culturing and immortalizing cells from other types of amphibians.

  9. Variation in the loss of O6-methylguanine-DNA methyltransferase during immortalization of human fibroblasts.

    Science.gov (United States)

    Green, M H; Karran, P; Lowe, J E; Priestley, A; Arlett, C F; Mayne, L

    1990-01-01

    We have examined O6-methylguanine-DNA methyltransferase (MT) activity in four human fibroblast cell lines during immortalization. Transfection of primary fibroblasts with the plasmid pSV3gpt or pSV3neo, which encode the SV40 large T antigen, confers a transformed phenotype but not immediate immortality. After a period of growth (pre-crisis) the cells enter a quiescent phase (crisis) from which an immortal clone of cells eventually grows out. From measurements of MT activity in extracts of cells taken at different defined stages of the immortalization process, we conclude that the establishment of a Mex- (MT-deficient) cell population is not specifically associated with cellular transformation or with any particular stage of immortalization. It appears that in different cell populations the change from Mex+ to Mex- may occur at different times during the immortalization process and that the change may be very abrupt.

  10. A Transcriptomic and Epigenomic Comparison of Fetal and Adult Human Cardiac Fibroblasts Reveals Novel Key Transcription Factors in Adult Cardiac Fibroblasts

    Directory of Open Access Journals (Sweden)

    Malin K.B. Jonsson, PhD

    2016-12-01

    Full Text Available Cardiovascular disease remains the number one global cause of death and presents as multiple phenotypes in which the interplay between cardiomyocytes and cardiac fibroblasts (CFs has become increasingly highlighted. Fetal and adult CFs influence neighboring cardiomyocytes in different ways. Thus far, a detailed comparison between the two is lacking. Using a genome-wide approach, we identified and validated 2 crucial players for maintaining the adult primary human CF phenotype. Knockdown of these factors induced significant phenotypical changes, including senescence and reduced collagen gene expression. These may now represent novel therapeutic targets against deleterious functions of CFs in adult cardiovascular disease.

  11. Immortalization of Werner syndrome and progeria fibroblasts

    International Nuclear Information System (INIS)

    Saito, H.; Moses, R.E.

    1991-01-01

    Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents

  12. Coupling of cytoskeleton functions for fibroblast locomotion

    DEFF Research Database (Denmark)

    Couchman, J R; Lenn, M; Rees, D A

    1985-01-01

    caused visible protrusions in projected positions at the leading edge. We conclude that fibroblast locomotion may be driven coordinately by a common set of motility mechanisms and that this coordination may be lost as a result of physical or pharmacological disturbance. Taking our evidence with results...... from other Laboratories, we propose the following cytoskeleton functions. (i) Protrusive activity, probably based on solation--gelation cycles of the actin based cytoskeleton and membrane recycling which provides cellular and membrane components for streaming through the cell body to the leading edge...

  13. Differences in motility pattern between human buccal fibroblasts and periodontal and skin fibroblasts

    DEFF Research Database (Denmark)

    Lepekhin, Eugene; Grøn, Birgitte; Berezin, Vladimir

    2002-01-01

    at these sites can be explained by differences in the motile behavior of their respective fibroblast populations. The migratory characteristics were studied in a two-dimensional culture system. The migration of single cells was time-lapse video recorded at intervals of 15 min for a period of 6 h using a computer...

  14. File list: Oth.Emb.10.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.10.AllAg.Embryonic_palates mm9 TFs and others Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.10.AllAg.Embryonic_palates.bed ...

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    Lifescience Database Archive (English)

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  17. File list: Pol.Emb.05.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: Pol.Emb.20.AllAg.Embryonic_palates [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.20.AllAg.Embryonic_palates mm9 RNA polymerase Embryo Embryonic palates http...://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.20.AllAg.Embryonic_palates.bed ...

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  20. File list: Pol.Emb.20.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  1. File list: Unc.Emb.05.AllAg.Embryonic_pancreas [Chip-atlas[Archive

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  6. File list: Unc.Emb.50.AllAg.Embryonic_pancreas [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  7. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    International Nuclear Information System (INIS)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

    2011-01-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  8. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

    2011-07-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  9. Influx mechanisms in the embryonic and adult rat choroid plexus

    DEFF Research Database (Denmark)

    Saunders, Norman R; Dziegielewska, Katarzyna M; Møllgård, Kjeld

    2015-01-01

    The transcriptome of embryonic and adult rat lateral ventricular choroid plexus, using a combination of RNA-Sequencing and microarray data, was analyzed by functional groups of influx transporters, particularly solute carrier (SLC) transporters. RNA-Seq was performed at embryonic day (E) 15 and a...

