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  1. Cell cycle regulation of embryonic stem cells and mouse embryonic fibroblasts lacking functional Pax7.

    PubMed

    Czerwinska, Areta M; Nowacka, Joanna; Aszer, Magdalena; Gawrzak, Sylwia; Archacka, Karolina; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Jańczyk-Ilach, Katarzyna; Koblowska, Marta; Ciemerych, Maria A; Grabowska, Iwona

    2016-11-01

    The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells. In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be executed by modulating the expression of positive cell cycle regulators, such as cyclin E.

  2. Cell cycle regulation of embryonic stem cells and mouse embryonic fibroblasts lacking functional Pax7

    PubMed Central

    Czerwinska, Areta M.; Nowacka, Joanna; Aszer, Magdalena; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Jańczyk-Ilach, Katarzyna; Ciemerych, Maria A.; Grabowska, Iwona

    2016-01-01

    ABSTRACT The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells. In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be executed by modulating the expression of positive cell cycle regulators, such as cyclin E. PMID:27610933

  3. Constitutive Smad signaling and Smad-dependent collagen gene expression in mouse embryonic fibroblasts lacking peroxisome proliferator-activated receptor-{gamma}

    SciTech Connect

    Ghosh, Asish K Wei, Jun; Wu, Minghua; Varga, John

    2008-09-19

    Transforming growth factor-{beta} (TGF-{beta}), a potent inducer of collagen synthesis, is implicated in pathological fibrosis. Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is a nuclear hormone receptor that regulates adipogenesis and numerous other biological processes. Here, we demonstrate that collagen gene expression was markedly elevated in mouse embryonic fibroblasts (MEFs) lacking PPAR-{gamma} compared to heterozygous control MEFs. Treatment with the PPAR-{gamma} ligand 15d-PGJ{sub 2} failed to down-regulate collagen gene expression in PPAR-{gamma} null MEFs, whereas reconstitution of these cells with ectopic PPAR-{gamma} resulted in their normalization. Compared to control MEFs, PPAR-{gamma} null MEFs displayed elevated levels of the Type I TGF-{beta} receptor (T{beta}RI), and secreted more TGF-{beta}1 into the media. Furthermore, PPAR-{gamma} null MEFs showed constitutive phosphorylation of cellular Smad2 and Smad3, even in the absence of exogenous TGF-{beta}, which was abrogated by the ALK5 inhibitor SB431542. Constitutive Smad2/3 phosphorylation in PPAR-{gamma} null MEFs was associated with Smad3 binding to its cognate DNA recognition sequences, and interaction with coactivator p300 previously implicated in TGF-{beta} responses. Taken together, these results indicate that loss of PPAR-{gamma} in MEFs is associated with upregulation of collagen synthesis, and activation of intracellular Smad signal transduction, due, at least in part, to autocrine TGF-{beta} stimulation.

  4. Myosin synthesis in embryonic chicken fibroblasts

    PubMed Central

    1979-01-01

    The rate of constitutive myosin synthesis was measured in cultures of replicating embryonic chicken skin fibroblasts by pulse labeling with [3H]leucine. These cells synthesized the 200,000-dalton heavy chain of myosin (MHC) at a rate of 3.2 x 10(3) molecules/cell/min. Additionally, an independent estimate of the MHC synthesis rate needed to maintain a constant level of constitutive MHC/cell was calculated from total protein content, percentage MHC, fibroblast doubling time, and MHC half- life. This calculated rate of approximately 2.9 x 10(3) molecules/cell/min was in close agreement with the measured rate. By comparison, the synthesis rate of myofibrillar MHC in fully activated muscle cell cultures was approximately 2.9 x 10(4) molecules/nucleus/min. PMID:479285

  5. [The comparison of biologic character between mouse embryonic fibroblast and human embryonic fibroblast].

    PubMed

    Zhang, Yi; Zhao, Liansan; Wang, Chengxiao; Lei, Binjun

    2003-06-01

    To evaluate the feasibility of using human embryonic fibroblast(HEF) as feeder layer in the culture of human embryonic stem(ES) cells in vitro, we investigated the morphology, the sensitivity to 0.25% trypsin, the growth curve and cell cycle of HEF with DMEM(low glucose) +10% FBS used as culture medium, and then we compared HEF with mouse embryonic fibroblast (MEF). The results showed that both HEF and MEF are adherent cells in vitro, and HEF has longer life span and better growth ability than MEF. In room temperature, HEF is more sensitive to 0.25% trypsin. Our research suggested that HEF can be used as feeder layer in culture of ES cells. HEF has longer service life than MEF and is worthy to be studied further.

  6. Epigenetic analysis of bovine parthenogenetic embryonic fibroblasts.

    PubMed

    Kaneda, Masahiro; Takahashi, Masashi; Yamanaka, Ken-Ichi; Saito, Koji; Taniguchi, Masanori; Akagi, Satoshi; Watanabe, Shinya; Nagai, Takashi

    2017-08-19

    Although more than 100 imprinted genes have already been identified in the mouse and human genomes, little is known about genomic imprinting in cattle. For a better understanding of these genes in cattle, parthenogenetically activated bovine blastocysts were transferred to recipient cows to obtain parthenotes, and fibroblasts derived from a Day 40 (Day 0 being the day of parthenogenetic activation) parthenogenetic embryo (BpEFs) were successfully obtained. Bovine embryonic fibroblasts (BEFs) were also isolated from a normal fertilized embryo obtained from an artificially inseminated cow. The expression of imprinted genes was analyzed by RT-PCR. Paternally expressed genes (PEGs) in mouse (viz., IGF2, PEG3, ZAC1, NDN, DLK1, SGCE, and PEG10) were expressed in BEFs, but not in BpEFs, suggesting that these genes are also imprinted in cattle. However, other PEGs in mouse (viz., IMPACT, MAGEL2, SNRPN, and PEG1/MEST) were expressed in both BEFs and BpEFs. These genes may not be imprinted in BEFs. The expression of seven maternally expressed genes in mouse was also analyzed, and only CDKN1C was not expressed in BpEFs. The DNA methylation patterns of repetitive elements (Satellite I, Satellite II, alpha-satellite, and Art2) were not different between the BEFs and BpEFs; however, the differentially methylated region (DMR) of paternally methylated H19 was hypomethylated, whereas those of maternally methylated PEG3 and PEG10 were hypermethylated in BpEFs, as expected. The methylation of the SNRPN DMR was not different between the BEFs and BpEFs, in accordance with the SNRPN expression levels in both cell types. The XIST gene, which is essential for X chromosome inactivation in females, was expressed in BpEFs, whereas its DMR was half-methylated, suggesting that X chromosome inactivation is normal in these cells. Microarray analysis was also applied to identify novel PEGs that should be expressed only in BEFs but not in BpEFs. More than 300 PEG candidate genes, including IGF2

  7. Evaluating biotoxicity with fibroblasts derived from human embryonic stem cells.

    PubMed

    Wang, Xiaoying; Li, Shenglin; Cao, Tong; Fu, Xin; Yu, Guangyan

    2012-09-01

    To investigate the use of differentiated fibroblasts from human embryonic stem cells as a cellular model for cytotoxicity and genotoxicity screening. The EBf-H9 cells were derived from human embryonic stem cells (H9) via embryonic body (EB) and treated with Sodium fluoride (NaF) and Formaldehyde (FA). Proliferation, specific gene and protein expression and karyotype of cells were analyzed by MTT assay, RT-PCR, immunocytochemistry and karyotype analysis, respectively. Cytotoxicity was detected by MTT assay and flow cytometry, and genotoxicity was studied by micronucleus test (MNT), sister chromatid exchange (SCE) and comet assay. EBf-H9s were spindle-shaped with a diploid karyotype. They expressed the fibroblast markers prolyl 4-hydroxylase β and vimentin but did not express Oct-4 and Sox-2, and decreased expression of Nanog. The proliferation of EBf-H9 and murine L929 cells was inhibited by sodium fluoride (NaF) and formaldehyde (FA), and the cell cycle was arrested in different phases with the treatments. In genotoxicity assays with NaF and FA, positive responses were detected in human EBf-H9s comparable to those in the murine L929 cell line. EBf-H9 may be a suitable new cell source for toxicity research on biomaterials and other agents. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells.

    PubMed

    Wei, Quan-Xiang; van der Hoeven, Franciscus; Hollstein, Monica; Odell, Adam F

    2012-05-17

    This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform' mouse, which allows site-specific integration of plasmid DNA into the Trp53 locus. Simple PCR protocols identify correctly targeted clones and immunoblots verify re-expression of the protein. We also present protocol modifications needed for efficient recovery of MEF clones expressing p53 constructs that retain wild-type function, including growth at low (3%) oxygen and transient downregulation of p53 regulators to forestall cell senescence of primary MEFs. A library of cell lines expressing various p53 mutants derived from the same population of primary fibroblasts or platform ES cells can be acquired and screened in less than 1 month.

  9. Differentiation of Human Embryonic Stem Cells on Periodontal Ligament Fibroblasts.

    PubMed

    Elçin, Y Murat; İnanç, Bülend; Elçin, A Eser

    2016-01-01

    Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types makes them one of the potential cell sources in cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering is an approach to reconstitute the ectomesenchymally derived alveolar bone, periodontal ligament apparatus, and cementum tissues lost as a result of periodontal diseases. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with contemporary regenerative procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Further elucidation of developmental mechanisms associated with tooth formation may also contribute to valuable knowledge based upon which the future therapies can be designed. Protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue represent an initial step in developing hESC-based experimental and tissue engineering strategies. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.

  10. Transgenic Mice Overexpressing Methionine Sulfoxide Reductase A: Characterization of Embryonic Fibroblasts

    PubMed Central

    Zhao, Hang; Kim, Geumsoo; Liu, Chengyu; Levine, Rodney L.

    2012-01-01

    Methionine residues in protein can be oxidized by reactive oxygen species to generate methionine sulfoxide. Aerobic organisms have methionine sulfoxide reductases capable of reducing methionine sulfoxide back to methionine. Methionine sulfoxide reductase A acts on the S-epimer of methionine sulfoxide, and it is known that altering its cellular level by genetic ablation or overexpression has notable effects on resistance to oxidative stress and on lifespan in species from microorganisms to animals. In mammals, the enzyme is present both in the cytosol and mitochondria, and this study was undertaken to assess the contribution of each subcellular compartment’s reductase activity to resistance against oxidative stresses. Non-transgenic mouse embryonic fibroblasts lack methionine sulfoxide reductase A activity, providing a convenient cell type to determine the effect of expression of the enzyme in each compartment. We created transgenic mice with methionine sulfoxide reductase A targeted to the cytosol, mitochondria, or both and studied embryonic fibroblasts derived from each line. Unexpectedly, none of the transgenic cells gained resistance to a variety of oxidative stresses even though the expressed enzymes were catalytically active when assayed in vitro. Noting that activity in vivo requires thioredoxin and thioredoxin reductase, we determined the levels of these proteins in the fibroblasts and found that they were very low in both the non-transgenic and transgenic cells. We conclude that overexpression of methionine sulfoxide reductase A did not confer resistance to oxidative stress because the cells lacked other proteins required to constitute a functional methionine sulfoxide reduction system. PMID:20510353

  11. Recombinant Rabbit Leukemia Inhibitory Factor and Rabbit Embryonic Fibroblasts Support the Derivation and Maintenance of Rabbit Embryonic Stem Cells

    PubMed Central

    Xue, Fei; Ma, Yinghong; Chen, Y. Eugene; Zhang, Jifeng; Lin, Tzu-An; Chen, Chien-Hong; Lin, Wei-Wen; Roach, Marsha; Ju, Jyh-Cherng; Yang, Lan; Du, Fuliang

    2012-01-01

    Abstract The rabbit is a classical experimental animal species. A major limitation in using rabbits for biomedical research is the lack of germ-line-competent rabbit embryonic stem cells (rbESCs). We hypothesized that the use of homologous feeder cells and recombinant rabbit leukemia inhibitory factor (rbLIF) might improve the chance in deriving germ-line-competent rbES cells. In the present study, we established rabbit embryonic fibroblast (REF) feeder layers and synthesized recombinant rbLIF. We derived a total of seven putative rbESC lines, of which two lines (M5 and M23) were from culture Condition I using mouse embryonic fibroblasts (MEFs) as feeders supplemented with human LIF (hLIF) (MEF+hLIF). Another five lines (R4, R9, R15, R21, and R31) were derived from Condition II using REFs as feeder cells supplemented with rbLIF (REF+rbLIF). Similar derivation efficiency was observed between these two conditions (8.7% vs. 10.2%). In a separate experiment with 2×3 factorial design, we examined the effects of feeder cells (MEF vs. REF) and LIFs (mLIF, hLIF vs. rbLIF) on rbESC culture. Both Conditions I and II supported satisfactory rbESC culture, with similar or better population doubling time and colony-forming efficiency than other combinations of feeder cells with LIFs. Rabbit ESCs derived and maintained on both conditions displayed typical ESC characteristics, including ESC pluripotency marker expression (AP, Oct4, Sox2, Nanog, and SSEA4) and gene expression (Oct4, Sox2, Nanog, c-Myc, Klf4, and Dppa5), and the capacity to differentiate into three primary germ layers in vitro. The present work is the first attempt to establish rbESC lines using homologous feeder cells and recombinant rbLIF, by which the rbESCs were derived and maintained normally. These cell lines are unique resources and may facilitate the derivation of germ-line-competent rbESCs. PMID:22775411

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

    SciTech Connect

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

    2009-02-13

    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.

  13. Metabolomic Analysis of Mouse Embryonic Fibroblast Cells in Response to Autophagy Induced by Acute Starvation

    PubMed Central

    Shen, Sensen; Weng, Rui; Li, Linnan; Xu, Xinyuan; Bai, Yu; Liu, Huwei

    2016-01-01

    Autophagy-related protein 7 (Atg7) is essential in the formation of the autophagophore and is indispensable for autophagy induction. Autophagy will exist in lower level or even be blocked in cells without Atg7. Even though the possible signaling pathways of Atg7 have been proposed, the metabolomic responses under acute starvation in cells with and without Atg7 have not been elucidated. This study therefore was designed and aimed to reveal the metabolomics of Atg7-dependent autophagy through metabolomic analysis of Atg7−/− mouse embryonic fibroblast cells (MEFs) and wild-type MEFs along with the starvation time. 30 significantly altered metabolites were identified in response to nutrient stress, which were mainly associated with amino acid, energy, carbohydrate, and lipid metabolism. For the wild-type MEFs, the induction of autophagy protected cell survival with some up-regulated lipids during the first two hours’ starvation, while the subsequent apoptosis resulted in the decrease of cell viability after four hours’ starvation. For the Atg7−/− MEFs, apoptosis perhaps led to the deactivation of tricarboxylic acid (TCA) cycle due to the lack of autophagy, which resulted in the immediate drop of cellular viability under starvation. These results contributed to the metabolomic study and provided new insights into the mechanism associated with Atg7-dependent autophagy. PMID:27703171

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

    SciTech Connect

    Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith , E-Mail: Judith.hall@ncl.ac.uk

    2005-09-16

    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.

  15. FADD null mouse embryonic fibroblasts undergo apoptosis after photosensitization with the silicon phthalocyanine Pc 4.

    PubMed

    Nagy, B; Yeh, W C; Mak, T W; Chiu, S M; Separovic, D

    2001-01-01

    Oxidative stress, such as photodynamic therapy with the silicon phthalocyanine Pc 4 (Pc 4-PDT), can induce apoptosis and tumor necrosis factor alpha (TNF) production. TNF receptors, as well as other death receptors, have been implicated in stress-induced apoptosis. To assess directly the role of FADD, a death receptor-associated protein, in induction of apoptosis post-Pc 4-PDT, embryonic fibroblasts from FADD knock out (k/o) and wild-type (wt) mice were used. Pc 4-PDT induced casp-3 activation and apoptosis in both cell types. In the presence of zVAD, a pancaspase inhibitor, Pc 4-PDT-induced apoptosis was abrogated in both cell lines. Fumonisin B1 (FB), an inhibitor of ceramide synthase, had no effect on apoptosis after Pc 4-PDT in either cell line. Similar to Pc 4-PDT, exogenous C6-ceramide bypassed FADD deficiency and induced zVAD-sensitive apoptosis. In contrast to Pc 4 photosensitization, TNF did not induce either apoptosis or ceramide accumulation in FADD k/o cells. In the absence of FADD deficiency, TNF-induced apoptosis was zVAD-sensitive and FB-insensitive. Induced ceramide levels remained elevated after cotreatment with TNF and zVAD in FADD wt cells. Taken together, these data provide genetic evidence for a lack of FADD requirement in Pc 4-PDT- or C6-ceramide-induced apoptosis. FB-sensitive ceramide production accompanies, but does not suffice, for apoptosis after Pc 4 photosensitization or TNF.

  16. Genotoxicity of alpha particles in human embryonic skin fibroblasts

    SciTech Connect

    Chen, D.J.; Strniste, G.F.; Tokita, N.

    1984-11-01

    Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to ..cap alpha.. particles from /sup 238/ Pu and 250 kVp X rays. The survival curves resulting from exposure to ..cap alpha.. particles are exponential. The mean lethal dose, D/sub 0/, is approximately 1.3 Gy for X rays and 0.25 Gy for ..cap alpha.. particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for ..cap alpha.. particles whereas the X-ray-induced mutation data were better fitted by a quadratic function. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in cells exposed to ..cap alpha.. particles than to X rays.

  17. Effect of spermine synthase deficiency on polyamine biosynthesis and content in mice and embryonic fibroblasts, and the sensitivity of fibroblasts to 1,3-bis-(2-chloroethyl)-N-nitrosourea.

    PubMed Central

    Mackintosh, C A; Pegg, A E

    2000-01-01

    Mutant Gy male mice, which have previously been described as having disruption of the phosphate-regulating Phex gene and a spermine synthase gene [Meyer, Henley, Meyer, Morgan, McDonald, Mills and Price (1998) Genomics, 48, 289-295; Lorenz, Francis, Gempel, Böddrich, Josten, Schmahl and Schmidt (1998) Hum. Mol. Genet. 7, 541-547], as well as mutant Hyp male mice, which have disruption of the Phex gene only, were examined along with their respective normal male littermates. Biochemical analyses of extracts of brains, hearts and livers of 5-week-old mice showed that Gy males lacked any significant spermine synthase activity as well as spermine content. Organs of Gy males had a higher spermidine content. This was caused not only by the lack of conversion of spermidine into spermine, but also because of compensatory increases in the activities of other polyamine biosynthetic enzymes. Gy males were half the body weight of their normal male littermates at weaning age. Hyp males, however, were no different in size when compared with their controls. High mortality of Gy males occurs by weaning age and this mortality was shown to be largely post-natal. Embryonic fibroblasts were isolated from Gy males and their normal male littermates and were similarly shown to lack any significant spermine synthase activity as well as spermine content. The lack of spermine, however, had no significant effect on the growth of immortalized fibroblasts or of primary fibroblast cultures. Similarly, there was no difference in the time of senescence of primary fibroblast cultures from Gy males compared with cultures derived from normal male littermates. However, the lack of spermine did increase the sensitivity of immortalized fibroblasts to killing by the chloroethylating agent 1, 3-bis(2-chloroethyl)-N-nitrosourea. Therefore both the Gy male mice and derived embryonic fibroblasts provide valuable models to study the importance of spermine and spermine synthase, without the use of inhibitors

  18. Short exposure to collagenase and coculture with mouse embryonic pancreas improve human dermal fibroblast culture.

    PubMed

    Pandamooz, Sareh; Hadipour, Abbas; Akhavan-Niaki, Haleh; Pourghasem, Mohsen; Abedian, Zeinab; Ardekani, Ali Motevallizadeh; Golpour, Monireh; Hassan, Zuhair Mohammad; Mostafazadeh, Amrollah

    2012-01-01

    The dermal fibroblast as a major component of connective tissue has attracted much attention in the past few years, and application of these very fast growing cells in several fields has been intensively studied. Isolating dermal fibroblasts is an appropriate way to expand these fast growing cells in vitro. Although using a dissociated fibroblast culture method is more convenient than skin explant culture, its enzymatic digestion is critical because a large number of cells can be lost over prolonged exposure to collagenase. This study was performed to increase the number of viable cells after digestion of fresh human foreskin of donors aged from 1 to 3 months with collagenase and also by to design a coculture system for resuscitation of the injured fibroblast. Our results demonstrate that we can maximize cell yield while maintaining cell viability by cutting the specimens into very small pieces (1-2 mm³) after removing the epidermal layer with dispase II and also by collecting released cells every 20 Min subsequent to digesting the dermal layer with collagenase. Moreover, our data strongly indicate that coculturing of isolated fibroblasts with embryonic pancreas explants can enhance the rate of proliferation in cultured fibroblasts.

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

    PubMed

    Bayer, Monika L; Yeung, Chin-Yan C; Kadler, Karl E; Qvortrup, Klaus; Baar, Keith; Svensson, René B; Magnusson, S Peter; Krogsgaard, Michael; Koch, Manuel; Kjaer, Michael

    2010-06-01

    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 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 along the axis of tension. The fibrils had a homogeneous narrow diameter that was similar to collagen fibrils occurring in embryonic tendon. Immunostaining showed colocalization of collagen type I with collagen III, XII and XIV. A fibronectin network was formed in parallel with the collagen, and fibroblasts stained positive for integrin alpha(5). Finally, the presence of cell extensions into the extracellular space with membrane-enclosed fibrils in fibripositors indicated characteristics of embryonic tendon. We conclude that mature human tendon fibroblasts retain an intrinsic capability to perform collagen fibrillogenesis similar to that of developing tendon, which implies that the hormonal/mechanical milieu, rather than intrinsic cellular function, inhibits regenerative potential in mature tendon. (c) 2010 Elsevier Ltd. All rights reserved.

  20. T-cell intracellular antigen (TIA)-proteins deficiency in murine embryonic fibroblasts alters cell cycle progression and induces autophagy.

    PubMed

    Sánchez-Jiménez, Carmen; Izquierdo, José M

    2013-01-01

    Mice lacking either T-cell intracellular antigen 1 (TIA1) or TIA1 related/like protein (TIAR/TIAL1) show high rates of embryonic lethality, suggesting a relevant role for these proteins during embryonic development. However, intrinsic molecular and cellular consequences of either TIA1 or TIAR deficiency remain poorly defined. By using genome-wide expression profiling approach, we demonstrate that either TIA1 or TIAR inactivation broadly alter normal development-associated signalling pathways in murine embryonic fibroblasts (MEF). Indeed, these analyses highlighted alterations of cytokine-cytokine and ECM-receptor interactions and Wnt, MAPK, TGF-beta dependent signalling pathways. Consistent with these results, TIA1 and TIAR knockout (KO) MEF show reduced rates of cell proliferation, cell cycle progression delay and increased cell size. Furthermore, TIA-proteins deficiency also caused metabolic deficiencies, increased ROS levels and DNA damage, promoting a gentle rise of cell death. Concomitantly, high rates of autophagy were detected in both TIA1 and TIAR KO MEF with induction of the formation of autophagosomes, as evidenced by the up-regulation of the LC3B protein, and autolysosomes, measured by colocalization of LC3B and LAMP1, as a survival mechanism attempt. Taken together, these observations support that TIA proteins orchestrate a transcriptome programme to activate specific developmental decisions. This program is likely to contribute to mouse physiology starting at early stages of the embryonic development. TIA1/TIAR might function as cell sensors to maintain homeostasis and promote adaptation/survival responses to developmental stress.

  1. T-cell Intracellular Antigen (TIA)-Proteins Deficiency in Murine Embryonic Fibroblasts Alters Cell Cycle Progression and Induces Autophagy

    PubMed Central

    Sánchez-Jiménez, Carmen; Izquierdo, José M.

    2013-01-01

    Mice lacking either T-cell intracellular antigen 1 (TIA1) or TIA1 related/like protein (TIAR/TIAL1) show high rates of embryonic lethality, suggesting a relevant role for these proteins during embryonic development. However, intrinsic molecular and cellular consequences of either TIA1 or TIAR deficiency remain poorly defined. By using genome-wide expression profiling approach, we demonstrate that either TIA1 or TIAR inactivation broadly alter normal development-associated signalling pathways in murine embryonic fibroblasts (MEF). Indeed, these analyses highlighted alterations of cytokine-cytokine and ECM-receptor interactions and Wnt, MAPK, TGF-beta dependent signalling pathways. Consistent with these results, TIA1 and TIAR knockout (KO) MEF show reduced rates of cell proliferation, cell cycle progression delay and increased cell size. Furthermore, TIA-proteins deficiency also caused metabolic deficiencies, increased ROS levels and DNA damage, promoting a gentle rise of cell death. Concomitantly, high rates of autophagy were detected in both TIA1 and TIAR KO MEF with induction of the formation of autophagosomes, as evidenced by the up-regulation of the LC3B protein, and autolysosomes, measured by colocalization of LC3B and LAMP1, as a survival mechanism attempt. Taken together, these observations support that TIA proteins orchestrate a transcriptome programme to activate specific developmental decisions. This program is likely to contribute to mouse physiology starting at early stages of the embryonic development. TIA1/TIAR might function as cell sensors to maintain homeostasis and promote adaptation/survival responses to developmental stress. PMID:24086455

  2. miR-194 functions as a novel modulator of cellular senescence in mouse embryonic fibroblasts.

    PubMed

    Xu, Shun; Zhang, Bing; Zhu, Yanmei; Huang, Haijiao; Yang, Wenping; Huang, Haiyong; Zheng, Hui-Ling; Liu, Xinguang

    2017-03-01

    MicroRNA-194 (miR-194), a typical p53 responsive miRNA, serves as a tumor suppressor similar as p53, and has been demonstrated to play an anti-proliferation role in various human cancers. In spite of the pivotal role of p53 during aging process, the knowledge of miR-194's contribution to cellular senescence is limited. We herein sought to explore the role of miR-194 in the replicative senescence and stress-induced senescence of mouse embryonic fibroblasts. Our results unraveled that, compared to young cells, miR-194 is highly expressed in senescent cells, and extra expression of miR-194 significantly triggers the replicative senescence of MEFs and H2 O2 -induced senescence of NIH/3T3 cells, while inhibition of miR-194 exhibited the opposite effect. We further unveiled that DNMT3A was a direct and authentic target of miR-194, which has been reported to be closely associated with cellular senescence. Taken together, our data suggest that miR-194 may significantly promote the development of cellular senescence in mouse embryonic fibroblasts, which potentially occurs through inhibiting the DNMT3A expression.

  3. Embryonic fibroblast motility and orientation can be influenced by physiological electric fields

    PubMed Central

    1984-01-01

    Epithelial layers in developing embryos are known to drive ion currents through themselves that will, in turn, generate small electric fields within the embryo. We hypothesized that the movement of migratory embryonic cells might be guided by such fields, and report here that embryonic quail somite fibroblast motility can be strongly influenced by small DC electric fields. These cells responded to such fields in three ways: (a) The cells migrated towards the cathodal end of the field by extending lamellipodia in that direction. The threshold field strength for this galvanotaxis was between 1 and 10 mV/mm when the cells were cultured in plasma. (b) The cells oriented their long axes perpendicular to the field lines. The threshold field strength for this response for a 90-min interval in the field was 150 mV/mm in F12 medium and between 50 and 100 mV/mm in plasma. (c) The cells elongated under the influence of field strengths of 400 mV/mm and greater. These fibroblasts were therefore able to detect a voltage gradient at least as low as 0.2 mV across their width. Electric fields of at least 10- fold larger in magnitude than this threshold field have been detected in vivo in at least one vertebrate thus far, so we believe that these field effects encompass a physiological range. PMID:6707093

  4. Telomere-associated factor expression in replicative senescence of human embryonic lung fibroblasts.

    PubMed

    Du, H; Yang, L; Xu, X-Y; Hai, L; Han, Y-Q; Shi, Y-X

    2015-08-10

    The objective of this study was to find the key regulatory molecules in the cell senescence process through observing the expression of telomere-associated factor during the normal cell replicative senescence process. Based on the established cell replicative senescence model, reverse transcription-polymerase chain reaction and western blot analyses were used to detect telomere-associated factor expression at the mRNA and protein levels, including that of human telomere binding protein 1, tankyrase 1, telomerase RNA, telomere protection protein 1 (POT1), and p53 during the process of human embryonic lung fibroblast replicative senescence. The results showed that transcription of human telomere binding protein 1 did not change with cell senescence, whereas the protein expression of human telomere binding protein 1 increased gradually and then decreased rapidly; there was no change in the mRNA and protein expression of POT1; with the replicative senescence of human embryonic lung fibroblasts, expression of POT1 decreased gradually; TRF1 showed an increasing trend with cell senescence; and p53 protein expression did not change. Together, the results from this study suggest that human telomere binding protein 1, POT1, and TRF1 played important roles in cell senescence.

  5. Lack of endothelial cell survivin causes embryonic defects in angiogenesis, cardiogenesis, and neural tube closure.

    PubMed

    Zwerts, Femke; Lupu, Florea; De Vriese, Astrid; Pollefeyt, Saskia; Moons, Lieve; Altura, Rachel A; Jiang, Yuying; Maxwell, Patrick H; Hill, Peter; Oh, Hideyasu; Rieker, Claus; Collen, Désiré; Conway, Simon J; Conway, Edward M

    2007-06-01

    We explored the physiologic role of endothelial cell apoptosis during development by generating mouse embryos lacking the inhibitor of apoptosis protein (IAP) survivin in endothelium. This was accomplished by intercrossing survivin(lox/lox) mice with mice expressing cre recombinase under the control of the endothelial cell specific tie1 promoter (tie1-cre mice). Lack of endothelial cell survivin resulted in embryonic lethality. Mutant embryos had prominent and diffuse hemorrhages from embryonic day 9.5 (E9.5) and died before E13.5. Heart development was strikingly abnormal. Survivin-null endocardial lineage cells could not support normal epithelial-mesenchymal transformation (EMT), resulting in hypoplastic endocardial cushions and in utero heart failure. In addition, 30% of mutant embryos had neural tube closure defects (NTDs) that were not caused by bleeding or growth retardation, but were likely due to alterations in the release of soluble factors from endothelial cells that otherwise support neural stem cell proliferation and neurulation. Thus, regulation of endothelial cell survival, and maintenance of vascular integrity by survivin are crucial for normal embryonic angiogenesis, cardiogenesis, and neurogenesis.

  6. Golgi Disruption and Early Embryonic Lethality in Mice Lacking USO1

    PubMed Central

    Kim, Susie; Hill, Adele; Warman, Matthew L.; Smits, Patrick

    2012-01-01

    Golgins are a family of long rod-like proteins characterized by the presence of central coiled-coil domains. Members of the golgin family have important roles in membrane trafficking, where they function as tethering factors that capture transport vesicles and facilitate membrane fusion. Golgin family members also have essential roles in maintaining the organization of the Golgi apparatus. Knockdown of individual golgins in cultured cells resulted in the disruption of the Golgi structure and the dispersal of Golgi marker proteins throughout the cytoplasm. However, these cellular phenotypes have not always been recapitulated in vivo. For example, embryonic development proceeds much further than expected and Golgi disruption was observed in only a subset of cell types in mice lacking the ubiquitously expressed golgin GMAP-210. Cell-type specific functional compensation among golgins may explain the absence of global cell lethality when a ubiquitously expressed golgin is missing. In this study we show that functional compensation does not occur for the golgin USO1. Mice lacking this ubiquitously expressed protein exhibit disruption of Golgi structure and early embryonic lethality, indicating that USO1 is indispensable for early embryonic development. PMID:23185636

  7. Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-{beta} responsiveness

    SciTech Connect

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

    2008-03-10

    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-{beta} (TGF-{beta}) induces CCN2 expression in mesenchymal cells. Because CCN2 can enhance profibrotic responses elicited by TGF-{beta}, it has been proposed that CCN2 functions as an essential downstream signaling mediator for TGF-{beta}. To explore this notion, we characterized TGF-{beta}-induced activation of fibroblasts from CCN2-null (CCN2{sup -/-}) mouse embryos. Methods: The regulation of CCN2 expression was examined in vivo in a model of fibrosis induced by bleomycin. Cellular TGF-{beta} signal transduction and regulation of collagen gene expression were examined in CCN2{sup -/-} 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{sup -/-} MEFs was markedly reduced compared to wild type MEFs, TGF-{beta}-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{sup -/-} 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-{beta}-induced myofibroblast transdifferentiation, it appears to be dispensable for Smad-dependent stimulation of collagen and extracellular matrix synthesis in murine embryonic fibroblasts.

  8. Alteration of ganglioside synthesis by GM3 synthase knockout in murine embryonic fibroblasts.

    PubMed

    Shevchuk, Nikolai A; Hathout, Yetrib; Epifano, Olga; Su, Yan; Liu, Yihui; Sutherland, Margaret; Ladisch, Stephan

    2007-09-01

    To probe the functions of membrane gangliosides, the availability of ganglioside-depleted cells would be a valuable resource. To attempt to identify a useful genetic model of ganglioside depletion, we assessed ganglioside metabolism in murine GM3 synthase (GM3S)-/- knockout primary embryonic fibroblasts (MEF), because normal fibroblast gangliosides (GM3, GM2, GM1, and GD1a), all downstream products of GM3S, should be absent. We found that heterozygote MEF (GM3S+/-) did have a 36% reduced content of qualitatively normal gangliosides (7.0+/-0.8 nmol LBSA/mg cell protein; control: 11+/-1.6 nmol). However, two unexpected findings characterized the homozygous (GM3-/-) MEF. Despite complete knockout of GM3S, (i) GM3-/- MEF retained substantial ganglioside content (21% of normal or 2.3+/-1.1 nmol) and (ii) these gangliosides were entirely different from those of wild type MEF by HPTLC. Mass spectrometry identified them as GM1b, GalNAc-GM1b, and GD1alpha, containing both N-acetyl and N-glycolylneuraminic acid and diverse ceramide structures. All are products of the 0 pathway of ganglioside synthesis, not normally expressed in fibroblasts. The results suggest that complete, but not partial, inhibition of GM3 synthesis results in robust activation of an alternate pathway that may compensate for the complete absence of the products of GM3S.

  9. Undifferentiated State Induced by Rb-p53 Double Inactivation in Mouse Thyroid Neuroendocrine Cells and Embryonic Fibroblasts.

    PubMed

    Kitajima, Shunsuke; Kohno, Susumu; Kondoh, Atsushi; Sasaki, Nobunari; Nishimoto, Yuuki; Li, Fengkai; Abdallah Mohammed, Mohammed Salah; Muranaka, Hayato; Nagatani, Naoko; Suzuki, Misa; Kido, Yukiharu; Takahashi, Chiaki

    2015-05-01

    Retinoblastoma tumor suppressor protein (RB) is inactivated more frequently during tumor progression than during tumor initiation. However, its exact role in controlling the malignant features associated with tumor progression is poorly understood. We established in vivo and in vitro models to investigate the undifferentiated state induced by Rb inactivation. Rb heterozygous mice develop well-differentiated thyroid medullary carcinoma. We found that additional deletion of Trp53, without change in lineage, converted these Rb-deficient tumors to a poorly differentiated type associated with higher self-renewal activity. Freshly prepared mouse embryonic fibroblasts (MEFs) of Rb(-/-) ; Trp53(-/-) background formed stem cell-like spheres that expressed significant levels of embryonic genes despite of lacking the ability to form colonies on soft agar or tumors in immune-deficient mice. This suggested that Rb-p53 double inactivation resulted in an undifferentiated status but without carcinogenic conversion. We next established Rb(-/-) ; N-ras(-/-) MEFs that harbored a spontaneous carcinogenic mutation in Trp53. These cells (RN6), in an Rb-dependent manner, efficiently generated spheres that expressed very high levels of embryonic genes, and appeared to be carcinogenic. We then screened an FDA-approved drug library to search for agents that suppressed the spherogenic activity of RN6 cells. Data revealed that RN6 cells were sensitive to specific agents including ones those are effective against cancer stem cells. Taken together, all these findings suggest that the genetic interaction between Rb and p53 is a critical determinant of the undifferentiated state in normal and tumor cells.

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

    PubMed Central

    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

  11. Fibroblast growth factor rescues brain endothelial cells lacking presenilin 1 from apoptotic cell death following serum starvation

    PubMed Central

    Gama Sosa, Miguel A.; De Gasperi, Rita; Hof, Patrick R.; Elder, Gregory A.

    2016-01-01

    Presenilin 1 (Psen1) is important for vascular brain development and is known to influence cellular stress responses. To understand the role of Psen1 in endothelial stress responses, we investigated the effects of serum withdrawal on wild type (wt) and Psen1−/− embryonic brain endothelial cells. Serum starvation induced apoptosis in Psen1−/− cells but did not affect wt cells. PI3K/AKT signaling was reduced in serum-starved Psen1−/− cells, and this was associated with elevated levels of phospho-p38 consistent with decreased pro-survival AKT signaling in the absence of Psen1. Fibroblast growth factor (FGF1 and FGF2), but not vascular endothelial growth factor (VEGF) rescued Psen1−/− cells from serum starvation induced apoptosis. Inhibition of FGF signaling induced apoptosis in wt cells under serum withdrawal, while blocking γ-secretase activity had no effect. In the absence of serum, FGF2 immunoreactivity was distributed diffusely in cytoplasmic and nuclear vesicles of wt and Psen1−/− cells, as levels of FGF2 in nuclear and cytosolic fractions were not significantly different. Thus, sensitivity of Psen1−/− cells to serum starvation is not due to lack of FGF synthesis but likely to effects of Psen1 on FGF release onto the cell surface and impaired activation of the PI3K/AKT survival pathway. PMID:27443835

  12. Toxic and DNA damaging effects of a functionalized fullerene in human embryonic lung fibroblasts.

    PubMed

    Ershova, E S; Sergeeva, V A; Chausheva, A I; Zheglo, D G; Nikitina, V A; Smirnova, T D; Kameneva, L V; Porokhovnik, L N; Kutsev, S I; Troshin, P A; Voronov, I I; Khakina, E A; Veiko, N N; Kostyuk, S V

    2016-07-01

    Water-soluble fullerenes have been studied as potential nanovectors and therapeutic agents, but their possible toxicity is of concern. We have studied the effects of F-828, a soluble fullerene [C60] derivative, on diploid human embryonic lung fibroblasts (HELFs) in vitro. F-828 causes complex time-dependent changes in ROS levels. Inhibition of Nox4 activity by plumbagin blocks F-828-dependent ROS elevation. F-828 induces DNA breaks, as measured by the comet assay and γH2AX expression, and the activities of the transcription factors NF-kB and p53 increase. F-828 concentrations>25μM are cytotoxic; cell death occurs by necrosis. Expression levels of TGF-β, RHOA, RHOC, ROCK1, and SMAD2 increase following exposure to F-828. Our results raise the possibility that fullerene F-828 may induce pulmonary fibrosis in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed

    Rossner, Pavel; Rossnerova, Andrea; Beskid, Olena; Tabashidze, Nana; Libalova, Helena; Uhlirova, Katerina; Topinka, Jan; Sram, Radim J

    2014-01-01

    In order to evaluate the ability of a representative polycyclic aromatic hydrocarbon (PAH) and PAH-containing complex mixtures to induce double strand DNA breaks (DSBs) and repair of damaged DNA in human embryonic lung fibroblasts (HEL12469 cells), we investigated the effect of benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5μm (PM2.5) on nonhomologous DNA end joining (NHEJ) and induction of stable chromosome aberrations (CAs). PM2.5 was collected in winter and summer 2011 in two Czech cities differing in levels and sources of air pollutants. The cells were treated for 24h with the following concentrations of tested chemicals: B[a]P: 1μM, 10μM, 25μM; EOMs: 1μg/ml, 10μg/ml, 25μg/ml. We tested several endpoints representing key steps leading from DSBs to the formation of CAs including histone H2AX phosphorylation, levels of proteins Ku70, Ku80 and XRCC4 participating in NHEJ, in vitro ligation activity of nuclear extracts of the HEL12469 cells and the frequency of stable CAs assessed by whole chromosome painting of chromosomes 1, 2, 4, 5, 7 and 17 using fluorescence in situ hybridization. Our results show that 25μM of B[a]P and most of the tested doses of EOMs induced DSBs as indicated by H2AX phosphorylation. DNA damage was accompanied by induction of XRCC4 expression and an increased frequency of CAs. Translocations most frequently affected chromosome 7. We observed only a weak induction of Ku70/80 expression as well as ligation activity of nuclear extracts. In summary, our data suggest the induction of DSBs and NHEJ after treatment of human embryonic lung fibroblasts with B[a]P and complex mixtures containing PAHs. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. ACTION ON FIBROBLASTS OF THE PROTEIN FRACTION OF EMBRYONIC TISSUE EXTRACT

    PubMed Central

    Baker, Lillian E.; Carrel, Alexis

    1926-01-01

    The above experiments indicate that the growth-stimulating substance found in embryonic tissue extract, which has been responsible for the continuous growth of fibroblasts in vitro for 14 years, is either protein in nature or closely associated with the protein of the extract and adsorbed by it. If any specific hormone responsible for cell division is present, it is united to the protein or carried along with it in its first precipitation. It seems probable that the tissues utilize this protein for the nitrogen which they build into protoplasm. Whether it is first hydrolyzed before adsorption by the tissues has not been ascertained as yet. It has been shown in other experiments reported in the following paper that the amino acids of the tissue juice do not suffice for the growth of fibroblasts and that hydrolyzed tissue juice is toxic in the same way that a too concentrated mixture of amino acids is toxic. The results of the foregoing experiments may be summarized as follows: 1. Fractionation of embryo tissue juice has shown that it is the protein fraction that contains the activating substance. 2. Tissues continue to grow for a long time in the protein of the extract precipitated by CO2 and at a rate approximately equal to that in the original extract diluted to the same nitrogen concentration. 3. The non-protein nitrogen gives slight stimulation to growth. 4. Purification of the protein by repeated precipitation destroys its growth-promoting properties, but whether this is due to a denaturing of the protein,—which occurs very readily,—or to loss of some substance possibly an enzyme attached to it, has not been ascertained. 5. Preparations of purified proteins from embryonic tissue and egg white have shown no marked nutritive or stimulating action. A number of other pure substances have been tried without effect. PMID:19869191

  15. Evidence for the involvement of fibroblast growth factor 10 in lipofibroblast formation during embryonic lung development.

    PubMed

    Al Alam, Denise; El Agha, Elie; Sakurai, Reiko; Kheirollahi, Vahid; Moiseenko, Alena; Danopoulos, Soula; Shrestha, Amit; Schmoldt, Carole; Quantius, Jennifer; Herold, Susanne; Chao, Cho-Ming; Tiozzo, Caterina; De Langhe, Stijn; Plikus, Maksim V; Thornton, Matthew; Grubbs, Brendan; Minoo, Parviz; Rehan, Virender K; Bellusci, Saverio

    2015-12-01

    Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10(+) progenitor cells, in vivo knockdown of Fgfr2b ligand activity and reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development.

  16. Evidence for the involvement of fibroblast growth factor 10 in lipofibroblast formation during embryonic lung development

    PubMed Central

    Al Alam, Denise; El Agha, Elie; Sakurai, Reiko; Kheirollahi, Vahid; Moiseenko, Alena; Danopoulos, Soula; Shrestha, Amit; Schmoldt, Carole; Quantius, Jennifer; Herold, Susanne; Chao, Cho-Ming; Tiozzo, Caterina; De Langhe, Stijn; Plikus, Maksim V.; Thornton, Matthew; Grubbs, Brendan; Minoo, Parviz; Rehan, Virender K.; Bellusci, Saverio

    2015-01-01

    Lipid-containing alveolar interstitial fibroblasts (lipofibroblasts) are increasingly recognized as an important component of the epithelial stem cell niche in the rodent lung. Although lipofibroblasts were initially believed merely to assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for survival and growth of epithelial stem cells during adulthood. Despite increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10+ progenitor cells, in vivo knockdown of Fgfr2b ligand activity and reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development. PMID:26511927

  17. Histone H3.3 regulates mitotic progression in mouse embryonic fibroblasts.

    PubMed

    Ors, Aysegul; Papin, Christophe; Favier, Bertrand; Roulland, Yohan; Dalkara, Defne; Ozturk, Mehmet; Hamiche, Ali; Dimitrov, Stefan; Padmanabhan, Kiran

    2017-08-01

    H3.3 is a histone variant that marks transcription start sites as well as telomeres and heterochromatic sites on the genome. The presence of H3.3 is thought to positively correlate with the transcriptional status of its target genes. Using a conditional genetic strategy against H3.3B, combined with short hairpin RNAs against H3.3A, we essentially depleted all H3.3 gene expression in mouse embryonic fibroblasts. Following nearly complete loss of H3.3 in the cells, our transcriptomic analyses show very little impact on global gene expression or on the localization of histone variant H2A.Z. Instead, fibroblasts displayed slower cell growth and an increase in cell death, coincident with large-scale chromosome misalignment in mitosis and large polylobed or micronuclei in interphase cells. Thus, we conclude that H3.3 may have an important under-explored additional role in chromosome segregation, nuclear structure, and the maintenance of genome integrity.

  18. Early tissue patterning recreated by mouse embryonic fibroblasts in a three-dimensional environment.

    PubMed

    Quintana, Lluís; Muiños, Teresa Fernández; Genove, Elsa; Del Mar Olmos, María; Borrós, Salvador; Semino, Carlos E

    2009-01-01

    Cellular self-organization studies have been mainly focused on models such as Volvox, the slime mold Dictyostelium discoideum, and animal (metazoan) embryos. Moreover, animal tissues undergoing regeneration also exhibit properties of embryonic systems such as the self-organization process that rebuilds tissue complexity and function. We speculated that the recreation in vitro of the biological, biophysical, and biomechanical conditions similar to those of a regenerative milieu could elicit the intrinsic capacity of differentiated cells to proceed to the development of a tissue-like structure. Here we show that, when primary mouse embryonic fibroblasts are cultured in a soft nanofiber scaffold, they establish a cellular network that causes an organized cell contraction,proliferation, and migration that ends in the formation of a symmetrically bilateral structure with a distinct central axis. A subset of mesodermal genes (brachyury, Sox9, Runx2) is upregulated during this morphogenetic process. The expression of brachyury was localized first at the central axis, extending then to both sides of the structure. The spontaneous formation of cartilage-like tissue mainly at the paraxial zone followed expression ofSox9 and Runx2. Because cellular self-organization is an intrinsic property of the tissues undergoing development,this model could lead to new ways to consider tissue engineering and regenerative medicine.

  19. Early Tissue Patterning Recreated by Mouse Embryonic Fibroblasts in a Three-Dimensional Environment

    PubMed Central

    Quintana, Lluís; Muiños, Teresa Fernández; Genové, Elsa; Del Mar Olmos, María; Borrós, Salvador

    2009-01-01

    Cellular self-organization studies have been mainly focused on models such as Volvox, the slime mold Dictyostelium discoideum, and animal (metazoan) embryos. Moreover, animal tissues undergoing regeneration also exhibit properties of embryonic systems such as the self-organization process that rebuilds tissue complexity and function. We speculated that the recreation in vitro of the biological, biophysical, and biomechanical conditions similar to those of a regenerative milieu could elicit the intrinsic capacity of differentiated cells to proceed to the development of a tissue-like structure. Here we show that, when primary mouse embryonic fibroblasts are cultured in a soft nanofiber scaffold, they establish a cellular network that causes an organized cell contraction, proliferation, and migration that ends in the formation of a symmetrically bilateral structure with a distinct central axis. A subset of mesodermal genes (brachyury, Sox9, Runx2) is upregulated during this morphogenetic process. The expression of brachyury was localized first at the central axis, extending then to both sides of the structure. The spontaneous formation of cartilage-like tissue mainly at the paraxial zone followed expression of Sox9 and Runx2. Because cellular self-organization is an intrinsic property of the tissues undergoing development, this model could lead to new ways to consider tissue engineering and regenerative medicine. PMID:19025338

  20. Myogenic Differentiation of Mouse Embryonic Stem Cells That Lack a Functional Pax7 Gene

    PubMed Central

    Czerwinska, Areta M.; Grabowska, Iwona; Archacka, Karolina; Bem, Joanna; Swierczek, Barbara; Helinska, Anita; Streminska, Wladyslawa; Fogtman, Anna; Iwanicka-Nowicka, Roksana; Koblowska, Marta

    2016-01-01

    The transcription factor Pax7 plays a key role during embryonic myogenesis and sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Overexpression of Pax7 has been shown to promote the myogenic differentiation of pluripotent stem cells. However, the effects of the absence of functional Pax7 in differentiating embryonic stem cells (ESCs) have not yet been directly tested. Herein, we studied mouse stem cells that lacked a functional Pax7 gene and characterized the differentiation of these stem cells under conditions that promoted the derivation of myoblasts in vitro. We analyzed the expression of myogenic factors, such as myogenic regulatory factors and muscle-specific microRNAs, in wild-type and mutant cells. Finally, we compared the transcriptome of both types of cells and did not find substantial differences in the expression of genes related to the regulation of myogenesis. As a result, we showed that the absence of functional Pax7 does not prevent the in vitro myogenic differentiation of ESCs. PMID:26649785

  1. Mitochondrial bioenergetics and drug-induced toxicity in a panel of mouse embryonic fibroblasts with mitochondrial DNA single nucleotide polymorphisms

    SciTech Connect

    Pereira, Claudia V.; Oliveira, Paulo J.; Will, Yvonne; Nadanaciva, Sashi

    2012-10-15

    Mitochondrial DNA (mtDNA) variations including single nucleotide polymorphisms (SNPs) have been proposed to be involved in idiosyncratic drug reactions. However, current in vitro and in vivo models lack the genetic diversity seen in the human population. Our hypothesis is that different cell strains with distinct mtDNA SNPs may have different mitochondrial bioenergetic profiles and may therefore vary in their response to drug-induced toxicity. Therefore, we used an in vitro system composed of four strains of mouse embryonic fibroblasts (MEFs) with mtDNA polymorphisms. We sequenced mtDNA from embryonic fibroblasts isolated from four mouse strains, C57BL/6J, MOLF/EiJ, CZECHII/EiJ and PERA/EiJ, with the latter two being sequenced for the first time. The bioenergetic profile of the four strains of MEFs was investigated at both passages 3 and 10. Our results showed that there were clear differences among the four strains of MEFs at both passages, with CZECHII/EiJ having a lower mitochondrial robustness when compared to C57BL/6J, followed by MOLF/EiJ and PERA/EiJ. Seven drugs known to impair mitochondrial function were tested for their effect on the ATP content of the four strains of MEFs in both glucose- and galactose-containing media. Our results showed that there were strain-dependent differences in the response to some of the drugs. We propose that this model is a useful starting point to study compounds that may cause mitochondrial off-target toxicity in early stages of drug development, thus decreasing the number of experimental animals used. -- Highlights: ► mtDNA SNPs may be linked to individual predisposition to drug-induced toxicity. ► CZECHII/EiJ and PERA/EiJ mtDNA was sequenced for the first time in this study. ► Strain-dependent mitochondrial capacity differences were measured. ► Strain-dependent differences in response to mitochondrial toxicants were observed.

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

    PubMed

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27857169

  4. Expression profiling of mouse embryonic fibroblasts with a deletion in the helicase domain of the Werner Syndrome gene homologue treated with hydrogen peroxide

    PubMed Central

    2010-01-01

    Background Werner Syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for WS encodes a DNA helicase/exonuclease protein believed to affect different aspects of transcription, replication, and/or DNA repair. In addition to genomic instability, human WS cells exhibit oxidative stress. In this report, we have examined the impact of exogenous hydrogen peroxide on the expression profile of mouse embryonic fibroblasts lacking part of the helicase domain of the WRN homologue (here referred to as WrnΔhel/Δhel). Results WrnΔhel/Δhel mutant mouse embryonic fibroblasts exhibit increased oxidative stress. This was reflected by increased intracellular reactive oxygen species (ROS), increased oxidative damage in genomic DNA, changes in ATP/ADP ratios, and a disruption of the inner mitochondrial transmembrane potential when compared to wild type mouse embryonic fibroblasts. Expression profile analyses of hydrogen peroxide-treated wild type cells have indicated significant decreases in the expression of genes involved in mitosis, glycolysis, fatty acid metabolism, nucleic acid metabolism, and cell cycle control, as well as protein modification and stability. Such decreases in these biological processes were not observed in hydrogen peroxide-treated WrnΔhel/Δhel cells. Importantly, untreated WrnΔhel/Δhel cells already exhibited down regulation of several biological processes decreased in wild type cells that had been treated with hydrogen peroxide. Conclusion Expression profiling of WrnΔhel/Δhel mutant cells revealed a very different response to exogenous addition of hydrogen peroxide in culture compared to wild type cells. This is due in part to the fact that WrnΔhel/Δhel mutant cells already exhibited a modest chronic intracellular oxidative stress. PMID:20175907

  5. Absence of AMPKα2 accelerates cellular senescence via p16 induction in mouse embryonic fibroblasts.

    PubMed

    Ding, Ye; Chen, Jie; Okon, Imoh Sunday; Zou, Ming-Hui; Song, Ping

    2016-02-01

    Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK), an energy gauge and redox sensor, delays aging process. However, the molecular mechanisms by which AMPKα isoform regulates cellular senescence remain largely unknown. The aim of this study was to determine if AMPKα deletion contributes to the accelerated cell senescence by inducing p16(INK4A) (p16) expression thereby arresting cell cycle. The markers of cellular senescence, cell cycle proteins, and reactive oxygen species (ROS) were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J), AMPKα1, or AMPKα2 homozygous deficient (AMPKα1(-/-), AMPKα2(-/-)) mice by Western blot and cellular immunofluorescence staining, as well as immunohistochemistry (IHC) in skin tissue of young and aged mice. Deletion of AMPKα2, the minor isoform of AMPKα, but not AMPKα1 in high-passaged MEFs led to spontaneous cell senescence demonstrated by accumulation of senescence-associated-β-galactosidase (SA-β-gal) staining and foci formation of heterochromatin protein 1 homolog gamma (HP1γ). It was shown here that AMPKα2 deletion upregulates cyclin-dependent kinase (CDK) inhibitor, p16, which arrests cell cycle. Furthermore, AMPKα2 null cells exhibited elevated ROS production. Interestingly, knockdown of HMG box-containing protein 1 (HBP1) partially blocked the cellular senescence of AMPKα2-deleted MEFs via the reduction of p16. Finally, dermal cells senescence, including fibroblasts senescence evidenced by the staining of p16, HBP1, and Ki-67, in the skin of aged AMPKα2(-/-) mice was enhanced when compared with that in wild type mice. Taken together, our results suggest that AMPKα2 isoform plays a fundamental role in anti-oxidant stress and anti-senescence.

  6. Lack of fibroblast growth factor 21 accelerates metabolic liver injury characterized by steatohepatities in mice

    PubMed Central

    Liu, Xingkai; Zhang, Ping; Martin, Robert C; Cui, Guozhen; Wang, Guangyi; Tan, Yi; Cai, Lu; Lv, Guoyue; Li, Yan

    2016-01-01

    Fibroblast growth factor 21 (FGF21) concentrations are increased in human subjects who either have type 2 diabetes or nonalcoholic fatty liver disease (NAFLD). While excessive fat in the liver promotes the release of pro-inflammatory cytokines, NAFLD progresses from steatosis to non alcoholic steatohepatitis (NASH), a more aggressive form of hepatic damage, and lastly toward cirrhosis and HCC. In our previous study, loss of FGF21 is associated with hyper-proliferation, aberrant p53, and HCC development in diabetes mice. In this study, we proposed to investigate the liver metabolic disorders by diabetes and the potential roles of FGF21 played in NASH and potential carcinogenetic transformation of HCC. NASH was induced in FGF21 knockout (FGF21KO) mice by streptozotocin administration or fed with high fat diet (HFD). The pathological transformation of steatohepatities as well as parameters of inflammation, lipid metabolism, cellular events, mesenchymal-epithelial transition (MET) and Wnt/β-catenin signaling was determined in the FGF21 KO diabetic mice and HFD fed mice. We found that mice lacking the FGF21 gene are more prone to develop NASH. A compromised microenvironment of NASH, which could facilitate the HCC carcinogenetic transformation, was found in FGF21 KO mice under metabolic disorders by diabetes and HFD feeding. This study provided further evidence that lack of FGF21 worsened the metabolic disorders in NASH and could render a tumor microenvironment for HCC initiation and progression in the liver of diabetes mice. PMID:27293995

  7. Neuronal and astrocyte dysfunction diverges from embryonic fibroblasts in the Ndufs4fky/fky mouse

    PubMed Central

    Bird, Matthew J.; Wijeyeratne, Xiaonan W.; Komen, Jasper C.; Laskowski, Adrienne; Ryan, Michael T.; Thorburn, David R.; Frazier, Ann E.

    2014-01-01

    Mitochondrial dysfunction causes a range of early-onset neurological diseases and contributes to neurodegenerative conditions. The mechanisms of neurological damage however are poorly understood, as accessing relevant tissue from patients is difficult, and appropriate models are limited. Hence, we assessed mitochondrial function in neurologically relevant primary cell lines from a CI (complex I) deficient Ndufs4 KO (knockout) mouse (Ndufs4fky/fky) modelling aspects of the mitochondrial disease LS (Leigh syndrome), as well as MEFs (mouse embryonic fibroblasts). Although CI structure and function were compromised in all Ndufs4fky/fky cell types, the mitochondrial membrane potential was selectively impaired in the MEFs, correlating with decreased CI-dependent ATP synthesis. In addition, increased ROS (reactive oxygen species) generation and altered sensitivity to cell death were only observed in Ndufs4fky/fky primary MEFs. In contrast, Ndufs4fky/fky primary isocortical neurons and primary isocortical astrocytes displayed only impaired ATP generation without mitochondrial membrane potential changes. Therefore the neurological dysfunction in the Ndufs4fky/fky mouse may partly originate from a more severe ATP depletion in neurons and astrocytes, even at the expense of maintaining the mitochondrial membrane potential. This may provide protection from cell death, but would ultimately compromise cell functionality in neurons and astrocytes. Furthermore, RET (reverse electron transfer) from complex II to CI appears more prominent in neurons than MEFs or astrocytes, and is attenuated in Ndufs4fky/fky cells. PMID:25312000

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

    SciTech Connect

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

    2011-06-10

    Highlights: {yields} Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. {yields} This process is independent of endogenous ROS production. {yields} 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{sub 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.

  9. SILAC based protein profiling data of MKK3 knockout mouse embryonic fibroblasts

    PubMed Central

    Srivastava, Anup; Shinn, Amanda S.; Lam, TuKiet T.; Lee, Patty J.; Mannam, Praveen

    2016-01-01

    This data article reports changes in the phospho and total proteome of MKK3 knock out (MKK3−/−) mouse embryonic fibroblasts (MEFs). The dataset generated highlights the changes at protein level which can be helpful for understanding targets of the MAP kinase signaling pathway. Data was collected after TiO2-based phosphopeptide enrichment of whole cell lysate at baseline condition with bottom-up SILAC-based LC MS/MS quantitative mass spectrometry. We report all the proteins and peptides identified and quantified in MKK3−/− and WT MEFs. The altered pathways in MKK3−/− MEFs were analyzed by Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.7) and Ingenuity Pathway Analysis (IPA) and are presented as a table and graph, respectively. The data reported here is related to the published work [1]. All the associated mass spectrometry data has been deposited in the Yale Protein Expression Database (YPED) with the web-link to the data: http://yped.med.yale.edu/repository/ViewSeriesMenu.do;jsessionid=6A5CB07543D8B529FAE8C3FCFE29471D?series_id=5044&series_name=MMK3+Deletion+in+MEFs. PMID:26977448

  10. Morphology, cytoskeletal organization, and myosin dynamics of mouse embryonic fibroblasts cultured on nanofibrillar surfaces.

    PubMed

    Ahmed, Ijaz; Ponery, Abdul S; Nur-E-Kamal, Alam; Kamal, Jabeen; Meshel, Adam S; Sheetz, Michael P; Schindler, Melvin; Meiners, Sally

    2007-07-01

    Growth of cells in tissue culture is generally performed on two-dimensional (2D) surfaces composed of polystyrene or glass. Recent work, however, has shown that such 2D cultures are incomplete and do not adequately represent the physical characteristics of native extracellular matrix (ECM)/basement membrane (BM), namely dimensionality, compliance, fibrillarity, and porosity. In the current study, a three-dimensional (3D) nanofibrillar surface composed of electrospun polyamide nanofibers was utilized to mimic the topology and physical structure of ECM/BM. Additional chemical cues were incorporated into the nanofibrillar matrix by coating the surfaces with fibronectin, collagen I, or laminin-1. Results from the current study show an enhanced response of primary mouse embryonic fibroblasts (MEFs) to culture on nanofibrillar surfaces with more dramatic changes in cell spreading and reorganization of the cytoskeleton than previously observed for established cell lines. In addition, the cells cultured on nanofibrillar and 2D surfaces exhibited differential responses to the specific ECM/BM coatings. The localization and activity of myosin II-B for MEFs cultured on nanofibers was also compared. A dynamic redistribution of myosin II-B was observed within membrane protrusions. This was previously described for cells associated with nanofibers composed of collagen I but not for cells attached to 2D surfaces coated with monomeric collagen. These results provide further evidence that nanofibrillar surfaces offer a significantly different environment for cells than 2D substrates.

  11. Fibroblast growth factor 13 is essential for neural differentiation in Xenopus early embryonic development.

    PubMed

    Nishimoto, Satoko; Nishida, Eisuke

    2007-08-17

    In Xenopus embryonic development, the MEK5-ERK5 pathway, one of the MAPK pathways, lies downstream of SoxD and upstream of Xngnr1 in a signaling pathway regulating neural differentiation. It remains unclear, however, how the MEK5-ERK5 pathway is regulated in Xenopus neural development. As SoxD is a transcription factor, we hypothesized that some growth factor should be induced by SoxD and activate the MEK5-ERK5 pathway. As the expression level of fibroblast growth factor 13 (FGF13) is increased by SoxD, we analyzed the function of FGF13 in neural development. Knockdown of FGF13 with antisense morpholino-oligonucleotides (MOs) results in the reduced head structure and inhibition of neural differentiation. FGF13 MOs inhibit the SoxD-induced expression of Xngnr1 and the Xngnr1-induced expression of NeuroD, suggesting that FGF13 is necessary both upstream and downstream of Xngnr1 in neural differentiation. In addition, FGF13 MOs inhibit the activation of the MEK5-ERK5 pathway by dominant-negative bone morphogenetic protein receptor, a mimicker of neural inducers, indicating that FGF13 is involved in the activation of the MEK5-ERK5 pathway. Together, these results identify a role of FGF13 in Xenopus neural differentiation.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. In vitro myogenic and adipogenic differentiation model of genetically engineered bovine embryonic fibroblast cell lines.

    PubMed

    Yin, Jinlong; Jin, Xun; Beck, Samuel; Kang, Dong Ho; Hong, Zhongshan; Li, Zhehu; Jin, Yongcheng; Zhang, Qiankun; Choi, Yun-Jaie; Kim, Sung-Chan; Kim, Hyunggee

    2010-02-01

    Our current understanding of muscle and adipose tissue development has been largely restricted to the study of murine myogenic and adipogenic cell lines, since attempts to establish these cell lines from other species have met with only limited success. Here we report that a spontaneously immortalized bovine embryonic fibroblast cell line (BEFS) undergoes differentiation into adipogenic or myogenic lineages when ectopically transduced with PPARgamma2 (an adipogenic lineage determinant) or MyoD (a myogenic lineage determinant) and grown in adipogenic and myogenic differentiation culture media (ADCM and MDCM, respectively). We also found that PPARgamma2-overexpressing BEFS cells (BEFS-PPARgamma2) grown in ADCM with or without the PPARgamma2 ligand, troglitazone, preferentially differentiate into adipogenic cells in the presence of ectopic MyoD expression. Ectopic expression of PPARgamma2 in the inducible MyoD-overepxressing BEFS cells (BEFS-TetOn-MyoD) completely suppresses myogenic differentiation and leads to a significant increase in adipogenic differentiation, suggesting that the adipogenic differentiation program might be dominant. Therefore, BEFS, BEFS-PPARgamma2, and BEFS-TetOn-MyoD would be a valuable biological model for understanding a fundamental principle underlying myogenic and adipogenic development, and for isolating various genetic and chemical factors that enable muscle and adipocyte differentiation.

  14. Egg drop syndrome virus enters duck embryonic fibroblast cells via clathrin-mediated endocytosis.

    PubMed

    Huang, Jingjing; Tan, Dan; Wang, Yang; Liu, Caihong; Xu, Jiamin; Wang, Jingyu

    2015-12-02

    Previous studies of egg drop syndrome virus (EDSV) is restricted to serological surveys, disease diagnostics, and complete viral genome analysis. Consequently, the infection characteristics and entry routes of EDSV are poorly understood. Therefore, we aimed to explore the entry pathway of EDSV into duck embryonic fibroblast (DEF) cells as well as the infection characteristics and proliferation of EDSV in primary DEF and primary chicken embryo liver (CEL) cells. Transmission electron microscopy revealed that the virus triggered DEF cell membrane invagination as early as 10 min post-infection and that integrated endocytic vesicles formed at 20 min post-infection. The virus yield in EDSV-infected DEF cells treated with chlorpromazine (CPZ), sucrose, methyl-β-cyclodextrin (MβCD), or NH4Cl was measured by quantitative real-time PCR. Compared with the mock treatment, CPZ and sucrose greatly inhibited the production of viral progeny in a dose-dependent manner, while MβCD treatment did not result in a significant difference. Furthermore, NH4Cl had a strong inhibitory effect on the production of EDSV progeny. In addition, indirect immunofluorescence demonstrated that virus particles clustered on the surface of DEF cells treated with CPZ or sucrose. These results indicate that EDSV enters DEF cells through clathrin-mediated endocytosis followed by a pH-dependent step, which is similar to the mechanism of entry of human adenovirus types 2 and 5. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    PubMed

    Kristiansen, Troels Zakarias; Nielsen, Mogens Møller; Blagoev, Blagoy; Pandey, Akhilesh; Mann, Matthias

    2004-08-31

    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 as a KIAA clone (KIAA 0229) from the Kazusa DNA Research Institute which maintains the HUGE protein database--a database devoted to the analysis of long cDNA clones encoding large proteins (>50 kDa). Odin has been demonstrated to cause downregulation of c-Fos promoter activity and to inhibit PDGF-induced mitogenesis in cell lines. To further investigate the role of Odin in growth factor receptor signaling and to elucidate its biological function in vivo, we have generated mice deficient in Odin by gene targeting. Odin-deficient mice do not display any obvious phenotype, and histological examination of the kidney, lung and liver does not show any major abnormalities as compared to wild-type controls. However, mouse embryonic fibroblasts (MEFs) generated from Odin-deficient mice exhibit a hyperproliferative phenotype compared to wild-type-derived MEFs, consistent with its role as a negative regulator of growth factor receptor signaling. Our results confirm that although Odin expression in mice is not essential for any major developmental pathway, it could play a significant functional role to negatively regulate growth factor receptor signaling pathways.

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

    PubMed

    Popov, Anton L; Popova, Nelly R; Selezneva, Irina I; Akkizov, Azamat Y; Ivanov, Vladimir K

    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(-3)М-10(-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.

  17. Suppression of oxidative phosphorylation in mouse embryonic fibroblast cells deficient in apurinic/apyrimidinic endonuclease

    PubMed Central

    Suganya, Rangaswamy; Chakraborty, Anirban; Miriyala, Sumitra; Hazra, Tapas K.; Izumi, Tadahide

    2015-01-01

    The mammalian apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is an essential DNA repair/gene regulatory protein. Decrease of APE1 in cells by inducible shRNA knockdown or by conditional gene knockout caused apoptosis. Here we succeeded in establishing a unique mouse embryonic fibroblast (MEF) line expressing APE1 at a level far lower than those achieved with shRNA knockdown. The cells, named MEFla (MEFlowAPE1), were hypersensitive to methyl methanesulfonate (MMS), and showed little activity for repairing AP-sites and MMS induced DNA damage. While these results were consistent with the essential role of APE1 in repair of AP sites, the MEFla cells grew normally and the basal activation of poly(ADP-ribose) polymerases in MEFla was lower than that in the wild-type MEF (MEFwt), indicating the low DNA damage stress in MEFla under the normal growth condition. Oxidative phosphorylation activity in MEFla was lower than in MEFwt, while the glycolysis rates in MEFla were higher than in MEFwt. In addition, we observed decreased intracellular oxidative stress in MEFla. These results suggest that cells with low APE1 reversibly suppress mitochondrial respiration and thereby reduce DNA damage stress and increases the cell viability. PMID:25645679

  18. Biocompatibility of microbially reduced graphene oxide in primary mouse embryonic fibroblast cells.

    PubMed

    Gurunathan, Sangiliyandi; Han, Jae Woong; Eppakayala, Vasuki; Kim, Jin-Hoi

    2013-05-01

    Graphene nanosheet is a one-atom thick planar sheet of sp(2)-bonded carbon atoms, which are densely packed in a honeycomb crystal lattice, attracting tremendous attention from both fundamental research and industrial applications. The synthesis of graphene from graphene oxide (GO) using a biological method is one of the important topics in the areas of nanotechnology, because graphene-based nanomaterials have potential applications. A green, simple and non-toxic method for preparing graphene using biomass of Pseudomonas aeruginosa as the reducing reagent is proposed. The resulting microbially reduced graphene oxide (M-rGO) was characterized using a range of analytical techniques. UV-visible spectroscopy confirms the transition of graphene oxide to graphene. Fourier transform infrared spectroscopy (FT-IR) was used to study the changes in surface functionalities. X-ray diffraction (XRD) and high resolution scanning electron microscopy (SEM) were used to investigate the crystalline nature and the morphologies of prepared graphene respectively. Furthermore, the biocompatibility of the M-rGO was investigated using primary mouse embryonic fibroblast (PMEF) cells. The present study suggests that the M-rGO has significant biocompatibility for PMEF cells, even at a high concentration of 100 μg ml(-1). Therefore, the proposed safe and green method confers the M-rGO with a great potential for various biomedical applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Similarities in Gene Expression Profiles during In Vitro Aging of Primary Human Embryonic Lung and Foreskin Fibroblasts.

    PubMed

    Marthandan, Shiva; Priebe, Steffen; Baumgart, Mario; Groth, Marco; Cellerino, Alessandro; Guthke, Reinhard; Hemmerich, Peter; Diekmann, Stephan

    2015-01-01

    Replicative senescence is of fundamental importance for the process of cellular aging, since it is a property of most of our somatic cells. Here, we elucidated this process by comparing gene expression changes, measured by RNA-seq, in fibroblasts originating from two different tissues, embryonic lung (MRC-5) and foreskin (HFF), at five different time points during their transition into senescence. Although the expression patterns of both fibroblast cell lines can be clearly distinguished, the similar differential expression of an ensemble of genes was found to correlate well with their transition into senescence, with only a minority of genes being cell line specific. Clustering-based approaches further revealed common signatures between the cell lines. Investigation of the mRNA expression levels at various time points during the lifespan of either of the fibroblasts resulted in a number of monotonically up- and downregulated genes which clearly showed a novel strong link to aging and senescence related processes which might be functional. In terms of expression profiles of differentially expressed genes with age, common genes identified here have the potential to rule the transition into senescence of embryonic lung and foreskin fibroblasts irrespective of their different cellular origin.

  20. Similarities in Gene Expression Profiles during In Vitro Aging of Primary Human Embryonic Lung and Foreskin Fibroblasts

    PubMed Central

    Marthandan, Shiva; Priebe, Steffen; Groth, Marco; Cellerino, Alessandro; Guthke, Reinhard; Hemmerich, Peter; Diekmann, Stephan

    2015-01-01

    Replicative senescence is of fundamental importance for the process of cellular aging, since it is a property of most of our somatic cells. Here, we elucidated this process by comparing gene expression changes, measured by RNA-seq, in fibroblasts originating from two different tissues, embryonic lung (MRC-5) and foreskin (HFF), at five different time points during their transition into senescence. Although the expression patterns of both fibroblast cell lines can be clearly distinguished, the similar differential expression of an ensemble of genes was found to correlate well with their transition into senescence, with only a minority of genes being cell line specific. Clustering-based approaches further revealed common signatures between the cell lines. Investigation of the mRNA expression levels at various time points during the lifespan of either of the fibroblasts resulted in a number of monotonically up- and downregulated genes which clearly showed a novel strong link to aging and senescence related processes which might be functional. In terms of expression profiles of differentially expressed genes with age, common genes identified here have the potential to rule the transition into senescence of embryonic lung and foreskin fibroblasts irrespective of their different cellular origin. PMID:26339636

  1. Nucleotide excision repair is not induced in human embryonic lung fibroblasts treated with environmental pollutants.

    PubMed

    Rossner, Pavel; Mrhalkova, Andrea; Uhlirova, Katerina; Spatova, Milada; Rossnerova, Andrea; Libalova, Helena; Schmuczerova, Jana; Milcova, Alena; Topinka, Jan; Sram, Radim J

    2013-01-01

    The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (-S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (-S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced.

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

    PubMed Central

    Rossner, Pavel; Spatova, Milada; Rossnerova, Andrea; Libalova, Helena; Schmuczerova, Jana; Milcova, Alena; Topinka, Jan; Sram, Radim J.

    2013-01-01

    The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (–S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (–S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced. PMID:23894430

  3. Ascorbic acid extends replicative life span of human embryonic fibroblast by reducing DNA and mitochondrial damages.

    PubMed

    Hwang, Won-Sang; Park, Seong-Hoon; Kim, Hyun-Seok; Kang, Hong-Jun; Kim, Min-Ju; Oh, Soo-Jin; Park, Jae-Bong; Kim, Jaebong; Kim, Sung Chan; Lee, Jae-Yong

    2007-01-01

    Ascorbic acid has been reported to extend replicative life span of human embryonic fibroblast (HEF). Since the detailed molecular mechanism of this phenomenon has not been investigated, we attempted to elucidate. Continuous treatment of HEF cells with ascorbic acid (at 200 microM) from 40 population doubling (PD) increased maximum PD numbers by 18% and lowered SA-beta-gal positive staining, an aging marker, by 2.3 folds, indicating that ascorbic acid extends replicative life span of HEF cells. Ascorbic acid treatment lowered DCFH by about 7 folds and Rho123 by about 70%, suggesting that ascorbic acid dramatically decreased ROS formation. Ascorbic acid also increased aconitase activity, a marker of mitochondrial aging, by 41%, indicating that ascorbic acid treatment restores age-related decline of mitochondrial function. Cell cycle analysis by flow cytometry revealed that ascorbic acid treatment decreased G1 population up to 12%. Further western blot analysis showed that ascorbic acid treatment decreased levels of p53, phospho-p53 at ser 15, and p21, indicating that ascorbic acid relieved senescence-related G1 arrest. Analysis of AP (apurinic/apyrimidinic) sites showed that ascorbic acid treatment decreased AP site formation by 35%. We also tested the effect of hydrogen peroxide treatment, as an additional oxidative stress. Continuous treatment of 20 microM of hydrogen peroxide from PD 40 of HEF cells resulted in premature senescence due to increased ROS level, and increased AP sites. Taken together, the results suggest that ascorbic acid extends replicative life span of HEF cells by reducing mitochondrial and DNA damages through lowering cellular ROS.

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

    PubMed Central

    2013-01-01

    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. PMID:24059222

  5. Bioenergetic and autophagic control by Sirt3 in response to nutrient deprivation in mouse embryonic fibroblasts.

    PubMed

    Liang, Qiuli; Benavides, Gloria A; Vassilopoulos, Athanassios; Gius, David; Darley-Usmar, Victor; Zhang, Jianhua

    2013-09-01

    Sirt3 (sirtuin 3) is an NAD-dependent deacetylase localized to mitochondria. Sirt3 expression is increased in mouse muscle and liver by starvation, which could protect against the starvation-dependent increase in oxidative stress and protein damage. Damaged proteins and organelles depend on autophagy for removal and this is critical for cell survival, but the role of Sirt3 is unclear. To examine this, we used Sirt3-KO (knockout) mouse embryonic fibroblast cells, and found that, under basal conditions, Sirt3-KO cells exhibited increased autophagy flux compared with WT (wild-type) cells. In response to nutrient deprivation, both WT and KO cells exhibited increased basal and ATP-linked mitochondrial respiration, indicating an increased energy demand. Both cells exhibited lower levels of phosphorylated mTOR (mammalian target of rapamycin) and higher autophagy flux, with KO cells exhibiting lower maximal mitochondrial respiration and reserve capacity, and higher levels of autophagy than WT cells. KO cells exhibit higher phospho-JNK (c-Jun N-terminal kinase) and phospho-c-Jun than WT cells under starvation conditions. However, inhibition of JNK activity in Sirt3-KO cells did not affect LC3-I (light chain 3-I) and LC3-II levels, indicating that Sirt3-regulated autophagy is independent of the JNK pathway. Caspase 3 activation and cell death are significantly higher in Sirt3-KO cells compared with WT cells in response to nutrient deprivation. Inhibition of autophagy by chloroquine exacerbated cell death in both WT and Sirt3-KO cells, and by 3-methyadenine exacerbated cell death in Sirt3-KO cells. These data suggest that nutrient deprivation-induced autophagy plays a protective role in cell survival, and Sirt3 decreases the requirement for enhanced autophagy and improves cellular bioenergetics.

  6. Hernia fibroblasts lack β-estradiol induced alterations of collagen gene expression

    PubMed Central

    2006-01-01

    Background Estrogens are reported to increase type I and type III collagen deposition and to regulate Metalloproteinase 2 (MMP-2) expression. These proteins are reported to be dysregulated in incisional hernia formation resulting in a significantly decreased type I to III ratio. We aimed to evaluate the β-estradiol mediated regulation of type I and type III collagen genes as well as MMP-2 gene expression in fibroblasts derived from patients with or without history of recurrent incisional hernia disease. We compared primary fibroblast cultures from male/female subjects without/without incisional hernia disease. Results Incisional hernia fibroblasts (IHFs) revealed a decreased type I/III collagen mRNA ratio. Whereas fibroblasts from healthy female donors responded to β-estradiol, type I and type III gene transcription is not affected in fibroblasts from males or affected females. Furthermore β-estradiol had no influence on the impaired type I to III collagen ratio in fibroblasts from recurrent hernia patients. Conclusion Our results suggest that β-estradiol does not restore the imbaired balance of type I/III collagen in incisional hernia fibroblasts. Furthermore, the individual was identified as an independent factor for the β-estradiol induced alterations of collagen gene expression. The observation of gender specific β-estradiol-dependent changes of collagen gene expression in vitro is of significance for future studies of cellular response. PMID:17010202

  7. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells.

    PubMed

    Hovatta, Outi; Mikkola, Milla; Gertow, Karin; Strömberg, Anne-Marie; Inzunza, José; Hreinsson, Julius; Rozell, Björn; Blennow, Elisabeth; Andäng, Michael; Ahrlund-Richter, Lars

    2003-07-01

    Human embryonic stem (hES) cell lines were first cultured using fetal mouse fibroblasts as feeder cells. To avoid feeders and to reduce the amount of xeno-components, Matrigel- and laminin-coated dishes, and conditioned mouse feeder cell medium have been used, and hES cells have also been cultured on human fetal muscle and skin, and adult Fallopian tube epithelial cells. We used post-natal, commercially available human foreskin fibroblasts as feeder cells. Inner cell masses (ICM) were isolated from five supernumerary blastocysts, obtained as donations from couples undergoing IVF treatment. Two ICM showed continuous growth. One line, HS181, has been in culture for 41 weeks with a doubling time of 24-36 h. It continues to express stem cell markers alkaline phosphatase, Oct-4, stage-specific embryonic antigen (SSEA)-4 and tumour-related antigen (TRA)-1-60. The karyotype is 46,XX. Pluripotency was demonstrated by teratoma formation in immunodeficient mice. In high-density cultures, spontaneous differentiation to beating cells and neuron-like cells was seen. The second line, HS207, was cultured for 9 weeks and cryopreserved, as were samples of line HS181. Both lines began to grow after thawing. We used successfully human foreskin fibroblasts as feeder cells for derivation and continued undifferentiated growth of hES cells. These feeder cells are convenient for IVF units, because no fetal human tissues or tissue from operations are needed.

  8. Hmga1 null mouse embryonic fibroblasts display downregulation of spindle assembly checkpoint gene expression associated to nuclear and karyotypic abnormalities

    PubMed Central

    Pierantoni, Giovanna Maria; Conte, Andrea; Rinaldo, Cinzia; Tornincasa, Mara; Gerlini, Raffaele; Valente, Davide; Izzo, Antonella; Fusco, Alfredo

    2016-01-01

    ABSTRACT The High Mobility Group A1 proteins (HMGA1) are nonhistone chromatinic proteins with a critical role in development and cancer. We have recently reported that HMGA1 proteins are able to increase the expression of spindle assembly checkpoint (SAC) genes, thus impairing SAC function and causing chromosomal instability in cancer cells. Moreover, we found a significant correlation between HMGA1 and SAC genes expression in human colon carcinomas. Here, we report that mouse embryonic fibroblasts null for the Hmga1 gene show downregulation of Bub1, Bub1b, Mad2l1 and Ttk SAC genes, and present several features of chromosomal instability, such as nuclear abnormalities, binucleation, micronuclei and karyotypic alterations. Interestingky, also MEFs carrying only one impaired Hmga1 allele present karyotypic alterations. These results indicate that HMGA1 proteins regulate SAC genes expression and, thereby, genomic stability also in embryonic cells. PMID:26889953

  9. Lack of complementation in somatic cell hybrids between fibroblasts from patients with different forms of cystinosis

    SciTech Connect

    Pellett, O.L.; Smith, M.L.; Greene, A.A.; Schneider, J.A. )

    1988-05-01

    Cystinosis is an autosomal recessive disease in which three clinical forms are recognized: infantile nephropathic, with renal tubular damage by 1 year of age and progressive glomerular insufficiency; intermediate, with tubular and glomerular insufficiency beginning at a later age; benign, with no kidney damage. Skin fibroblasts cultured from patients with all types of cystinosis show increased intralysosomal free (nonprotein) cystine; however, fibroblasts from heterozygotes have normal free-cystine values. To determine whether genetic complementation occurs between the different forms, somatic cell hybrids were constructed between cells from a patient with infantile nephropathic cystinosis and cells from patients with other types of cystinosis. If complementation occurred, the hybrids would be expected to have normal cystine levels. To construct hybrid cells, a universal parent cell type (TG1-neo), which was hypoxanthine/aminopterin/thymidine (HAT) sensitive and G418 resistant was constructed from an infantile nephropathic cystinosis fibroblast strain. Polyethylene glycol fusion of TG1-neo with other cells that are not HAT sensitive or G418 resistant allowed for selection of hybrid cells in a medium containing HAT and the aminoglycoside G418. As indicated by elevated cystine levels, complementation did not occur between TG1-neo and two different benign cystinosis strains, an intermediate cystinosis strain, or another nephropathic cystinosis cell strain. When a normal fibroblast strain was fused with TG1-neo, all 15 hybrid clones studied contained normal amounts of intracellular free cystine.