  10. Pathways in pluripotency and differentiation of embryonic cells

    NARCIS (Netherlands)

    du Puy, L.

    2010-01-01

    Pluripotency - the potential to differentiate into derivatives of the three embryonic germ layers endoderm, ectoderm and mesoderm - is the main characteristic of embryonic stem (ES) cells. ES cells are derived from the inner cell mass (ICM) of a pre-implantation blastocyst and can self-renew

  11. Embryonic muscle development of Convoluta pulchra (Turbellaria-acoelomorpha, platyhelminthes).

    Science.gov (United States)

    Ladurner, P; Rieger, R

    2000-06-15

    We studied the embryonic development of body-wall musculature in the acoel turbellarian Convoluta pulchra by fluorescence microscopy using phalloidin-bound stains for F-actin. During stage 1, which we define as development prior to 50% of the time between egg-laying and hatching, actin was visible only in zonulae adhaerentes of epidermal cells. Subsequent development of muscle occurred in two distinct phases: first, formation of an orthogonal grid of early muscles and, second, differentiation of other myoblasts upon this grid. The first elements of the primary orthogonal muscle grid appeared as short, isolated, circular muscle fibers (stage 2; 50% developmental time), which eventually elongated to completely encircle the embryo (stage 3; at 60% of total developmental time). The first primary longitudinal fibers appeared later, along with some new primary circular fibers, by 60-63% of total developmental time (stage 4). From 65 to 100% of total developmental time (stages 5 to 7), secondary fibers, using primary fibers as templates, arose; the number of circular and longitudinal muscles thus increased, and at the same time parenchymal muscles began appearing. Hatchlings (stage 8) possessed about 25 circular and 30 longitudinal muscles as well as strong parenchymal muscles. The remarkable feature of the body wall of many adult acoel flatworms is that longitudinal muscles bend medially and cross each other behind the level of the mouth. We found that this development starts shortly after the appearance of the ventral mouth opening within the body wall muscle grid. The adult organization of the body-wall musculature consists of a grid of several hundred longitudinal and circular fibers and a few diagonal muscles. Musculature of the reproductive organs developed after hatching. Thus, extensive myogenesis must occur also during postembryonic development. Comparison between the turbellarians and the annelids suggests that formation of a primary orthogonal muscle grid and

  12. Toll-like receptor 9 mediated responses in cardiac fibroblasts.

    Directory of Open Access Journals (Sweden)

    Ingrid Kristine Ohm

    Full Text Available Altered cardiac Toll-like receptor 9 (TLR9 signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β. Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088 or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

  13. Photobiomodulation changes type 1 collagen gene expression by pulp fibroblasts

    Science.gov (United States)

    Lourenço Ribeiro Vitor, Luciana; Tavares Oliveira Prado, Mariel; Lourenço Neto, Natalino; Cardoso de Oliveira, Rodrigo; Ferreira Santos, Carlos; Moreira Machado, Maria Aparecida Andrade; Marchini Oliveira, Thais

    2018-06-01

    This study aimed to evaluate type 1 collagen (COL1) gene expression by human pulp fibroblasts from primary teeth (HPF) after the variation of photobiomodulation (PBM) parameters. HPF were obtained from a biorepository, used at 4th passage, and irradiated (InGaAlP—660 nm) varying the power and application time according to the following groups: G1: 1.2 J cm‑2–05 mW–10 s G2: 2.5 J cm‑2–05 mW–20 s G3: 3.7 J cm‑2–05 mW–30 s G4: 5.0 J cm‑2–05 mW–40 s G5: 6.2 J cm‑2–05 mW–50 s G6: 2.5 J cm‑2–10 mW–10 s G7: 3.7 J cm‑2–15 mW–10 s G8: 5.0 J cm‑2–20 mW–10 s G9: 6.2 J cm‑2–25 mW–10 s. The control group (G10) was not irradiated and maintained with DMEM  +  10% SFB. RT-PCR was used to evaluate COL1 gene expression at 6, 12, and 24 h after irradiation. Intra- and intergroup comparisons were performed by two-way ANOVA followed by Tukey test (p  differences among periods (p  differences (p  >  0.05). The energy densities from 2.5 to 5 J cm‑2, regardless of the variation in PBM parameters, biomodulated the COL1 gene expression. At the energy density of 6.2 J cm‑2, longer application time and smaller power changed the pattern of COL1 gene expression by pulp fibroblasts from human primary teeth.

  14. Characterization of Tetraploid Somatic Cell Nuclear Transfer-Derived Human Embryonic Stem Cells.

    Science.gov (United States)

    Shin, Dong-Hyuk; Lee, Jeoung-Eun; Eum, Jin Hee; Chung, Young Gie; Lee, Hoon Taek; Lee, Dong Ryul

    2017-12-01

    Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

  15. Mapping the stem cell state: eight novel human embryonic stem and embryonal carcinoma cell antibodies

    DEFF Research Database (Denmark)

    Wright, A; Andrews, N; Bardsley, K

    2011-01-01

    The antigenic profile of human embryonic stem (ES) and embryonal carcinoma (EC) cells has served as a key element of their characterization, with a common panel of surface and intracellular markers now widely used. Such markers have been used to identify cells within the 'undifferentiated state...... of reactivity for all antibodies against both ES and EC cells, suggesting that these markers will afford recognition of unique sub-states within the undifferentiated stem cell compartment....... and EC cells, and herein describe their characterization. The reactivity of these antibodies against a range of cell lines is reported, as well as their developmental regulation, basic biochemistry and reactivity in immunohistochemistry of testicular germ cell tumours. Our data reveal a range...

  16. Rac inhibition reverses the phenotype of fibrotic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Shi-wen Xu

    Full Text Available BACKGROUND: Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce alpha-smooth muscle actin (alpha-SMA, type I collagen and CCN2 (connective tissue growth factor, CTGF. The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies. METHODS AND FINDINGS: Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766. CONCLUSION: Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.

  17. Mesenchymal stem cells induce dermal fibroblast responses to injury

    International Nuclear Information System (INIS)

    Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

    2010-01-01

    Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

  18. Human embryonic stem cells: preclinical perspectives

    Directory of Open Access Journals (Sweden)

    Sarda Kanchan

    2008-01-01

    Full Text Available Abstract Human embryonic stem cells (hESCs have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

  19. Epigenetic control of embryonic stem cell fate

    DEFF Research Database (Denmark)

    Christophersen, Nicolaj Strøyer; Helin, Kristian

    2010-01-01

    Embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and are pluripotent, as they are able to differentiate into all cell types of the adult organism. Once established, the pluripotent ES cells can be maintained under defined culture conditions, but can also...... be induced rapidly to differentiate. Maintaining this balance of stability versus plasticity is a challenge, and extensive studies in recent years have focused on understanding the contributions of transcription factors and epigenetic enzymes to the "stemness" properties of these cells. Identifying...... the molecular switches that regulate ES cell self-renewal versus differentiation can provide insights into the nature of the pluripotent state and enhance the potential use of these cells in therapeutic applications. Here, we review the latest models for how changes in chromatin methylation can modulate ES cell...

  20. Embryonic stem cells in pig and cattle

    DEFF Research Database (Denmark)

    Maddox-Hyttel, Poul; Wolf, Xenia Asbæk; Rasmussen, Mikkel Aabech

    2007-01-01

    Porcine and bovine cell lines derived from the inner cell mass (ICM) or epiblasts of blastocysts have been maintained over extended periods of time and characterized by morphology, identification of some stem cell markers and, in few cases, by production of chimaeric offspring. However, germ line...... transmission in chimaeras has never been obtained. Due to this incomplete characterization of the cell lines, the expression embryonic stem (ES)-like cells is presently used in pig and cattle. The ICM or epiblast can be isolated from the blastocyst by whole blastocyst culture, mechanical isolation......, or immunosurgery, and they are generally cultured on feeder cells. The resulting ES-like cells may be differentiated in vivo by chimaera and teratoma formation or in vitro by embryoid body formation and monolayer induction. It is likely that more well characterized and stable porcine and bovine ES cell lines...

  1. Ionizing radiation-induced phosphorylation of RPA p34 is deficient in ataxia telangiectasia and reduced in aged normal fibroblasts

    International Nuclear Information System (INIS)