  10. Yolk sac angiogenic defect and intra-embryonic apoptosis in mice lacking the Ets-related factor TEL.

    PubMed Central

    Wang, L C; Kuo, F; Fujiwara, Y; Gilliland, D G; Golub, T R; Orkin, S H

    1997-01-01

    The TEL gene, which is frequently rearranged in human leukemias of both myeloid and lymphoid origin, encodes a member of the Ets family of transcription factors. The TEL gene is widely expressed throughout embryonic development and in the adult. To determine the requirement for the TEL gene product in development we generated TEL knockout mice (TEL-/-) by gene targeting in embryonic stem cells. TEL-/- mice are embryonic lethal and die between E10.5-11.5 with defective yolk sac angiogenesis and intra-embryonic apoptosis of mesenchymal and neural cells. Two-thirds of TEL-deficient yolk sacs at E9.5 lack vitelline vessels, yet possess capillaries, indicative of normal vasculogenesis. Vitelline vessels regress by E10.5 in the remaining TEL-/- yolk sacs. Hematopoiesis at the yolk sac stage, however, appears unaffected in TEL-/- embryos. Our findings demonstrate that TEL is required for maintenance of the developing vascular network in the yolk sac and for survival of selected cell types within the embryo proper. PMID:9250681

  11. Generation of mouse chimeras with high contribution of tetraploid embryonic stem cells and embryonic stem cell-fibroblast hybrid cells.

    PubMed

    Matveeva, Natalia M; Kizilova, Elena A; Serov, Oleg L

    2015-01-01

    The in vitro long-term cultivation of embryonic stem (ES) cells derived from pre-implantation embryos offers the unique possibility of combining ES cells with pre-implantation embryos to generate chimeras, thus facilitating the creation of a bridge between in vitro and in vivo investigations. Genomic manipulation using ES cells and homologous recombination is one of the most outstanding scientific achievements, resulting in the generation of animals with desirable genome modifications. As such, the generation of ES cells with different ploidy via cell fusion also deserves much attention because this approach allows for the production of chimeras that contain somatic cells with various ploidy. Therefore, this is a powerful tool that can be used to study the role of polyploidy in the normal development of mammals.

  12. TGFbeta and TGFalpha, antagonistic effect in vitro on extracellular matrix accumulation by chick skin fibroblasts at two distinct embryonic stages.

    PubMed

    Locci, P; Baroni, T; Lilli, C; Martinese, D; Marinucci, L; Bellocchio, S; Calvitti, M; Becchetti, E

    1999-03-01

    ECM macromolecules create a specific environment that participates in the control of cell proliferation and differentiation during embryogenesis. Quantitative and qualitative alterations in the ECM may depend on several growth factors that modify cell metabolism. Since transforming growth factor beta (TGFbeta) and alpha (TGFalpha) are abundantly expressed during embryonic development in organs in which epithelial-mesenchymal interactions occur, the aim of this study was to determine: a) the effect of TGFbeta on the phenotype of 7 and 14 day chick embryo back skin (CEBS) fibroblasts by evaluating the neosynthesis of GAG, collagen and fibronectin; b) whether TGFalpha and TGFbeta production, in particular TGFbeta3 and TGFbeta4, and the number of TGFbeta receptors change during these two stages of embryonic development. The results show that the neosynthesis of ECM macromolecules, tested using radiolabelled precursors, is increased by TGFbeta. The growth factor generally favours cellular accumulation more than secretion. As far as GAG is concerned, TGFbeta has a greater stimulatory effect on sulphated GAG than on HA. Specific bioassay shows that TGFbeta3 and TGFbeta4 activity is higher in 7 day than 14 day CEBS fibroblasts. Moreover, TGFbeta3 and TGFbeta4 mRNA expression is increased in the first stages of development. Instead, the level of TGFalpha increases in successive developmental stages. Since TGFalpha stimulates the synthesis and secretion of HA, and HA binds and inactivates TGFbeta, the greater quantity of HA in 14 day fibroblasts may contribute to reducing the TGFbeta effect. Overall our data suggest that the production of TGFbeta and TGFalpha are age-dependent and that the balance between the two growth factors may be a mechanism for controlling skin differentiation.

  13. Embryonic stem cell/fibroblast hybrid cells with near-tetraploid karyotype provide high yield of chimeras.

    PubMed

    Kruglova, A A; Kizilova, E A; Zhelezova, A I; Gridina, M M; Golubitsa, A N; Serov, O L

    2008-12-01

    Ten primary clones of hybrid cells were produced by the fusion of diploid embryonic stem (ES) cells, viz., line E14Tg2aSc4TP6.3 marked by green fluorescent protein (GFP), with diploid embryonic or adult fibroblasts derived from DD/c mice. All the hybrid clones had many characteristics similar to those of ES cells and were positive for GFP. Five hybrid clones having ploidy close to tetraploidy (over 80% of cells had 76-80 chromosomes) were chosen for the generation of chimeras via injection into C57BL blastocysts. These hybrid clones also contained microsatellites marking all ES cell and fibroblast chromosomes judging from microsatellite analysis. Twenty chimeric embryos at 11-13 days post-conception were obtained after injection of hybrid cells derived from two of three clones. Many embryos showed a high content of GFP-positive descendents of the tested hybrid cells. Twenty one adult chimeras were generated by the injection of hybrid cells derived from three clones. The contribution of GFP-labeled hybrid cells was significant and comparable with that of diploid E14Tg2aSc4TP6.3 cells. Cytogenetic and microsatellite analyses of cell cultures derived from chimeric embryos or adults indicated that the initial karyotype of the tested hybrid cells remained stable during the development of the chimeras, i.e., the hybrid cells were mainly responsible for the generation of the chimeras. Thus, ES cell/fibroblast hybrid cells with near-tetraploid karyotype are able to generate chimeras at a high rate, and many adult chimeras contain a high percentage of descendants of the hybrid cells.

  14. Brief exposure to small molecules allows induction of mouse embryonic fibroblasts into neural crest-like precursors.

    PubMed

    Takayama, Yuzo; Wakabayashi, Tamami; Kushige, Hiroko; Saito, Yutaka; Shibuya, Yoichiro; Shibata, Shinsuke; Akamatsu, Wado; Okano, Hideyuki; Kida, Yasuyuki S

    2017-02-01

    In this study, we propose a novel method for inducing neuronal cells by briefly exposing them to small-molecule cocktails in a step-by-step manner. Global gene expression analysis with immunohistochemical staining and calcium flux assays reveal the generation of neurons from mouse embryonic fibroblasts. In addition, time-lapse imaging of neural precursor-specific enhancer expression and global gene expression analyses show that the neurons are generated by passing through a neural crest-like precursor stage. Consistent with these results, the neural crest-like cells are able to differentiate into neural crest lineage cells, such as sympathetic neurons, adipocytes, osteocytes, and smooth muscle cells. Therefore, these results indicate that brief exposure to chemical compounds could expand and induce a substantial multipotent cell population without viral transduction. © 2017 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

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

    SciTech Connect

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

    1986-06-20

    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.

  16. [Study on characteristic spectrum of ingredients from different species Cordyceps and its anti-fibrotic activity on human embryonic fibroblasts].

    PubMed

    Si, Nan; Peng, Bo; Zhao, Hai-Yu; Wang, Hong-Jie; Yang, Jian; Li, Jian-Rong; Sun, Wei; Bian, Bao-Lin

    2016-07-01

    In this study, 10 samples of parasites, cursive, and the whole from six different species of Cordyceps were determined and compared by HPLC and LC-MS methods. Uridine, adenosine, and cordycepin were selected as the main evaluation index. The anti-fibrotic activity of different species Cordyceps extracts was observed using in vitro TGF-β1-induced ECM accumulation in human embryonic fibroblasts CCC-ESF-1. The results demonstrated that the number of atoms and hyphae ingredients of different species showed little difference, however, the content distribution of each component has obvious significance. The in vitro anti-fibrotic activities of different species were as follow: Cordyceps flower > Cicada Cordyceps (Cicada flower)> Silkworm Cordyceps> Tussah Cordyceps>natural Cordyceps. Our preliminary data could serve as reference for the discovery of artificial alternatives of natural Cordyceps. Copyright© by the Chinese Pharmaceutical Association.

  17. N-Acetyl-L-cysteine enhances apoptosis through inhibition of nuclear factor-kappaB in hypoxic murine embryonic fibroblasts.

    PubMed

    Qanungo, Suparna; Wang, Mi; Nieminen, Anna-Liisa

    2004-11-26

    In this study, we investigated the role of reduced glutathione (GSH) and nuclear factor-kappaB (NFkappaB) in hypoxia-induced apoptosis. Hypoxia caused p53-dependent apoptosis in murine embryonic fibroblasts transfected with Ras and E1A. N-Acetyl-l-cysteine (NAC) but not other antioxidants, such as the vitamin E analog trolox and epigallocatechin-3-gallate, enhanced hypoxia-induced caspase-3 activation and apoptosis. NAC also enhanced hypoxia-induced apoptosis in two human cancer cell lines, MIA PaCa-2 pancreatic cancer cells and A549 lung carcinoma cells. In murine embryonic fibroblasts, all three antioxidants blocked hypoxia-induced reactive oxygen species formation. NAC did not enhance hypoxia-induced cytochrome c release but did enhance poly-(ADP ribose) polymerase cleavage, indicating that NAC acted at a post-mitochondrial level. NAC-mediated enhancement of apoptosis was mimicked by incubating cells with GSH monoester, which increased intracellular GSH similarly to NAC. Hypoxia promoted degradation of an inhibitor of kappaB(IkappaBalpha), NFkappaB-p65 translocation into the nucleus, NFkappaB binding to DNA, and subsequent transactivation of NFkappaB, which increased X chromosome-linked inhibitor of apoptosis protein levels. NAC failed to block degradation by IkappaBalpha and sequestration of the p65 subunit of NFkappaB to the nucleus. However, NAC did abrogate hypoxia-induced NFkappaB binding to DNA, NFkappaB-dependent gene expression, and induction of X chromosome-linked inhibitor of apoptosis protein. In conclusion, NAC enhanced hypoxic apoptosis by a mechanism apparently involving GSH-dependent suppression of NFkappaB transactivation.

  18. The role of hypoxia inducible factor 1alpha in cobalt chloride induced cell death in mouse embryonic fibroblasts.

    PubMed

    Vengellur, A; LaPres, J J

    2004-12-01

    Cobalt has been widely used in the treatment of anemia and as a hypoxia mimic in cell culture and it is known to activate hypoxic signaling by stabilizing the hypoxia inducible transcription factor 1alpha (HIF1alpha). However, cobalt exposure can lead to tissue and cellular toxicity. These studies were conducted to determine the role of HIF1alpha in mediating cobalt-induced toxicity. Mouse embryonic fibroblasts (MEFs) that were null for the HIF1alpha protein were used to show that HIF1alpha protein plays a major role in mediating cobalt-induced cytotoxicity. Previous work from our lab and others has shown that two BH3 domain containing cell death genes, BNip3 and NIX, are targets of hypoxia signaling. These experiments document that BNip3 and NIX expression is HIF1alpha-dependent, and cobalt induces their expression in a time and dose dependent manner. In addition, their expression is correlated with an increase in BNIP3 and NIX protein. Characteristically, the elevated level of BNIP3 was correlated with an increased presence of chromatin condensation, one marker for cell injury. Interestingly, this increased chromosomal condensation was not coupled to caspase-3 activation as usually seen in a typical apoptotic response. These results show that HIF1alpha is playing a major role in mediating cobalt-induced toxicity in mouse embryonic fibroblasts and may offer a possible mechanism for the underlying pathology of injuries seen in workers exposed to environmental contaminants that can influence the hypoxia signaling system, such as cobalt.

  19. Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells.

    PubMed

    Inzunza, José; Gertow, Karin; Strömberg, Marie A; Matilainen, Eija; Blennow, Elisabeth; Skottman, Heli; Wolbank, Susanne; Ahrlund-Richter, Lars; Hovatta, Outi

    2005-04-01

    Derivation and culture of human embryonic stem cells (hESCs) without animal-derived material would be optimal for cell transplantation. We derived two new hES (HS293 and HS306) and 10 early cell lines using serum replacement (SR) medium instead of conventional fetal calf serum and human foreskin fibroblasts as feeder cells. Line HS293 has been in continuous culture, with a passage time of 5-8 days, since October 2003 and is at passage level 56. Line HS306 has been cultured since February 2004, now at passage 41. The lines express markers of pluripotent hESCs (Oct-4, SSEA-4, TRA-1-60, TRA-1-81, GCTM-2, and alkaline phosphatase). The pluripotency has been shown in embryoid bodies in vitro, and the pluripotency of line 293 has also been shown in vivo by teratoma formation in severe combined immunodeficiency/beige mice. The karyotype of HS293 is 46,XY, and that of HS306 is 46,XX. Ten more early lines have been derived under similar conditions since September 2004. We conclude that hESC lines can be successfully derived using SR medium and postnatal human fibroblasts as feeder cells. This is a step toward xeno-free conditions and facilitates the use of these cells in transplantation.

  20. Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment

    PubMed Central

    Sekinaka, Tamotsu; Hayashi, Yohei; Noce, Toshiaki; Niwa, Hitoshi; Matsui, Yasuhisa

    2016-01-01

    Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs. PMID:27608931

  1. Docking protein SNT1 is a critical mediator of fibroblast growth factor signaling during Xenopus embryonic development.

    PubMed

    Akagi, Keiko; Kyun Park, Eui; Mood, Kathleen; Daar, Ira O

    2002-03-01

    The docking protein SNT1/FRS2 (fibroblast growth factor receptor substrate 2) is implicated in the transmission of extracellular signals from several growth factor receptors to the mitogen-activated protein (MAP) kinase signaling cascade, but its biological function during development is not well characterized. Here, we show that the Xenopus homolog of mammalian SNT1/FRS-2 (XSNT1) plays a critical role in the appropriate formation of mesoderm-derived tissue during embryogenesis. XSNT1 has an expression pattern that is quite similar to the fibroblast growth factor receptor-1 (FGFR1) during Xenopus development. Ectopic expression of XSNT1 markedly enhanced the embryonic defects induced by an activated FGF receptor, and increased the MAP kinase activity as well as the expression of a mesodermal marker in response to FGF receptor signaling. A loss-of-function study using antisense XSNT1 morpholino oligonucleotides (XSNT-AS) shows severe malformation of trunk and posterior structures. Moreover, XSNT-AS disrupts muscle and notochord formation, and inhibits FGFR-induced MAP kinase activation. In ectodermal explants, XSNT-AS blocks FGFR-mediated induction of mesoderm and the accompanying elongation movements. Our results indicate that XSNT1 is a critical mediator of FGF signaling and is required for early Xenopus development.

  2. Molecular evidence on the protective effect of ellagic acid on phosalone-induced senescence in rat embryonic fibroblast cells.

    PubMed

    Baeeri, Maryam; Momtaz, Saeideh; Navaei-Nigjeh, Mona; Niaz, Kamal; Rahimifard, Mahban; Ghasemi-Niri, Seyedeh Farnaz; Sanadgol, Nima; Hodjat, Mahshid; Sharifzadeh, Mohammad; Abdollahi, Mohammad

    2017-02-01

    Salient evidence testifies the link between organophosphorus (OPs) exposure and the formation of free radical oxidants; and it is well accepted that free radicals are one of the basic concerns of senescence. To show the oxidative features of phosalone (PLN) as a key member of OPs, to induce senescence in rat embryonic fibroblast (REF) cells and to demonstrate the beneficial effects of the known antioxidant ellagic acid (EA) in diminishing the PLN-induced toxic effects, the levels of cell viability, oxidative stress markers, inflammatory cytokines, telomerase activity, and the expression of the genes related to senescence were investigated. Our results lend support to the hypothesis that PLN enhances the entire premature senescence parameters of REF cells. This accounts for the mechanistic approval of the role of OPs in induction of senescence in rat fibroblasts. Moreover, incorporation of EA diminished PLN toxicity mainly through suppression of p38 and p53 at gene and protein levels, and tempered the inflammation factors (TNF-α, IL-1β, IL-6 and NF-κB), which further affected cell division. Analysis of cell cycle showed that the percentage of G0/G1 arrest, in REF cells treated by EA was elevated as compared to control and PLN treated cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Pleiotropic functions of fibroblast growth factor signaling in embryonic mammary gland development.

    PubMed

    Kim, Eun-Jung; Jung, Han-Sung; Lu, Pengfei

    2013-06-01

    The mammary gland is an ectodermal appendage and a defining feature of mammals. Consistent with it being a recent evolutionary novelty, many of the molecules essential for the ontogeny and morphogenesis of various vertebrate organs, including those in the fibroblast growth factor (FGF) signaling pathway, are co-opted for induction, maintenance and morphogenesis of the mammary glands. Understanding the mechanism whereby FGF signaling regulates the fundamental cell behavior during normal mammary gland develop may facilitate determination of the consequences of its deregulation during breast cancer progression.

  4. AICAR induces Bax/Bak-dependent apoptosis through upregulation of the BH3-only proteins Bim and Noxa in mouse embryonic fibroblasts.

    PubMed

    González-Gironès, Diana M; Moncunill-Massaguer, Cristina; Iglesias-Serret, Daniel; Cosialls, Ana M; Pérez-Perarnau, Alba; Palmeri, Claudia M; Rubio-Patiño, Camila; Villunger, Andreas; Pons, Gabriel; Gil, Joan

    2013-08-01

    5-Aminoimidazole-4-carboxamide (AICA) riboside (AICAR) is a nucleoside analogue that is phosphorylated to 5-amino-4-imidazolecarboxamide ribotide (ZMP), which acts as an AMP mimetic and activates AMP-activated protein kinase (AMPK). It has been recently described that AICAR triggers apoptosis in chronic lymphocytic leukemia (CLL) cells, and its mechanism of action is independent of AMPK as well as p53. AICAR-mediated upregulation of the BH3-only proteins BIM and NOXA correlates with apoptosis induction in CLL cells. Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the mechanism of AICAR-induced apoptosis. ZMP formation was required for AICAR-induced apoptosis, though direct Ampk activation with A-769662 failed to induce apoptosis in MEFs. AICAR potently induced apoptosis in Ampkα1 (-/-) /α2 (-/-) MEFs, demonstrating an Ampk-independent mechanism of cell death activation. In addition, AICAR acts independently of p53, as MEFs lacking p53 also underwent apoptosis normally. Notably, MEFs lacking Bax and Bak were completely resistant to AICAR-induced apoptosis, confirming the involvement of the mitochondrial pathway in its mechanism of action. Apoptosis was preceded by ZMP-dependent but Ampk-independent modulation of the mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2. Bim protein levels were accumulated upon AICAR treatment of MEFs, suggesting its role in the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa displayed high resistance to AICAR. These findings support the notion that MEFs are a useful system to further dissect the mechanism of AICAR-induced apoptosis.

  5. The effect of aloe vera on the expression of wound healing factors (TGFβ1 and bFGF) in mouse embryonic fibroblast cell: In vitro study.

    PubMed

    Hormozi, Maryam; Assaei, Raheleh; Boroujeni, Mandana Beigi

    2017-04-01

    Aloe vera (A.v) have been used traditionally for topical treatment of wounds and burns in different countries for centuries, but the mechanism of this effect is not well understood. Various growth factors are implicated in the process of wound healing. Among the different growth factors involved in the process, TGFβ1 and bFGF are the most importantly expressed in fibroblast cells. The aim of this study was to evaluate the effect of A.v on the expression of angiogenesis growth factors in mouse embryonic fibroblast cells. We exposed mouse embryonic fibroblast cells to different concentrations of A.v (50, 100 and 150μg/ml) at two different time of 12 and 24h. Fibroblast cell without A.v treatment serves as the control. The expression of TGFβ1and bFGF was measured by real time-polymerase chain reaction (real-time-PCR) and enzyme-linked immunosorbent assay (ELISA) at the level of gene and protein. We observed that A.v gel at first up-regulated the expression of TGFβ1 and bFGF, but, these genes were later repressed after a particular time. Our results demonstrated that A.v was dose-dependent and time-dependent on the expression of bFGF and TGFβ1 in fibroblast cell in vitro. This mechanism can be employed in the prospective treatment of physical lesion. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

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

    PubMed

    Kuo, Shiu-Ming; Burl, Lana R; Hu, Zihua

    2012-01-01

    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 discussed.

  7. Expression profiling and pathway analysis of Krüppel-like factor 4 in mouse embryonic fibroblasts

    PubMed Central

    Hagos, Engda G; Ghaleb, Amr M; Kumar, Amrita; Neish, Andrew S; Yang, Vincent W

    2011-01-01

    Background: Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor with diverse regulatory functions in proliferation, differentiation, and development. KLF4 also plays a role in inflammation, tumorigenesis, and reprogramming of somatic cells to induced pluripotent stem (iPS) cells. To gain insight into the mechanisms by which KLF4 regulates these processes, we conducted DNA microarray analyses to identify differentially expressed genes in mouse embryonic fibroblasts (MEFs) wild type and null for Klf4. Methods: Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. Differentially expressed genes were subjected to the Database for Annotation, Visualization and Integrated Discovery (DAVID). The microarray data were also interrogated with the Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) for pathway identification. Results obtained from the microarray analysis were confirmed by Western blotting for select genes with biological relevance to determine the correlation between mRNA and protein levels. Results: One hundred and sixty three up-regulated and 88 down-regulated genes were identified that demonstrated a fold-change of at least 1.5 and a P-value < 0.05 in Klf4-null MEFs compared to wild type MEFs. Many of the up-regulated genes in Klf4-null MEFs encode proto-oncogenes, growth factors, extracellular matrix, and cell cycle activators. In contrast, genes encoding tumor suppressors and those involved in JAK-STAT signaling pathways are down-regulated in Klf4-null MEFs. IPA and GSEA also identified various pathways that are regulated by KLF4. Lastly, Western blotting of select target genes confirmed the changes revealed by microarray data. Conclusions: These data are not only consistent with previous functional studies of KLF4's role in tumor suppression and somatic cell reprogramming, but also revealed novel target genes that mediate KLF4's

  8. Characterization of Endogenous Avian Leukosis Viruses in Chicken Embryonic Fibroblast Substrates Used in Production of Measles and Mumps Vaccines

    PubMed Central

    Johnson, Jeffrey A.; Heneine, Walid

    2001-01-01

    Previous findings of low levels of reverse transcriptase (RT) activity in chick cell-derived measles and mumps vaccines showed this activity to be associated with virus particles containing RNA of both subgroup E endogenous avian leukosis viruses (ALV-E) and endogenous avian viruses (EAV). These particles originate from chicken embryonic fibroblast (CEF) substrates used for propagating vaccine strains. To better characterize vaccine-associated ALV-E, we examined the endogenous ALV proviruses (ev loci) present in a White Leghorn CEF substrate pool by restriction fragment length polymorphism. Five ev loci were detected, ev-1, ev-3, ev-6, ev-18, andev-19. Both ev-18 and ev-19 can express infectious ALV-E, while ev-1, ev-3, and ev-6 are defective. We analyzed the full-length sequence of ev-1 and identified an adenosine insertion within the pol RT-β region at position 5026, which results in a truncated RT-β and integrase. We defined the 1,692-bp deletion in the gag-pol region of ev-3, and we found that in ev-6, sequences from the 5′ long terminal repeat to the 5′ pol region were absent. Based on the sequences of the ev loci, RT-PCR assays were developed to examine expression of ALV-E particles (EV) in CEF supernatants. Both ev-1- and ev-3-like RNA sequences were identified, as well as two other RNA sequences with intact pol regions, presumably of ev-18 and ev-19 origin. Inoculation of susceptible quail fibroblasts with CEF culture supernatants from both 5-azacytidine-induced and noninduced CEF led to ALV infection, confirming the presence of infectious ALV-E. Our data demonstrate that both defective and nondefective ev loci can be present in CEF vaccine substrates and suggest that both ev classes may contribute to the ALV present in vaccines. PMID:11264350

  9. In vitro cultivation of Herpetosoma trypanosomes on embryonic fibroblasts and in semidefined cell-free medium.

    PubMed

    Mohamed, H A; Maraghi, S; Wallbanks, K R; Molyneux, D H

    1988-06-01

    Microtus agrestis embryo fibroblasts (MAEF) support the survival and multiplication at 37 C of the mammalian multiplicative forms of the Herpetosoma trypanosomes Trypanosoma microti, T. evotomys, T. musculi, and T. lewisi passaged from cultures on Schneider's Drosophila medium and of T. grosi from Grace's medium. MAEF layers with parasites were maintained for a period of over 5 mo. A semidefined cell-free medium also supported the multiplication (at 37 C) of the mammalian forms of the same trypanosome species, passaged directly from Schneider's Drosophila medium or Grace's medium, without their prior culture on cell lines. Reproductive stages were observed in cultures; T. microti and T. evotomys produced nests of dividing amastigotes from which trypomastigotes developed in the medium supernatant. Trypanosoma lewisi, T. musculi, and T. grosi divided initially as epimastigotes, which then transformed to bloodstream trypomastigotes. Multiplication of trypomastigotes was also observed. These methods of reproduction are the same as those reported in the respective mammalian hosts.

  10. Isolation of influenza virus in human lung embryonated fibroblast cells (MRC-5) from clinical samples.

    PubMed Central

    de Oña, M; Melón, S; de la Iglesia, P; Hidalgo, F; Verdugo, A F

    1995-01-01

    Ninety-four pharyngeal swab samples corresponding to 94 patients with suspected influenza virus infection were inoculated in Madin-Darby canine kidney (MDCK) cells, the conventional cell system for the isolation of influenza virus, and in fibroblastic human embryo lung (MRC-5) cells, a cell system less commonly used for this purpose but one frequently used in clinical virology laboratories. Both cell preparations were treated with trypsin. Influenza virus was recovered from 15% of the samples inoculated in MDCK cells and from 18% of those inoculated in MRC-5 cells. The use of MRC-5 cells can simplify the search for respiratory viruses and would assist in the rapid detection of influenza virus during new epidemics. PMID:7665680

  11. A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts

    NASA Astrophysics Data System (ADS)

    Pandian, Ganesh N.; Nakano, Yusuke; Sato, Shinsuke; Morinaga, Hironobu; Bando, Toshikazu; Nagase, Hiroki; Sugiyama, Hiroshi

    2012-07-01

    Cellular reprogramming involves profound alterations in genome-wide gene expression that is precisely controlled by a hypothetical epigenetic code. Small molecules have been shown to artificially induce epigenetic modifications in a sequence independent manner. Recently, we showed that specific DNA binding hairpin pyrrole-imidazole polyamides (PIPs) could be conjugated with chromatin modifying histone deacetylase inhibitors like SAHA to epigenetically activate certain pluripotent genes in mouse fibroblasts. In our steadfast progress to improve the efficiency of SAHA-PIPs, we identified a novel compound termed, δ that could dramatically induce the endogenous expression of Oct-3/4 and Nanog. Genome-wide gene analysis suggests that in just 24 h and at nM concentration, δ induced multiple pluripotency-associated genes including Rex1 and Cdh1 by more than ten-fold. δ treated MEFs also rapidly overcame the rate-limiting step of epithelial transition in cellular reprogramming by switching ``'' the complex transcriptional gene network.

  12. Gene profiling of embryonic skeletal muscle lacking type I ryanodine receptor Ca(2+) release channel.

    PubMed

    Filipova, Dilyana; Walter, Anna M; Gaspar, John A; Brunn, Anna; Linde, Nina F; Ardestani, Mostafa A; Deckert, Martina; Hescheler, Jürgen; Pfitzer, Gabriele; Sachinidis, Agapios; Papadopoulos, Symeon

    2016-02-01

    In mature skeletal muscle, the intracellular Ca(2+) concentration rises dramatically upon membrane depolarization, constituting the link between excitation and contraction. This process requires Ca(2+) release from the sarcoplasmic reticulum via the type 1 ryanodine receptor (RYR1). However, RYR1's potential roles in muscle development remain obscure. We used an established RyR1- null mouse model, dyspedic, to investigate the effects of the absence of a functional RYR1 and, consequently, the lack of RyR1-mediated Ca(2+) signaling, during embryogenesis. Homozygous dyspedic mice die after birth and display small limbs and abnormal skeletal muscle organization. Skeletal muscles from front and hind limbs of dyspedic fetuses (day E18.5) were subjected to microarray analyses, revealing 318 differentially expressed genes. We observed altered expression of multiple transcription factors and members of key signaling pathways. Differential regulation was also observed for genes encoding contractile as well as muscle-specific structural proteins. Additional qRT-PCR analysis revealed altered mRNA levels of the canonical muscle regulatory factors Six1, Six4, Pax7, MyoD, MyoG and MRF4 in mutant muscle, which is in line with the severe developmental retardation seen in dyspedic muscle histology analyses. Taken together, these findings suggest an important non-contractile role of RyR1 or RYR1-mediated Ca(2+) signaling during muscle organ development.

  13. 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

    SciTech Connect

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

    1988-12-01

    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.

  14. Skp2 promotes adipocyte differentiation via a p27{sup Kip1}-independent mechanism in primary mouse embryonic fibroblasts

    SciTech Connect

    Okada, Mitsuru; Sakai, Tamon; Nakamura, Takehiro; Tamamori-Adachi, Mimi; Kitajima, Shigetaka; Matsuki, Yasushi; Watanabe, Eijiro; Hiramatsu, Ryuji; Sakaue, Hiroshi Kasuga, Masato

    2009-02-06

    Skp2, the substrate-binding subunit of an SCF ubiquitin ligase complex, is a key regulator of cell cycle progression that targets substrates for degradation by the 26S proteasome. We have now shown that ablation of Skp2 in primary mouse embryonic fibroblasts (MEFs) results both in impairment of adipocyte differentiation and in the accumulation of the cyclin-dependent kinase inhibitor p27{sup Kip1}, a principal target of the SCF{sup Skp2} complex. Genetic ablation of p27{sup Kip1} in MEFs promoted both lipid accumulation and adipocyte-specific gene expression. However, depletion of p27{sup Kip1} by adenovirus-mediated RNA interference failed to correct the impairment of adipocyte differentiation in Skp2{sup -/-} MEFs. In contrast, troglitazone, a high-affinity ligand for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), largely restored lipid accumulation and PPAR{gamma} gene expression in Skp2{sup -/-} MEFs. Our data suggest that Skp2 plays an essential role in adipogenesis in MEFs in a manner that is at least in part independent of regulation of p27{sup Kip1} expression.

  15. Specific fixation of bovine brain and retinal acidic and basic fibroblast growth factors to mouse embryonic eye basement membranes

    SciTech Connect

    Jeanny, J.C.; Fayein, N.; Courtois, Y. ); Moenner, M.; Chevallier, B.; Barritault, D. )

    1987-07-01

    The labeling pattern of mouse embryonic eye frozen sections incubated with radioiodinated brain acidic and basic fibroblasts growth factors (aFGF and bFGF) was investigated by autoradiography. Both growth factors bind to basement membranes in a dose-dependent way, with a higher affinity for bFGF. Similar data were obtained with eye-derived growth factors (EDGF), the retinal forms of FGF. There was a heterogeneity in the affinity of the various basement membranes toward these growth factors. The specificity of the growth factor-basement membrane interaction was demonstrated by the following experiments: (i) an excess of unlabeled growth factor displaced the labeling; (ii) unrelated proteins with different isoelectric points did not modify the labeling; and (iii) iodinated EGF or PDGF did not label basement membrane. In order to get a better understanding of the nature of this binding, the authors performed the incubation of the frozen sections with iodinated FGFs preincubated with various compounds. These results demonstrate that FGFs bind specifically to basement membranes, probably on the polysaccharidic part of the proteoheparan sulfate, and suggest that this type of interaction may be a general feature of the mechanism of action of these growth factors.

  16. Inhibitory effect of peroxiredoxin II (Prx II) on Ras-ERK-NFkappaB pathway in mouse embryonic fibroblast (MEF) senescence.

    PubMed

    Han, Ying-Hao; Kwon, Jeong-Hoon; Yu, Dae-Yeul; Moon, Eun-Yi

    2006-11-01

    Intracellular reactive oxygen species (ROS) were attenuated by the expression of peroxiredoxin II (Prx II). Cellular senescence as judged by senescence-associated (SA)-beta-galactosidase (Gal) positive cell formation was increased in Prx II-deficient mouse embryonic fibroblast (MEF). Ras expression was increased following passages. The level of Ras expression was higher in Prx II-/- MEF than wild type MEF. ERK activity was also augmented by the deletion of Prx II. SA-beta-Gal-positive cell formation was reduced by PD98059, ERK inhibitor. Activated nuclear transcription factor, nuclear factor-kappaB (NFkappaB) by the deletion of Prx II was inhibited by the treatment with PD98059. In contrast, no changes in SA-beta-Gal-positive cell formation were detected by NFkappaB inhibitor, N-alpha-tosyl-L-phenylalanyl chloromethyl ketone (TPCK). Collectively, results suggest that Prx II deletion activate Ras-ERK-NFkappaB pathways and cellular senescence in Prx II-/- MEF cells was mediated by ERK activation but not by NFkappaB activation.

  17. Export-deficient monoubiquitinated PEX5 triggers peroxisome removal in SV40 large T antigen-transformed mouse embryonic fibroblasts

    PubMed Central

    Nordgren, Marcus; Francisco, Tânia; Lismont, Celien; Hennebel, Lore; Brees, Chantal; Wang, Bo; Van Veldhoven, Paul P; Azevedo, Jorge E; Fransen, Marc

    2015-01-01

    Peroxisomes are ubiquitous cell organelles essential for human health. To maintain a healthy cellular environment, dysfunctional and superfluous peroxisomes need to be selectively removed. Although emerging evidence suggests that peroxisomes are mainly degraded by pexophagy, little is known about the triggers and molecular mechanisms underlying this process in mammalian cells. In this study, we show that PEX5 proteins fused to a bulky C-terminal tag trigger peroxisome degradation in SV40 large T antigen-transformed mouse embryonic fibroblasts. In addition, we provide evidence that this process is autophagy-dependent and requires monoubiquitination of the N-terminal cysteine residue that marks PEX5 for recycling. As our findings also demonstrate that the addition of a bulky tag to the C terminus of PEX5 does not interfere with PEX5 monoubiquitination but strongly inhibits its export from the peroxisomal membrane, we hypothesize that such a tag mimics a cargo protein that cannot be released from PEX5, thus keeping monoubiquitinated PEX5 at the membrane for a sufficiently long time to be recognized by the autophagic machinery. This in turn suggests that monoubiquitination of the N-terminal cysteine of peroxisome-associated PEX5 not only functions to recycle the peroxin back to the cytosol, but also serves as a quality control mechanism to eliminate peroxisomes with a defective protein import machinery. PMID:26086376

  18. Export-deficient monoubiquitinated PEX5 triggers peroxisome removal in SV40 large T antigen-transformed mouse embryonic fibroblasts.

    PubMed

    Nordgren, Marcus; Francisco, Tânia; Lismont, Celien; Hennebel, Lore; Brees, Chantal; Wang, Bo; Van Veldhoven, Paul P; Azevedo, Jorge E; Fransen, Marc

    2015-01-01

    Peroxisomes are ubiquitous cell organelles essential for human health. To maintain a healthy cellular environment, dysfunctional and superfluous peroxisomes need to be selectively removed. Although emerging evidence suggests that peroxisomes are mainly degraded by pexophagy, little is known about the triggers and molecular mechanisms underlying this process in mammalian cells. In this study, we show that PEX5 proteins fused to a bulky C-terminal tag trigger peroxisome degradation in SV40 large T antigen-transformed mouse embryonic fibroblasts. In addition, we provide evidence that this process is autophagy-dependent and requires monoubiquitination of the N-terminal cysteine residue that marks PEX5 for recycling. As our findings also demonstrate that the addition of a bulky tag to the C terminus of PEX5 does not interfere with PEX5 monoubiquitination but strongly inhibits its export from the peroxisomal membrane, we hypothesize that such a tag mimics a cargo protein that cannot be released from PEX5, thus keeping monoubiquitinated PEX5 at the membrane for a sufficiently long time to be recognized by the autophagic machinery. This in turn suggests that monoubiquitination of the N-terminal cysteine of peroxisome-associated PEX5 not only functions to recycle the peroxin back to the cytosol, but also serves as a quality control mechanism to eliminate peroxisomes with a defective protein import machinery.

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

    SciTech Connect

    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.

  20. Metabolism of 6-nitrobenzo(a)pyrene by hamster embryonic fibroblasts and its interaction with nuclear macromolecules

    SciTech Connect

    Tong, S.; Selkirk, J.K.

    1983-01-01

    Incubation of 6-nitrobenzo(a)pyrene (6-nitroBaP) with hamster embryonic fibroblasts led to formation of both organic solvent-soluble and water-soluble products. High-pressure liquid chromatographic analysis of organic solvent-soluble extracellular metabolites showed the predominant presence of dihydrodiols, with only small amounts of phenolic products. This differed from microsomal metabolism, using hepatic preparations from 3-methylcholanthrene-pretreated rats, where a major phenolic peak was obtained. Subsequent treatment of aqueous layer with ..beta..-glucuronidase, however, revealed that most of the phenols were associated with glucuronic acid to form water-soluble products. Interaction of 6-nitroBaP with nuclear macromolecules from HEF was also studied. The chemical interacted with both DNA and RNA, but the specific activity was highest with nuclear proteins. This binding profile was found to be similar to that when benzo(a)pyrene was used, although the affinity toward protein binding was slightly higher for 6-nitroBaP. 23 references, 6 figures, 2 tables.

  1. Low-Level Laser Therapy Activates NF-kB via Generation of Reactive Oxygen Species in Mouse Embryonic Fibroblasts

    PubMed Central

    Huang, Ying-Ying; Tomkinson, Elizabeth M.; Sharma, Sulbha K.; Kharkwal, Gitika B.; Saleem, Taimur; Mooney, David; Yull, Fiona E.; Blackwell, Timothy S.; Hamblin, Michael R.

    2011-01-01

    Background Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear. Methodology/Principal Findings 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/cm2 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. Conclusion 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. PMID:21814580

  2. Loss of STAT3 in Mouse Embryonic Fibroblasts Reveals Its Janus-Like Actions on Mitochondrial Function and Cell Viability

    PubMed Central

    Zouein, Fouad A.; Duhé, Roy J.; Arany, Istvan; Shirey, Kristin; Hosler, Jonathan P.; Liu, Huiling; Saad, Iman; Kurdi, Mazen; Booz, George W.

    2014-01-01

    STAT3 has been implicated in mitochondrial function; however, the physiological relevance of this action is not established. Here we studied the importance of STAT3 to the cellular response to stimuli, TNFα and serum deprivation, which increase mitochondrial reactive oxygen species (ROS) formation. Experiments were performed using wild type (WT) and STAT3 knockout (KO) mouse embryonic fibroblasts (MEF). Both WT and STAT3 KO MEF expressed similar levels of tumor necrosis factor receptor 1 (TNFR1) and exhibited comparable IκBα degradation with TNFα. However, in the absence of STAT3 nuclear accumulation of NFκB p65 with TNFα was attenuated and induction of the survival protein c-FLIPL was eliminated. Nonetheless, WT MEF were more sensitive to TNFα-induced death which was attributed to necrosis. Deletion of STAT3 decreased ROS formation induced by TNFα and serum deprivation. STAT3 deletion was associated with lower levels of complex I and rates of respiration. Relative to WT cells, mitochondria of STAT3 KO cells released significantly more cytochrome c in response to oxidative stress and had greater caspase 3 cleavage due to serum deprivation. Our findings are consistent with STAT3 being important for mitochondrial function and cell viability by ensuring mitochondrial integrity and the expression of pro-survival genes. PMID:24548419

  3. Loss of STAT3 in mouse embryonic fibroblasts reveals its Janus-like actions on mitochondrial function and cell viability.

    PubMed

    Zouein, Fouad A; Duhé, Roy J; Arany, Istvan; Shirey, Kristin; Hosler, Jonathan P; Liu, Huiling; Saad, Iman; Kurdi, Mazen; Booz, George W

    2014-03-01

    STAT3 has been implicated in mitochondrial function; however, the physiological relevance of this action is not established. Here we studied the importance of STAT3 to the cellular response to stimuli, TNFα and serum deprivation, which increase mitochondrial reactive oxygen species (ROS) formation. Experiments were performed using wild type (WT) and STAT3 knockout (KO) mouse embryonic fibroblasts (MEF). Both WT and STAT3 KO MEF expressed similar levels of tumor necrosis factor receptor 1 (TNFR1) and exhibited comparable IκBα degradation with TNFα. However, in the absence of STAT3 nuclear accumulation of NFκB p65 with TNFα was attenuated and induction of the survival protein c-FLIPL was eliminated. Nonetheless, WT MEF were more sensitive to TNFα-induced death which was attributed to necrosis. Deletion of STAT3 decreased ROS formation induced by TNFα and serum deprivation. STAT3 deletion was associated with lower levels of complex I and rates of respiration. Relative to WT cells, mitochondria of STAT3 KO cells released significantly more cytochrome c in response to oxidative stress and had greater caspase 3 cleavage due to serum deprivation. Our findings are consistent with STAT3 being important for mitochondrial function and cell viability by ensuring mitochondrial integrity and the expression of pro-survival genes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Validation of a commercially available anti-REDD1 antibody using RNA interference and REDD1-/- mouse embryonic fibroblasts.