    Xinbo Cheng; Nge Cheong; Ya Wang; Iliakis, George

    1996-01-01

    Replication protein A (RPA, also called human single stranded DNA binding protein, hSSB) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair and recombination. Phosphorylation of RPA p34 subunit is observed after exposure of cells to radiation and other DNA damaging agents, which implicates the protein not only in repair but also in the regulation of replication on damaged DNA template. Here, we show that the phosphorylation observed in RPA p34 after exposure to ionizing radiation, X- or γ-rays, is reduced and occurs later in primary fibroblasts from patients suffering from ataxia telangiectasia (AT), as compared to normal fibroblasts. We also show that in primary normal human fibroblasts, radiation-induced phosphorylation of RPA p34 is 'age'-dependent and decreases significantly as cultures senesce. Radiation-induced phosphorylation of RPA p34 is nearly absent in non-cycling cells, while the expression of p21 cip1/waf1/sdi1 remains inducible. The results demonstrate a growth-stage and culture-age dependency in radiation-induced RPA p34 phosphorylation, and suggest the operation of a signal transduction pathway that is inactivated in senescing or quiescent fibroblasts and defective in AT cells

  2. Control of fibroblast fibronectin expression and alternative splicing via the PI3K/Akt/mTOR pathway

    International Nuclear Information System (INIS)

    White, Eric S.; Sagana, Rommel L.; Booth, Adam J.; Yan, Mei; Cornett, Ashley M.; Bloomheart, Christopher A.; Tsui, Jessica L.; Wilke, Carol A.; Moore, Bethany B.; Ritzenthaler, Jeffrey D.; Roman, Jesse; Muro, Andres F.

    2010-01-01

    Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-β, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten -/- fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten -/- cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten -/- cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.

  3. Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

    Science.gov (United States)

    Imsoonthornruksa, Sumeth; Pruksananonda, Kamthorn; Parnpai, Rangsun; Rungsiwiwut, Ruttachuk; Ketudat-Cairns, Mariena

    2015-01-01

    To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins. © 2015 S. Karger AG, Basel.

  4. Generation of HEXA-deficient hiPSCs from fibroblasts of a Tay-Sachs disease patient.

    Science.gov (United States)

    Liu, Zhong; Zhao, Rui

    2016-09-01

    Human iPSC line TSD-01-hiPSC was generated from fibroblasts of a patient with infantile Tay-Sachs disease (TSD). The patient is compound heterozygous at the HEXA gene by carrying a 1278insTATC allele and an IVS12+1G>C allele. STEMCCA lentivirus, which expresses OCT4, SOX2, KLF4, and c-MYC from a polycistronic transcript, were used for reprogramming. TSD-01-hiPSC express pluripotency markers such as OCT4, SOX2, NANOG, Tra-1-60, and alkaline phosphatase, and can differentiate into tissues from all the three embryonic germ layers. This TSD patient-derived hiPSC line may serve as a valuable in vitro tool for disease modeling and drug test. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Generation of HEXA-deficient hiPSCs from fibroblasts of a Tay-Sachs disease patient

    Directory of Open Access Journals (Sweden)

    Zhong Liu

    2016-09-01

    Full Text Available Human iPSC line TSD-01-hiPSC was generated from fibroblasts of a patient with infantile Tay-Sachs disease (TSD. The patient is compound heterozygous at the HEXA gene by carrying a 1278insTATC allele and an IVS12+1G>C allele. STEMCCA lentivirus, which expresses OCT4, SOX2, KLF4, and c-MYC from a polycistronic transcript, were used for reprogramming. TSD-01-hiPSC express pluripotency markers such as OCT4, SOX2, NANOG, Tra-1-60, and alkaline phosphatase, and can differentiate into tissues from all the three embryonic germ layers. This TSD patient-derived hiPSC line may serve as a valuable in vitro tool for disease modeling and drug test.

  6. Human embryonic stem cells and microenvironment

    Directory of Open Access Journals (Sweden)

    Banu İskender

    2014-09-01

    Full Text Available Human embryonic stem cells (hESCs possess a great potential in the field of regenerative medicine by their virtue of pluripotent potential with indefinite proliferation capabilities. They can self renew themselves and differentiate into three embryonic germ layers. Although they are conventionally grown on mitotically inactivated mouse feeder cells, there are in vitro culture systems utilizing feeder cells of human origin in order to prevent cross-species contamination. Recently established in vitro culture systems suggested that direct interaction with feeder cells is not necessary but rather attachment to a substrate is required to ensure long-term, efficient hESC culture in vitro. This substrate is usually composed of a mixture of extracellular matrix components representing in vivo natural niche. In hESC biology, the mechanism of interaction of hESCs with extracellular matrix molecules remained insufficiently explored area of research due to their transient nature of interaction with the in vivo niche. However, an in vitro culture system established using extracellular matrix molecules may provide a safer alternative to culture systems with feeder cells while paving the way to Good Manufacturing Practice-GMP production of hESCs for therapeutic purposes. Therefore, it is essential to study the interaction of extracellular matrix molecules with hESCs in order to standardize in vitro culture systems for large-scale production of hESCs in a less labor-intensive way. This would not only provide valuable information regarding the mechanisms that control pluripotency but also serve to dissect the molecular signaling pathways of directed differentiation for prospective therapeutic applications in the future. J Clin Exp Invest 2014; 5 (3: 486-495