    PubMed

    Grainger, Deborah L; Kutzler, Lydia; Rannels, Sharon L; Kimball, Scot R

    2016-01-01

    REDD1 is a transcriptional target gene of p53 and HIF-1, and an inhibitor of mTOR (mechanistic target of rapamycin) complex 1 (mTORC1)-signaling through PP2A-dependent interaction, making it an important convergence point of both tumor suppression and cell growth pathways. In accordance with this positioning, REDD1 levels are transcriptionally upregulated in response to a variety of cellular stress factors such as nutrient deprivation, hypoxia and DNA damage. In the absence of such conditions, and in particular where growth factor signaling is activated, REDD1 expression is typically negligible; therefore, it is necessary to induce REDD1 prior to experimentation or detection in model systems. Here, we evaluated the performance of a commercially available polyclonal antibody recognizing REDD1 by Western blotting in the presence of thapsigargin, a pharmacological inducer of ER stress well known to upregulate REDD1 protein expression. Further, REDD1 antibody specificity was challenged in HEK-293 cells in the presence of RNA interference and with a REDD1 (-/-) mouse embryonic fibroblast knockout cell line. Results showed reproducibility and specificity of the antibody, which was upheld in the presence of thapsigargin treatment. We conclude that this antibody can be used to reliably detect REDD1 endogenous expression in samples of both human and mouse origin.

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

    SciTech Connect

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

    2009-06-05

    Akt/PKB plays a pivotal role in cell survival and proliferation. Previously, we reported that UV-irradiation induces extensive cell death in Akt2{sup -/-} mouse embryonic fibroblasts (MEFs) while Akt1{sup -/-} MEFs show cell cycle arrest. Here, we find that Akt1{sup -/-} MEFs exhibit phenotypic changes characteristics of senescence upon UV-irradiation. An enlarged and flattened morphology, a reduced cell proliferation and an increased senescence-associated {beta}-galactosidase (SA {beta}-gal) staining indicate that Akt1{sup -/-} MEFs undergo premature senescence after UV-irradiation. Restoring Akt1 expression in Akt1{sup -/-} MEFs suppressed SA {beta}-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{sup -/-} MEFs, suggesting that UV light induces premature senescence in Akt1{sup -/-} 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.

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

    PubMed Central

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

    2011-01-01

    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 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 micronuclei-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. PMID:21412778

  7. Mouse embryonic fibroblasts accumulate differentially on titanium surfaces treated with nanosecond laser pulses.

    PubMed

    Radmanesh, Mitra; Ektesabi, Amin M; Wyatt, Rachael A; Crawford, Bryan D; Kiani, Amirkianoosh

    2016-10-01

    Biomaterial engineering, specifically in bone implant and osseointegration, is currently facing a critical challenge regarding the response of cells to foreign objects and general biocompatibility of the materials used in the production of these implants. Using the developing technology of the laser surface treatment, this study investigates the effects of the laser repetition rate (frequency) on cell distribution across the surface of the titanium substrates. The main objective of this research is building a fundamental understanding of how cells interact with treated titanium and how different treatments affect cell accumulation. Cells respond differently to surfaces treated with different frequency lasers. The results of this research identify the influence of frequency on surface topography properties and oxidation of titanium, and their subsequent effects on the pattern of cell accumulation on its surface. Despite increased oxidation in laser-treated regions, the authors observe that fibroblast cells prefer untreated titanium to laser-treated regions, except the regions treated with 25 kHz pulses, which become preferentially colonized after 72 h.

  8. Proteasome-mediated degradation of integral inner nuclear membrane protein emerin in fibroblasts lacking A-type lamins

    SciTech Connect

    Muchir, Antoine; Massart, Catherine; Engelen, Baziel G. van; Lammens, Martin; Bonne, Gisele; Worman, Howard J. . E-mail: hjw14@columbia.edu

    2006-12-29

    We previously identified and characterized a homozygous LMNA nonsense mutation leading to the absence of A-type lamins in a premature neonate who died at birth. We show here that the absence of A-type lamins is due to degradation of the aberrant mRNA transcript with a premature termination codon. In cultured fibroblasts from the subject with the homozygous LMNA nonsense mutation, there was a decreased steady-state expression of the integral inner nuclear membrane proteins emerin and nesprin-1{alpha} associated with their mislocalization to the bulk endoplasmic reticulum and a hyperphosphorylation of emerin. To determine if decreased emerin expression occurred post-translationally, we treated cells with a selective proteasome inhibitor and observed an increase in expression. Our results show that mislocalization of integral inner nuclear membrane proteins to the endoplasmic reticulum in human cells lacking A-type lamins leads to their degradation and provides the first evidence that their degradation is mediated by the proteasome.

  9. Exogenous Fibroblast Growth Factor-10 Induces Cystic Lung Development with Altered Target Gene Expression in the Presence of Heparin in Cultures of Embryonic Rat Lung

    PubMed Central

    Hashimoto, Shuichi; Nakano, Hiroshi; Suguta, Yuko; Irie, Seiko; Jianhua, Luo; Katyal, Sikardar L.

    2012-01-01

    Objectives Signaling by fibroblast growth factor (FGF) receptor (FGFR) 2IIIb regulates branching morphogenesis in the mammalian lung. FGFR2IIIb is primarily expressed in epithelial cells, whereas its ligands, FGF-10 and keratinocyte growth factor (KGF; FGF-7), are expressed in mesenchymal cells. FGF-10 null mice lack lungs, whereas KGF null animals have normal lung development, indicating that FGF-10 regulates lung branching morphogenesis. In this study, we determined the effects of FGF-10 on lung branching morphogenesis and accompanying gene expression in cultures of embryonic rat lungs. Methods Embryonic day 14 rat lungs were cultured with FGF-10 (0–250 ng/ml) in the absence or presence of heparin (30 ng/ml) for 4 days. Gene expression profiles were analyzed by Affymetrix microchip array including pathway analysis. Some of these genes, functionally important in FGF-10 signaling, were further analyzed by Northern blot, real-time PCR, in situ hybridization and immunohistochemistry. Results Exogenous FGF-10 inhibited branching and induced cystic lung growth only in cultures containing heparin. In total, 252 upregulated genes and 164 downregulated genes were identified, and these included Spry1 (Sprouty-1), Spry2 (Sprouty-2), Spred-1, Bmp4 (bone morphogenetic protein-4, BMP-4), Shh(sonic hedgehog, SHH), Pthlh (parathyroid hormone-related protein, PTHrP), Dusp6 (MAP kinase phosphatase-3, MKP-3) and Clic4 (chloride intracellular channel-4, CLIC-4) among the upregulated genes and Igf1 (insulin-like growth factor-1, IGF-1), Tcf21 (POD), Gyg1 (glycogenin 1), Sparc (secreted protein acidic and rich in cysteine, SPARC), Pcolce (procollagen C-endopeptidase enhancer protein, Pro CEP) and Lox (lysyl oxidase) among the downregulated genes. Gsk3β and Wnt2, which are involved in canonical Wnt signaling, were up- and downregulated, respectively. Conclusions Unlike FGF-7, FGF-10 effects on lung branching morphogenesis are heparin-dependent. Sprouty-2, BMP-4, SHH, IGF-1, SPARC

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

    NASA Astrophysics Data System (ADS)

    Chen, Aaron Chih-Hao; Arany, Praveen R.; Huang, Ying-Ying; Tomkinson, Elizabeth M.; Saleem, Taimur; Yull, Fiona E.; Blackwell, Timothy S.; Hamblin, Michael R.

    2009-02-01

    Despite over forty years of investigation on low-level light therapy (LLLT), the fundamental mechanisms underlying photobiomodulation 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 NFkB was observed for fluences higher than 0.003 J/cm2. NF-kB activation by laser was detectable at 1-hour time point. Moreover, we demonstrated that laser phosphorylated both IKK α/β and NF-kB 15 minutes after irradiation, which implied that laser activates NF-kB via phosphorylation of IKK α/β. Suspecting mitochondria as the source of NF-kB activation signaling pathway, we demonstrated that laser increased both intracellular reactive oxygen species (ROS) by fluorescence microscopy with dichlorodihydrofluorescein and ATP synthesis by luciferase assay. Mitochondrial inhibitors, such as antimycin A, rotenone and paraquat increased ROS and NF-kB activation but had no effect on ATP. The ROS quenchers N-acetyl-L-cysteine and ascorbic acid abrogated laser-induced NF-kB and ROS but not ATP. These results suggested that ROS might play an important role in the signaling pathway of laser induced NF-kB activation. However, the western blot showed that antimycin A, a mitochondrial inhibitor, did not activate NF-kB via serine phosphorylation of IKK α/β as the laser did. On the other hand, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that light also upregulates mitochondrial respiration. ATP upregulation reached a maximum at 0.3 J/cm2 or higher. We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive transcription factor NF-kB by generating ROS as signaling molecules.

  11. Mouse embryonic fibroblasts (MEF) exhibit a similar but not identical phenotype to bone marrow stromal stem cells (BMSC).

    PubMed

    Saeed, Hamid; Taipaleenmäki, Hanna; Aldahmash, Abdullah M; Abdallah, Basem M; Kassem, Moustapha

    2012-06-01

    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 reported. Utilizing standard in vitro and in vivo assays we performed a side-by-side comparison of MEF and BMSC to determine their ability to differentiate into mesoderm-type cells. BMSC were isolated from 8-10 weeks old mouse bone marrow by plastic adherence. MEF were established by trypsin/EDTA digestion from E13.5 embryos after removing heads and viscera, followed by plastic adherence. Compared to BMSC, MEF exhibited telomerase activity and improved cell proliferation as assessed by q-PCR based TRAP assay and cell number quantification, respectively. FACS analysis revealed that MEF exhibited surface markers characteristic of the BMSC: Sca-1(+), CD73(+), CD105(+), CD29(+), CD44(+), CD106(+), CD11b(-), and CD45(-). In contrast to BMSC, ex vivo osteoblast (OB) differentiation of MEF exhibited a less mature osteoblastic phenotype (less alkaline phosphatase, collagen type I and osteocalcin) as assessed 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/tricalcium phosphate, in immune deficient mice. In conclusion, MEF contain a population of stem cells that behave in ex vivo and in vivo assays, similar but not identical, to BMSC. Due to their enhanced cell growth, they may represent a good alternative for BMSC in studying molecular mechanisms of stem cell commitment and differentiation to osteoblasts, adipocytes and chondrocytes.

  12. Elf5 is an epithelium-specific, fibroblast growth factor-sensitive transcription factor in the embryonic lung.

    PubMed

    Metzger, David E; Xu, Yan; Shannon, John M

    2007-05-01

    Fibroblast growth factor (FGF) signaling has been shown to be essential for many aspects of normal lung development. To determine epithelial targets of FGF signaling, we cultured embryonic day (E) 11.5 mouse lungs for 24 hr in the presence or absence of the FGF receptor antagonist SU5402, which inhibited branching morphogenesis. Affymetrix gene chip analysis of treated and control epithelia identified several genes regulated by FGF signaling, including Elf5, a member of the Epithelial-specific Ets family of transcription factors. SU5402 reduced Elf5 expression in mesenchyme-free cultures of E12.5 epithelium, demonstrating that the inhibition was direct. In situ hybridization revealed that Elf5 had a dynamic pattern of expression during lung development. We found that expression of Elf5 was induced by FGF7 and FGF10, ligands that primarily bind FGFR2b. To further define the pathways by which FGFs activate Elf5 expression, we cultured E11.5 lung tips in the presence of compounds to inhibit FGF receptors (SU5402), PI3-Kinase/Akt-mediated signaling (LY294002), and MAP Kinase/Erk-mediated signaling (U0126). We found that SU5402 and LY294002 significantly reduced Elf5 expression, whereas U0126 had no effect. LY294002 also reduced Elf5 expression in cultures of purified epithelium. Finally, pAkt was coexpressed with Elf5 in the proximal epithelial airways of E17.5 lungs. These results demonstrate that Elf5 is an FGF-sensitive transcription factor in the lung with a dynamic pattern of expression and that FGF regulation of Elf5 by means of FGFR2b occurs through the PI3-Kinase/Akt pathway.

  13. Innate Immune Response of Human Embryonic Stem Cell-Derived Fibroblasts and Mesenchymal Stem Cells to Periodontopathogens

    PubMed Central

    Seneviratne, Chaminda Jayampath

    2016-01-01

    Periodontitis involves complex interplay of bacteria and host immune response resulting in destruction of supporting tissues of the tooth. Toll-like receptors (TLRs) play a role in recognizing microbial pathogens and eliciting an innate immune response. Recently, the potential application of multipotent stem cells and pluripotent stem cells including human embryonic stem cells (hESCs) in periodontal regenerative therapy has been proposed. However, little is known about the impact of periodontopathogens on hESC-derived progenies. This study investigates the effects of heat-killed periodontopathogens, namely, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, on TLR and cytokine expression profile of hESC-derived progenies, namely, fibroblasts (hESC-Fib) and mesenchymal stem cells (hESC-MSCs). Additionally, the serotype-dependent effect of A. actinomycetemcomitans on hESC-derived progenies was explored. Both hESC-Fib and hESC-MSCs constitutively expressed TLR-2 and TLR-4. hESC-Fib upon exposure to periodontopathogens displayed upregulation of TLRs and release of cytokines (IL-1β, IL-6, and IL-8). In contrast, hESC-MSCs were largely nonresponsive to bacterial challenge, especially in terms of cytokine production. Further, exposure of hESC-Fib to A. actinomycetemcomitans serotype c was associated with higher IL-8 production than serotype b. In contrast, the hESC-MSCs displayed no serotype-dependent response. Differential response of the two hESC progenies implies a phenotype-dependent response to periodontopathogens and supports the concept of immunomodulatory properties of MSCs. PMID:27642305

  14. All-trans retinoic acid shifts rosiglitazone-induced adipogenic differentiation to osteogenic differentiation in mouse embryonic fibroblasts.

    PubMed

    Shao, Ying; Chen, Qian-Zhao; Zeng, Yu-Hua; Li, Yang; Ren, Wen-Yan; Zhou, Lin-Yun; Liu, Rong-Xin; Wu, Ke; Yang, Jun-Qing; Deng, Zhong-Liang; Yu, Yu; Sun, Wen-Juan; He, Bai-Cheng

    2016-12-01

    Rosiglitazone (RSG) is a potent drug used in the treatment of insulin resistance; however, it is associated with marked skeletal toxicity. RSG-induced osteoporosis may contribute to the promotion of adipogenic differentiation at the expense of osteogenic differentiation in bone marrow stromal cells. The aim of this study was to investigate whether RSG-induced bone toxicity can be reversed by combined treatment with all-trans retinoic acid (ATRA). We examined different osteogenic markers in mouse embryonic fibroblasts (MEFs) following treatment with RSG, ATRA, or RSG and ATRA in combination. We examined the effects of RSG and/or ATRA on ectopic bone formation, and dissected the possible molecular mechanisms underlying this process. We found that ATRA or RSG both induced alkaline phosphatase (ALP) activity in the MEFs, and that the ATRA-induced ALP activity was enhanced by RSG and vice versa. However, only the combination of RSG and ATRA increased the expression of osteopontin and osteocalcin, promoted matrix mineralization, and induced ectopic ossification in MEFs. Mechanistically, we found that the osteogenic differentiation induced by the combination of RSG and ATRA may be mediated partly by suppressing RSG-induced adipogenic differentiation and activating bone morphogenetic protein (BMP)/Smad signaling. On the whole, our findings demonstrate that RSG in combination with ATRA promotes the commitment of MEFs to the osteoblast lineage. Thus, the combination of these two agents may prove to be a promising and novel therapeutic regimen for insulin resistance without skeletal toxicity. It may also be a better strategy with which to prevent RSG-induced osteoporosis.

  15. All-trans retinoic acid shifts rosiglitazone-induced adipogenic differentiation to osteogenic differentiation in mouse embryonic fibroblasts

    PubMed Central

    Shao, Ying; Chen, Qian-Zhao; Zeng, Yu-Hua; Li, Yang; Ren, Wen-Yan; Zhou, Lin-Yun; Liu, Rong-Xin; Wu, Ke; Yang, Jun-Qing; Deng, Zhong-Liang; Yu, Yu; Sun, Wen-Juan; He, Bai-Cheng

    2016-01-01

    Rosiglitazone (RSG) is a potent drug used in the treatment of insulin resistance; however, it is associated with marked skeletal toxicity. RSG-induced osteoporosis may contribute to the promotion of adipogenic differentiation at the expense of osteogenic differentiation in bone marrow stromal cells. The aim of this study was to investigate whether RSG-induced bone toxicity can be reversed by combined treatment with all-trans retinoic acid (ATRA). We examined different osteogenic markers in mouse embryonic fibroblasts (MEFs) following treatment with RSG, ATRA, or RSG and ATRA in combination. We examined the effects of RSG and/or ATRA on ectopic bone formation, and dissected the possible molecular mechanisms underlying this process. We found that ATRA or RSG both induced alkaline phosphatase (ALP) activity in the MEFs, and that the ATRA-induced ALP activity was enhanced by RSG and vice versa. However, only the combination of RSG and ATRA increased the expression of osteopontin and osteocalcin, promoted matrix mineralization, and induced ectopic ossification in MEFs. Mechanistically, we found that the osteogenic differentiation induced by the combination of RSG and ATRA may be mediated partly by suppressing RSG-induced adipogenic differentiation and activating bone morphogenetic protein (BMP)/Smad signaling. On the whole, our findings demonstrate that RSG in combination with ATRA promotes the commitment of MEFs to the osteoblast lineage. Thus, the combination of these two agents may prove to be a promising and novel therapeutic regimen for insulin resistance without skeletal toxicity. It may also be a better strategy with which to prevent RSG-induced osteoporosis. PMID:27779644

  16. Cytotoxicity of various chemicals and mycotoxins in fresh primary duck embryonic fibroblasts: a comparison to HepG2 cells.

    PubMed

    Chen, Xi; Murdoch, Rhonda; Shafer, Daniel J; Ajuwon, Kolapo M; Applegate, Todd J

    2016-11-01

    To screen cost-effectively the overall toxicity of a sample, particularly in the case of food and feed ingredient quality control, a sensitive bioassay is necessary. With the wide variety of cytotoxicity assays, performance comparison between assays using different cells has become of interest. Fresh primary duck embryonic fibroblasts (DEF) were hypothesized to be a sensitive tool for in vitro cytotoxicity screening; cell viability of DEF in response to various cytotoxins was determined and compared with response of HepG2 cells. The IC50 values by the alamar blue assay in the DEF cells had a high correlation (R(2)  = 0.96) with those obtained in HepG2 cells. Within the same toxin, primary DEF yielded significantly lower IC50 values than that obtained from HepG2 cells using the MTT and alamar blue assay. Additionally, primary DEF responded to all mycotoxins tested using the alamar blue assay, while HepG2 was less sensitive, particularly at short exposure times. The estimated IC50 for aflatoxin B1 , fumonisins B1 and deoxynivalenol in DEF after 72 h incubation were 3.69, 4.19 and 1.26 μg ml(-1) , respectively. Results from the current study suggest that primary DEF are more sensitive to cytotoxins and mycotoxins compared to HepG2, and thus may have great potential as an effective tool for cytotoxicity assessment. The question remains whether in vitro IC50 values can accurately predict in vivo toxicity; however, the current study accentuates the need for further attention to identify sensitive cell models for in vitro cytotoxicity screening and subsequent exploration of species-specific prediction models for in vivo toxicity. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Production and validation of a good manufacturing practice grade human fibroblast line for supporting human embryonic stem cell derivation and culture

    PubMed Central

    2012-01-01

    Introduction The development of reproducible methods for deriving human embryonic stem cell (hESC) lines in compliance with good manufacturing practice (GMP) is essential for the development of hESC-based therapies. Although significant progress has been made toward the development of chemically defined conditions for the maintenance and differentiation of hESCs, efficient derivation of new hESCs requires the use of fibroblast feeder cells. However, GMP-grade feeder cell lines validated for hESC derivation are not readily available. Methods We derived a fibroblast cell line (NclFed1A) from human foreskin in compliance with GMP standards. Consent was obtained to use the cells for the production of hESCs and to generate induced pluripotent stem cells (iPSCs). We compared the line with a variety of other cell lines for its ability to support derivation and self-renewal of hESCs. Results NclFed1A supports efficient rates (33%) of hESC colony formation after explantation of the inner cell mass (ICM) of human blastocysts. This compared favorably with two mouse embryonic fibroblast (MEF) cell lines. NclFed1A also compared favorably with commercially available foreskin fibroblasts and MEFs in promoting proliferation and pluripotency of a number of existing and widely used hESCs. The ability of NclFed1A to maintain self-renewal remained undiminished for up to 28 population doublings from the master cell bank. Conclusions The human fibroblast line Ncl1Fed1A, produced in compliance with GMP standards and qualified for derivation and maintenance of hESCs, is a useful resource for the advancement of progress toward hESC-based therapies in regenerative medicine. PMID:22472092

  18. Cytotoxicity, oxidative stress, apoptosis and the autophagic effects of silver nanoparticles in mouse embryonic fibroblasts.

    PubMed

    Lee, Yu-Hsuan; Cheng, Fong-Yu; Chiu, Hui-Wen; Tsai, Jui-Chen; Fang, Chun-Yong; Chen, Chun-Wan; Wang, Ying-Jan

    2014-05-01

    With the advancement of nanotechnology, nanomaterials have been comprehensively applied in our modern society. However, the hazardous impacts of nanoscale particles on organisms have not yet been thoroughly clarified. Currently, there exist numerous approaches to perform toxicity tests, but common and reasonable bio-indicators for toxicity evaluations are lacking. In this study, we investigated the effects of silver nanoparticles (AgNPs) on NIH 3T3 cells to explore the potential application of these nanoparticles in consumer products. Our results demonstrated that AgNPs were taken up by NIH 3T3 cells and localized within the intracellular endosomal compartments. Exposure to AgNPs is a potential source of oxidative stress, which leads to the induction of reactive oxygen species (ROS), the up-regulation of Heme oxygenase 1 (HO-1) expression, apoptosis and autophagy. Interestingly, AgNPs induced morphological and biochemical markers of autophagy in NIH 3T3 cells and induced autophagosome formation, as evidenced by transmission electron microscopic analysis, the formation of microtubule-associated protein-1 light chain-3 (LC3) puncta and the expression of LC3-II protein. Thus, autophagy activation may be a key player in the cellular response against nano-toxicity.

  19. Disrupting the IL-4 gene rescues mice homozygous for the tight-skin mutation from embryonic death and diminishes TGF-β production by fibroblasts

    PubMed Central

    Kodera, Takao; McGaha, Tracy L.; Phelps, Robert; Paul, William E.; Bona, Constantin A.

    2002-01-01

    The TSK/TSK mutation is embryonic lethal; embryos have been reported to die at 7–8 days of gestational age. Crossing TSK/+, IL-4+/− mice revealed that disrupting one or both IL-4 alleles allowed survival of 29 and 47%, respectively, of TSK/TSK mice. These mice failed to develop cutaneous hyperplasia but did exhibit the emphysema that is found in TSK/+ mice. We showed that IL-4 stimulation of fibroblasts increased the level of transforming growth factor-β (TGF-β) mRNA and that lungs of TSK/+, IL-4−/− mice had substantially less TGF-β mRNA than lungs of TSK/+, IL-4+/+ mice. Thus IL-4 seems to regulate the expression of TGF-β in fibroblasts, providing an explanation for the absence of cutaneous hyperplasia in TSK/+, IL-4Rα−/− and TSK/+, TGF-β+/− mice. PMID:11891315

  20. Cultures of human embryonic stem cells: serum replacement medium or serum-containing media and the effect of basic fibroblast growth factor.

    PubMed

    Koivisto, Heidi; Hyvärinen, Marjukka; Strömberg, Anne-Marie; Inzunza, Jose; Matilainen, Eija; Mikkola, Milla; Hovatta, Outi; Teerijoki, Heli

    2004-09-01

    Human embryonic stem (hES) cells have traditionally been cultured in medium containing fetal calf serum (FCS) and mouse fibroblasts as feeder cells. The use of animal derived materials carries a risk of transmitting animal pathogens, and they are not optimal in cultures aimed at cell transplantation in humans. This technical study aiming at facilitating IVF units to establish new hES cell lines, has systematically compared the non-differentiated growth of the hES cell line HS237, originally derived and thereafter cultured using human foreskin fibroblasts as feeder cells, by culturing it in media containing serum replacement (SR; 10, 15, 20%), FCS, and human serum. In addition, optimal concentrations of insulin-transferrin-selenium (ITS) mixture and the effect of basic fibroblast growth factor (bFGF) have also been studied. Cellular growth was monitored daily and maintenance of their non-differentiated character was studied using antibodies against TRA-1-60, TRA-1-81 and SSEA-4 and expression of Oct-4. The hES cells proliferated fastest when 20% of SR was used. In human serum-containing medium, the cells underwent extensive spontaneous differentiation within a few passages. The FCS supported the non-differentiated growth poorly. Basic fibroblast growth factor supported non-differentiated growth, the highest concentration (8 ng/ml) giving the best result, while ITS was not beneficial.

  1. Selenium promotes adipogenic determination and differentiation of chicken embryonic fibroblasts with regulation of genes involved in fatty acid uptake, triacylglycerol synthesis and lipolysis.

    PubMed

    Hassan, Aishlin; Ahn, Jinsoo; Suh, Yeunsu; Choi, Young Min; Chen, Paula; Lee, Kichoon

    2014-08-01

    Selenium (Se) has been utilized in the differentiation of primary pig and rat preadipocytes, indicating that it may have proadipogenic potential; however, some studies have also demonstrated that Se has antiadipogenic activity. In this study, chicken embryonic fibroblasts (CEFs) were used to investigate the role of Se in adipogenesis in vitro and in ovo. Se supplementation increased lipid droplet accumulation and inhibited proliferation of cultured CEFs isolated from 6-day-old embryos dose-dependently. This suggests that Se may play a role in cell cycle inhibition, thereby promoting the differentiation of fibroblasts to adipocytes. Se did not stimulate adipogenic differentiation of CEFs isolated from 9- to 12-day-old embryos, implying a permissive stage of adipogenic determination by Se at earlier embryonic ages. Microarray analysis comparing control and Se treatments on CEFs from 6-day-old embryos and confirmatory analysis by quantitative real-time polymerase chain reaction revealed that genes involved in adipocyte determination and differentiation, fatty acid uptake and triacylglycerol synthesis were up-regulated. In addition, up-regulation of an anti-lipolytic G0/G1 switch gene 2 and down-regulation of a prolipolytic monoglyceride lipase may lead to inhibition of lipolysis by Se. Both osteogenic and myogenic genes were down-regulated, and several genes related to oxidative stress response during adipogenesis were up-regulated. In ovo injection of Se at embryonic day 8 increased adipose tissue mass by 30% and caused adipocyte hypertrophy in 17-day-old chicken embryos, further supporting the proadipogenic role of Se during the embryonic development of chickens. These results suggest that Se plays a significant role in several mechanisms related to adipogenesis.

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

    PubMed Central

    2011-01-01

    Background 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 (Rb-/- MEFs). Findings Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination 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 of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like) adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment. Conclusion In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte differentiation of Rb

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

    PubMed

    Hakim-Weber, Robab; Krogsdam, Anne-M; Jørgensen, Claus; Fischer, Maria; Prokesch, Andreas; Bogner-Strauss, Juliane G; Bornstein, Stefan R; Hansen, Jacob B; Madsen, Lise; Kristiansen, Karsten; Trajanoski, Zlatko; Hackl, Hubert

    2011-05-26

    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 (Rb-/- MEFs). Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination 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 of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like) adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment. In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte differentiation of Rb-/- MEFs could be identified. These

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

    PubMed Central

    2012-01-01

    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 (associated

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

    PubMed

    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.

  6. Fibroblast growth factor receptors in in vitro and in vivo chondrogenesis: relating tissue engineering using adult mesenchymal stem cells to embryonic development.

    PubMed

    Hellingman, Catharine A; Koevoet, Wendy; Kops, Nicole; Farrell, Eric; Jahr, Holger; Liu, Wei; Baatenburg de Jong, Robert J; Frenz, Dorothy A; van Osch, Gerjo J V M

    2010-02-01

    Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb development, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is followed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1-3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR expression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation.

  7. Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation.

    PubMed

    Tremblay, K D; Dunn, N R; Robertson, E J

    2001-09-01

    The Smad proteins are important intracellular mediators of the transforming growth factor beta (TGFbeta) family of secreted growth factors. Smad1 is an effector of signals provided by the bone morphogenetic protein (BMP) sub-group of TGFbeta molecules. To understand the role of Smad1 in mouse development, we have generated a Smad1 loss-of-function allele using homologous recombination in ES cells. Smad1-/- embryos die by 10.5 dpc because they fail to connect to the placenta. Mutant embryos are first recognizable by 7.0 dpc, owing to a characteristic localized outpocketing of the visceral endoderm at the posterior embryonic/extra-embryonic junction, accompanied by a dramatic twisting of the epiblast and nascent mesoderm. Chimera analysis reveals that these two defects are attributable to a requirement for Smad1 in the extra-embryonic tissues. By 7.5 dpc, Smad1-deficient embryos show a marked impairment in allantois formation. By contrast, the chorion overproliferates, is erratically folded within the extra-embryonic space and is impeded in proximal migration. BMP signals are known to be essential for the specification and proliferation of primordial germ cells. We find a drastic reduction of primordial germ cells in Smad1-deficient embryos, suggesting an essential role for Smad1-dependent signals in primordial germ cell specification. Surprisingly, despite the key involvement of BMP signaling in tissues of the embryo proper, Smad1-deficient embryos develop remarkably normally. An examination of the expression domains of Smad1, Smad5 and Smad8 in early mouse embryos show that, while Smad1 is uniquely expressed in the visceral endoderm at 6.5 dpc, in other tissues Smad1 is co-expressed with Smad5 and/or Smad8. Collectively, these data have uncovered a unique function for Smad1 signaling in coordinating the growth of extra-embryonic structures necessary to support development within the uterine environment.

  8. Induction of 6-thioguanine-resistant mutations by rat-liver homogenate (S9)-activated promutagens in human embryonic skin fibroblasts.

    PubMed

    Chen, D J; Okinaka, R T; Strniste, G F; Barnhart, B J

    1982-03-01

    Most normal human fibroblasts grown in culture do not metabolize promutagens/procarcinogens. Thus screening assays employing normal human fibroblasts have only been successful for direct-acting chemical mutagens and various radiations. In this report we describe a mutation assay (HGPRT locus) employing a normal human embryonic skin fibroblast and a rat-liver homogenate (S9) mixture. 3 model promutagens, benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3MC), and dimethylnitrosamine (DMN) have been utilized in these studies. In addition to discussing conditions for optimizing the response of this assay, our results indicate that at constant amount of S9 protein concentration, there exists a linear correlation between mutagenicity and dose. At 50% survival, the mutant frequencies induced by B[a]P and 3MC (5 micrograms/ml) are 60 and 30 times the background mutant frequency, respectively. Similarly, at 50% survival, DMN (5 mg/ml) induced 6-TGr mutant frequencies are 25-fold over the background frequency. The increase in cytotoxicity resulting from exposure of cells to these 'activated' chemicals is also a linear dose response. At high S9 concentrations a deactivation or detoxification phenomenon occurs. However, the mutagenic efficiency of S9-activated chemicals when plotted as the number of induced mutations versus log survival is unaffected by the deactivating capacity of S9 proteins. This study demonstrates a quantitative mutation assay using an early passage human culture with an exogenous rat-liver microsomal preparation providing activating enzymes.

  9. In vitro exposure to very low-level laser modifies expression level of extracellular matrix protein RNAs and mitochondria dynamics in mouse embryonic fibroblasts.

    PubMed

    Giuliani, Alessandro; Lorenzini, Luca; Alessandri, Marco; Torricella, Roberta; Baldassarro, Vito Antonio; Giardino, Luciana; Calzà, Laura

    2015-03-24

    Low-level lasers working at 633 or 670 nm and emitting extremely low power densities (Ultra Low Level Lasers - ULLL) exert an overall effect of photobiostimulation on cellular metabolism and energy balance. In previous studies, it was demonstrated that ULLL pulsed emission mode regulates neurite elongation in vitro and exerts protective action against oxidative stress. In this study the action of ULLL supplied in both pulsed and continuous mode vs continuous LLL on fibroblast cultures (Mouse Embryonic Fibroblast-MEF) was tested, focusing on mitochondria network and the expression level of mRNA encoding for proteins involved in the cell-matrix adhesion. It was shown that ULLL at 670 nm, at extremely low average power output (0.21 mW/ cm(2)) and dose (4.3 mJ/ cm(2)), when dispensed in pulsed mode (PW), but not in continuous mode (CW) supplied at both at very low (0.21 mW/cm(2)) and low levels (500 mW/cm(2)), modifies mitochondria network dynamics, as well as expression level of mRNA encoding for selective matrix proteins in MEF, e.g. collagen type 1α1 and integrin α5. We suggest that pulsatility, but not energy density, is crucial in regulating expression level of collagen I and integrin α5 in fibroblasts by ULLL.

  10. Appl1 and Appl2 are Expendable for Mouse Development But Are Essential for HGF-Induced Akt Activation and Migration in Mouse Embryonic Fibroblasts.

    PubMed

    Tan, Yinfei; Xin, Xiaoban; Coffey, Francis J; Wiest, David L; Dong, Lily Q; Testa, Joseph R

    2016-05-01

    Although Appl1 and Appl2 have been implicated in multiple cellular activities, we and others have found that Appl1 is dispensable for mouse embryonic development, suggesting that Appl2 can substitute for Appl1 during development. To address this possibility, we generated conditionally targeted Appl2 mice. We found that ubiquitous Appl2 knockout (Appl2-/-) mice, much like Appl1-/- mice, are viable and grow normally to adulthood. Intriguingly, when Appl1-/- mice were crossed with Appl2-/- mice, we found that homozygous Appl1;Appl2 double knockout (DKO) animals are also viable and grossly normal with regard to reproductive potential and postnatal growth. Appl2-null and DKO mice were found to exhibit altered red blood cell physiology, with erythrocytes from these mice generally being larger and having a more irregular shape than erythrocytes from wild type mice. Although Appl1/2 proteins have been previously shown to have a very strong interaction with phosphatidylinositol-3 kinase (Pi3k) in thymic T cells, Pi3k-Akt signaling and cellular differentiation was unaltered in thymocytes from Appl1;Appl2 (DKO) mice. However, Appl1/2-null mouse embryonic fibroblasts exhibited defects in HGF-induced Akt activation, migration, and invasion. Taken together, these data suggest that Appl1 and Appl2 are required for robust HGF cell signaling but are dispensable for embryonic development and reproduction. © 2015 Wiley Periodicals, Inc.

  11. Fundamentals of ultrananocrystallie diamond (UNCD) thin films as biomaterials for developmental biology : embryonic fibroblasts growth on the surface of (UNCD) films.

    SciTech Connect

    Shi, B.; Jin, Q.; Chen, L.; Auciello, O.

    2008-09-13

    Ultrananocrystalline diamond (UNCD) films possess numerous valuable good physical, chemical and mechanical properties, making UNCD an excellent material for implantable biodevices. However, one very important property required for biomaterials i.e., biocompatibility has not been studied for UNCD. In this research, biocompatible UNCD films were synthesized. It was found that UNCD film coated substrates can dramatically promote the growth of mouse embryonic fibroblasts (MEFs), while the uncoated substrates inhabit cell attachment. Through analyzing the microstructure and the surface chemistry of UNCD, the mechanisms of cell growth on UNCD were investigated. Given the unique properties of UNCD on inertness and toughness, the results consolidate UNCD film as the leading coating candidate for the next generation of medical implanted devices.

  12. Signalling Through Retinoic Acid Receptors is Required for Reprogramming of Both Mouse Embryonic Fibroblast Cells and Epiblast Stem Cells to Induced Pluripotent Stem Cells.

    PubMed

    Yang, Jian; Wang, Wei; Ooi, Jolene; Campos, Lia S; Lu, Liming; Liu, Pentao

    2015-05-01

    We previously demonstrated that coexpressing retinoic acid (RA) receptor gamma and liver receptor homolog-1 (LRH1 or NR5A2) with OCT4, MYC, KLF4, and SOX2 (4F) rapidly reprograms mouse embryonic fibroblast cells (MEFs) into induced pluripotent stem cells (iPSCs). Here, we further explore the role of RA in reprogramming and report that the six factors (6F) efficiently and directly reprogram MEFs into integration-free iPSCs in defined medium (N2B27) in the absence of feeder cells. Through genetic and chemical approaches, we find that RA signalling is essential, in a highly dose-sensitive manner, for MEF reprogramming. The removal of exogenous RA from N2B27, the inhibition of endogenous RA synthesis or the expression of a dominant-negative form of RARA severely impedes reprogramming. By contrast, supplementing N2B27 with various retinoids substantially boosts reprogramming. In addition, when coexpressed with LRH1, RA receptors (RARs) can promote reprogramming in the absence of both exogenous and endogenously synthesized RA. Remarkably, the reprogramming of epiblast stem cells into embryonic stem cell-like cells also requires low levels of RA, which can modulate Wnt signalling through physical interactions of RARs with β-catenin. These results highlight the important functions of RA signalling in reprogramming somatic cells and primed stem cells to naïve pluripotency. Stem Cells 2015;33:1390-1404. © 2014 AlphaMed Press.

  13. Glucocorticoids facilitate the stable transformation of embryonal rat fibroblasts by a polyomavirus large tumor antigen-deficient mutant.

    PubMed Central

    Martens, I; Nilsson, M; Magnusson, G; Linder, S

    1988-01-01

    The addition of glucocorticoids to the growth medium could substitute for the expression of the polyomavirus large tumor antigen in the transformation of rat fibroblasts in vitro. After transfection with a large tumor antigen-deficient mutant of polyomavirus, pbc1051, high-frequency permanent transformation was observed, if the cells were grown in medium containing dexamethasone. Growth of pbc1051-transfected rat fibroblasts was strictly dependent on the presence of glucocorticoids during the initial phase of transformation. In the second phase, the growth of pbc1051-transfected cells was stimulated by dexamethasone, but the hormone was not essential for growth. After approximately 10 weeks in culture, pbc1051-transfected cells had progressed to hormone independent growth. Rat embryo cells transfected with wild-type polyomavirus DNA had the second phase in which growth was stimulated by glucocorticoid, and after this phase growth was steroid independent. Addition of glucocorticoids to rat fibroblasts transfected with a plasmid encoding only the middle-sized tumor antigen resulted in only a weak stimulation of growth. In contrast, embryo cells transfected with a plasmid containing the human homologue of the cellular T24 Ha-ras gene linked to murine sarcoma virus and simian virus 40 enhancers could be efficiently established as cell lines in medium supplemented with glucocorticoids. The data suggest that, in the transformation of primary rodent cells by polyomavirus, the activity of large tumor antigen can be substituted for by stimulating normal cellular functions with dexamethasone. Images PMID:2840668

  14. EFFECT OF THE AMINO ACIDS AND DIALYZABLE CONSTITUENTS OF EMBRYONIC TISSUE JUICE ON THE GROWTH OF FIBROBLASTS

    PubMed Central

    Baker, Lillian E.; Carrel, Alexis

    1926-01-01

    The ultrafilterable constituents of embryonic tissue extract are unable to support cell life in vitro. They stimulate cell migration and possibly multiplication, without increasing the mass of the tissue. Embryonic tissue extract freed from amino acids by dialysis still retains a considerable part of its growth-promoting properties. The area of growth of tissues in embryonic tissue extract free from amino acids is appreciably less than that with the whole extract, probably owing to the denaturation of part of the protein, or perhaps to the inactivation or loss of an enzyme. The addition of either the ultrafilterable components or an artificial mixture of amino acids to this dialyzed extract increases the area of cell migration but does not restore all the activity lost on dialysis. The observed differences in growth of tissue, due to the addition or removal of dialyzable and ultrafilterable constituents of the extract, prove that the amino acids produce a more active cell migration and possibly multiplication, but no building up of new protoplasm. PMID:19869192

  15. Reduced mitotic activity at the periphery of human embryonic stem cell colonies cultured in vitro with mitotically-inactivated murine embryonic fibroblast feeder cells.

    PubMed

    Heng, Boon Chin; Cao, Tong; Liu, Hua; Rufaihah, Abdul Jalil

    2005-01-01

    This study attempted to investigate whether different levels of mitotic activity exist within different physical regions of a human embryonic stem (hES) cell colony. Incorporation of 5-bromo-2-deoxyuridine (BrdU) within newly-synthesized DNA, followed by immunocytochemical staining was used as a means of detecting mitotically-active cells within hES colonies. The results showed rather surprisingly that the highest levels of mitotic activity are primarily concentrated within the central regions of hES colonies, whereas the peripheral regions exhibited reduced levels of cellular proliferation. Two hypothetical mechanisms are therefore proposed for hES colony growth and expansion. Firstly, it is envisaged that the less mitotically-active hES cells at the periphery of the colony are continually migrating outwards, thereby providing space for newly-divided daughter cells within the more mitotically-active central region of the hES colony. Secondly, it is proposed that the newly-divided hES cells within the central region of the colony somehow migrate to the outer periphery. This could possibly explain why the periphery of hES colonies are less mitotically-active, since there would obviously be an extended time-lag before newly-divided daughter cells are ready again for the next cell division. Further investigations need to be carried out to characterize the atypical mechanisms by which hES colonies grow and expand in size.

  16. Lack of evidence for low-LET radiation induced bystander response in normal human fibroblasts and colon carcinoma cells

    SciTech Connect

    Marianne B. Sowa; Wilfried Goetz; Janet E. Baulch; Dinah N. Pyles; Jaroslaw Dziegielewski; Susannah Yovino; Andrew R. Snyder; Sonia M. de Toledo; Edouard I. Azzam; William F. Morgan

    2008-06-30

    Purpose: To investigate radiation induced bystander responses and to determine the role of gap junction intercellular communication and the radiation environment in propagating this response. Materials and Methods: We use medium transfer and targeted irradiation to examine radiation induced bystander effects in primary human fibroblast (AG1522) and human colon carcinoma (RKO36) cells. We examined the effect of variables such as gap junction intercellular communication, linear energy transfer (LET), and the role of the radiation environment in non-targeted responses. Endpoints included clonogenic survival, micronucleus formation and foci formation at histone 2AX over doses ranging from 10 to 100 cGy. Results: The results show no evidence of a low-LET radiation induced bystander response for the endpoints of clonogenic survival and induction of DNA damage. Nor do we see evidence of a high-LET, Fe ion radiation (1 GeV/n) induced bystander effect. However, direct comparison for 3.2 MeV α-particle exposures showed a statistically significant medium transfer bystander effect for this high-LET radiation. Conclusions: From our results, it is evident that there are many confounding factors influencing bystander responses as reported in the literature. Our observations reflect the inherent variability in biological systems and the difficulties in extrapolating from in vitro models to radiation risks in humans.

  17. Enhanced bradykinin-stimulated phospholipase C activity in murine embryonic stem cells lacking the G-protein alphaq-subunit.

    PubMed Central

    Ricupero, D A; Polgar, P; Taylor, L; Sowell, M O; Gao, Y; Bradwin, G; Mortensen, R M

    1997-01-01

    The gene coding for the G-protein alphaq subunit was interrupted by homologous recombination in murine embryonic stem cells (alphaq-null ES cells) as detected by Southern analysis and reverse-transcriptase PCR. The bradykinin (BK) B2 receptor was stably transfected into wild-type (WT) alphai-2-null and alphaq-null ES cells. The B2 receptor bound BK with high affinity and mobilized Ca2+. BK also activated phospholipase C (PLC), as determined by total inositol phosphate (IP) accumulation in a Bordetella pertussis toxin- and genistein-insensitive manner. In WT and alphai-2-null ES cells, BK increased IP levels approx. 4-fold above baseline. Most interestingly, in alphaq-null ES cells, BK increased IP accumulation approx. 9-fold above baseline. Re-expression of alphaq in alphaq-null ES cells resulted in normalization of the BK-stimulated IP accumulation (4-fold above baseline). These results suggest that the B2 receptor activates PLC through more than one member of the Gq family. Additionally, the absence of alphaq alters the kinetics of IP generation, which may reflect intrinsic characteristics of individual members of the Gq family or a decreased susceptibility to heterologous regulation in the alphaq-null ES cells, thus allowing for a more sustained generation of IP. PMID:9581559

  18. Homeobox, Wnt, and Fibroblast Growth Factor Signaling is Augmented During Alveogenesis in Mice Lacking Superoxide Dismutase 3, Extracellular.

    PubMed

    Thimraj, Tania A; Birru, Rahel L; Mitra, Ankita; Schulz, Holger; Leikauf, George D; Ganguly, Koustav

    2017-04-01

    Superoxide dismutase 3, extracellular (SOD3) polymorphisms have been implicated in reduced pulmonary function development and altered risk for chronic obstructive pulmonary disease. We previously reported that gene-targeted Sod3-/- mice have impaired lung function and human SOD3 variants are associated with reduced pulmonary function in children. Reduced lung SOD3 levels were reported in mice with lower lung function with the greatest difference occurring during alveogenesis phase [postnatal (P) days 14-28]. Interactions between homeobox (HOX), wingless-type MMTV integration site member (WNT), and fibroblast growth factor (FGF) signaling govern complex developmental processes in several organs. A subset of HOX family members, HOXA5 and HOXB5, is expressed in the developing lung. Therefore, in this study we assessed the transcript expression of these family members and their downstream targets in Sod3-/- mice during alveogenesis (P14). In the lung of Sod3-/- mice, Hoxa5 and Hoxb5 increased. These transcription factors regulate WNT gene expression and were accompanied by increases in their downstream targets Wnt2 and Wnt5A, canonical and noncanonical WNT members, respectively. The WNT signaling target, lymphoid enhancer binding factor 1 (Lef1), also increased along with its downstream targets Fgf2, Fgf7, and Fgf10 in the lungs of Sod3-/- mice. Due to limited knowledge on the role of FGF2 in lung development, we further examined FGF2 protein and found increased levels in the bronchial and alveolar type II epithelial cells of Sod3-/- mice compared to age-matched controls. Thus, our findings suggest that deficient management of extracellular superoxide can lead to altered lung developmental signaling during alveogenesis in mice.

  19. Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts

    PubMed Central

    Ghayad, Sandra E.; Rammal, Ghina; Ghamloush, Farah; Basma, Hussein; Nasr, Rihab; Diab-Assaf, Mona; Chelala, Claude; Saab, Raya

    2016-01-01

    Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes. PMID:27853183

  20. Exosomes derived from embryonal and alveolar rhabdomyosarcoma carry differential miRNA cargo and promote invasion of recipient fibroblasts.

    PubMed

    Ghayad, Sandra E; Rammal, Ghina; Ghamloush, Farah; Basma, Hussein; Nasr, Rihab; Diab-Assaf, Mona; Chelala, Claude; Saab, Raya

    2016-11-17

    Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, which exists in oncoprotein PAX-FOXO1 fusion positive and fusion negative subtypes, with the fusion-positive RMS being characterized by a more aggressive clinical behavior. Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and have been implicated in metastatic progression through paracrine signaling. We characterized exosomes secreted by a panel of 5 RMS cell lines. Expression array analysis showed that, for both fusion-positive and fusion-negative cells, exosome miRNA clustered well together and to a higher extent than cellular miRNA. While enriched miRNA in exosomes of fusion-negative RMS cells were distinct from those of fusion-positive RMS cells, the most significant predicted disease and functions in both groups were related to processes relevant to cancer and tissue remodelling. Functionally, we found that RMS-derived exosomes exerted a positive effect on cellular proliferation of recipient RMS cells and fibroblasts, induced cellular migration and invasion of fibroblasts, and promoted angiogenesis. These findings show that RMS-derived exosomes enhance invasive properties of recipient cells, and that exosome content of fusion-positive RMS is different than that of fusion-negative RMS, possibly contributing to the different metastatic propensity of the two subtypes.

  1. Maintenance of human embryonic stem cells in media conditioned by human mesenchymal stem cells obviates the requirement of exogenous basic fibroblast growth factor supplementation.

    PubMed

    Sánchez, Laura; Gutierrez-Aranda, Iván; Ligero, Gertrudis; Martín, Miguel; Ayllón, Verónica; Real, Pedro J; Ramos-Mejía, Verónica; Bueno, Clara; Menendez, Pablo

    2012-05-01

    Despite the improvements in the human embryonic stem cell (hESC) culture systems, very similar conditions to those used to maintain hESCs on mouse feeders are broadly applied to culture methods based on human feeders. Indeed, basic fibroblast growth factor (bFGF), a master hESC-sustaining factor, is still added in nearly all medium formulations for hESC propagation. Human foreskin fibroblasts (HFFs) and mesenchymal stem cells (MSCs) used as feeders have recently been reported to support hESC growth without exogenous bFGF. However, whether hESCs may be maintained undifferentiated without exogenous bFGF using media conditioned (CM) by human feeders remains elusive. We hypothesize that HFFs and hMSCs are likely to be functionally different and therefore the mechanisms by which HFF-CM and MSC-CM support undifferentiated growth of hESCs may differ. We have thus determined whether HFF-CM and/or MSC-CM sustain feeder-free undifferentiated growth of hESC without exogenous supplementation of bFGF. We report that hMSCs synthesize higher levels of endogenous bFGF than HFFs. Accordingly and in contrast to HFF-CM, MSC-CM produced without the addition of exogenous bFGF supports hESC pluripotency and culture homeostasis beyond 20 passages without the need of bFGF supplementation. hESCs maintained without exogenous bFGF in MSC-CM retained hESC morphology and expression of pluripotency surface markers and transcription factors, formed teratomas, and showed spontaneous and lineage-directed in vitro differentiation capacity. Our data indicate that MSC-CM, but not HFF-CM, provides microenvironment cues supporting feeder-free long-term maintenance of pluripotent hESCs and obviates the requirement of exogenous bFGF at any time.

  2. Comparison of cell cycle components, apoptosis and cytoskeleton-related molecules and therapeutic effects of flavopiridol and geldanamycin on the mouse fibroblast, lung cancer and embryonic stem cells.

    PubMed

    Aktug, Huseyin; Acikgoz, Eda; Uysal, Aysegul; Oltulu, Fatih; Oktem, Gulperi; Yigitturk, Gurkan; Demir, Kenan; Yavasoglu, Altug; Bozok Cetintas, Vildan

    2016-09-01

    Similarities and differences in the cell cycle components, apoptosis and cytoskeleton-related molecules among mouse skin fibroblast cells (MSFs), mouse squamous cell lung carcinomas (SqCLCs) and mouse embryonic stem cells (mESCs) are important determinants of the behaviour and differentiation capacity of these cells. To reveal apoptotic pathways and to examine the distribution and the role of cell cycle-cell skeleton comparatively would necessitate tumour biology and stem cell biology to be assessed together in terms of oncogenesis and embryogenesis. The primary objectives of this study are to investigate the effects of flavopiridol, a cell cycle inhibitor, and geldanamycin, a heat shock protein inhibitor on mouse somatic, tumour and embryonic stem cells, by specifically focusing on alterations in cytoskeletal proteins, cell polarity and motility as well as cell cycle regulators. To meet these objectives, expression of several genes, cell cycle analysis and immunofluorescence staining of intracellular cytoskeletal molecules were performed in untreated and flavopiridol- or geldanamycin-treated cell lines. Cytotoxicity assays showed that SqCLCs are more sensitive to flavopiridol than MSFs and mESCs. Keratin-9 and keratin-2 expressions increased dramatically whereas cell cycle regulatory genes decreased significantly in the flavopiridol-treated MSFs. Flavopiridol-treated SqCLCs displayed a slight increase in several cell cytoskeleton regulatory genes as well as cell cycle regulatory genes. However, gene expression profiles of mESCs were not affected after flavopiridol treatment except the Cdc2a. Cytotoxic concentrations of geldanamycin were close to each other for all cell lines. Cdkn1a was the most increased gene in the geldanamycin-treated MSFs. However, expression levels of cell cytoskeleton-associated genes were increased dramatically in the geldanamycin-treated SqCLCs. Our results revealing differences in molecular mechanisms between embryogenesis and

  3. Basic fibroblast growth factor is critical to reprogramming buffalo (Bubalus bubalis) primordial germ cells into embryonic germ stem cell-like cells.

    PubMed

    Wang, Caizhu; Deng, Yanfei; Chen, Feng; Zhu, Peng; Wei, Jingwei; Luo, Chan; Lu, Fenghua; Yang, Sufang; Shi, Deshun

    2017-03-15

    Primordial germ cells (PGCs) are destined to form gametes in vivo, and they can be reprogrammed into pluripotent embryonic germ (EG) cells in vitro. Buffalo PGC have been reported to be reprogrammed into EG-like cells, but the identities of the major signaling pathways and culture media involved in this derivation remain unclear. Here, the effects of basic fibroblast growth factor (bFGF) and downstream signaling pathways on the reprogramming of buffalo PGCs into EG-like cells were investigated. Results showed bFGF to be critical to buffalo PGCs to dedifferentiate into EG-like cells (20 ng/mL is optimal) with many characteristics of pluripotent stem cells, including alkaline phosphatase (AP) activity, expression of pluripotency marker genes such as OCT4, NANOG, SOX2, SSEA-1, CDH1, and TRA-1-81, and the capacity to differentiate into all three embryonic germ layers. After chemically inhibiting pathways or components downstream of bFGF, data showed that inhibition of the PI3K/AKT pathway led to significantly lower EG cell derivation, while inhibition of P53 activity resulted in an efficiency of EG cell derivation comparable to that in the presence of bFGF. These results suggest that the role of bFGF in PGC-derived EG-like cell generation is mainly due to the activation of the PI3K/AKT/P53 pathway, in particular, the inhibition of P53 function. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Continuous production of tissue plasminogen activator (t-PA) by human embryonic lung diploid fibroblast, IMR-90 cells, using a ceramic bed reactor.

    PubMed

    Mitsuda, S; Matsuda, Y; Kobayashi, N; Suzuki, A; Itagaki, Y; Kumazawa, E; Higashio, K; Kawanishi, G

    1991-05-01

    Ceramic pieces composed of 99.5% Al2O3, 3 to 6 mm long, were found to be a good matrix for growth of the human embryonic lung diploid fibroblast, IMR-90 cells. The tissue plasminogen activator (t-PA) was secreted in DME medium containing proteose peptone as a t-PA inducer. In addition, production of t-PA was enhanced by increasing extracellular CaCl2, from 3.6 to 5.4 mM. In order to eliminate negative feed-back control caused by t-PA produced and thus raise productivity, perfusion cultivation was performed using a ceramic-packed bed column, with a recirculating vessel. The recirculating vessel was used to mix fresh medium with spent medium, and to control dissolved oxygen concentrations in the extracellular environment by stirring. In continuous production using the packed bed column with 2 kg of ceramics (phi = H = 150 mm), increasing dilution rate to 0.5 day-1 could reduce product inhibition at 3-4 x 10(5) cells/ml. Cellular productivity of 560 IU/10(6) cells/day was obtained over 40 days and corresponded to the volumetric productivity of 183 IU/ml/day.

  5. Potent Effects of Flavonoid Nobiletin on Amplitude, Period, and Phase of the Circadian Clock Rhythm in PER2::LUCIFERASE Mouse Embryonic Fibroblasts

    PubMed Central

    Shinozaki, Ayako; Misawa, Kenichiro; Ikeda, Yuko; Haraguchi, Atsushi; Kamagata, Mayo; Tahara, Yu; Shibata, Shigenobu

    2017-01-01

    Flavonoids are natural polyphenols that are widely found in plants. The effects of flavonoids on obesity and numerous diseases such as cancer, diabetes, and Alzheimer’s have been well studied. However, little is known about the relationships between flavonoids and the circadian clock. In this study, we show that continuous or transient application of flavonoids to the culture medium of embryonic fibroblasts from PER2::LUCIFERASE (PER2::LUC) mice induced various modifications in the circadian clock amplitude, period, and phase. Transient application of some of the tested flavonoids to cultured cells induced a phase delay of the PER2::LUC rhythm at the down slope phase. In addition, continuous application of the polymethoxy flavonoids nobiletin and tangeretin increased the amplitude and lengthened the period of the PER2::LUC rhythm. The nobiletin-induced phase delay was blocked by co-treatment with U0126, an ERK inhibitor. In summary, among the tested flavonoids, polymethoxy flavones increased the amplitude, lengthened the period, and delayed the phase of the PER2::LUC circadian rhythm. Therefore, foods that contain polymethoxy flavones may have beneficial effects on circadian rhythm disorders and jet lag. PMID:28152057

  6. Disruption of O-GlcNAc cycling by deletion of O-GlcNAcase (Oga/Mgea5) changed gene expression pattern in mouse embryonic fibroblast (MEF) cells.

    PubMed

    Keembiyehetty, Chithra

    2015-09-01

    Adding a single O-GlcNAc moiety to a Ser/Thr molecule of a protein by O-GlcNAc transferase and transiently removing it by O-GlcNAcase is referred to as O-GlcNAc cycling (or O-GlcNAcylation). This O-GlcNAc modification is sensitive to nutrient availability and also shows cross talk with phosphorylation signaling, affecting downstream targets. A mouse model system was developed and evaluated to show genome wide transcriptional changes associated with disruption of O-GlcNAc cycling. Mouse embryonic fibroblast cells derived from O-GlcNAcase (Oga) knock out (KO), heterozygous (Het) and wild type (WT) embryos were used for an Affymetrix based microarray. Results are deposited in GEO dataset GSE52721. Data reveals that Oga KO MEFs had 2534 transcripts differentially expressed at 1.5 fold level while Oga heterozygous MEFs had 959 transcripts changed compared to WT MEFs. There were 1835 transcripts differentially expressed at 1.5 fold Het versus WT comparison group. Gene ontology analysis indicated differentially expressed genes enriched in metabolic, growth, and cell proliferation categories.

  7. A high G418-resistant neo(R) transgenic mouse and mouse embryonic fibroblast (MEF) feeder layers for cytotoxicity and gene targeting in vivo and in vitro.

    PubMed

    Aubrecht, Jiri; Goad, Mary E P; Czopik, Agnieszka K; Lerner, Charles P; Johnson, Kevin A; Simpson, Elizabeth M; Schiestl, Robert H

    2011-10-01

    Aminoglycoside antibiotics have been in use since 1944 with the discovery of streptomycin. The aim of this study was to derive a new, highly resistant multicopy neo(R) transgenic mouse strain, named TgN3Ems, by random insertion of the plasmid, pPGKneobpA, and compare the level of drug resistance of wild-type and transgenic mice in vivo and corresponding primary mouse embryonic fibroblasts (MEFs) in vitro to a model neomycin analog, G418. The expression neoR in transgenic animals caused a 5-fold increase in the approximate lethal dose of G418, compared to wild type. No adverse pathological changes were found for the transgenic mice treated with G418, as they all died within minutes after injection. In contrast, the G418 treatment of wild-type mice resulted in a marked liver and kidney toxicity detected microscopically and via increases of serum biomarkers for liver and kidney damage. In addition, there was a mild bone marrow and lymphoid depletion. In in vitro studies, the transgenic MEFs survived 20-fold higher G418 levels, compared to the wild-type MEF cells. Therefore, TgN3Ems transgenic mice could be used as a source of G418-resistant feeder cells for gene targeting. Since the expression of drug-resistance genes in transgenic animals confers resistance to toxicity, the TgN3Ems mice might serve as a tool applicable in drug design.

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

    PubMed Central

    Motohashi, Tsutomu; Watanabe, Natsuki; Nishioka, Masahiro; Nakatake, Yuhki; Yulan, Piao; Mochizuki, Hiromi; Kawamura, Yoshifumi; Ko, Minoru S. H.; Goshima, Naoki; Kunisada, Takahiro

    2016-01-01

    ABSTRACT 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. PMID:26873953

  9. Double-strand break repair deficiency in NONO knockout murine embryonic fibroblasts and compensation by spontaneous upregulation of the PSPC1 paralog.

    PubMed

    Li, Shuyi; Li, Zhentian; Shu, Feng-Jue; Xiong, Hairong; Phillips, Andrew C; Dynan, William S

    2014-09-01

    NONO, SFPQ and PSPC1 make up a family of proteins with diverse roles in transcription, RNA processing and DNA double-strand break (DSB) repair. To understand long-term effects of loss of NONO, we characterized murine embryonic fibroblasts (MEFs) from knockout mice. In the absence of genotoxic stress, wild-type and mutant MEFs showed similar growth rates and cell cycle distributions, and the mutants were only mildly radiosensitive. Further investigation showed that NONO deficiency led to upregulation of PSPC1, which replaced NONO in a stable complex with SFPQ. Knockdown of PSPC1 in a NONO-deficient background led to severe radiosensitivity and delayed resolution of DSB repair foci. The DNA-dependent protein kinase (DNA-PK) inhibitor, NU7741, sensitized wild-type and singly deficient MEFs, but had no additional effect on doubly deficient cells, suggesting that NONO/PSPC1 and DNA-PK function in the same pathway. We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes. Of 12 genes tested, none were downregulated, and several were upregulated. Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.

  10. The role of COX-2 in mediating the effect of PTEN on BMP9 induced osteogenic differentiation in mouse embryonic fibroblasts.

    PubMed

    Huang, Jun; Yuan, Shuang-Xue; Wang, Dong-Xu; Wu, Qiu-Xiang; Wang, Xing; Pi, Chang-Jun; Zou, Xiang; Chen, Liang; Ying, Liang-Jun; Wu, Ke; Yang, Jun-Qing; Sun, Wen-Juan; Deng, Zhong-Liang; He, Bai-Cheng

    2014-12-01

    Mouse embryonic fibroblasts (MEFs) are multi-potent progenitor cells (MPCs), can differentiate into different lineages, such as osteogenic, and adipogenic. PTEN, a tumor suppressor, may be involved in regulating bone development through interacting with COX-2. BMP9, the most potent osteogenic BMPs, can up-regulate COX-2 in MPCs. Whether PTEN is involved in BMP9 induced osteogenic differentiation in MPCs remains unknown. The goal of this investigation is to identify the effect of PTEN on BMP9-induced osteogenic differentiation in MPCs and dissect the possible mechanism underlay this. We found that BMP9 down-regulates PTEN, and PTEN inhibitor (VO) effectively increases different osteogenic markers induced by BMP9 in MEFs. Exogenous expression of PTEN inhibits BMP9 induced ectopic bone formation apparently. Mechanistically, we found that VO can enhance BMP9 induced BMPs/Smads signaling prominently without no substantial effects on cell cycle. Further analysis indicates that VO can promote BMP9-induced expression of COX-2 in MEFs, which can be eliminated by PI3K inhibitor. Additionally, COX-2 knockdown abolishes the effect of VO on BMP9-induced ALP activities in MEFs. Our findings suggest that PTEN plays an important role in regulating BMP9 induced osteogenic differentiation in MPCs, which may be mediated by PTEN/PI3K/Akt signaling to modulate the expression of COX-2.

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

    PubMed

    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 (H2) 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 H2 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 H2, further supporting the idea that H2 acts as a signaling molecule to serve various physiological functions. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. CG hypomethylation in Lsh-/- mouse embryonic fibroblasts is associated with de novo H3K4me1 formation and altered cellular plasticity.

    PubMed

    Yu, Weishi; Briones, Victorino; Lister, Ryan; McIntosh, Carl; Han, Yixing; Lee, Eunice Y; Ren, Jianke; Terashima, Minoru; Leighty, Robert M; Ecker, Joseph R; Muegge, Kathrin

    2014-04-22

    DNA methylation patterns are established in early embryogenesis and are critical for cellular differentiation. To investigate the role of CG methylation in potential enhancer formation, we assessed H3K4me1 modification in murine embryonic fibroblasts (MEFs) derived from the DNA methylation mutant Lsh(-/-) mice. We report here de novo formation of putative enhancer elements at CG hypomethylated sites that can be dynamically altered. We found a subset of differentially enriched H3K4me1 regions clustered at neuronal lineage genes and overlapping with known cis-regulatory elements present in brain tissue. Reprogramming of Lsh(-/-) MEFs into induced pluripotent stem (iPS) cells leads to increased neuronal lineage gene expression of premarked genes and enhanced differentiation potential of Lsh(-/-) iPS cells toward the neuronal lineage pathway compared with WT iPS cells in vitro and in vivo. The state of CG hypomethylation and H3K4me1 enrichment is partially maintained in Lsh(-/-) iPS cells. The acquisition of H3K27ac and activity of subcloned fragments in an enhancer reporter assay indicate functional activity of several of de novo H3K4me1-marked sequences. Our results suggest a functional link of H3K4me1 enrichment at CG hypomethylated sites, enhancer formation, and cellular plasticity.

  13. Potent Effects of Flavonoid Nobiletin on Amplitude, Period, and Phase of the Circadian Clock Rhythm in PER2::LUCIFERASE Mouse Embryonic Fibroblasts.

    PubMed

    Shinozaki, Ayako; Misawa, Kenichiro; Ikeda, Yuko; Haraguchi, Atsushi; Kamagata, Mayo; Tahara, Yu; Shibata, Shigenobu

    2017-01-01

    Flavonoids are natural polyphenols that are widely found in plants. The effects of flavonoids on obesity and numerous diseases such as cancer, diabetes, and Alzheimer's have been well studied. However, little is known about the relationships between flavonoids and the circadian clock. In this study, we show that continuous or transient application of flavonoids to the culture medium of embryonic fibroblasts from PER2::LUCIFERASE (PER2::LUC) mice induced various modifications in the circadian clock amplitude, period, and phase. Transient application of some of the tested flavonoids to cultured cells induced a phase delay of the PER2::LUC rhythm at the down slope phase. In addition, continuous application of the polymethoxy flavonoids nobiletin and tangeretin increased the amplitude and lengthened the period of the PER2::LUC rhythm. The nobiletin-induced phase delay was blocked by co-treatment with U0126, an ERK inhibitor. In summary, among the tested flavonoids, polymethoxy flavones increased the amplitude, lengthened the period, and delayed the phase of the PER2::LUC circadian rhythm. Therefore, foods that contain polymethoxy flavones may have beneficial effects on circadian rhythm disorders and jet lag.

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

    PubMed

    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.

  15. Age dependency of the metabolic conversion of polyamines into amino acids in IMR-90 human embryonic lung diploid fibroblasts

    SciTech Connect

    Chen, K.Y.; Chang, Z.

    1986-07-01

    When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000-dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000-dalton protein. The authors have investigated these two polyamine-associated biochemical events in IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL). They found that (1) the metabolic labeling of the 18,000-dalton protein was about two-fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six-fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4-fold higher in the young cells as compared to the old cells, their data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR-90 cells was about eight-fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000-dalton protein were also observed in cell strains derived from patients with premature aging disease.

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

    PubMed Central

    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

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

    PubMed

    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 < 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. 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.

  18. Elevated Nuclear and Cytoplasmic FTY720-Phosphate in Mouse Embryonic Fibroblasts Suggests the Potential for Multiple Mechanisms in FTY720-Induced Neural Tube Defects.

    PubMed

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

    2016-03-01

    FTY720 (fingolimod) is a U.S. Food and Drug Administration-approved drug to treat relapsing remitting multiple sclerosis. FTY720 treatment in pregnant inbred LM/Bc mice results in approximately 60% of embryos having a neural tube defect (NTD). Sphingosine kinases (Sphk1, Sphk2) phosphorylate FTY720 in vivo to form the bioactive metabolite FTY720-1-phosphate (FTY720-P). Cytoplasmic FTY720-P is an agonist for 4 of the 5 sphingosine-1-phosphate (S1P) receptors (S1P1, 3-5) and can also act as a functional antagonist of S1P1, whereas FTY720-P generated in the nucleus inhibits histone deacetylases (HDACs), leading to increased histone acetylation. This study demonstrates that treatment of LM/Bc mouse embryonic fibroblasts (MEFs) with FTY720 results in a significant accumulation of FTY720-P in both the cytoplasmic and nuclear compartments. Elevated nuclear FTY720-P is associated with decreased HDAC activity and increased histone acetylation at H3K18 and H3K23 in LM/Bc MEFs. Treatment of LM/Bc MEFs with FTY720 and a selective Sphk2 inhibitor, ABC294640, significantly reduces the amount of FTY720-P that accumulates in the nucleus. The data provide insight into the relative amounts of FTY720-P generated in the nuclear versus cytoplasmic subcellular compartments after FTY720 treatment and the specific Sphk isoforms involved. The results of this study suggest that FTY720-induced NTDs may involve multiple mechanisms, including: (1) sustained and/or altered S1P receptor activation and signaling by FTY720-P produced in the cytoplasm and (2) HDAC inhibition and histone hyperacetylation by FTY720-P generated in the nucleus that could lead to epigenetic changes in gene regulation.

  19. Dehydrodiconiferyl alcohol isolated from Cucurbita moschata shows anti-adipogenic and anti-lipogenic effects in 3T3-L1 cells and primary mouse embryonic fibroblasts.

    PubMed

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

    2012-03-16

    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.

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

    PubMed Central

    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

  1. A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures.

    PubMed

    Kawabe, Keiko; Tateyama, Daiki; Toyoda, Hidenao; Kawasaki, Nana; Hashii, Noritaka; Nakao, Hiromi; Matsumoto, Shogo; Nonaka, Motohiro; Matsumura, Hiroko; Hirose, Yoshinori; Morita, Ayaha; Katayama, Madoka; Sakuma, Makoto; Kawasaki, Nobuko; Furue, Miho Kusuda; Kawasaki, Toshisuke

    2013-03-01

    We have generated a monoclonal antibody (R-10G) specific to human induced pluripotent stem (hiPS)/embryonic stem (hES) cells by using hiPS cells (Tic) as an antigen, followed by differential screening of mouse hybridomas with hiPS and human embryonal carcinoma (hEC) cells. Upon western blotting with R-10G, hiPS/ES cell lysates gave a single but an unusually diffuse band at a position corresponding to >250 kDa. The antigen protein was isolated from the induced pluripotent stem (iPS) cell lysates with an affinity column of R-10G. The R-10G positive band was resistant to digestion with peptide N-glycanase F (PNGase F), neuraminidase, fucosidase, chondrotinase ABC and heparinase mix, but it disappeared almost completely on digestion with keratanase, keratanase II and endo-β-galactosidase, indicating that the R-10G epitope is a keratan sulfate. The carrier protein of the R-10G epitope was identified as podocalyxin by liquid chromatography/mass spectrometry (LC/MS/MS) analysis of the R-10G positive-protein band material obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R-10G epitope is a type of keratan sulfate with some unique properties. (1) The epitope is expressed only on hiPS/ES cells, i.e. not on hEC cells, unlike those recognized by the conventional hiPS/ES marker antibodies. (2) The epitope is a type of keratan sulfate lacking oversulfated structures and is not immunologically cross-reactive with high-sulfated keratan sulfate. (3) The R-10G epitope is distributed heterogeneously on hiPS cells, suggesting that a single colony of undifferentiated hiPS cells consists of different cell subtypes. Thus, R-10G is a novel antibody recognizing hiPS/ES cells, and should be a new molecular probe for disclosing the roles of glycans on these cells.

  2. Sensitivity to methylmercury toxicity is enhanced in oxoguanine glycosylase 1 knockout murine embryonic fibroblasts and is dependent on cellular proliferation capacity

    SciTech Connect

    Ondovcik, Stephanie L.; Tamblyn, Laura; McPherson, John Peter; Wells, Peter G.

    2013-07-01

    Methylmercury (MeHg) is a persistent environmental contaminant with potent neurotoxic action for which the underlying molecular mechanisms remain to be conclusively delineated. Our objectives herein were twofold: first, to corroborate our previous findings of an increased sensitivity of spontaneously-immortalized oxoguanine glycosylase 1-null (Ogg1{sup −/−}) murine embryonic fibroblasts (MEFs) to MeHg through generation of Simian virus 40 (SV40) large T antigen-immortalized wild-type and Ogg1{sup −/−} MEFs; and second, to determine whether MeHg toxicity is proliferation-dependent. As with the spontaneously-immortalized cells used previously, the SV40 large T antigen-immortalized cells exhibited similar tendencies to undergo MeHg-initiated cell cycle arrest, with increased sensitivity in the Ogg1{sup −/−} MEFs as measured by clonogenic survival and DNA damage. Compared to exponentially growing cells, those seeded at a higher density exhibited compromised proliferation, which proved protective against MeHg-mediated cell cycle arrest and induction of DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), and by its functional confirmation by micronucleus assessment. This enhanced sensitivity of Ogg1{sup −/−} MEFs to MeHg toxicity using discrete SV40 immortalization corroborates our previous studies, and suggests a novel role for OGG1 in minimizing MeHg-initiated DNA lesions that trigger replication-associated DSBs. Furthermore, proliferative capacity may determine MeHg toxicity in vivo and in utero. Accordingly, variations in cellular proliferative capacity and interindividual variability in repair activity may modulate the risk of toxicological consequences following MeHg exposure. - Highlights: • SV40 large T antigen-immortalized Ogg1{sup −/−} cells are more sensitive to MeHg. • Sensitivity to MeHg is dependent on cellular proliferation capacity. • OGG1 maintains genomic

  3. The therapeutic effect of extracellular superoxide dismutase (EC-SOD) mouse embryonic fibroblast (MEF) on collagen-induced arthritis (CIA) mice.

    PubMed

    Yu, Dong Hoon; Kim, Myoung Ok; Kim, Sung Hyun; Shin, Mi Jung; Kim, Bong Soo; Kim, Hei Jung; Lee, Sang Ryeul; Lee, Sang Gyu; Yoo, Seung-Ah; Kim, Wan Uk; Hyun, Byung Hwa; Park, Young Sik; Kim, Tae Yoon; Ryoo, Zae Young

    2008-01-01

    Rheumatoid arthritis is a chronic inflammatory disease. The generation of reactive oxygen species (ROS) within an inflamed joint has been suggested as playing a significant pathogenic role. Extracellular superoxide dismutase (EC-SOD) is a major scavenger enzyme of ROS, which has received growing attention for its therapeutic potential. To investigate the therapeutic effect of EC-SOD in mice with collagen-induced arthritis (CIA), we used mouse embryonic fibroblast (MEF) of transgenic mice that overexpresses EC-SOD on the skin by using hK14 promoter. DBA/1 mice that had been treated with bovine type II collagen were administrated subcutaneous injections of EC-SOD transgenic MEF (each at 1.4 x 10(60 cells) on days 28, 35, and 42 after primary immunization. To test EC-SOD activity, blood samples were collected in each group on day 49. The EC-SOD activity was nearly 1.5-fold higher in the transgenic MEF-treated group than in the nontransgenic MEF-treated group (p < 0.05). The severity of arthritis in mice was scored in a double-blind manner, with each paw being assigned a separate clinical score. The severity of arthritis in EC-SOD transgenic MEF-treated mice was significantly suppressed in the arthritic clinical score (p < 0.05). To investigate the alteration of cytokine levels, ELISA was used to measure blood samples. Levels of IL-1beta and TNF-alpha were reduced in the transgenic MEF-treated group (p < 0.05). Abnormalities of the joints were examined by H&E staining. There were no signs of inflammation except for mild hyperplasia of the synovium in the transgenic MEF-treated group. The proliferation of CII-specific T cells was lower in the transgenic MEF-treated mice than in those in the other groups. The transfer of EC-SOD transgenic MEF has shown a therapeutic effect in CIA mice and this approach may be a safer and more effective form of therapy for rheumatoid arthritis.

  4. The vector-related influences of autophagic microRNA delivery by Lipofectamine 2000 and polyethylenimine 25K on mouse embryonic fibroblast cells.

    PubMed

    Lin, Chia-Wei; Jan, Ming-Shiou; Kuo, Jung-Hua Steven

    2017-04-01

    Despite the greater potential for clinical applications of autophagic microRNA (miRNA) delivery, the vector-related effects of such delivery on cells have not been fully explored. In this study, autophagic mmu-miR-494-3p (miR-494) in mouse embryonic fibroblast (MEF) cells was selected as a cargo miRNA, and two commonly used non-viral carriers (Lipofectamine 2000 (Lipo) and polyethylenimine 25K (PEI)), were used as delivery vectors to mechanistically elucidate its vector-related effects. The cellular uptake, nuclear localization, and quantitative miR-494 levels of the complexes of miR-494 with Lipo (miR-494 lipoplexes) were lower than those of the complexes of miR-494 with PEI (miR-494 polyplexes) in MEF cells. The indicator of autophagic activity (LC3 (microtubule-associated protein 1 light chain 3)-II/LC3-I ratio) in cells treated with miR-494 lipoplexes was higher than that in cells treated with miR-494 polyplexes. Lipo alone and PEI alone induced slight increases in the quantitative levels of miR-494 in cells, but Lipo resulted in higher gene and protein expressions of target Igf1, higher LC3-II/LC3-I ratios, and higher autophagosome formation than PEI. We also demonstrated that the delivery of miR-494 by Lipo was more involved in apoptotic caspase-3 pathways than such delivery by PEI. By applying knock-out atg5 gene in MEF cells, we found that autophagy played a protective role in cell survival and also affected cellular uptake, the quantitative level of miR-494, and target gene Igf1 regulation of delivery systems. Taken together, these results indicate that there are different degrees of responses in MEF cells for autophagic miR-494 delivery through the use of Lipo or PEI vectors that also induce autophagy in cells. Therefore, Lipo and PEI vectors cannot be treated as inert molecules, and their effects must be known and evaluated when they are used in autophagic miRNA delivery systems. Most importantly, understanding these vector-related effects on cells will

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

    PubMed Central

    Xiong, Xing-dong; Jung, Hwa Jin; Gombar, Saurabh; Park, Jung Yoon; Zhang, Chun-long; Zheng, Huiling; Ruan, Jie; Li, Jiang-bin; Kaeberlein, Matt; Kennedy, Brian K.; Zhou, Zhongjun; Liu, Xinguang; Suh, Yousin

    2015-01-01

    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 ×106 reads from WT MEFs and 16.5 × 106 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 putative 3′UTR target-site. Consistently, expression levels of miR-365 were found to inversely correlate with endogenous Rasd1 levels. These findings suggest that miR-365 is down-regulated in Zmpste24-/- MEFs and acts as a novel negative regulator of Rasd1. Our comprehensive miRNA data provide a resource to study gene

  6. The absence of expression of the three isoenzymes of the inositol 1,4,5-trisphosphate 3-kinase does not prevent the formation of inositol pentakisphosphate and hexakisphosphate in mouse embryonic fibroblasts.

    PubMed

    Leyman, Alexandre; Pouillon, Valérie; Bostan, Alionka; Schurmans, Stéphane; Erneux, Christophe; Pesesse, Xavier

    2007-07-01

    The activation of phospholipase C leads to the formation of both I(1,4,5)P(3) and diacylglycerol (DAG). I(1,4,5)P(3) can be metabolized by dephosphorylation catalyzed by Type I I(1,4,5)P(3) 5-phosphatase and by enzymatic phosphorylation to various inositol phosphates. This last step is catalyzed by three mammalian isoenzymes that specifically phosphorylate the 3-phosphate position of the inositol ring Itpka, Itpkb and Itpkc and a less specific enzyme Ipmk (or inositol multikinase) that phosphorylates I(1,4,5)P(3) at the D-3 and D-6 positions. This study was performed in mice cells in order to understand the synthetic pathway of IP5 and IP6 following PLC stimulation and possible link with Itpk activity. Mouse embryonic fibroblasts (MEF) were prepared from Itpkb(-/-) Itpkc(-/-) mice. Western blot and RT-PCR analysis show that the cells do not express Itpka. In contrast, they do express Ipmk. The cells still produce IP5 and IP6. Our data show that the absence of expression of the three isoenzymes of Itpk does not prevent the formation of IP5 and IP6, at least in mouse embryonic fibroblasts. The nuclear Ipmk plays therefore a critical role in the metabolism of I(1,4,5)P(3) and production of highly phosphorylated IP5 and IP6.

  7. Cord blood Lin(-)CD45(-) embryonic-like stem cells are a heterogeneous population that lack self-renewal capacity.

    PubMed

    Alvarez-Gonzalez, Cesar; Duggleby, Richard; Vagaska, Barbora; Querol, Sergio; Gomez, Susana G; Ferretti, Patrizia; Madrigal, Alejandro

    2013-01-01

    Human umbilical cord blood (hUCB) has been proposed to contain not only haematopoietic stem cells, but also a rare pluripotent embryonic-like stem cell (ELSc) population that is negative for hematopoietic markers (Lin(-)CD45(-)) and expresses markers typical of pluripotent cells. The aim of this work was to isolate, characterise and expand this ELSc fraction from hUCB, as it may provide a valuable cell source for regenerative medicine applications. We found that we could indeed isolate a Lin(-)CD45(-) population of small cells (3-10 µm diameter) with a high nucleus to cytoplasm ratio that expressed the stem cell markers CD34 and CXCR4. However, in contrast to some previous reports, this fraction was not positive for CD133. Furthermore, although these cells expressed transcripts typical of pluripotent cells, such as SOX2, OCT3/4, and NANOG, they were not able to proliferate in any of the culture media known to support stem cell growth that we tested. Further analysis of the Lin(-)CD45(-) population by flow cytometry showed the presence of a Lin(-)CD45(-)Nestin(+) population that were also positive for CD34 (20%) but negative for CXCR4. These data suggest that the Lin(-)CD45(-) stem cell fraction present in the cord blood represents a small heterogeneous population with phenotypic characteristics of stem cells, including a Lin(-)CD45(-)Nestin(+) population not previously described. This study also suggests that heterogeneity within the Lin(-)CD45(-) cell fraction is the likely explanation for differences in the hUCB cell populations described by different groups that were isolated using different methods. These populations have been widely called "embryonic-like stem cell" on the basis of their phenotypical similarity to embryonic stem cells. However, the fact they do not seem to be able to self-renew casts some doubt on their identity, and warns against defining them as "embryonic-like stem cell" at this stage.

  8. 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.

    PubMed

    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.

  9. Lack of Phenotypical and Morphological Evidences of Endothelial to Hematopoietic Transition in the Murine Embryonic Head during Hematopoietic Stem Cell Emergence

    PubMed Central

    Iizuka, Kazuhide; Yokomizo, Tomomasa; Watanabe, Naoki; Tanaka, Yosuke; Osato, Motomi; Takaku, Tomoiku; Komatsu, Norio

    2016-01-01

    During mouse ontogeny, hematopoietic cells arise from specialized endothelial cells, i.e., the hemogenic endothelium, and form clusters in the lumen of arterial vessels. Hemogenic endothelial cells have been observed in several embryonic tissues, such as the dorsal aorta, the placenta and the yolk sac. Recent work suggests that the mouse embryonic head also produces hematopoietic stem cells (HSCs)/progenitors. However, a histological basis for HSC generation in the head has not yet been determined because the hematopoietic clusters and hemogenic endothelium in the head region have not been well characterized. In this study, we used whole-mount immunostaining and 3D confocal reconstruction techniques to analyze both c-Kit+ hematopoietic clusters and Runx1+ hemogenic endothelium in the whole-head vasculature. The number of c-Kit+ hematopoietic cells was 20-fold less in the head arteries than in the dorsal aorta. In addition, apparent nascent hematopoietic cells, which are characterized by a “budding” structure and a Runx1+ hemogenic endothelium, were not observed in the head. These results suggest that head HSCs may not be or are rarely generated from the endothelium in the same manner as aortic HSCs. PMID:27227884

  10. Effects of DNA methyltransferase inhibitor RG108 on methylation in buffalo adult fibroblasts and subsequent embryonic development following somatic cell nuclear transfer.