  7. MSX-1 gene expression and regulation in embryonic palatal tissue.

    Science.gov (United States)

    Nugent, P; Greene, R M

    1998-01-01

    The palatal cleft seen in Msx-1 knock-out mice suggests a role for this gene in normal palate development. The cleft is presumed secondary to tooth and jaw malformations, since in situ hybridization suggests that Msx-1 mRNA is not highly expressed in developing palatal tissue. In this study we demonstrate, by Northern blot analysis, the expression of Msx-1, but not Msx-2, in the developing palate and in primary cultures of murine embryonic palate mesenchymal cells. Furthermore, we propose a role for Msx-1 in retinoic acid-induced cleft palate, since retinoic acid inhibits Msx-1 mRNA expression in palate mesenchymal cells. We also demonstrate that transforming growth factor beta inhibits Msx-1 mRNA expression in palate mesenchymal cells, with retinoic acid and transforming growth factor beta acting synergistically when added simultaneously to these cells. These data suggest a mechanistic interaction between retinoic acid, transforming growth factor beta, and Msx-1 in the etiology of retinoic acid-induced cleft palate.

  8. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease

    Science.gov (United States)

    Gourdie, Robert G.; Dimmeler, Stefanie; Kohl, Peter

    2016-01-01

    Our understanding of cardiac fibroblast functions has moved beyond their roles in heart structure and extracellular matrix generation, and now includes contributions to paracrine, mechanical and electrical signalling during ontogenesis and normal cardiac activity. Fibroblasts have central roles in pathogenic remodelling during myocardial ischaemia, hypertension and heart failure. As key contributors to scar formation, they are crucial for tissue repair after interventions including surgery and ablation. Novel experimental approaches targeting cardiac fibroblasts are promising potential therapies for heart disease. Indeed, several existing drugs act, at least partially, through effects on cardiac connective tissue. This Review outlines the origins and roles of fibroblasts in cardiac development, homeostasis and disease; illustrates the involvement of fibroblasts in current and emerging clinical interventions; and identifies future targets for research and development. PMID:27339799

  9. The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

    Science.gov (United States)

    Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi

    2017-05-01

    Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.

  10. A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells

    International Nuclear Information System (INIS)

    Liu Yanxia; Song Zhihua; Zhao Yang; Qin Han; Cai Jun; Zhang Hong; Yu Tianxin; Jiang Siming; Wang Guangwen; Ding Mingxiao; Deng Hongkui

    2006-01-01

    Traditionally, undifferentiated human embryonic stem cells (hESCs) are maintained on mouse embryonic fibroblast (MEF) cells or on matrigel with an MEF-conditioned medium (CM), which hampers the clinical applications of hESCs due to the contamination by animal pathogens. Here we report a novel chemical-defined medium using DMEM/F12 supplemented with N2, B27, and basic fibroblast growth factor (bFGF) [termed NBF]. This medium can support prolonged self-renewal of hESCs. hESCs cultured in NBF maintain an undifferentiated state and normal karyotype, are able to form embryoid bodies in vitro, and differentiate into three germ layers and extraembryonic cells. Furthermore, we find that hESCs cultured in NBF possess a low apoptosis rate and a high proliferation rate compared with those cultured in MEF-CM. Our findings provide a novel, simplified chemical-defined culture medium suitable for further therapeutic applications and developmental studies of hESCs

  11. Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity: EFFECTS ON EMBRYO SURVIVAL.

    Science.gov (United States)