    PubMed

    Sun, H L; Meng, L N; Zhao, X; Jiang, J R; Liu, Q Y; Shi, D S; Lu, F H

    2016-09-02

    Buffalo are characteristic livestock of the Guangxi Zhuang Autonomous Region of China, but their low reproductive capacity necessitates the use of somatic cell nuclear transfer (SCNT). We investigated the effects of RG108 on DNA methylation in buffalo adult fibroblasts, and on subsequent SCNT embryo development. RG108 treatment (0, 5, 10, 20, and 100 mM) had no effect on cell morphology, viability, or karyotype (2n = 48), and cell growth followed a typical "S" curve. Immunohistochemistry showed that relative DNA methylation gradually decreased as RG108 concentration increased, and was significantly lower in the 20 and 100 mM groups compared to the 0, 5, and 10 mM treatments (0.94 ± 0.03 and 0.92 ± 0.05 vs 1.0 ± 0.02, 0.98 ± 0.05, and 0.98 ± 0.09, respectively; P < 0.05). Quantitative polymerase chain reaction revealed that DNMT1 gene expression of fibroblasts administered 10, 20, and 100 mM RG108 was significantly lower than those in the 0 and 5 mM groups (0.2 ± 0.05, 0.18 ± 0.07, and 0.3 ± 0.09 vs 1.0 ± 0.12 and 1.4 ± 0.12, respectively; P < 0.05). Treatment with 20 mM RG108 resulted in the lowest expression levels. Fibroblasts incubated with 20 mM RG108 for 72 h were used as donor cells to generate SCNT embryos. A greater number of such embryos developed into blastocysts compared to the non-treated group (28.9 ± 3.9 vs 15.3 ± 3.4%; P < 0.05). RG108 treatment can modify DNA methylation in buffalo adult fibroblasts and promote development of subsequent SCNT embryos.

  11. Lack of effects on key cellular parameters of MRC-5 human lung fibroblasts exposed to 370 mT static magnetic field

    PubMed Central

    Romeo, Stefania; Sannino, Anna; Scarfì, Maria Rosaria; Massa, Rita; d’Angelo, Raffaele; Zeni, Olga

    2016-01-01

    The last decades have seen increased interest toward possible adverse effects arising from exposure to intense static magnetic fields. This concern is mainly due to the wider and wider applications of such fields in industry and clinical practice; among them, Magnetic Resonance Imaging (MRI) facilities are the main sources of exposure to static magnetic fields for both general public (patients) and workers. In recent investigations, exposures to static magnetic fields have been demonstrated to elicit, in different cell models, both permanent and transient modifications in cellular endpoints critical for the carcinogenesis process. The World Health Organization has therefore recommended in vitro investigations as important research need, to be carried out under strictly controlled exposure conditions. Here we report on the absence of effects on cell viability, reactive oxygen species levels and DNA integrity in MRC-5 human foetal lung fibroblasts exposed to 370 mT magnetic induction level, under different exposure regimens. Exposures have been performed by using an experimental apparatus designed and realized for operating with the static magnetic field generated by permanent magnets, and confined in a magnetic circuit, to allow cell cultures exposure in absence of confounding factors like heating or electric field components. PMID:26762783

  12. Lack of effects on key cellular parameters of MRC-5 human lung fibroblasts exposed to 370 mT static magnetic field.

    PubMed

    Romeo, Stefania; Sannino, Anna; Scarfì, Maria Rosaria; Massa, Rita; d'Angelo, Raffaele; Zeni, Olga

    2016-01-14

    The last decades have seen increased interest toward possible adverse effects arising from exposure to intense static magnetic fields. This concern is mainly due to the wider and wider applications of such fields in industry and clinical practice; among them, Magnetic Resonance Imaging (MRI) facilities are the main sources of exposure to static magnetic fields for both general public (patients) and workers. In recent investigations, exposures to static magnetic fields have been demonstrated to elicit, in different cell models, both permanent and transient modifications in cellular endpoints critical for the carcinogenesis process. The World Health Organization has therefore recommended in vitro investigations as important research need, to be carried out under strictly controlled exposure conditions. Here we report on the absence of effects on cell viability, reactive oxygen species levels and DNA integrity in MRC-5 human foetal lung fibroblasts exposed to 370 mT magnetic induction level, under different exposure regimens. Exposures have been performed by using an experimental apparatus designed and realized for operating with the static magnetic field generated by permanent magnets, and confined in a magnetic circuit, to allow cell cultures exposure in absence of confounding factors like heating or electric field components.

  13. Lack of effects on key cellular parameters of MRC-5 human lung fibroblasts exposed to 370 mT static magnetic field

    NASA Astrophysics Data System (ADS)

    Romeo, Stefania; Sannino, Anna; Scarfì, Maria Rosaria; Massa, Rita; D’Angelo, Raffaele; Zeni, Olga

    2016-01-01

    The last decades have seen increased interest toward possible adverse effects arising from exposure to intense static magnetic fields. This concern is mainly due to the wider and wider applications of such fields in industry and clinical practice; among them, Magnetic Resonance Imaging (MRI) facilities are the main sources of exposure to static magnetic fields for both general public (patients) and workers. In recent investigations, exposures to static magnetic fields have been demonstrated to elicit, in different cell models, both permanent and transient modifications in cellular endpoints critical for the carcinogenesis process. The World Health Organization has therefore recommended in vitro investigations as important research need, to be carried out under strictly controlled exposure conditions. Here we report on the absence of effects on cell viability, reactive oxygen species levels and DNA integrity in MRC-5 human foetal lung fibroblasts exposed to 370 mT magnetic induction level, under different exposure regimens. Exposures have been performed by using an experimental apparatus designed and realized for operating with the static magnetic field generated by permanent magnets, and confined in a magnetic circuit, to allow cell cultures exposure in absence of confounding factors like heating or electric field components.

  14. Lack of WDR36 leads to preimplantation embryonic lethality in mice and delays the formation of small subunit ribosomal RNA in human cells in vitro.

    PubMed

    Gallenberger, Martin; Meinel, Dominik M; Kroeber, Markus; Wegner, Michael; Milkereit, Philipp; Bösl, Michael R; Tamm, Ernst R

    2011-02-01

    Mutations in WD repeat domain 36 gene (WDR36) play a causative role in some forms of primary open-angle glaucoma, a leading cause of blindness worldwide. WDR36 is characterized by the presence of multiple WD40 repeats and shows homology to Utp21, an essential protein component of the yeast small subunit (SSU) processome required for maturation of 18S rRNA. To clarify the functional role of WDR36 in the mammalian organism, we generated and investigated mutant mice with a targeted deletion of Wdr36. In parallel experiments, we used RNA interference to deplete WDR36 mRNA in mouse embryos and cultured human trabecular meshwork (HTM-N) cells. Deletion of Wdr36 in the mouse caused preimplantation embryonic lethality, and essentially similar effects were observed when WDR36 mRNA was depleted in mouse embryos by RNA interference. Depletion of WDR36 mRNA in HTM-N cells caused apoptotic cell death and upregulation of mRNA for BAX, TP53 and CDKN1A. By immunocytochemistry, staining for WDR36 was observed in the nucleolus of cells, which co-localized with that of nucleolar proteins such as nucleophosmin and PWP2. In addition, recombinant and epitope-tagged WDR36 localized to the nucleolus of HTM-N cells. By northern blot analysis, a substantial decrease in 21S rRNA, the precursor of 18S rRNA, was observed following knockdown of WDR36. In addition, metabolic-labeling experiments consistently showed a delay of 18S rRNA maturation in WDR36-depleted cells. Our results provide evidence that WDR36 is an essential protein in mammalian cells which is involved in the nucleolar processing of SSU 18S rRNA.

  15. Cell competition in mouse NIH3T3 embryonic fibroblasts is controlled by the activity of Tead family proteins and Myc.

    PubMed

    Mamada, Hiroshi; Sato, Takashi; Ota, Mitsunori; Sasaki, Hiroshi

    2015-02-15

    Cell competition is a short-range communication originally observed in Drosophila. Relatively little is known about cell competition in mammals or in non-epithelial cells. Hippo signaling and its downstream transcription factors of the Tead family, control cell proliferation and apoptosis. Here, we established an in vitro model system that shows cell competition in mouse NIH3T3 embryo fibroblast cells. Co-culture of Tead-activity-manipulated cells with normal (wild-type) cells caused cell competition. Cells with reduced Tead activity became losers, whereas cells with increased Tead activity became super-competitors. Tead directly regulated Myc RNA expression, and cells with increased Myc expression also became super-competitors. At low cell density, cell proliferation required both Tead activity and Myc. At high cell density, however, reduction of either Tead activity or Myc was compensated for by an increase in the other, and this increase was sufficient to confer 'winner' activity. Collectively, NIH3T3 cells have cell competition mechanisms similar to those regulated by Yki and Myc in Drosophila. Establishment of this in vitro model system should be useful for analyses of the mechanisms of cell competition in mammals and in fibroblasts. © 2015. Published by The Company of Biologists Ltd.

  16. [Rosiglitazone inhibited the transformation and synthesis of collagen in rats' embryonic lung fibroblasts through the modulation of plasminogen activator inhibitor-1].

    PubMed

    Zhang, Yanping; Bai, Linlin; Ma, Lihua; Zhao, Jing; Li, Na; Li, Wenbin

    2016-03-01

    To investigate the role of plasminogen activator inhibitor-1 (PAI-1) in the inhibition of rosiglitazone on the transformation and collagen synthesis of rats' embryo lung fibroblasts and to examine the signal pathways in the process. Fibroblasts from rats' embryo lung tissues were divided into 3 groups: Control, Rosiglitazone (Rosiglitazone 30 mmol/L) and PAI-1 (Rosiglitazone 30 mmol/L and PAI-1 20 mmol/L) groups. The fibroblasts were collected at 24, 48 and 72 h, and stored at -80 °C. RT-PCR was used to determine the expressions of collagen type-1 and type-3 at 24 h. Western blot analysis was used to determine the expressions of PAI-1, a-smooth muscle actin (α-SMA), p-AKT and p-ERK at 24, 48 and 72 h. There was a significant decrease in the protein expression of PAI-1 in the Rosiglitazone group (0.732±0.015, 0.583±0.005, 0.762±0.032) at 24, 48 and 72 h compared with the Control group (1.116±0.046). There was a significant increase in the protein expression in the PAI-1 group (0.923±0.042, 1.024±0.009, 1.070±0.011) compared with the Rosiglitazone group (F=78.609, P<0.01). The α-SMA protein expressions were significantly reduced in the Rosiglitazone group (0.209±0.012, 0.280±0.140, 0.254±0.025) compared with the Control group (0.340±0.026), while the expressions were significantly increased in the PAI-1 group (0.386±0.042, 0.400±0.037, 0.385±0.026) compared with the Rosiglitazone group (F=35.009, P<0.01). The collagen type-1 (1.065±0.004) and type-3 (1.282±0.001) mRNA expressions were significantly reduced in the Rosiglitazone group compared with the Control group (1.279±0.013, 1.690±0.005), while the expressions were significantly increased in the PAI-1 groups (1.390±0.029, 1.350±0.044) compared with the Rosiglitazone group (type-1: F=12.429, P<0.01; type-3: F=127.456, P<0.01). The expressions of p-AKT showed no differences among the 3 groups, but there were significant differences in the expressions of p-ERK in the Rosiglitazone group (0.288

  17. Combined Effects of High-Dose Bisphenol A and Oxidizing Agent (KBrO3) on Cellular Microenvironment, Gene Expression, and Chromatin Structure of Ku70-deficient Mouse Embryonic Fibroblasts

    PubMed Central

    Gassman, Natalie R.; Coskun, Erdem; Jaruga, Pawel; Dizdaroglu, Miral; Wilson, Samuel H.

    2016-01-01

    Background: Exposure to bisphenol A (BPA) has been reported to alter global gene expression, induce epigenetic modifications, and interfere with complex regulatory networks of cells. In addition to these reprogramming events, we have demonstrated that BPA exposure generates reactive oxygen species and promotes cellular survival when co-exposed with the oxidizing agent potassium bromate (KBrO3). Objectives: We determined the cellular microenvironment changes induced by co-exposure of BPA and KBrO3 versus either agent alone. Methods: Ku70-deficient cells were exposed to 150 μM BPA, 20 mM KBrO3, or co-exposed to both agents. Four and 24 hr post-damage initiation by KBrO3, with BPA-only samples timed to coincide with these designated time points, we performed whole-genome microarray analysis and evaluated chromatin structure, DNA lesion load, glutathione content, and intracellular pH. Results: We found that 4 hr post-damage initiation, BPA exposure and co-exposure transiently condensed chromatin compared with untreated and KBrO3-only treated cells; the transcription of DNA repair proteins was also reduced. At this time point, BPA exposure and co-exposure also reduced the change in intracellular pH observed after treatment with KBrO3 alone. Twenty-four hours post-damage initiation, BPA-exposed cells showed less condensed chromatin than cells treated with KBrO3 alone; the intracellular pH of the co-exposed cells was significantly reduced compared with untreated and KBrO3-treated cells; and significant up-regulation of DNA repair proteins was observed after co-exposure. Conclusion: These results support the induction of an adaptive response by BPA co-exposure that alters the microcellular environment and modulates DNA repair. Further work is required to determine whether BPA induces similar DNA lesions in vivo at environmentally relevant doses; however, in the Ku70-deficient mouse embryonic fibroblasts, exposure to a high dose of BPA was associated with changes in the

  18. Multiple roles of Activin/Nodal, bone morphogenetic protein, fibroblast growth factor and Wnt/β-catenin signalling in the anterior neural patterning of adherent human embryonic stem cell cultures

    PubMed Central

    Lupo, Giuseppe; Novorol, Claire; Smith, Joseph R.; Vallier, Ludovic; Miranda, Elena; Alexander, Morgan; Biagioni, Stefano; Pedersen, Roger A.; Harris, William A.

    2013-01-01

    Several studies have successfully produced a variety of neural cell types from human embryonic stem cells (hESCs), but there has been limited systematic analysis of how different regional identities are established using well-defined differentiation conditions. We have used adherent, chemically defined cultures to analyse the roles of Activin/Nodal, bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wnt/β-catenin signalling in neural induction, anteroposterior patterning and eye field specification in hESCs. We show that either BMP inhibition or activation of FGF signalling is required for effective neural induction, but these two pathways have distinct outcomes on rostrocaudal patterning. While BMP inhibition leads to specification of forebrain/midbrain positional identities, FGF-dependent neural induction is associated with strong posteriorization towards hindbrain/spinal cord fates. We also demonstrate that Wnt/β-catenin signalling is activated during neural induction and promotes acquisition of neural fates posterior to forebrain. Therefore, inhibition of this pathway is needed for efficient forebrain specification. Finally, we provide evidence that the levels of Activin/Nodal and BMP signalling have a marked influence on further forebrain patterning and that constitutive inhibition of these pathways represses expression of eye field genes. These results show that the key mechanisms controlling neural patterning in model vertebrate species are preserved in adherent, chemically defined hESC cultures and reveal new insights into the signals regulating eye field specification. PMID:23576785

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

    PubMed

    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.

  20. Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line

    PubMed Central

    Czyrek, Aleksandra; Basinska, Katarzyna; Trynda, Justyna; Skaradzińska, Aneta; Siudzińska, Anna; Marycz, Krzysztof

    2015-01-01

    Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs) and Balb/3T3 fibroblast at concentration of 1 mM, 5 mM, and 10 mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2) signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10 mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5 mM and increased after 10 mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure. PMID:26064951

  1. Cellular pharmacokinetics of telavancin, a novel lipoglycopeptide antibiotic, and analysis of lysosomal changes in cultured eukaryotic cells (J774 mouse macrophages and rat embryonic fibroblasts)

    PubMed Central

    Barcia-Macay, Maritza; Mouaden, Fatima; Mingeot-Leclercq, Marie-Paule; Tulkens, Paul M.; Van Bambeke, Françoise

    2008-01-01

    Background Telavancin is a lipoglycopeptide with multiple mechanisms of action that include membrane-destabilizing effects towards bacterial cells. It shows bactericidal activity against forms of Staphylococcus aureus (phagolysosomal infection) with different resistance phenotypes [methicillin-resistant S. aureus, vancomycin-intermediate S. aureus or vancomycin-resistant S. aureus]. We examine here the uptake, efflux and intracellular distribution of telavancin in eukaryotic cells as well as its potential to induce lysosomal changes (in comparison with vancomycin and oritavancin). Methods J774 macrophages and rat embryo fibroblasts were exposed for up to 24 and 72 h to telavancin (5–90 mg/L). The following studies were performed: measurement of 14C-labelled telavancin cellular uptake and subcellular distribution (cell fractionation), determination of pericellular membrane integrity (lactate dehydrogenase release), electron microscopy with morphometric analysis of changes in lysosome size and determination of total phospholipid and cholesterol content. Results The uptake of telavancin proceeded linearly as a function of time and concentration in both cell types (clearance rate of ∼10 mL/g of protein/h). Efflux (macrophages) was ∼5.7-fold slower. Telavancin subcellular distribution was superimposable on that of a lysosomal marker (N-acetyl-β-hexosaminidase). It did not cause an increase in the release of lactate dehydrogenase and did not induce significant increases in total phospholipid or cholesterol content. It caused only mild morphological lysosomal alterations (similar to vancomycin and much less than oritavancin by morphometric analysis). Conclusions Telavancin is taken up by eukaryotic cells and localizes in lysosomes, causing mild morphological alterations without evidence of lipid metabolism alterations. These data support our observations that telavancin is active against intracellular S. aureus. PMID:18375379

  2. Disruption of fibroblast growth factor receptor signaling in bovine cumulus-oocyte complexes during in vitro maturation reduces subsequent embryonic development.

    PubMed

    Zhang, K; Ealy, A D

    2012-05-01

    Several fibroblast growth factors (FGF) mediate folliculogenesis and oogenesis in cattle but it is unclear whether FGFs are required during the final stages of oocyte maturation. The objectives of this work were to determine whether blocking FGF receptor (FGFR) activity during in vitro maturation (IVM) affects oocyte fertilization and embryo development; examine changes in FGFR transcript profiles in cumulus cells and oocytes during IVM; and evaluate whether gonadotropins modulate FGFR transcript abundance during IVM. In the first set of studies, bovine cumulus-oocyte complexes (COCs) were matured in the presence of one of two FGFR kinase inhibitors (SU5402 or PD173074). After maturation, COCs were washed and cultured without inhibitors. Inhibitors did not affect cleavage rates but the percentage of ≥ 8-cell embryos at d 3 and blastocysts at d 7 and d 8 postfertilization were decreased when COCs were matured with either inhibitor. Profiles of FGFR mRNA variants were examined in cumulus cells and oocytes separated either immediately before (0 h) or at 6 or 21 h after beginning IVM. In cumulus cells, increases in R1b, R2b, and R2c abundance were detected when cultured in the absence of follicle-stimulating hormone (FSH). Supplementing FSH (1 or 25 μM) increased the abundance of R1b, R1c, R2b, and R2c. In oocytes, no time- or FSH-dependent changes in FGFR transcript abundance were detected. These observations implicate FGFs as crucial components of bovine oocyte competency and indicate that FSH augments FGFR mRNA abundance in cumulus cells during the final stages of oocyte maturation.

  3. Site-specific interactions of neurotrophin-3 and fibroblast growth factor (FGF2) in the embryonic development of the mouse cochlear nucleus.

    PubMed

    Hossain, Waheeda A; D'Sa, Chrystal; Morest, D Kent

    2006-08-01

    Neurotrophins and FGF2 contribute to formation of the cochlea, but their roles in cochlear nucleus development are unknown. The effects of these factors may differ in the cochlea and cochlear nucleus, which may influence each other's development. It is important to analyze the effects of these factors on cellular structures at well-defined steps in the normal morphogenetic sequence. The present study used immunohistochemistry to localize factors in situ and to test hypotheses about their roles in an in vitro model. Specific antibody staining revealed that TrkC, the NT3 receptor, is present in neural precursors prior to embryonic day E11 until after birth. NT3 appeared in precursor cells during migration (E13-E15) and disappeared at birth. TrkC and NT3 occurred in the same structures, including growing axons, terminals, and their synaptic targets. Thus, NT3 tracks the migration routes and the morphogenetic sequences within a window defined by TrkC. In vitro, the cochlear nucleus anlage was explanted from E11 embryos. Cultures were divided into groups fed with defined medium, with or without FGF2, BDNF, and NT3 supplements, alone or in combinations, for 7 days. When neuroblasts migrated and differentiated, immunostaining was used for locating NT3 and TrkC in the morphogenetic sequence, bromodeoxyuridine for proliferation, and synaptic vesicle protein for synaptogenesis. By time-lapse imaging and quantitative measures, the results support the hypothesis that FGF2 promotes proliferation and migration. NT3 interacts with FGF2 and BDNF to promote neurite outgrowth, fasciculation, and synapse formation. Factors and receptors localize to the structural sites undergoing critical changes.

  4. 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*

    PubMed Central

    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

    2014-01-01

    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. PMID:24398673

  5. The absence of Ser389 phosphorylation in p53 affects the basal gene expression level of many p53-dependent genes and alters the biphasic response to UV exposure in mouse embryonic fibroblasts.

    PubMed

    Bruins, Wendy; Bruning, Oskar; Jonker, Martijs J; Zwart, Edwin; van der Hoeven, Tessa V; Pennings, Jeroen L A; Rauwerda, Han; de Vries, Annemieke; Breit, Timo M

    2008-03-01

    Phosphorylation is important in p53-mediated DNA damage responses. After UV irradiation, p53 is phosphorylated specifically at murine residue Ser389. Phosphorylation mutant p53.S389A cells and mice show reduced apoptosis and compromised tumor suppression after UV irradiation. We investigated the underlying cellular processes by time-series analysis of UV-induced gene expression responses in wild-type, p53.S389A, and p53(-/-) mouse embryonic fibroblasts. The absence of p53.S389 phosphorylation already causes small endogenous gene expression changes for 2,253, mostly p53-dependent, genes. These genes showed basal gene expression levels intermediate to the wild type and p53(-/-), possibly to readjust the p53 network. Overall, the p53.S389A mutation lifts p53-dependent gene repression to a level similar to that of p53(-/-) but has lesser effect on p53-dependently induced genes. In the wild type, the response of 6,058 genes to UV irradiation was strictly biphasic. The early stress response, from 0 to 3 h, results in the activation of processes to prevent the accumulation of DNA damage in cells, whereas the late response, from 12 to 24 h, relates more to reentering the cell cycle. Although the p53.S389A UV gene response was only subtly changed, many cellular processes were significantly affected. The early response was affected the most, and many cellular processes were phase-specifically lost, gained, or altered, e.g., induction of apoptosis, cell division, and DNA repair, respectively. Altogether, p53.S389 phosphorylation seems essential for many p53 target genes and p53-dependent processes.

  6. cDNA sequence of a human skeletal muscle ADP/ATP translocator: lack of a leader peptide, divergence from a fibroblast translocator cDNA, and coevolution with mitochondrial DNA genes

    SciTech Connect

    Neckelmann, N.; Li, K.; Wade, R.P.; Shuster, R.; Wallace, D.C.

    1987-11-01

    The authors have characterized a 1400-nucleotide cDNA for the human skeletal muscle ADP/ATP translocator. The deduced amino acid sequence is 94% homologous to the beef heart ADP/ATP translocator protein and contains only a single additional amino-terminal methionine. This implies that the human translocator lacks an amino-terminal targeting peptide, a conclusion substantiated by measuring the molecular weight of the protein synthesized in vitro. A 1400-nucleotide transcript encoding the skeletal muscle translocator was detected on blots of total RNA from human heart, kidney, skeletal muscle, and HeLa cells by hybridization with oligonucleotide probes homologous to the coding region and 3' noncoding region of the cDNA. However, the level of this mRNA varied substantially among tissues. Comparison of our skeletal muscle translocator sequence with that of a recently published human fibroblast translocator cognate revealed that the two proteins are 88% identical and diverged about 275 million years ago. Hence, tissues vary both in the level of expression of individual translocator genes and in differential expression of cognate translocator genes. Comparison of the base substitution rates of the ADP/ATP translocator and the oxidative phosphorylation genes encoded by mitochondrial DNA revealed that the mitochondrial DNA genes fix 10 times more synonymous substitutions and 12 times more replacement substitutions; yet, these nuclear and cytoplasmic respiration genes experience comparable evolutionary constraints. This suggest that the mitochondrial DNA genes are highly prone to deleterious mutations.

  7. Neurons generated from APP/APLP1/APLP2 triple knockout embryonic stem cells behave normally in vitro and in vivo: lack of evidence for a cell autonomous role of the amyloid precursor protein in neuronal differentiation.

    PubMed

    Bergmans, Bruno A; Shariati, S Ali M; Habets, Ron L P; Verstreken, Patrik; Schoonjans, Luc; Müller, Ulrike; Dotti, Carlos G; De Strooper, Bart

    2010-03-31

    Alzheimer's disease amyloid precursor protein (APP) has been implicated in many neurobiologic processes, but supporting evidence remains indirect. Studies are confounded by the existence of two partially redundant APP homologues, APLP1 and APLP2. APP/APLP1/APLP2 triple knockout (APP tKO) mice display cobblestone lissencephaly and are perinatally lethal. To circumvent this problem, we generated APP triple knockout embryonic stem (ES) cells and differentiated these to APP triple knockout neurons in vitro and in vivo. In comparison with wild-type (WT) ES cell-derived neurons, APP tKO neurons formed equally pure neuronal cultures, had unaltered in vitro migratory capacities, had a similar acquisition of polarity, and were capable of extending long neurites and forming active excitatory synapses. These data were confirmed in vivo in chimeric mice with APP tKO neurons expressing the enhanced green fluorescent protein (eGFP) present in a WT background brain. The results suggest that the loss of the APP family of proteins has no major effect on these critical neuronal processes and that the apparent multitude of functions in which APP has been implicated might be characterized by molecular redundancy. Our stem cell culture provides an excellent tool to circumvent the problem of lack of viability of APP/APLP triple knockout mice and will help to explore the function of this intriguing protein further in vitro and in vivo.

  8. FGF-16 is required for embryonic heart development

    PubMed Central

    Lu, Shun Yan; Sheikh, Farah; Sheppard, Patricia C.; Fresnoza, Agnes; Duckworth, Mary Lynn; Detillieux, Karen A.; Cattini, Peter A.

    2016-01-01

    Fibroblast growth factor 16 (FGF-16) expression has previously been detected in mouse heart at mid-gestation in the endocardium and epicardium, suggesting a role in embryonic heart development. More specifically, exogenously applied FGF-16 has been shown to stimulate growth of embryonic myocardial cells in tissue explants. We have generated mice lacking FGF-16 by targeting the Fgf16 locus on the X chromosome. Elimination of Fgf16 expression resulted in embryonic death as early as day 11.5 (E11.5). External abnormalities, including hemorrhage in the heart and ventral body region as well as facial defects, began to appear in null embryos from E11.5. Morphological analysis of FGF-16 null hearts revealed cardiac defects including chamber dilation, thinning of the atrial and ventricular walls, and poor trabeculation, which were visible at E10.5 and more pronounced at E11.5. These findings indicate FGF-16 is required for embryonic heart development in mid-gestation through its positive effect on myocardial growth. PMID:18565327

  9. Embryonic cerebrospinal fluid collaborates with the isthmic organizer to regulate mesencephalic gene expression.

    PubMed

    Parada, Carolina; Martín, Cristina; Alonso, María I; Moro, José A; Bueno, David; Gato, Angel

    2005-11-01

    Early in development, the behavior of neuroepithelial cells is controlled by several factors acting in a developmentally regulated manner. Recently it has been shown that diffusible factors contained within embryonic cerebrospinal fluid (CSF) promote neuroepithelial cell survival, proliferation, and neurogenesis in mesencephalic explants lacking any known organizing center. In this paper, we show that mesencephalic and mesencephalic+isthmic organizer explants cultured only with basal medium do not express the typically expressed mesencephalic or isthmic organizer genes analyzed (otx2 and fgf8, respectively) and that mesencephalic explants cultured with embryonic CSF-supplemented medium do effect such expression, although they exhibit an altered pattern of gene expression, including ectopic shh expression domains. Other trophic sources that are able to maintain normal neuroepithelial cell behavior, i.e., fibroblast growth factor-2, fail to activate this ectopic shh expression. Conversely, the expression pattern of the analyzed genes in mesencephalic+isthmic organizer explants cultured with embryonic cerebrospinal fluid-supplemented medium mimics the pattern for control embryos developed in ovo. We demonstrate that embryonic CSF collaborates with the isthmic organizer in regulation of the expression pattern of some characteristic neuroectodermal genes during early stages of central nervous system (CNS) development, and we suggest that this collaboration is not restricted to the maintenance of neuroepithelial cell survival. Data reported in this paper corroborate the hypothesis that factors contained within embryonic CSF contribute to the patterning of the CNS during early embryonic development.

  10. Lack of CAK complex accumulation at DNA damage sites in XP-B and XP-B/CS fibroblasts reveals differential regulation of CAK anchoring to core TFIIH by XPB and XPD helicases during nucleotide excision repair

    PubMed Central

    Zhu, Qianzheng; Wani, Gulzar; Sharma, Nidhi; Wani, Altaf

    2012-01-01

    Transcription factor II H (TFIIH) is composed of core TFIIH and Cdk-activating kinase (CAK) complexes. Besides transcription, TFIIH also participates in nucleotide excision repair (NER), verifying DNA lesions through its helicase components XPB and XPD. The assembly state of TFIIH is known to be affected by truncation mutations in Xeroderma pigmentosum group G/Cockayne syndrome (XP-G/CS). Here, we showed that CAK component MAT1 was rapidly recruited to UV-induced DNA damage sites, co-localizing with core TFIIH component p62, and dispersed from the damage sites upon completion of DNA repair. While the core TFIIH-CAK association remained intact, MAT1 failed to accumulate at DNA damage sites in fibroblasts harboring XP-B or XP-B/CS mutations. Nevertheless, MAT1, XPD and XPC as well as XPG were able to accumulate at damage sites in XP-D fibroblasts, in which the core TFIIH-CAK association also remained intact. Interestingly, XPG recruitment was impaired in XP-B/CS fibroblasts derived from patients with mild phenotype, but persisted in XP-B/CS fibroblasts from severely affected patients resulting in a nonfunctional preincision complex. An examination of steady-state levels of RNA polymerase II (RNAPII) indicated that UV-induced RNAPII phosphorylation was dramatically reduced in XP-B/CS fibroblasts. These results demonstrated that the CAK rapidly disassociates from the core TFIIH upon assembly of nonfunctional preincision complex in XP-B and XP-B/CS cells. The persistency of nonfunctional preincision complex correlates with the severity exhibited by XP-B patients. The results suggest that XPB and XPD helicases differentially regulate the anchoring of CAK to core TFIIH during damage verification step of NER. PMID:23083890

  11. Cardiac fibroblast in development and wound healing.

    PubMed

    Deb, Arjun; Ubil, Eric

    2014-05-01

    Cardiac fibroblasts are the most abundant cell type in the mammalian heart and comprise approximately two-thirds of the total number of cardiac cell types. During development, epicardial cells undergo epithelial-mesenchymal-transition to generate cardiac fibroblasts that subsequently migrate into the developing myocardium to become resident cardiac fibroblasts. Fibroblasts form a structural scaffold for the attachment of cardiac cell types during development, express growth factors and cytokines and regulate proliferation of embryonic cardiomyocytes. In post natal life, cardiac fibroblasts play a critical role in orchestrating an injury response. Fibroblast activation and proliferation early after cardiac injury are critical for maintaining cardiac integrity and function, while the persistence of fibroblasts long after injury leads to chronic scarring and adverse ventricular remodeling. In this review, we discuss the physiologic function of the fibroblast during cardiac development and wound healing, molecular mediators of activation that could be possible targets for drug development for fibrosis and finally the use of reprogramming technologies for reversing scar. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium." Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. PIWI Proteins Are Dispensable for Mouse Somatic Development and Reprogramming of Fibroblasts into Pluripotent Stem Cells

    PubMed Central

    Cheng, Ee-Chun; Kang, Dongwan; Wang, Zhong; Lin, Haifan

    2014-01-01

    PIWI proteins play essential and conserved roles in germline development, including germline stem cell maintenance and meiosis. Because germline regulators such as OCT4, NANOG, and SOX2 are known to be potent factors that reprogram differentiated somatic cells into induced pluripotent stem cells (iPSCs), we investigated whether the PIWI protein family is involved in iPSC production. We find that all three mouse Piwi genes, Miwi, Mili, and Miwi2, are expressed in embryonic stem cells (ESCs) at higher levels than in fibroblasts, with Mili being the highest. However, mice lacking all three Piwi genes are viable and female fertile, and are only male sterile. Furthermore, embryonic fibroblasts derived from Miwi/Mili/Miwi2 triple knockout embryos can be efficiently reprogrammed into iPS cells. These iPS cells expressed pluripotency markers and were capable of differentiating into all three germ layers in teratoma assays. Genome-wide expression profiling reveals that the triple knockout iPS cells are very similar to littermate control iPS cells. These results indicate that PIWI proteins are dispensable for direct reprogramming of mouse fibroblasts. PMID:25238487

  13. Extinction of Oct-3/4 gene expression in embryonal carcinoma [times] fibroblast somatic cell hybrids is accompanied by changes in the methylation status, chromatin structure, and transcriptional activity of the Oct-3/4 upstream region

    SciTech Connect

    Ben-Shushan, E.; Pikarsky, E.; Klar, A.; Bergman, Y. )

    1993-02-01

    The OCT-3/4 gene provides an excellent model system with which to study the extinction phenomenon in somatic cell hybrids. The molecular mechanism that underlies the extinction of a tissue-specific transcription factor in somatic cell hybrides is evaluated and compared with its down-regulation in retinoic acid treated embryonal carcinoma cells. This study draws a connection between the shutdown of OCT-3/4 expression in retinoic acid (RA)-differentiated embryonal carcinoma (EC) cells and its extinction in hybrid cells. This repression of OCT-3/4 expression is achieved through changes in the methylation status, chromatin structure, and transcriptional activity of the OCT-3/4 upstream regulatory region. 59 refs.

  14. Herpesvirus-Associated Lymphadenitis Distorts Fibroblastic Reticular Cell Microarchitecture and Attenuates CD8 T Cell Responses to Neurotropic Infection in Mice Lacking the STING-IFNα/β Defense Pathways.

    PubMed

    Royer, Derek J; Conrady, Christopher D; Carr, Daniel J J

    2016-09-15

    Type I IFN (IFN-α/β)-driven immune responses to acute viral infection are critical to counter replication and prevent dissemination. However, the mechanisms underlying host resistance to HSV type 1 (HSV-1) are incompletely understood. In this study, we show that mice with deficiencies in IFN-α/β signaling or stimulator of IFN genes (STING) exhibit exacerbated neurovirulence and atypical lymphotropic dissemination of HSV-1 following ocular infection. Synergy between IFN-α/β signaling and efficacy of early adaptive immune responses to HSV-1 were dissected using bone marrow chimeras and adoptive cell transfer approaches to profile clonal expansion, effector function, and recruitment of HSV-specific CD8(+) T cells. Lymphotropic viral dissemination was commensurate with abrogated CD8(+) T cell responses and pathological alterations of fibroblastic reticular cell networks in the draining lymph nodes. Our results show that resistance to HSV-1 in the trigeminal ganglia during acute infection is conferred in part by STING and IFN-α/β signaling in both bone marrow-derived and -resident cells, which coalesce to support a robust HSV-1-specific CD8(+) T cell response. Copyright © 2016 by The American Association of Immunologists, Inc.

  15. Herpesvirus-associated lymphadenitis distorts fibroblastic reticular cell microarchitecture and attenuates CD8 T cell responses to neurotropic infection in mice lacking the STING-IFNα/β defense pathways1

    PubMed Central

    Royer, Derek J.; Conrady, Christopher D.; Carr, Daniel J.J.

    2016-01-01

    Type I interferon (IFNα/β)-driven immune responses to acute viral infection are critical to counter replication and prevent dissemination. However, the mechanisms underlying host resistance to herpes simplex virus type 1 (HSV-1) are incompletely understood. Here we show that mice with deficiencies in IFNα/β signaling or STING exhibit exacerbated neurovirulence and atypical lymphotropic dissemination of HSV-1 following ocular infection. Synergy between IFNα/β signaling and efficacy of early adaptive immune responses to HSV-1 were dissected using bone marrow chimeras and adoptive cell transfer approaches to profile clonal expansion, effector function, and recruitment of HSV-specific CD8+ T cells. Lymphotropic viral dissemination was commensurate with abrogated CD8+ T cell responses and pathological alterations of fibroblastic reticular cell (FRC) networks in the draining lymph nodes. Our results show that resistance to HSV-1 in the TG during acute infection is conferred in part by STING and IFNα/β-signaling in both bone marrow-derived and resident cells, which coalesce to support a robust HSV-1-specific CD8+ T cell response. PMID:27511736

  16. Embryonic Stem Cells: Isolation, Characterization and Culture

    NASA Astrophysics Data System (ADS)

    Amit, Michal; Itskovitz-Eldor, Joseph

    Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

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

    PubMed

    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.

  18. Vitamin K2 Biosynthetic Enzyme, UBIAD1 Is Essential for Embryonic Development of Mice

    PubMed Central

    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. PMID:25127365

  19. Myocardial fibroblast-matrix interactions and potential therapeutic targets.

    PubMed

    Goldsmith, Edie C; Bradshaw, Amy D; Zile, Michael R; Spinale, Francis G

    2014-05-01

    The cardiac extracellular matrix (ECM) is a dynamic structure, adapting to physiological and pathological stresses placed on the myocardium. Deposition and organization of the matrix fall under the purview of cardiac fibroblasts. While often overlooked compared to myocytes, fibroblasts play a critical role in maintaining ECM homeostasis under normal conditions and in response to pathological stimuli assume an activated, myofibroblast phenotype associated with excessive collagen accumulation contributing to impaired cardiac function. Complete appreciation of fibroblast function is hampered by the lack of fibroblast-specific reagents and the heterogeneity of fibroblast precursors. This is further complicated by our ability to dissect the role of myofibroblasts versus fibroblasts in myocardial in remodeling. This review highlights critical points in the regulation of collagen deposition by fibroblasts, the current panel of molecular tools used to identify fibroblasts and the role of fibroblast-matrix interactions in fibroblast function and differentiation into the myofibroblast phenotype. The clinical potential of exploiting differences between fibroblasts and myofibroblasts and using them to target specific fibroblast populations is also discussed. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium." Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Ganglioside synthase knockout in oncogene-transformed fibroblasts depletes gangliosides and impairs tumor growth.

    PubMed

    Liu, Y; Yan, S; Wondimu, A; Bob, D; Weiss, M; Sliwinski, K; Villar, J; Notario, V; Sutherland, M; Colberg-Poley, A M; Ladisch, S

    2010-06-03

    Biologically active membrane gangliosides, expressed and released by many human tumors, are hypothesized to significantly impact tumor progression. Lack of a model of complete and specific tumor ganglioside depletion in vivo, however, has hampered elucidation of their role. Here, we report the creation of a novel, stable, genetically induced tumor cell system resulting in specific and complete blockade of ganglioside synthesis. Wild-type (WT) and GM3 synthase/GM2 synthase double knockout (DKO) murine embryonic fibroblasts were transformed using amphotropic retrovirus-transduced oncogenes (pBABE-c-Myc(T58A)+H-RasG12V). The transformed cells, WT(t) and DKO(t) respectively, evidenced comparable integrated copy numbers and oncogene expression. Ganglioside synthesis was completely blocked in the DKO(t) cells, importantly without triggering an alternate pathway of ganglioside synthesis. Ganglioside depletion (to <0.5 nmol/10(7) cells from 9 to 11 nmol/10(7) WT(t) or untransfected normal fibroblasts) did not adversely affect cell proliferation kinetics but did reduce cell migration on fibronectin-coated wells, consistent with our previous observations in ganglioside-depleted normal human fibroblasts. Strikingly, despite similar oncogene expression and growth kinetics, DKO(t) cells evidenced significantly impaired tumor growth in syngeneic immunocompetent mice, underscoring the pivotal role of tumor cell gangliosides and providing an ideal system for probing their mechanisms of action in vivo.

  1. Ganglioside synthase knockout in oncogene-transformed fibroblasts depletes gangliosides and impairs tumor growth

    PubMed Central

    Liu, Yihui; Yan, Su; Wondimu, Assefa; Bob, Daniel; Weiss, Michael; Sliwinski, Konrad; Villar, Joaquín; Notario, Vicente; Sutherland, Margaret; Colberg-Poley, Anamaris M.; Ladisch, Stephan

    2010-01-01

    Biologically active membrane gangliosides, expressed and released by many human tumors, are hypothesized to significantly impact tumor progression. Lack of a model of complete and specific tumor ganglioside depletion in vivo, however, has hampered elucidation of their role. Here we report the creation of a novel, stable, genetically induced tumor cell system resulting in specific and complete blockade of ganglioside synthesis. Wild type (WT) and GM3 synthase/GM2 synthase double knockout (DKO) murine embryonic fibroblasts were transformed using amphotropic retrovirus-transduced oncogenes (pBABE-c-MycT58A+H-RasG12V). The transformed cells, WTt and DKOt respectively, evidenced comparable integrated copy numbers and oncogene expression. Ganglioside synthesis was completely blocked in the DKOt cells, importantly without triggering an alternate pathway of ganglioside synthesis. Ganglioside depletion (to <0.5 nmol/107 cells from 9-11 nmol/107 WTt or untransfected normal fibroblasts) did not adversely affect cell proliferation kinetics but did reduce cell migration on fibronectin-coated wells, consistent with our previous observations in ganglioside-depleted normal human fibroblasts. Strikingly, despite similar oncogene expression and growth kinetics, DKOt cells evidenced significantly impaired tumor growth in syngeneic immunocompetent mice, underscoring the pivotal role of tumor cell gangliosides and providing an ideal system for probing their mechanisms of action in vivo. PMID:20305696

  2. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

    PubMed Central

    Akopian, Veronika; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J.; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B.; Ludwig, Tenneille E.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi

    2010-01-01

    There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

  3. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells.

    PubMed

    Akopian, Veronika; Andrews, Peter W; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B; Ludwig, Tenneille E; McKay, Ronald D G; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K W; Pera, Martin F; Rossant, Janet; Stacey, Glyn N; Suemori, Hirofumi

    2010-04-01

    There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.

  4. Mitochondrial fission factor Drp1 is essential for embryonic development and synapse formation in mice.

    PubMed

    Ishihara, Naotada; Nomura, Masatoshi; Jofuku, Akihiro; Kato, Hiroki; Suzuki, Satoshi O; Masuda, Keiji; Otera, Hidenori; Nakanishi, Yae; Nonaka, Ikuya; Goto, Yu-Ichi; Taguchi, Naoko; Morinaga, Hidetaka; Maeda, Maki; Takayanagi, Ryoichi; Yokota, Sadaki; Mihara, Katsuyoshi

    2009-08-01

    Mitochondrial morphology is dynamically controlled by a balance between fusion and fission. The physiological importance of mitochondrial fission in vertebrates is less clearly defined than that of mitochondrial fusion. Here we show that mice lacking the mitochondrial fission GTPase Drp1 have developmental abnormalities, particularly in the forebrain, and die after embryonic day 12.5. Neural cell-specific (NS) Drp1(-/-) mice die shortly after birth as a result of brain hypoplasia with apoptosis. Primary culture of NS-Drp1(-/-) mouse forebrain showed a decreased number of neurites and defective synapse formation, thought to be due to aggregated mitochondria that failed to distribute properly within the cell processes. These defects were reflected by abnormal forebrain development and highlight the importance of Drp1-dependent mitochondrial fission within highly polarized cells such as neurons. Moreover, Drp1(-/-) murine embryonic fibroblasts and embryonic stem cells revealed that Drp1 is required for a normal rate of cytochrome c release and caspase activation during apoptosis, although mitochondrial outer membrane permeabilization, as examined by the release of Smac/Diablo and Tim8a, may occur independently of Drp1 activity.

  5. The tumor suppressor PML specifically accumulates at RPA/Rad51-containing DNA damage repair foci but is nonessential for DNA damage-induced fibroblast senescence.

    PubMed

    Münch, Sandra; Weidtkamp-Peters, Stefanie; Klement, Karolin; Grigaravicius, Paulius; Monajembashi, Shamci; Salomoni, Paolo; Pandolfi, Pier Paolo; Weißhart, Klaus; Hemmerich, Peter

    2014-05-01

    The PML tumor suppressor has been functionally implicated in DNA damage response and cellular senescence. Direct evidence for such a role based on PML knockdown or knockout approaches is still lacking. We have therefore analyzed the irradiation-induced DNA damage response and cellular senescence in human and mouse fibroblasts lacking PML. Our data show that PML nuclear bodies (NBs) nonrandomly associate with persistent DNA damage foci in unperturbed human skin and in high-dose-irradiated cell culture systems. PML bodies do not associate with transient γH2AX foci after low-dose gamma irradiation. Superresolution microscopy reveals that all PML bodies within a nucleus are engaged at Rad51- and RPA-containing repair foci during ongoing DNA repair. The lack of PML (i) does not majorly affect the DNA damage response, (ii) does not alter the efficiency of senescence induction after DNA damage, and (iii) does not affect the proliferative potential of primary mouse embryonic fibroblasts during serial passaging. Thus, while PML NBs specifically accumulate at Rad51/RPA-containing lesions and senescence-derived persistent DNA damage foci, they are not essential for DNA damage-induced and replicative senescence of human and murine fibroblasts.

  6. KDM6 Demethylase Independent Loss of Histone H3 Lysine 27 Trimethylation during Early Embryonic Development

    PubMed Central

    Shpargel, Karl B.; Starmer, Joshua; Yee, Della; Pohlers, Michael; Magnuson, Terry

    2014-01-01

    The early mammalian embryo utilizes histone H3 lysine 27 trimethylation (H3K27me3) to maintain essential developmental genes in a repressive chromatin state. As differentiation progresses, H3K27me3 is removed in a distinct fashion to activate lineage specific patterns of developmental gene expression. These rapid changes in early embryonic chromatin environment are thought to be dependent on H3K27 demethylases. We have taken a mouse genetics approach to remove activity of both H3K27 demethylases of the Kdm6 gene family, Utx (Kdm6a, X-linked gene) and Jmjd3 (Kdm6b, autosomal gene). Male embryos null for active H3K27 demethylation by the Kdm6 gene family survive to term. At mid-gestation, embryos demonstrate proper patterning and activation of Hox genes. These male embryos retain the Y-chromosome UTX homolog, UTY, which cannot demethylate H3K27me3 due to mutations in catalytic site of the Jumonji-C domain. Embryonic stem (ES) cells lacking all enzymatic KDM6 demethylation exhibit a typical decrease in global H3K27me3 levels with differentiation. Retinoic acid differentiations of these ES cells demonstrate loss of H3K27me3 and gain of H3K4me3 to Hox promoters and other transcription factors, and induce expression similar to control cells. A small subset of genes exhibit decreased expression associated with reduction of promoter H3K4me3 and some low-level accumulation of H3K27me3. Finally, Utx and Jmjd3 mutant mouse embryonic fibroblasts (MEFs) demonstrate dramatic loss of H3K27me3 from promoters of several Hox genes and transcription factors. Our results indicate that early embryonic H3K27me3 repression can be alleviated in the absence of active demethylation by the Kdm6 gene family. PMID:25101834

  7. Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally.

    PubMed

    Zhang, Yu; Kwon, Sohee; Yamaguchi, Teppei; Cubizolles, Fabien; Rousseaux, Sophie; Kneissel, Michaela; Cao, Chun; Li, Na; Cheng, Hwei-Ling; Chua, Katrin; Lombard, David; Mizeracki, Adam; Matthias, Gabriele; Alt, Frederick W; Khochbin, Saadi; Matthias, Patrick

    2008-03-01

    Posttranslational modifications play important roles in regulating protein structure and function. Histone deacetylase 6 (HDAC6) is a mostly cytoplasmic class II HDAC, which has a unique structure with two catalytic domains and a domain binding ubiquitin with high affinity. This enzyme was recently identified as a multisubstrate protein deacetylase that can act on acetylated histone tails, alpha-tubulin and Hsp90. To investigate the in vivo functions of HDAC6 and the relevance of tubulin acetylation/deacetylation, we targeted the HDAC6 gene by homologous recombination in embryonic stem cells and generated knockout mice. HDAC6-deficient mice are viable and fertile and show hyperacetylated tubulin in most tissues. The highest level of expression of HDAC6 is seen in the testis, yet development and function of this organ are normal in the absence of HDAC6. Likewise, lymphoid development is normal, but the immune response is moderately affected. Furthermore, the lack of HDAC6 results in a small increase in cancellous bone mineral density, indicating that this deacetylase plays a minor role in bone biology. HDAC6-deficient mouse embryonic fibroblasts show apparently normal microtubule organization and stability and also show increased Hsp90 acetylation correlating with impaired Hsp90 function. Collectively, these data demonstrate that mice survive well without HDAC6 and that tubulin hyperacetylation is not detrimental to normal mammalian development.

  8. ACTION ON FIBROBLASTS OF EXTRACTS OF HOMOLOGOUS AND HETEROLOGOUS TISSUES

    PubMed Central

    Carrel, Alexis; Ebeling, Albert H.

    1923-01-01

    Extracts of homologous adult tissues detemine an increase in the mass of pure cultures of chicken fibroblasts nourished thereon comparable to that resulting from embryonic tissue juice. But the effect of these extracts differs markedly from that of the latter, since cell multiplication does not continue indefinitely. After a few passages, the fibroblasts cultivated in adult tissue extracts grew more slowly than in Tyrode solution. The cytoplasm became dark and filled with fat granules, and death followed. It is possible that the tissues of adult animals contain, as does the serum, substances which are toxic for the homologous cells, and which progressively overcome the effect of the growth-activating substances. The effect of heterologous adult tissue extracts did not differ markedly from that of homologous tissues. The chicken connective tissue increased slightly in mass, but died sooner than the controls in Tyrode solution. By contrast, tissue juices derived from the embryos of mice, guinea pigs, and rabbits acted on chicken fibroblasts in the same manner as chick embryo juices. The increase in mass of the cultures was regular and rapid. They doubled in size every 48 or 72 hours, and the rate of growth did not decrease after 30 days. It appears that embryonic tissue juices are not necessarily toxic for heterologous fibroblasts, and that they can be used in the building up of protoplasm in the tissues of a different species. In experiments made long ago, the action of tissue juice was described as being specific. The premature death of the fibroblasts cultivated in heterologous juices at that time would now appear to have been due to spontaneous changes in the pH and the deterioration that even normal chick embryo juice at a pH of 7.8 undergoes spontaneously. In the recent experiments, when freshly prepared homologous and heterologous juices were used, their action on chicken fibroblasts in pure culture was identical. However, the fibroblasts produced in cultures

  9. Lacking "Lack": A Reply to Joldersma

    ERIC Educational Resources Information Center

    Marshall, James D.

    2007-01-01

    First I would like to thank Clarence Joldersma for his review of our "Poststructuralism, Philosophy, Pedagogy" (Marshall, 2004-PPP). In particular, I would thank him for his opening sentence: "[t]his book is a response to a lack." It is the notion of a lack, noted again later in his review, which I wish to take up mainly in this response. Rather…

  10. Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes

    PubMed Central

    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 iPS 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 with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. Conclusions We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature. PMID:26694224

  11. Comparison of American mink embryonic stem and induced pluripotent stem cell transcriptomes.

    PubMed

    Menzorov, Aleksei G; Matveeva, Natalia M; Markakis, Marios N; Fishman, Venyamin S; Christensen, Knud; Khabarova, Anna A; Pristyazhnyuk, Inna E; Kizilova, Elena A; Cirera, Susanna; Anistoroaei, Razvan; Serov, Oleg L

    2015-01-01

    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. We report the generation of 10 mink ES and 22 iPS 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 with cell types representing all three germ layers. Transcriptome analysis of mink embryonic fibroblasts (EF), two ES and two iPS cell lines allowed us to identify 11831 assembled contigs which were annotated. These led to a number of 6891 unique genes. Of these 3201 were differentially expressed between mink EF and ES cells. We analyzed expression levels of these genes in iPS cell lines. This allowed us to show that 80% of genes were correctly reprogrammed in iPS cells, whereas approximately 6% had an intermediate expression pattern, about 7% were not reprogrammed and about 5% had a "novel" expression pattern. We observed expression of pluripotency marker genes such as Oct4, Sox2 and Rex1 in ES and iPS cell lines with notable exception of Nanog. We had produced and characterized American mink ES and iPS cells. These cells were pluripotent by a number of criteria and iPS cells exhibited effective reprogramming. Interestingly, we had showed lack of Nanog expression and consider it as a species-specific feature.

  12. Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development

    PubMed Central

    Chen, Hsiuchen; Detmer, Scott A.; Ewald, Andrew J.; Griffin, Erik E.; Fraser, Scott E.; Chan, David C.

    2003-01-01

    Mitochondrial morphology is determined by a dynamic equilibrium between organelle fusion and fission, but the significance of these processes in vertebrates is unknown. The mitofusins, Mfn1 and Mfn2, have been shown to affect mitochondrial morphology when overexpressed. We find that mice deficient in either Mfn1 or Mfn2 die in midgestation. However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal. Embryonic fibroblasts lacking Mfn1 or Mfn2 display distinct types of fragmented mitochondria, a phenotype we determine to be due to a severe reduction in mitochondrial fusion. Moreover, we find that Mfn1 and Mfn2 form homotypic and heterotypic complexes and show, by rescue of mutant cells, that the homotypic complexes are functional for fusion. We conclude that Mfn1 and Mfn2 have both redundant and distinct functions and act in three separate molecular complexes to promote mitochondrial fusion. Strikingly, a subset of mitochondria in mutant cells lose membrane potential. Therefore, mitochondrial fusion is essential for embryonic development, and by enabling cooperation between mitochondria, has protective effects on the mitochondrial population. PMID:12527753

  13. Pericellular Versican Regulates the Fibroblast-Myofibroblast Transition

    PubMed Central

    Hattori, Noriko; Carrino, David A.; Lauer, Mark E.; Vasanji, Amit; Wylie, James D.; Nelson, Courtney M.; Apte, Suneel S.

    2011-01-01

    The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5−/− mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5−/− cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcanhdf/+) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5−/− cells resulted from increased PCM versican content, we generated Adamts5−/−;Vcanhdf/+ mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5−/− mice. In Adamts5−/− fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates. PMID:21828051

  14. Intramyocardial Fibroblast - Myocyte Communication

    PubMed Central

    Kakkar, Rahul; Lee, Richard T.

    2009-01-01

    Cardiac fibroblasts are emerging as key components of normal cardiac function as well as the response to stressors and injury. These most numerous cells of the heart interact with myocytes via paracrine mechanisms, alterations in extracellular matrix homeostasis, and direct cell-cell interactions. It is possible that they are a contributor to the inability of adult myocytes to proliferate, and may influence cardiac progenitor biology. Furthering our understanding of how cardiac fibroblast and myocytes interact may provide an avenue to novel treatments for heart failure prevention. This review discusses the most recent concepts in cardiac fibroblast-myocyte communication and areas of potential future research. PMID:20056945

  15. Fibroblast growth factor signaling during early vertebrate development.

    PubMed

    Böttcher, Ralph T; Niehrs, Christof

    2005-02-01

    Fibroblast growth factors (FGFs) have been implicated in diverse cellular processes including apoptosis, cell survival, chemotaxis, cell adhesion, migration, differentiation, and proliferation. This review presents our current understanding on the roles of FGF signaling, the pathways employed, and its regulation. We focus on FGF signaling during early embryonic processes in vertebrates, such as induction and patterning of the three germ layers as well as its function in the control of morphogenetic movements.

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

    PubMed

    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).

  17. Site-Specific Differentiation of Fibroblasts in Normal and Scleroderma Skin

    DTIC Science & Technology

    2010-06-01

    expression of one HOX lncRNA, termed nc-HOXC10, is correlated with fibrotic gene expression in fibroblasts. In our initial study, we found nc-HOXC10...anatomic expression pattern of Hox genes from embryonic development through adulthood. The ongoing Hox expression endowed fibroblasts with site...specific inductive activities that control the homeostasis and regeneration of epithelia throughout the body. The epigenetic memory of Hox genes depend on

  18. Embryotoxicity hazard assessment of cadmium and arsenic compounds using embryonic stem cells.

    PubMed

    Stummann, T C; Hareng, L; Bremer, S

    2008-10-30

    The Embryonic Stem Cell Test (EST) has been successfully validated as an in vitro method for detecting embryotoxicity, showing a good overall test accuracy of 78% [Genschow, E., Spielmann, H., Scholz, G., Seiler, A., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., 2002. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern. Lab. Anim. 30, 151-176]. Methylmercury was the only strong in vivo embryotoxicant falsely predicted as non-embryotoxic making the metal the most significant outlayer [Genschow, E., Spielmann, H., Scholz, G., Pohl, I., Seiler, A., Clemann, N., Bremer, S., Becker, K., 2004. Validation of the Embryonic Stem Cell Test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern. Lab. Anim. 32, 209-244]. The misclassification of methylmercury and the potential environmental exposure to developmental toxic heavy metals promoted our investigation of whether the EST applicability domain covers cadmium and arsenic compounds. The EST misclassified cadmium, arsenite and arsenate compounds as non-embryotoxic, even when including arsenic metabolites (methylarsonate, methylarsonous and dimethylarsinic). The reasons were the lack of higher cytotoxicity towards embryonic stem cells as compared to more mature cells (3T3 fibroblasts) or the absence of inhibition of cardiac differentiation by specific mechanisms rather than general cytotoxicity. Including EST data on heavy metals from the literature (lithium, methylmercury, trivalent chromium and hexavalent chromium) revealed that the test correctly predicted the embryotoxic potential of three out of the seven heavy metals, indicating an insufficient predictivity for such metals. Refinement of the EST prediction model and inclusion of additional toxicological endpoints could

  19. Cardiac Fibroblasts Regulate Sympathetic Nerve Sprouting and Neurocardiac Synapse Stability

    PubMed Central

    Mias, Céline; Coatrieux, Christelle; Denis, Colette; Genet, Gaël; Seguelas, Marie-Hélène; Laplace, Nathalie; Rouzaud-Laborde, Charlotte; Calise, Denis; Parini, Angelo; Cussac, Daniel; Pathak, Atul; Sénard, Jean-Michel; Galés, Céline

    2013-01-01

    Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic

  20. Human cytomegalovirus gene UL21a encodes a short-lived cytoplasmic protein and facilitates virus replication in fibroblasts.

    PubMed

    Fehr, Anthony R; Yu, Dong

    2010-01-01

    The human cytomegalovirus (HCMV) gene UL21a was recently annotated by its conservation in chimpanzee cytomegalovirus. Two large-scale mutagenic analyses showed that mutations in overlapping UL21a/UL21 resulted in a severe defect of virus growth in fibroblasts. Here, we characterized UL21a and demonstrated its role in HCMV infection. We mapped a UL21a-specific transcript of approximately 600 bp that was expressed with early kinetics. UL21a encoded pUL21a, a protein of approximately 15 kDa, which was unstable and localized predominantly to the cytoplasm during HCMV infection or when expressed alone. Interestingly, pUL21a was drastically stabilized in the presence of proteasome inhibitor MG132, but its instability was independent of a functional ubiquitin-mediated pathway, suggesting that pUL21a underwent proteasome-dependent, ubiquitin-independent degradation. A UL21a deletion virus was attenuated in primary human newborn foreskin fibroblasts (HFFs) and embryonic lung fibroblasts (MRC-5), whereas a marker-rescued virus and mutant viruses lacking the neighboring or overlapping genes UL20, UL21, or UL21.5-UL23 replicated at wild-type levels. The growth defect of UL21a-deficient virus in MRC-5 cells was more pronounced than that in HFFs. At a high multiplicity of infection, the UL21a deletion virus synthesized viral proteins with wild-type kinetics but had a two- to threefold defect in viral DNA replication. More importantly, although pUL21a was not detected in the virion, progeny virions produced by the mutant virus were approximately 10 times less infectious than wild-type virus, suggesting that UL21a is required for HCMV to establish efficient productive infection. We conclude that UL21a encodes a short-lived cytoplasmic protein and facilitates HCMV replication in fibroblasts.

  1. Autophagy is required for IL-2-mediated fibroblast growth

    SciTech Connect

    Kang, Rui; Tang, Daolin; Lotze, Michael T.; Zeh III, Herbert J.

    2013-02-15

    Autophagy is an evolutionarily conserved pathway responsible for delivery of cytoplasmic material into the lysosomal degradation pathway to enable vesicular exocytosis. Interleukin (IL)-2 is produced by T-cells and its activity is important for immunoregulation. Fibroblasts are an immune competent cell type, playing a critical role in wound healing, chronic inflammation, and tumor development. Although autophagy plays an important role in each of these processes, whether it regulates IL-2 activity in fibroblasts is unknown. Here, we show that autophagy is required for IL-2-induced cell growth in fibroblasts. IL-2 significantly induced autophagy in mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts. Autophagy inhibitors (e.g., 3-methylamphetamine and bafilomycin A1) or knockdown of ATG5 and beclin 1 blocked clinical grade IL-2-induced autophagy. Moreover, IL-2 induced HMGB1 cytoplasmic translocation in MEFs and promoted interaction between HMGB1 and beclin1, which is required for autophagy induction. Pharmacological and genetic inhibition of autophagy inhibited IL-2-induced cell proliferation and enhanced IL-2-induced apoptosis. These findings suggest that autophagy is an important pro-survival regulator for IL-2-induced cell growth in fibroblasts.

  2. The Dynamic Role of Cardiac Fibroblasts in Development and Disease

    PubMed Central

    Lajiness, Jacquelyn D; Conway, Simon J.

    2013-01-01

    Cardiac fibroblasts are the most abundant cell in the mammalian heart. While they have been historically overlooked in terms of functional contributions to development and physiology, cardiac fibroblasts are now front and center. They are currently recognized as key protagonists during both normal development and cardiomyopathy disease, and work together with cardiomyocytes through paracrine, structural, and potentially electrical interactions. However, the lack of specific biomarkers and their heterogeneous nature currently convolutes the study of this dynamic cell lineage; though, efforts to advance marker analysis and lineage mapping technologies are ongoing. These tools will help elucidate the functional significance of fibroblast-cardiomyocyte interactions in vivo and delineate the dynamic nature of normal and pathological cardiac fibroblasts. Since therapeutic promise lies in understanding the interface between developmental biology and the postnatal injury response, future studies to understand the divergent roles played by cardiac fibroblasts both in utero and following cardiac insult are essential. PMID:22878976

  3. Localisation of embryonic prealbumin in formalin-fixed human fetal and adult tissue.

    PubMed Central

    Gallon, M E; Reid, W A; McHardie, G A; Hardman, R; Smith, G D; Horne, C H; Kalashnikov, V V; Tatarinov, Y S

    1981-01-01

    The presence of embryonic prealbumin (EPA) has been confirmed in fetal fibroblasts, chondrocytes, and distal tubular epithelial cells by an indirect immunoperoxidase technique. EPA has often been found also in the stromal cells of benign and malignant mesodermal tumours, but not in the epithelial cells of benign and malignant epithelial tumours. That EPA is not an exclusive product of neoplastic mesodermal cells is demonstrated by our finding of EPA in fibroblasts of granulation tissue, irradiated fibroblasts, and in distal tubular epithelial cells of miscellaneous adult kidneys. Images PMID:7021602

  4. Dupuytren's Contracture: Fibroblast Contraction?

    PubMed Central

    Gabbiani, Giulio; Majno, Guido

    1972-01-01

    In 6 cases of Dupuytren's disease and 1 of Ledderhose's disease, the nodules of the palmar and plantar aponeurosis were examined by light and electron microscopy. The cells composing these nodules, presumably fibroblasts, showed three significant ultrastructural features: (1) a fibrillar system similar to that of smooth muscle cells; (2) nuclear deformations such as are found in contracted cells, the severest being recognizable by light microscopy (cross-banded nuclei); (3) cell-to-cell and cell-to-stroma attachments. Based on these data and on recent information about the biology of the fibroblasts, it is suggested that these cells are fibroblasts that have modulated into contractile cells (myofibroblasts), and that their contraction plays a role in the pathogenesis of the contracture observed clinically. ImagesFig 10Fig 5Fig 11Fig 6 and 7Fig 8Fig 1Fig 2Fig 9Fig 3Fig 4 PMID:5009249

  5. Embryonal cancers in Europe.

    PubMed

    Gatta, Gemma; Ferrari, Andrea; Stiller, Charles A; Pastore, Guido; Bisogno, Gianni; Trama, Annalisa; Capocaccia, Riccardo

    2012-07-01

    Embryonal cancers are a heterogeneous group of rare cancers which mainly occur in children and adolescents. The aim of the present study was to estimate the burden (incidence, prevalence, survival and proportion of cured) for the principal embryonal cancers in Europe (EU27), using population-based data from cancer registries (CRs) participating in RARECARE. We identified 3322 cases diagnosed from 1995 to 2002 (latest period for which data are available): 44% neuroblastoma, 35% nephroblastoma, 13% retinoblastoma and 6% hepatoblastoma. Very few cases of pulmonary blastoma (43 cases) and pancreatoblastoma (seven cases) were diagnosed. About 2000 new embryonal cancers were estimated every year in EU27, for an annual incidence rate of 4 per million (1.8 neuroblastoma, 1.4 nephroblastoma, and 0.5 retinoblastoma); 91% of cases occurred in patients under 15 years. Five-year relative survival for all embryonal cancers was 80% (99% retinoblastoma, 90% nephroblastoma, 71% hepatoblastoma and 68% neuroblastoma). Overall survival was lower in adolescents and adults than in those under 15 years. The cure rate was estimated at 80%. Slightly less than 40,000 persons were estimated alive in EU27 with a diagnosis of embryonal cancer in 2008. Nephroblastoma was the most prevalent (18,150 cases in EU27), followed by neuroblastoma (12,100), retinoblastoma (5200), hepatoblastoma (2700) and pulmonary blastoma (614). This is the first study to delineate the embryonal cancer burden in Europe by age, sex and European region. Survival/cure rate is generally high, but there are considerable gaps in our understanding of the natural histories of these rare diseases particularly in adults.

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

    EPA Science Inventory

    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...

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

    EPA Science Inventory

    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...

  8. Fourier transform infrared microspectroscopy reveals that tissue culture conditions affect the macromolecular phenotype of human embryonic stem cells.

    PubMed

    Cao, Julie; Ng, Elizabeth S; McNaughton, Don; Stanley, Edouard G; Elefanty, Andrew G; Tobin, Mark J; Heraud, Philip

    2013-07-21

    We employed Fourier transform infrared (FTIR) microspectroscopy to investigate the effects of different tissue culture environments on the FTIR spectra of undifferentiated human embryonic stem cells (hESCs) and their differentiated progeny. First we tested whether there were any possible spectral artifacts resulting from the use of transflectance measurements by comparing them with transmission measurements and found no evidence of these concluding that the lack of any differences resulted from the homogeneity of the dried cytospun cellular monolayers. We found that hESCs that were enzymatically passaged onto mouse embryonic fibroblasts (MEFs) in KOSR based hESC medium, hESCs enzymatically passaged onto Matrigel in mTESR medium and hESCs mechanically passaged onto MEFs in KOSR-based hESC medium, possessed unique FTIR spectroscopic signatures that reflect differences in their macromolecular chemistry. Further, these spectroscopic differences persisted even upon differentiation towards mesendodermal lineages. Our results suggest that FTIR microspectroscopy is a powerful, objective, measurement modality that complements existing methods for studying the phenotype of hESCs and their progeny, particularly changes induced by the cellular environment.

  9. Smg6/Est1 licenses embryonic stem cell differentiation via nonsense-mediated mRNA decay

    PubMed Central

    Li, Tangliang; Shi, Yue; Wang, Pei; Guachalla, Luis Miguel; Sun, Baofa; Joerss, Tjard; Chen, Yu-Sheng; Groth, Marco; Krueger, Anja; Platzer, Matthias; Yang, Yun-Gui; Rudolph, Karl Lenhard; Wang, Zhao-Qi

    2015-01-01

    Nonsense-mediated mRNA decay (NMD) is a post-transcriptional mechanism that targets aberrant transcripts and regulates the cellular RNA reservoir. Genetic modulation in vertebrates suggests that NMD is critical for cellular and tissue homeostasis, although the underlying mechanism remains elusive. Here, we generate knockout mice lacking Smg6/Est1, a key nuclease in NMD and a telomerase cofactor. While the complete loss of Smg6 causes mouse lethality at the blastocyst stage, inducible deletion of Smg6 is compatible with embryonic stem cell (ESC) proliferation despite the absence of telomere maintenance and functional NMD. Differentiation of Smg6-deficient ESCs is blocked due to sustained expression of pluripotency genes, normally repressed by NMD, and forced down-regulation of one such target, c-Myc, relieves the differentiation block. Smg6-null embryonic fibroblasts are viable as well, but are refractory to cellular reprograming into induced pluripotent stem cells (iPSCs). Finally, depletion of all major NMD factors compromises ESC differentiation, thus identifying NMD as a licensing factor for the switch of cell identity in the process of stem cell differentiation and somatic cell reprograming. PMID:25770585

  10. Intestinal lineage commitment of embryonic stem cells.

    PubMed

    Cao, Li; Gibson, Jason D; Miyamoto, Shingo; Sail, Vibhavari; Verma, Rajeev; Rosenberg, Daniel W; Nelson, Craig E; Giardina, Charles

    2011-01-01

    Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue.

  11. Use of murine embryonic stem cells in embryotoxicity assays: the embryonic stem cell test.

    PubMed

    Seiler, Andrea E M; Buesen, Roland; Visan, Anke; Spielmann, Horst

    2006-01-01

    The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body outgrowths compared to cytotoxic effects on murine ES cells and differentiated 3T3 fibroblasts. Through a number of prevalidation and validation studies, the EST has been demonstrated to be a reliable alternative method for embryotoxicity testing based on the most important mechanisms in embryotoxicity-cytotoxicity and differentiation--as well as on differences in sensitivity between differentiated and embryonic tissues. Improvements of the EST protocol using flow cytometry analysis showed that differential expression of sarcomeric myosin heavy chain and alpha-actinin proteins quantified under the influence of a test compound is a useful marker for detecting potential teratogenicity. The in vitro embryotoxicity test described in this chapter is rapid, simple, and sensitive and can be usefully employed as a component of the risk/hazard assessment process.

  12. Developmental heterogeneity of cardiac fibroblasts does not predict pathological proliferation and activation.

    PubMed

    Ali, Shah R; Ranjbarvaziri, Sara; Talkhabi, Mahmood; Zhao, Peng; Subat, Ali; Hojjat, Armin; Kamran, Paniz; Müller, Antonia M S; Volz, Katharina S; Tang, Zhaoyi; Red-Horse, Kristy; Ardehali, Reza

    2014-09-12

    Fibrosis is mediated partly by extracellular matrix-depositing fibroblasts in the heart. Although these mesenchymal cells are reported to have multiple embryonic origins, the functional consequence of this heterogeneity is unknown. We sought to validate a panel of surface markers to prospectively identify cardiac fibroblasts. We elucidated the developmental origins of cardiac fibroblasts and characterized their corresponding phenotypes. We also determined proliferation rates of each developmental subset of fibroblasts after pressure overload injury. We showed that Thy1(+)CD45(-)CD31(-)CD11b(-)Ter119(-) cells constitute the majority of cardiac fibroblasts. We characterized these cells using flow cytometry, epifluorescence and confocal microscopy, and transcriptional profiling (using reverse transcription polymerase chain reaction and RNA-seq). We used lineage tracing, transplantation studies, and parabiosis to show that most adult cardiac fibroblasts derive from the epicardium, a minority arises from endothelial cells, and a small fraction from Pax3-expressing cells. We did not detect generation of cardiac fibroblasts by bone marrow or circulating cells. Interestingly, proliferation rates of fibroblast subsets on injury were identical, and the relative abundance of each lineage remained the same after injury. The anatomic distribution of fibroblast lineages also remained unchanged after pressure overload. Furthermore, RNA-seq analysis demonstrated that Tie2-derived and Tbx18-derived fibroblasts within each operation group exhibit similar gene expression profiles. The cellular expansion of cardiac fibroblasts after transaortic constriction surgery was not restricted to any single developmental subset. The parallel proliferation and activation of a heterogeneous population of fibroblasts on pressure overload could suggest that common signaling mechanisms stimulate their pathological response. © 2014 American Heart Association, Inc.

  13. Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression.

    PubMed

    López-Rodríguez, Cristina; Antos, Christopher L; Shelton, John M; Richardson, James A; Lin, Fangming; Novobrantseva, Tatiana I; Bronson, Roderick T; Igarashi, Peter; Rao, Anjana; Olson, Eric N

    2004-02-24

    The transcription factor NFAT5/TonEBP, a member of the NFAT/Rel family of transcription factors, has been implicated in diverse cellular responses, including the response to osmotic stress, integrin-dependent cell migration, T cell activation, and the Ras pathway in Drosophila. To clarify the in vivo role of NFAT5, we generated NFAT5-null mice. Homozygous mutants were genetically underrepresented after embryonic day 14.5. Surviving mice manifested a progressive and profound atrophy of the kidney medulla with impaired activation of several osmoprotective genes, including those encoding aldose reductase, Na+/Cl--coupled betaine/gamma-aminobutyric acid transporter, and the Na+/myo-inositol cotransporter. The aldose reductase gene is controlled by a tonicity-responsive enhancer, which was refractory to hypertonic stress in fibroblasts lacking NFAT5, establishing this enhancer as a direct transcriptional target of NFAT5. Our findings demonstrate a central role for NFAT5 as a tonicity-responsive transcription factor required for kidney homeostasis and function.

  14. Loss of NFAT5 results in renal atrophy and lack of tonicity-responsive gene expression

    PubMed Central

    López-Rodríguez, Cristina; Antos, Christopher L.; Shelton, John M.; Richardson, James A.; Lin, Fangming; Novobrantseva, Tatiana I.; Bronson, Roderick T.; Igarashi, Peter; Rao, Anjana; Olson, Eric N.

    2004-01-01

    The transcription factor NFAT5/TonEBP, a member of the NFAT/Rel family of transcription factors, has been implicated in diverse cellular responses, including the response to osmotic stress, integrin-dependent cell migration, T cell activation, and the Ras pathway in Drosophila. To clarify the in vivo role of NFAT5, we generated NFAT5-null mice. Homozygous mutants were genetically underrepresented after embryonic day 14.5. Surviving mice manifested a progressive and profound atrophy of the kidney medulla with impaired activation of several osmoprotective genes, including those encoding aldose reductase, Na+/Cl–-coupled betaine/γ-aminobutyric acid transporter, and the Na+/myo-inositol cotransporter. The aldose reductase gene is controlled by a tonicity-responsive enhancer, which was refractory to hypertonic stress in fibroblasts lacking NFAT5, establishing this enhancer as a direct transcriptional target of NFAT5. Our findings demonstrate a central role for NFAT5 as a tonicity-responsive transcription factor required for kidney homeostasis and function. PMID:14983020

  15. Folate deficiency induces neurodegeneration and brain dysfunction in mice lacking uracil DNA glycosylase.

    PubMed

    Kronenberg, Golo; Harms, Christoph; Sobol, Robert W; Cardozo-Pelaez, Fernando; Linhart, Heinz; Winter, Benjamin; Balkaya, Mustafa; Gertz, Karen; Gay, Shanna B; Cox, David; Eckart, Sarah; Ahmadi, Michael; Juckel, Georg; Kempermann, Gerd; Hellweg, Rainer; Sohr, Reinhard; Hörtnagl, Heide; Wilson, Samuel H; Jaenisch, Rudolf; Endres, Matthias

    2008-07-09

    Folate deficiency and resultant increased homocysteine levels have been linked experimentally and epidemiologically with neurodegenerative conditions like stroke and dementia. Moreover, folate deficiency has been implicated in the pathogenesis of psychiatric disorders, most notably depression. We hypothesized that the pathogenic mechanisms include uracil misincorporation and, therefore, analyzed the effects of folate deficiency in mice lacking uracil DNA glycosylase (Ung-/-) versus wild-type controls. Folate depletion increased nuclear mutation rates in Ung-/- embryonic fibroblasts, and conferred death of cultured Ung-/- hippocampal neurons. Feeding animals a folate-deficient diet (FD) for 3 months induced degeneration of CA3 pyramidal neurons in Ung-/- but not Ung+/+ mice along with decreased hippocampal expression of brain-derived neurotrophic factor protein and decreased brain levels of antioxidant glutathione. Furthermore, FD induced cognitive deficits and mood alterations such as anxious and despair-like behaviors that were aggravated in Ung-/- mice. Independent of Ung genotype, FD increased plasma homocysteine levels, altered brain monoamine metabolism, and inhibited adult hippocampal neurogenesis. These results indicate that impaired uracil repair is involved in neurodegeneration and neuropsychiatric dysfunction induced by experimental folate deficiency.

  16. Gravity and embryonic development

    NASA Technical Reports Server (NTRS)

    Young, R. S.

    1976-01-01

    The relationship between the developing embryo (both plant and animal) and a gravitational field has long been contemplated. The difficulty in designing critical experiments on the surface of the earth because of its background of 1 g, has been an obstacle to a resolution of the problem. Biological responses to gravity (particularly in plants) are obvious in many cases; however, the influence of gravity as an environmental input to the developing embryo is not as obvious and has proven to be extremely difficult to define. In spite of this, over the years numerous attempts have been made using a variety of embryonic materials to come to grips with the role of gravity in development. Three research tools are available: the centrifuge, the clinostat, and the orbiting spacecraft. Experimental results are now available from all three sources. Some tenuous conclusions are drawn, and an attempt at a unifying theory of gravitational influence on embryonic development is made.

  17. Gravity and embryonic development

    NASA Technical Reports Server (NTRS)

    Young, R. S.

    1976-01-01

    The relationship between the developing embryo (both plant and animal) and a gravitational field has long been contemplated. The difficulty in designing critical experiments on the surface of the earth because of its background of 1 g, has been an obstacle to a resolution of the problem. Biological responses to gravity (particularly in plants) are obvious in many cases; however, the influence of gravity as an environmental input to the developing embryo is not as obvious and has proven to be extremely difficult to define. In spite of this, over the years numerous attempts have been made using a variety of embryonic materials to come to grips with the role of gravity in development. Three research tools are available: the centrifuge, the clinostat, and the orbiting spacecraft. Experimental results are now available from all three sources. Some tenuous conclusions are drawn, and an attempt at a unifying theory of gravitational influence on embryonic development is made.

  18. A toolbox to explore the mechanics of living embryonic tissues

    PubMed Central

    Campàs, Otger

    2016-01-01

    The sculpting of embryonic tissues and organs into their functional morphologies involves the spatial and temporal regulation of mechanics at cell and tissue scales. Decades of in vitro work, complemented by some in vivo studies, have shown the relevance of mechanical cues in the control of cell behaviors that are central to developmental processes, but the lack of methodologies enabling precise, quantitative measurements of mechanical cues in vivo have hindered our understanding of the role of mechanics in embryonic development. Several methodologies are starting to enable quantitative studies of mechanics in vivo and in situ, opening new avenues to explore how mechanics contributes to shaping embryonic tissues and how it affects cell behavior within developing embryos. Here we review the present methodologies to study the role of mechanics in living embryonic tissues, considering their strengths and drawbacks as well as the conditions in which they are most suitable. PMID:27061360

  19. A toolbox to explore the mechanics of living embryonic tissues.

    PubMed

    Campàs, Otger

    2016-07-01

    The sculpting of embryonic tissues and organs into their functional morphologies involves the spatial and temporal regulation of mechanics at cell and tissue scales. Decades of in vitro work, complemented by some in vivo studies, have shown the relevance of mechanical cues in the control of cell behaviors that are central to developmental processes, but the lack of methodologies enabling precise, quantitative measurements of mechanical cues in vivo have hindered our understanding of the role of mechanics in embryonic development. Several methodologies are starting to enable quantitative studies of mechanics in vivo and in situ, opening new avenues to explore how mechanics contributes to shaping embryonic tissues and how it affects cell behavior within developing embryos. Here we review the present methodologies to study the role of mechanics in living embryonic tissues, considering their strengths and drawbacks as well as the conditions in which they are most suitable. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. [Effects of different culture system of isolating and passage of sheep embryonic stem-like cells].

    PubMed

    Bai, Changming; Liu, Chousheng; Wang, Zhigang; Wang, Xinzhuang

    2008-07-01

    In this research, we use mouse embryonic fibroblasts as feeder layers. To eliminate the influence of serum and mouse embryonic stem cells (ESCs) conditioned medium (ESCCM) on self-renewal of sheep embryonic stem-like cells, knockout serum replacement (KSR) was used to replace serum, then supplanted with ESCCM for the isolation and cloning of sheep embryonic stem-like cells. We found when inner cell masses (ICMs) cultured in the control group with medium supplanted with fetal bovine serum (FBS), sheep ES-like cells could not survive for more than 3 passages. However, sheep embryonic stem-like cells could remain undifferentiated for 5 passages when cultured in the medium that FBS was substituted by KSR. The result indicates that KSR culture system was more suitable for the isolation and cloning of sheep embryonic stem-like cells compared to FBS culture system. Finally we applied medium with 15% KSR as basic medium supplanted with 40% ESCCM as a new culture system to isolate sheep embryonic stem-like cells, we found one embryonic stem-like cell line still maintained undifferentiating for 8 passages, which characterized with a normal and stable karyotype and high expression of alkaline phosphatase. These results suggest that it is suitable to culture sheep ICM in the new culture system with 15% KSR as basic medium and supplanted with 40% ESCCM, which indicated that mouse ES cells might secrete factors playing important roles in promoting sheep ES-like cells' self-renewal.

  1. Globin synthesis in hybrid cells constructed by transplantation of dormant avian erythrocyte nuclei into enucleated fibroblasts.

    PubMed Central

    Bruno, J; Reich, N; Lucas, J J

    1981-01-01

    The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14- to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer than 200 species of polypeptides were detected; the major polypeptides made at this time were identified as the alpha A-, alpha D-, and beta-globin chains. The dormant erythrocyte nuclei were next reactivated to transcriptional competence by transplantation into enucleated mouse or chicken embryo fibroblasts, with frequencies of cytoplast renucleation of about 50 and 90%, respectively. Since large numbers of hybrid cells could be constructed, a biochemical analysis was possible. Electrophoretic analysis of the [35S]methionine-labeled polypeptides made in the hybrid cell types showed that polypeptides having the mobilities of only two (alpha A and alpha D) of the three major adult globin chains were made as major constituents of the hybrid cells. However, analysis of 14C-amino acid-labeled polypeptides revealed that a beta-like polypeptide that lacked methionine was also synthesized in large amounts. This polypeptide was tentatively identified as the early embryonic globin species rho. Globin synthesis was detected as early as 3 h after nuclear transplantation and as late as 18 h, the last time measured in these experiments. It appeared that globin polypeptides made at very early times were translated at least partially from chicken messenger ribonucleic acid introduced into the hybrid cells during fusion, whereas those made at later times were translated primarily from newly synthesized globin messenger ribonucleic acid. The potential usefulness of this hybrid cell system in analyzing mechanisms regulating globin gene expression is discussed. Images PMID:7346715

  2. Direct conversion of human fibroblasts to dopaminergic neurons

    PubMed Central

    Pfisterer, Ulrich; Kirkeby, Agnete; Torper, Olof; Wood, James; Nelander, Jenny; Dufour, Audrey; Björklund, Anders; Lindvall, Olle; Jakobsson, Johan; Parmar, Malin

    2011-01-01

    Recent reports demonstrate that somatic mouse cells can be directly converted to other mature cell types by using combined expression of defined factors. Here we show that the same strategy can be applied to human embryonic and postnatal fibroblasts. By overexpression of the transcription factors Ascl1, Brn2, and Myt1l, human fibroblasts were efficiently converted to functional neurons. We also demonstrate that the converted neurons can be directed toward distinct functional neurotransmitter phenotypes when the appropriate transcriptional cues are provided together with the three conversion factors. By combining expression of the three conversion factors with expression of two genes involved in dopamine neuron generation, Lmx1a and FoxA2, we could direct the phenotype of the converted cells toward dopaminergic neurons. Such subtype-specific induced neurons derived from human somatic cells could be valuable for disease modeling and cell replacement therapy. PMID:21646515

  3. Generation of Corneal Keratocytes from Human Embryonic Stem Cells

    PubMed Central

    Hertsenberg, Andrew J.; Funderburgh, James L.