    Fresco, Victor M; Kern, Christine B; Mohammadi, Moosa; Twal, Waleed O

    2016-09-02

    Fibulin-1 (FBLN1) is a member of a growing family of extracellular matrix glycoproteins that includes eight members and is involved in cellular functions such as adhesion, migration, and differentiation. FBLN1 has also been implicated in embryonic heart and valve development and in the formation of neural crest-derived structures, including aortic arch, thymus, and cranial nerves. Fibroblast growth factor 8 (FGF8) is a member of a large family of growth factors, and its functions include neural crest cell (NCC) maintenance, specifically NCC migration as well as patterning of structures formed from NCC such as outflow tract and cranial nerves. In this report, we sought to investigate whether FBLN1 and FGF8 have cooperative roles in vivo given their influence on the development of the same NCC-derived structures. Surface plasmon resonance binding data showed that FBLN1 binds tightly to FGF8 and prevents its enzymatic degradation by ADAM17. Moreover, overexpression of FBLN1 up-regulates FGF8 gene expression, and down-regulation of FBLN1 by siRNA inhibits FGF8 expression. The generation of a double mutant Fbln1 and Fgf8 mice (Fbln1(-/-) and Fgf8(-/-)) showed that haplo-insufficiency (Fbln1(+/-) and Fgf8(+/-)) resulted in increased embryonic mortality compared with single heterozygote crosses. The mortality of the FGF8/Fbln1 double heterozygote embryos occurred between 14.5 and 16.5 days post-coitus. In conclusion, FBLN1/FGF8 interaction plays a role in survival of vertebrate embryos, and reduced levels of both proteins resulted in added mortality in utero The FBLN1/FGF8 interaction may also be involved in the survival of neural crest cell population during development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization

    International Nuclear Information System (INIS)

    Lawrence, J.B.; Singer, R.H.; Villnave, C.A.; Stein, J.L.; Stein, G.S.

    1988-01-01

    We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs

  13. Interplay between Selenium Levels and Replicative Senescence in WI-38 Human Fibroblasts: A Proteomic Approach

    Directory of Open Access Journals (Sweden)

    Ghania Hammad

    2018-01-01

    Full Text Available Selenoproteins are essential components of antioxidant defense, redox homeostasis, and cell signaling in mammals, where selenium is found in the form of a rare amino acid, selenocysteine. Selenium, which is often limited both in food intake and cell culture media, is a strong regulator of selenoprotein expression and selenoenzyme activity. Aging is a slow, complex, and multifactorial process, resulting in a gradual and irreversible decline of various functions of the body. Several cellular aspects of organismal aging are recapitulated in the replicative senescence of cultured human diploid fibroblasts, such as embryonic lung fibroblast WI-38 cells. We previously reported that the long-term growth of young WI-38 cells with high (supplemented, moderate (control, or low (depleted concentrations of selenium in the culture medium impacts their replicative lifespan, due to rapid changes in replicative senescence-associated markers and signaling pathways. In order to gain insight into the molecular link between selenium levels and replicative senescence, in the present work, we have applied a quantitative proteomic approach based on 2-Dimensional Differential in-Gel Electrophoresis (2D-DIGE to the study of young and presenescent cells grown in selenium-supplemented, control, or depleted media. Applying a restrictive cut-off (spot intensity ±50% and a p value < 0.05 to the 2D-DIGE analyses revealed 81 differentially expressed protein spots, from which 123 proteins of interest were identified by mass spectrometry. We compared the changes in protein abundance for three different conditions: (i spots varying between young and presenescent cells, (ii spots varying in response to selenium concentration in young cells, and (iii spots varying in response to selenium concentration in presenescent cells. Interestingly, a 72% overlap between the impact of senescence and selenium was observed in our proteomic results, demonstrating a strong interplay between

  14. Doubling potential of fibroblasts from different species after ionising radiation

    International Nuclear Information System (INIS)

    Macieira-Coelho, A.; Diatloff, C.; Malaise, E.

    1976-01-01

    It is stated that whereas chicken fibroblasts invariably die after a certain number of doublings in vitro, and this fact is never altered by chemical or physical agents, mouse fibroblasts invariably acquire spontaneously an infinite growth potential. In the human species fibroblasts never acquire spontaneously the capacity to divide for ever, although they can become permanent cell lines after treatment with certain viruses. This behaviour of fibroblasts in vitro has been attributed to different nutritional requirements. Experiments are described with human and mouse fibroblasts in which it was found that the response to ionising radiation matches the relative tendencies of the fibroblasts to yield permanent cell lines. Irradiation was commenced during the phase of active proliferation. Human fibroblast cultures irradiated with 100 R stopped dividing earlier than the controls, whereas cultures irradiated with 200, 300 and 500 R had the same lifespan as the control cultures. Cultures irradiated with 400 R showed the longest survival. With mouse fibroblasts the growth curves of the irradiated cells were of the same type as in the controls, but recovery occurred earlier. The results indicated that ionising radiation accelerates a natural phenomenon; in cells with a limited growth potential (chicken) it shortens the lifespan, whereas in cells that can acquire an unlimited growth potential (mouse) it accelerate