    2017-01-01

    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. PMID:26026882

  4. Chemically induced bidirectional differentiation of embryonal carcinoma cells in vitro.

    PubMed Central

    Speers, W. C.; Birdwell, C. R.; Dixon, F. J.

    1979-01-01

    N,N-dimethylacetamide, hexamethylene bisacetamide, and Polybrene induced rapid and extensive differentiation in vitro in an otherwise slowly differentiating subline of embryonal carcinoma cells. The type of differentiated cell induced was dependent on the spatial organization of the stem cells during drug treatment. In monalayer culture "epithelial" cells were produced exclusively. However, treatment of aggregated suspension cultures yielded predominantly "fibroblast-like" cells. The undifferentiated embryonal carcinoma cells and the two differentiated cell types were morphologically distinct when examined by light microscopy, scanning electron microscopy, and transmission electron microscopy; and they had differences in cell surface antigens. Both differential cell types produced large amounts of fibronectin, whereas the embryonal carcinoma cells produced only minimal amounts. This system provides a convenient way to induce relatively synchronous differentiation of embryonal carcinoma cells into specific differentiated cell types. Images Figure 5 Figure 6 Figure 1 Figure 2 Figure 3 Figure 4 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 PMID:507191

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

    PubMed Central

    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 (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. PMID:24146866

  6. MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

    PubMed

    Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

    2014-07-17

    Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming.

  7. Ornithine-δ-Aminotransferase Inhibits Neurogenesis During Xenopus Embryonic Development

    PubMed Central

    Peng, Ying; Cooper, Sandra K.; Li, Yi; Mei, Jay M.; Qiu, Shuwei; Borchert, Gregory L.; Donald, Steven P.; Kung, Hsiang-fu; Phang, James M.

    2015-01-01

    Purpose. In humans, deficiency of ornithine-δ-aminotransferase (OAT) results in progressive degeneration of the neural retina (gyrate atrophy) with blindness in the fourth decade. In this study, we used the Xenopus embryonic developmental model to study functions of the OAT gene on embryonic development. Methods. We cloned and sequenced full-length OAT cDNA from Xenopus oocytes (X-OAT) and determined X-OAT expression in various developmental stages of Xenopus embryos and in a variety of adult tissues. The phenotype, gene expression of neural developmental markers, and enzymatic activity were detected by gain-of-function and loss-of-function manipulations. Results. We showed that X-OAT is essential for Xenopus embryonic development, and overexpression of X-OAT produces a ventralized phenotype characterized by a small head, lack of axial structure, and defective expression of neural developmental markers. Using X-OAT mutants based on mutations identified in humans, we found that substitution of both Arg 180 and Leu 402 abrogated both X-OAT enzymatic activity and ability to modulate the developmental phenotype. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development. Conclusions. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development, but it is essential for Xenopus embryonic development. The Arg 180 and Leu 402 are crucial for these effects of the OAT molecule in development. PMID:25783604

  8. VAN method lacks validity

    NASA Astrophysics Data System (ADS)

    Jackson, David D.; Kagan, Yan Y.

    Varotsos and colleagues (the VAN group) claim to have successfully predicted many earthquakes in Greece. Several authors have refuted these claims, as reported in the May 27,1996, special issue of Geophysical Research Letters and a recent book, A Critical Review of VAN [Lighthill 1996]. Nevertheless, the myth persists. Here we summarize why the VAN group's claims lack validity.The VAN group observes electrical potential differences that they call “seismic electric signals” (SES) weeks before and hundreds of kilometers away from some earthquakes, claiming that SES are somehow premonitory. This would require that increases in stress or decreases in strength cause the electrical variations, or that some regional process first causes the electrical signals and then helps trigger the earthquakes. Here we adopt their notation SES to refer to the electrical variations, without accepting any link to the quakes.

  9. Fibroblast PER2 Circadian Rhythmicity Depends on Cell Density

    PubMed Central

    Noguchi, Takako; Wang, Lexie L.; Welsh, David K.

    2013-01-01

    Like neurons in the suprachiasmatic nucleus (SCN), the master circadian pacemaker in the brain, single fibroblasts can function as independent oscillators. In the SCN, synaptic and paracrine signaling among cells creates a robust, synchronized circadian oscillation, whereas there is no evidence for such integration in fibroblast cultures. However, interactions among single-cell fibroblast oscillators cannot be completely excluded, because fibroblasts were not isolated in previous work. In this study, we tested the autonomy of fibroblasts as single-cell circadian oscillators in high and low density culture, by single-cell imaging of cells from PER2::LUC circadian reporter mice. We found greatly reduced PER2::LUC rhythmicity in low density cultures, which could result from lack of either constitutive or rhythmic paracrine signals from neighboring fibroblasts. To discriminate between these two possibilities, we mixed PER2::LUC wild type (WT) cells with non-luminescent, non-rhythmic Bmal1−/− cells, so that density of rhythmic cells was low but overall cell density remained high. In this condition, WT cells showed clear rhythmicity similar to high density cultures. We also mixed PER2::LUC WT cells with non-luminescent, long period Cry2−/− cells. In this condition, WT cells showed a period no different from cells cultured with rhythmic WT cells or non-rhythmic Bmal1−/− cells. In previous work, we found that low K+ suppresses fibroblast rhythmicity, and we and others have found that either low K+ or low Ca2+ suppresses SCN rhythmicity. Therefore, we attempted to rescue rhythmicity of low density fibroblasts with high K+ (21 mM), high Ca2+ (3.6 mM), or conditioned medium. Conditioned medium from high density fibroblast cultures rescued rhythmicity of low density cultures, whereas high K+ or Ca2+ medium did not consistently rescue rhythmicity. These data suggest that fibroblasts require paracrine signals from adjacent cells for normal expression of rhythmicity

  10. A Critical Role of Mitochondrial Phosphatase Ptpmt1 in Embryogenesis Reveals a Mitochondrial Metabolic Stress-Induced Differentiation Checkpoint in Embryonic Stem Cells ▿

    PubMed Central

    Shen, Jinhua; Liu, Xia; Yu, Wen-Mei; Liu, Jie; Groot Nibbelink, Milou; Guo, Caiying; Finkel, Toren; Qu, Cheng-Kui

    2011-01-01

    Mitochondria are highly dynamic organelles that play multiple roles in cells. How mitochondria cooperatively modulate embryonic stem (ES) cell function during development is not fully understood. Global disruption of Ptpmt1, a mitochondrial Pten-like phosphatidylinositol phosphate (PIP) phosphatase, resulted in developmental arrest and postimplantation lethality. Ptpmt1−/− blastocysts failed to outgrow, and inner-cell-mass cells failed to thrive. Depletion of Ptpmt1 in conditional knockout ES cells decreased proliferation without affecting energy homeostasis or cell survival. Differentiation of Ptpmt1-depleted ES cells was essentially blocked. This was accompanied by upregulation of cyclin-dependent kinase inhibitors and a significant cell cycle delay. Reintroduction of wild-type but not of catalytically deficient Ptpmt1 C132S or truncated Ptpmt1 lacking the mitochondrial localization signal restored the differentiation capabilities of Ptpmt1 knockout ES cells. Intriguingly, Ptpmt1 is specifically important for stem cells, as ablation of Ptpmt1 in differentiated embryonic fibroblasts did not disturb cellular function. Further analyses demonstrated that oxygen consumption of Ptpmt1-depleted cells was decreased, while glycolysis was concomitantly enhanced. In addition, mitochondrial fusion/dynamics were compromised in Ptpmt1 knockout cells due to accumulation of PIPs. These studies, while establishing a crucial role for Ptpmt1 phosphatase in embryogenesis, reveal a mitochondrial metabolic stress-activated checkpoint in the control of ES cell differentiation. PMID:21986498

  11. MiR-25 Regulates Wwp2 and Fbxw7 and Promotes Reprogramming of Mouse Fibroblast Cells to iPSCs

    PubMed Central

    Lu, Dong; Davis, Matthew P. A.; Abreu-Goodger, Cei; Wang, Wei; Campos, Lia S.; Siede, Julia; Vigorito, Elena; Skarnes, William C.; Dunham, Ian; Enright, Anton J.; Liu, Pentao

    2012-01-01

    Background miRNAs are a class of small non-coding RNAs that regulate gene expression and have critical functions in various biological processes. Hundreds of miRNAs have been identified in mammalian genomes but only a small number of them have been functionally characterized. Recent studies also demonstrate that some miRNAs have important roles in reprogramming somatic cells to induced pluripotent stem cells (iPSCs). Methods We screened 52 miRNAs cloned in a piggybac (PB) vector for their roles in reprogramming of mouse embryonic fibroblast cells to iPSCs. To identify targets of miRNAs, we made Dgcr8-deficient embryonic stem (ES) cells and introduced miRNA mimics to these cells, which lack miRNA biogenesis. The direct target genes of miRNA were identified through global gene expression analysis and target validation. Results and conclusion We found that over-expressing miR-25 or introducing miR-25 mimics enhanced production of iPSCs. We identified a number of miR-25 candidate gene targets. Of particular interest were two ubiquitin ligases, Wwp2 and Fbxw7, which have been proposed to regulate Oct4, c-Myc and Klf5, respectively. Our findings thus highlight the complex interplay between miRNAs and transcription factors involved in reprogramming, stem cell self-renewal and maintenance of pluripotency. PMID:22912667

  12. Optical stimulation enables paced electrophysiological studies in embryonic hearts

    PubMed Central

    Wang, Yves T.; Gu, Shi; Ma, Pei; Watanabe, Michiko; Rollins, Andrew M.; Jenkins, Michael W.

    2014-01-01

    Cardiac electrophysiology plays a critical role in the development and function of the heart. Studies of early embryonic electrical activity have lacked a viable point stimulation technique to pace in vitro samples. Here, optical pacing by high-precision infrared stimulation is used to pace excised embryonic hearts, allowing electrophysiological parameters to be quantified during pacing at varying rates with optical mapping. Combined optical pacing and optical mapping enables electrophysiological studies in embryos under more physiological conditions and at varying heart rates, allowing detection of abnormal conduction and comparisons between normal and pathological electrical activity during development in various models. PMID:24761284

  13. The hallmarks of fibroblast ageing.

    PubMed

    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.

  14. Genome-wide analysis of AR binding and comparison with transcript expression in primary human fetal prostate fibroblasts and cancer associated fibroblasts.

    PubMed

    Nash, Claire; Boufaied, Nadia; Mills, Ian G; Franco, Omar E; Hayward, Simon W; Thomson, Axel A

    2017-05-05

    The androgen receptor (AR) is a transcription factor, and key regulator of prostate development and cancer, which has discrete functions in stromal versus epithelial cells. AR expressed in mesenchyme is necessary and sufficient for prostate development while loss of stromal AR is predictive of prostate cancer progression. Many studies have characterized genome-wide binding of AR in prostate tumour cells but none have used primary mesenchyme or stroma. We applied ChIPseq to identify genomic AR binding sites in primary human fetal prostate fibroblasts and patient derived cancer associated fibroblasts, as well as the WPMY1 cell line overexpressing AR. We identified AR binding sites that were specific to fetal prostate fibroblasts (7534), cancer fibroblasts (629), WPMY1-AR (2561) as well as those common among all (783). Primary fibroblasts had a distinct AR binding profile versus prostate cancer cell lines and tissue, and showed a localisation to gene promoter binding sites 1 kb upstream of the transcriptional start site, as well as non-classical AR binding sequence motifs. We used RNAseq to define transcribed genes associated with AR binding sites and derived cistromes for embryonic and cancer fibroblasts as well as a cistrome common to both. These were compared to several in vivo ChIPseq and transcript expression datasets; which identified subsets of AR targets that were expressed in vivo and regulated by androgens. This analysis enabled us to deconvolute stromal AR targets active in stroma within tumour samples. Taken together, our data suggest that the AR shows significantly different genomic binding site locations in primary prostate fibroblasts compared to that observed in tumour cells. Validation of our AR binding site data with transcript expression in vitro and in vivo suggests that the AR target genes we have identified in primary fibroblasts may contribute to clinically significant and biologically important AR-regulated changes in prostate tissue

  15. Generation of KCL038 clinical grade human embryonic stem cell line

    PubMed Central

    Miere, Cristian; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL038 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. 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 and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345799

  16. Generation of KCL031 clinical grade human embryonic stem cell line

    PubMed Central

    Jacquet, Laureen; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Hobbs, Carl; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL031 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. 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 and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro and in vivo assays. PMID:27345813

  17. Generation of KCL039 clinical grade human embryonic stem cell line

    PubMed Central

    Devito, Liani; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL039 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. 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 and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345806

  18. Generation of KCL037 clinical grade human embryonic stem cell line

    PubMed Central

    Miere, Cristian; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL037 human embryonic stem cell line was derived from a normal healthy blastocyst donated for research. 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 and under current Good Manufacturing Practice (cGMP) standards. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345800

  19. Episomal-based generation of an iPS cell line from human fetal foreskin fibroblasts.

    PubMed

    Matz, Peggy; Adjaye, James

    2016-01-01

    Human fetal foreskin fibroblasts (HFF1) were used to generate the iPSC line epiHFF1-B1 employing a combination of three episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC, and KLF4. Pluripotency was confirmed both in vivo and in vitro. The transcriptome profile of epiHFF1-B1 and the human embryonic stem cell line-H1 have a pearson correlation of 0.936.

  20. The Fibroblast Growth Factor signaling pathway

    PubMed Central

    Ornitz, David M; Itoh, Nobuyuki

    2015-01-01

    The signaling component of the mammalian Fibroblast Growth Factor (FGF) family is comprised of eighteen secreted proteins that interact with four signaling tyrosine kinase FGF receptors (FGFRs). Interaction of FGF ligands with their signaling receptors is regulated by protein or proteoglycan cofactors and by extracellular binding proteins. Activated FGFRs phosphorylate specific tyrosine residues that mediate interaction with cytosolic adaptor proteins and the RAS-MAPK, PI3K-AKT, PLCγ, and STAT intracellular signaling pathways. Four structurally related intracellular non-signaling FGFs interact with and regulate the family of voltage gated sodium channels. Members of the FGF family function in the earliest stages of embryonic development and during organogenesis to maintain progenitor cells and mediate their growth, differentiation, survival, and patterning. FGFs also have roles in adult tissues where they mediate metabolic functions, tissue repair, and regeneration, often by reactivating developmental signaling pathways. Consistent with the presence of FGFs in almost all tissues and organs, aberrant activity of the pathway is associated with developmental defects that disrupt organogenesis, impair the response to injury, and result in metabolic disorders, and cancer. © 2015 Wiley Periodicals, Inc. PMID:25772309

  1. DNA methylation programming and reprogramming in primate embryonic stem cells.

    PubMed

    Cohen, Netta Mendelson; Dighe, Vikas; Landan, Gilad; Reynisdóttir, Sigrún; Palsson, Arnar; Mitalipov, Shoukhrat; Tanay, Amos

    2009-12-01

    DNA methylation is an important epigenetic mechanism, affecting normal development and playing a key role in reprogramming epigenomes during stem cell derivation. Here we report on DNA methylation patterns in native monkey embryonic stem cells (ESCs), fibroblasts, and ESCs generated through somatic cell nuclear transfer (SCNT), identifying and comparing epigenome programming and reprogramming. We characterize hundreds of regions that are hyper- or hypomethylated in fibroblasts compared to native ESCs and show that these are conserved in human cells and tissues. Remarkably, the vast majority of these regions are reprogrammed in SCNT ESCs, leading to almost perfect correlation between the epigenomic profiles of the native and reprogrammed lines. At least 58% of these changes are correlated in cis to transcription changes, Polycomb Repressive Complex-2 occupancy, or binding by the CTCF insulator. We also show that while epigenomic reprogramming is extensive and globally accurate, the efficiency of adding and stripping DNA methylation during reprogramming is regionally variable. In several cases, this variability results in regions that remain methylated in a fibroblast-like pattern even after reprogramming.

  2. Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors

    PubMed Central

    Cheng, Hui; Ang, Heather Yin-Kuan; A. EL Farran, Chadi; Li, Pin; Fang, Hai Tong; Liu, Tong Ming; Kong, Say Li; Chin, Michael Lingzi; Ling, Wei Yin; Lim, Edwin Kok Hao; Li, Hu; Huber, Tara; Loh, Kyle M.; Loh, Yuin-Han; Lim, Bing

    2016-01-01

    Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into ‘induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase+ megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit+ hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors. PMID:27869129

  3. PDGFRalphaalpha signaling is regulated through the primary cilium in fibroblasts.

    PubMed

    Schneider, Linda; Clement, Christian A; Teilmann, Stefan C; Pazour, Gregory J; Hoffmann, Else K; Satir, Peter; Christensen, Søren T

    2005-10-25

    Recent findings show that cilia are sensory organelles that display specific receptors and ion channels, which transmit signals from the extracellular environment via the cilium to the cell to control tissue homeostasis and function. Agenesis of primary cilia or mislocation of ciliary signal components affects human pathologies, such as polycystic kidney disease and disorders associated with Bardet-Biedl syndrome. Primary cilia are essential for hedgehog ligand-induced signaling cascade regulating growth and patterning. Here, we show that the primary cilium in fibroblasts plays a critical role in growth control via platelet-derived growth factor receptor alpha (PDGFRalpha), which localizes to the primary cilium during growth arrest in NIH3T3 cells and primary cultures of mouse embryonic fibroblasts. Ligand-dependent activation of PDGFRalphaalpha is followed by activation of Akt and the Mek1/2-Erk1/2 pathways, with Mek1/2 being phosphorylated within the cilium and at the basal body. Fibroblasts derived from Tg737(orpk) mutants fail to form normal cilia and to upregulate the level of PDGFRalpha; PDGF-AA fails to activate PDGFRalphaalpha and the Mek1/2-Erk1/2 pathway. Signaling through PDGFRbeta, which localizes to the plasma membrane, is maintained at comparable levels in wild-type and mutant cells. We propose that ciliary PDGFRalphaalpha signaling is linked to tissue homeostasis and to mitogenic signaling pathways.

  4. How does the tubular embryonic heart work? Looking for the physical mechanism generating unidirectional blood flow in the valveless embryonic heart tube.

    PubMed

    Männer, Jörg; Wessel, Armin; Yelbuz, T Mesud

    2010-04-01

    The heart is the first organ to function in vertebrate embryos. The human heart, for example, starts beating around the 21st embryonic day. During the initial phase of its pumping action, the embryonic heart is seen as a pulsating blood vessel that is built up by (1) an inner endothelial tube lacking valves, (2) a middle layer of extracellular matrix, and (3) an outer myocardial tube. Despite the absence of valves, this tubular heart generates unidirectional blood flow. This fact poses the question how it works. Visual examination of the pulsating embryonic heart tube shows that its pumping action is characterized by traveling mechanical waves sweeping from its venous to its arterial end. These traveling waves were traditionally described as myocardial peristaltic waves. It has, therefore, been speculated that the tubular embryonic heart works as a technical peristaltic pump. Recent hemodynamic data from living embryos, however, have shown that the pumping function of the embryonic heart tube differs in several respects from that of a technical peristaltic pump. Some of these data suggest that embryonic heart tubes work as valveless "Liebau pumps." In the present study, a review is given on the evolution of the two above-mentioned theories of early cardiac pumping mechanics. We discuss pros and cons for both of these theories. We show that the tubular embryonic heart works neither as a technical peristaltic pump nor as a classic Liebau pump. The question regarding how the embryonic heart tube works still awaits an answer.

  5. Extensive determination of glycan heterogeneity reveals an unusual abundance of high mannose glycans in enriched plasma membranes of human embryonic stem cells.

    PubMed

    An, Hyun Joo; Gip, Phung; Kim, Jaehan; Wu, Shuai; Park, Kun Wook; McVaugh, Cheryl T; Schaffer, David V; Bertozzi, Carolyn R; Lebrilla, Carlito B

    2012-04-01

    Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine

  6. MRG15 Regulates Embryonic Development and Cell Proliferation

    PubMed Central

    Tominaga, Kaoru; Kirtane, Bhakti; Jackson, James G.; Ikeno, Yuji; Ikeda, Takayoshi; Hawks, Christina; Smith, James R.; Matzuk, Martin M.; Pereira-Smith, Olivia M.

    2005-01-01

    MRG15 is a highly conserved protein, and orthologs exist in organisms from yeast to humans. MRG15 associates with at least two nucleoprotein complexes that include histone acetyltransferases and/or histone deacetylases, suggesting it is involved in chromatin remodeling. To study the role of MRG15 in vivo, we generated knockout mice and determined that the phenotype is embryonic lethal, with embryos and the few stillborn pups exhibiting developmental delay. Immunohistochemical analysis indicates that apoptosis in Mrg15−/− embryos is not increased compared with wild-type littermates. However, the number of proliferating cells is significantly reduced in various tissues of the smaller null embryos compared with control littermates. Cell proliferation defects are also observed in Mrg15−/− mouse embryonic fibroblasts. The hearts of the Mrg15−/− embryos exhibit some features of hypertrophic cardiomyopathy. The increase in size of the cardiomyocytes is most likely a response to decreased growth of the cells. Mrg15−/− embryos appeared pale, and microarray analysis revealed that α-globin gene expression was decreased in null versus wild-type embryos. We determined by chromatin immunoprecipitation that MRG15 was recruited to the α-globin promoter during dimethyl sulfoxide-induced mouse erythroleukemia cell differentiation. These findings demonstrate that MRG15 has an essential role in embryonic development via chromatin remodeling and transcriptional regulation. PMID:15798182

  7. Fibroblast heterogeneity: more than skin deep.

    PubMed

    Sorrell, J Michael; Caplan, Arnold I

    2004-02-15

    Dermal fibroblasts are a dynamic and diverse population of cells whose functions in skin in many respects remain unknown. Normal adult human skin contains at least three distinct subpopulations of fibroblasts, which occupy unique niches in the dermis. Fibroblasts from each of these niches exhibit distinctive differences when cultured separately. Specific differences in fibroblast physiology are evident in papillary dermal fibroblasts, which reside in the superficial dermis, and reticular fibroblasts, which reside in the deep dermis. Both of these subpopulations of fibroblasts differ from the fibroblasts that are associated with hair follicles. Fibroblasts engage in fibroblast-epidermal interactions during hair development and in interfollicular regions of skin. They also play an important role in cutaneous wound repair and an ever-increasing role in bioengineering of skin. Bioengineered skin currently performs important roles in providing (1) a basic understanding of skin biology, (2) a vehicle for testing topically applied products and (3) a resource for skin replacement.

  8. Embryonic lethal phenotype reveals a function of TDG in maintaining epigenetic stability.

    PubMed

    Cortázar, Daniel; Kunz, Christophe; Selfridge, Jim; Lettieri, Teresa; Saito, Yusuke; MacDougall, Eilidh; Wirz, Annika; Schuermann, David; Jacobs, Angelika L; Siegrist, Fredy; Steinacher, Roland; Jiricny, Josef; Bird, Adrian; Schär, Primo

    2011-02-17

    Thymine DNA glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily of DNA repair enzymes. Owing to its ability to excise thymine when mispaired with guanine, it was proposed to act against the mutability of 5-methylcytosine (5-mC) deamination in mammalian DNA. However, TDG was also found to interact with transcription factors, histone acetyltransferases and de novo DNA methyltransferases, and it has been associated with DNA demethylation in gene promoters following activation of transcription, altogether implicating an engagement in gene regulation rather than DNA repair. Here we use a mouse genetic approach to determine the biological function of this multifaceted DNA repair enzyme. We find that, unlike other DNA glycosylases, TDG is essential for embryonic development, and that this phenotype is associated with epigenetic aberrations affecting the expression of developmental genes. Fibroblasts derived from Tdg null embryos (mouse embryonic fibroblasts, MEFs) show impaired gene regulation, coincident with imbalanced histone modification and CpG methylation at promoters of affected genes. TDG associates with the promoters of such genes both in fibroblasts and in embryonic stem cells (ESCs), but epigenetic aberrations only appear upon cell lineage commitment. We show that TDG contributes to the maintenance of active and bivalent chromatin throughout cell differentiation, facilitating a proper assembly of chromatin-modifying complexes and initiating base excision repair to counter aberrant de novo methylation. We thus conclude that TDG-dependent DNA repair has evolved to provide epigenetic stability in lineage committed cells.

  9. Cadherin-11 Promotes Invasive Behavior of Fibroblast-like Synoviocytes

    PubMed Central

    Kiener, Hans P.; Niederreiter, Birgit; Lee, David M.; Jimenez-Boj, Esther; Smolen, Josef S.; Brenner, Michael B.

    2009-01-01

    Objective To define the expression pattern of cadherin-11 in destructive pannus tissue of patients with rheumatoid arthritis and to determine if cadherin-11 expression in fibroblast-like synoviocytes controls their invasive capacity. Methods Cadherin-11 expression in rheumatoid synovial tissue was evaluated using immunohistochemistry. To examine the role of cadherin-11 in regulating the invasive behavior of fibroblast-like synoviocytes, we generated L-cell clones expressing wild-type cadherin-11, mutant cadherin-11, and empty vector transfected controls. The invasive capacity of L-cell transfectants and cultured fibroblast-like synoviocytes treated with a blocking cadherin-11-Fc protein or control immunoglobulin was determined in Matrigel invasion assays. Results Immunohistochemistry revealed that cadherin-11 is abundantly expressed in cells at the cartilage-pannus junction in rheumatoid synovitis. Invasion assays demonstrate a twofold increased invasive capacity of cadherin-11 transfected L-cells compared to L-cells transfected with E-cadherin or control vector. The invasive behavior of the L-cells stably transfected with a cadherin-11 construct that lacked the juxta-membrane cytoplasmic domain (cadherin-11 ΔJMD) was diminished to the level of vector control L-cells. Further, treatment with the cadherin-11-Fc fusion protein diminished the invasive capacity of fibroblast-like synoviocytes. Conclusion These in vitro studies implicate a role for cadherin-11 in promoting cell invasion and contribute insight into the invasive nature of fibroblast-like synoviocytes in chronic synovitis and rheumatoid arthritis. PMID:19404963

  10. Altered nuclear structure in myotonic dystrophy type 1-derived fibroblasts.

    PubMed

    Rodríguez, R; Hernández-Hernández, O; Magaña, J J; González-Ramírez, R; García-López, E S; Cisneros, B

    2015-02-01

    Myotonic dystrophy type 1 (DM1) is a multisystem genetic disorder caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. DMPK gene transcripts containing CUG expanded repeats accumulate in nuclear foci and ultimately cause altered splicing/gene expression of numerous secondary genes. The study of primary cell cultures derived from patients with DM1 has allowed the identification and further characterization of molecular mechanisms underlying the pathology in the natural context of the disease. In this study we show for the first time impaired nuclear structure in fibroblasts of DM1 patients. DM1-derived fibroblasts exhibited altered localization of the nuclear envelope (NE) proteins emerin and lamins A/C and B1 with concomitant increased size and altered shape of nuclei. Abnormal NE organization is more common in DM1 fibroblasts containing abundant nuclear foci, implying expression of the expanded RNA as determinant of nuclear defects. That transient expression of the DMPK 3' UTR containing 960 CTG but not with the 3' UTR lacking CTG repeats is sufficient to generate NE disruption in normal fibroblasts confirms the direct impact of mutant RNA on NE architecture. We also evidence nucleoli distortion in DM1 fibroblasts by immunostaining of the nucleolar protein fibrillarin, implying a broader effect of the mutant RNA on nuclear structure. In summary, these findings reveal that NE disruption, a hallmark of laminopathy disorders, is a novel characteristic of DM1.

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

    PubMed

    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

  12. A small set of extra-embryonic genes defines a new landmark for bovine embryo staging.

    PubMed

    Degrelle, Séverine A; Lê Cao, Kim-Anh; Heyman, Yvan; Everts, Robin E; Campion, Evelyne; Richard, Christophe; Ducroix-Crépy, Céline; Tian, X Cindy; Lewin, Harris A; Renard, Jean-Paul; Robert-Granié, Christèle; Hue, Isabelle

    2011-01-01

    Axis specification in mouse is determined by a sequence of reciprocal interactions between embryonic and extra-embryonic tissues so that a few extra-embryonic genes appear as 'patterning' the embryo. Considering these interactions as essential, but lacking in most mammals the genetically driven approaches used in mouse and the corresponding patterning mutants, we examined whether a molecular signature originating from extra-embryonic tissues could relate to the developmental stage of the embryo proper and predict it. To this end, we have profiled bovine extra-embryonic tissues at peri-implantation stages, when gastrulation and early neurulation occur, and analysed the subsequent expression profiles through the use of predictive methods as previously reported for tumour classification. A set of six genes (CALM1, CPA3, CITED1, DLD, HNRNPDL, and TGFB3), half of which had not been previously associated with any extra-embryonic feature, appeared significantly discriminative and mainly dependent on embryonic tissues for its faithful expression. The predictive value of this set of genes for gastrulation and early neurulation stages, as assessed on naive samples, was remarkably high (93%). In silico connected to the bovine orthologues of the mouse patterning genes, this gene set is proposed as a new trait for embryo staging. As such, this will allow saving the bovine embryo proper for molecular or cellular studies. To us, it offers as well new perspectives for developmental phenotyping and modelling of embryonic/extra-embryonic co-differentiation.

  13. [Fibroblast growth factor-2].

    PubMed

    Faitová, J

    2004-01-01

    Fibroblast growth factor-2 is a member of a large family of proteins that bind heparin and heparan sulfate and modulate the function of a wide range of cell types. FGF-2 occurs in several isoforms resulting from alternative initiations of traslation: an 18 kDa cytoplasmic isoform and four larger molecular weight nuclear isoforms (22, 22.5, 24 and 34 kDa). It acts mainly through a paracrine/autocrine mechanism involving high affinity transmembrane receptors and heparan sulfate proteoglycan low affinity receptors. It is expressed mostly in tissues of mesoderm and neuroectoderm origin, and plays an important role in mesoderm induction, stimulates the growth and development of the new blood vessels (angiogenesis), normal wound healing and tissue development. FGF-2 positively regulates hematopoiesis by acting on various cellular targets: stromal cells, early and committed hematopoietic progenitors and possibly some mature blood cells. FGF-2 is a potent hematopoietic growth factor that is likely to play an important role in physiological and pathological hematopoiesis.

  14. Prostacyclin analogs inhibit fibroblast migration.

    PubMed

    Kohyama, Tadashi; Liu, Xiangde; Kim, Hui Jung; Kobayashi, Tetsu; Ertl, Ronald F; Wen, Fu-Qiang; Takizawa, Hajime; Rennard, Stephen I

    2002-08-01

    The controlled accumulation of fibroblasts to sites of inflammation is crucial to effective tissue repair after injury. Either inadequate or excessive accumulation of fibroblasts could result in abnormal tissue function. Prostacyclin (PGI(2)) is a potent mediator in the coagulation and inflammatory processes. The aim of this study was to investigate the effect of PGI(2) on chemotaxis of human fetal lung fibroblasts (HFL-1). Using the blind well chamber technique, we found that the PGI(2) analog carbaprostacyclin (10(-6) M) inhibited HFL-1 chemotaxis to human plasma fibronectin (20 microg/ml) 58.0 +/- 13.2% (P < 0.05) and to platelet-derived growth factor (PDGF)-BB (10 ng/ml) 48.7 +/- 4.6% (P < 0.05). Checkerboard analysis demonstrated that carbaprostacyclin inhibits both directed and undirected migration. The inhibitory effect of the carbaprostacyclin was concentration dependent and blocked by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, suggesting that a cAMP-PKA pathway may be involved in the process. Two other PGI(2) analogs, ciprostene and dehydro-15-cyclohexyl carbaprostacyclin (both 10(-6) M), significantly inhibited fibroblast migration to fibronectin. In summary, PGI(2) appears to inhibit fibroblast chemotaxis to fibronectin and PDGF-BB. Such an effect may contribute to the regulation of fibroblasts in wound healing and could contribute to the pathogenesis of diseases characterized by abnormal tissue repair remodeling.

  15. Mitochondrial permeabilization without caspase activation mediates the increase of basal apoptosis in cells lacking Nrf2.

    PubMed

    Ariza, Julia; González-Reyes, José A; Jódar, Laura; Díaz-Ruiz, Alberto; de Cabo, Rafael; Villalba, José Manuel

    2016-06-01

    Nuclear factor E2-related factor-2 (Nrf2) is a cap'n'collar/basic leucine zipper (b-ZIP) transcription factor which acts as sensor of oxidative and electrophilic stress. Low levels of Nrf2 predispose cells to chemical carcinogenesis but a dark side of Nrf2 function also exists because its unrestrained activation may allow the survival of potentially dangerous damaged cells. Since Nrf2 inhibition may be of therapeutic interest in cancer, and a decrease of Nrf2 activity may be related with degenerative changes associated with aging, it is important to investigate how the lack of Nrf2 function activates molecular mechanisms mediating cell death. Murine Embryonic Fibroblasts (MEFs) bearing a Nrf2 deletion (Nrf2KO) displayed diminished cellular growth rate and shortened lifespan compared with wild-type MEFs. Basal rates of DNA fragmentation and histone H2A.X phosphorylation were higher in Nrf2KO MEFs, although steady-state levels of reactive oxygen species were not significantly increased. Enhanced rates of apoptotic DNA fragmentation were confirmed in liver and lung tissues from Nrf2KO mice. Apoptosis in Nrf2KO MEFs was associated with a decrease of Bcl-2 but not Bax levels, and with the release of the mitochondrial pro-apoptotic factors cytochrome c and AIF. Procaspase-9 and Apaf-1 were also increased in Nrf2KO MEFs but caspase-3 was not activated. Inhibition of XIAP increased death in Nrf2KO but not in wild-type MEFs. Mitochondrial ultrastructure was also altered in Nrf2KO MEFs. Our results support that Nrf2 deletion produces mitochondrial dysfunction associated with mitochondrial permeabilization, increasing basal apoptosis through a caspase-independent and AIF-dependent pathway.

  16. Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

    PubMed Central

    Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

    2012-01-01

    Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

  17. Differential effects on cell motility, embryonic stem cell self-renewal and senescence by diverse Src kinase family inhibitors

    SciTech Connect

    Tamm, Christoffer Galito, Sara Pijuan Anneren, Cecilia

    2012-02-15

    The Src family of non-receptor tyrosine kinases (SFKs) has been shown to play an intricate role in embryonic stem (ES) cell maintenance. In the present study we have focused on the underlying molecular mechanisms responsible for the vastly different effects induced by various commonly used SFK inhibitors. We show that several diverse cell types, including fibroblasts completely lacking SFKs, cannot undergo mitosis in response to SU6656 and that this is caused by an unselective inhibition of Aurora kinases. In contrast, PP2 and PD173952 block motility immediately upon exposure and forces cells to grow in dense colonies. The subsequent halt in proliferation of fibroblast and epithelial cells in the center of the colonies approximately 24 h post-treatment appears to be caused by cell-to-cell contact inhibition rather than a direct effect of SFK kinase inhibition. Interestingly, in addition to generating more homogenous and dense ES cell cultures, without any diverse effect on proliferation, PP2 and PD173652 also promote ES cell self-renewal by reducing the small amount of spontaneous differentiation typically observed under standard ES cell culture conditions. These effects could not be mirrored by the use of Gleevec, a potent inhibitor of c-Abl and PDGFR kinases that are also inhibited by PP2. -- Highlights: Black-Right-Pointing-Pointer SFK inhibitor SU6656 induces senescence in mouse ES cells. Black-Right-Pointing-Pointer SU6656 inhibits mitosis in a SFK-independent manner via cross-selectivity for Aurora kinases. Black-Right-Pointing-Pointer SFK inhibitor PP2 impairs cell motility in various cell lines, including mouse ES cells. Black-Right-Pointing-Pointer Ensuing impeded motility, PP2 inhibits proliferation of various cells lines except for mouse ES cells. Black-Right-Pointing-Pointer SFK inhibitors PP2 and PD173952 impede spontaneous differentiation in standard mouse ES culture maintenance.

  18. The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts.

    PubMed

    Hartmann, Dieter; de Strooper, Bart; Serneels, Lutgarde; Craessaerts, Katleen; Herreman, An; Annaert, Wim; Umans, Lieve; Lübke, Torben; Lena Illert, Anna; von Figura, Kurt; Saftig, Paul

    2002-10-01

    The metalloprotease ADAM 10 is an important APP alpha-secretase candidate, but in vivo proof of this is lacking. Furthermore, invertebrate models point towards a key role of the ADAM 10 orthologues Kuzbanian and sup-17 in Notch signalling. In the mouse, this function is, however, currently attributed to ADAM 17/TACE, while the role of ADAM 10 remains unknown. We have created ADAM 10-deficient mice. They die at day 9.5 of embryogenesis with multiple defects of the developing central nervous system, somites, and cardiovascular system. In situ hybridization revealed a reduced expression of the Notch target gene hes-5 in the neural tube and an increased expression of the Notch ligand dll-1, supporting an important role for ADAM 10 in Notch signalling in the vertebrates as well. Since the early lethality precluded the establishment of primary neuronal cultures, APPs alpha generation was analyzed in embryonic fibroblasts and found to be preserved in 15 out of 17 independently generated ADAM 10-deficient fibroblast cell lines, albeit at a quantitatively more variable level than in controls, whereas a severe reduction was found in only two cases. The variability was not due to differences in genetic background or to variable expression of the alternative alpha-secretase candidates ADAM 9 and ADAM 17. These results indicate, therefore, either a regulation between ADAMs on the post-translational level or that other, not yet known, proteases are able to compensate for ADAM 10 deficiency. Thus, the observed variability, together with recent reports on tissue-specific expression patterns of ADAMs 9, 10 and 17, points to the existence of tissue-specific 'teams' of different proteases exerting alpha-secretase activity.

  19. Host cis-Mediated Extinction of a Retrovirus Permissive for Expression in Embryonal Stem Cells during Differentiation

    PubMed Central

    Laker, Christine; Meyer, Johann; Schopen, Arndt; Friel, Jutta; Heberlein, Christoph; Ostertag, Wolfram; Stocking, Carol

    1998-01-01

    The use of retroviral vectors for gene transfer into animals has been severely hampered by the lack of provirus transcription in the early embryo and embryonic stem (ES) cells. This primary block in provirus expression is maintained in differentiated cells by a cis-acting mechanism that is not well characterized. Retroviral vectors based on the murine embryonal stem cell virus (MESV), which overcome the transcriptional block in ES cells, were constructed to investigate this secondary mechanism. These vectors transferred G418 resistance to ES cells with the same efficiency as to fibroblasts, but overall transcript levels were greatly reduced. A mosaic but stable expression pattern was observed when single cells from G418-resistant clones were replated in G418 or assayed for expression of LacZ or interleukin-3. The expression levels in independent clones were variable and correlated inversely with methylation. However, a second, more pronounced, block to transcription was found upon differentiation induction. Differentiation of the infected ES cells to cells permissive for retroviral expression resulted in repression and complete extinction of provirus expression. Extinction was not accompanied by increased levels of methylation. Provirus expression is thus regulated by two independent cis-acting mechanisms: (i) partial repression in the undifferentiated state, accompanied by increased methylation but compatible with long-term, low expression of retroviral genes, and (ii) total repression and extinction during early stages of differentiation, apparently independent of changes in methylation. These results indicate a time window early during the transition from an undifferentiated to a differentiated stage in which provirus expression is silenced. The mechanisms are presently unknown, but elucidation of these events will have an important impact on vector development for targeting stem cells and for gene therapy. PMID:9420232

  20. Derivation of Transgene-Free Rat Induced Pluripotent Stem Cells Approximating the Quality of Embryonic Stem Cells.

    PubMed

    Li, Shuping; Lan, He; Men, Hongsheng; Wu, Yuanyuan; Li, Ning; Capecchi, Mario R; Bryda, Elizabeth C; Wu, Sen

    2017-02-01

    Although a variety of reprogramming strategies have been reported to create transgene-free induced pluripotent stem (iPS) cells from differentiated cell sources, a fundamental question still remains: Can we generate safe iPS cells that have the full spectrum of features of corresponding embryonic stem (ES) cells? Studies in transgene-free mouse iPS cells have indicated a positive answer to this question. However, the reality is that no other species have a derived transgene-free iPS cell line that can truly mimic ES cell quality. Specifically, critical data for chimera formation and germline transmission are generally lacking. To date, the rat is the only species, other than the mouse, that has commonly recognized authentic ES cells that can be used for direct comparison with measure features of iPS cells. To help find the underlying reasons of the current inability to derive germline-competent ES/iPS cells in nonrodent animals, we first used optimized culture conditions to isolate and establish rat ES cell lines and demonstrated they are fully competent for chimeric formation and germline transmission. We then used episomal vectors bearing eight reprogramming genes to improve rat iPS (riPS) cell generation from Sprague-Dawley rat embryonic fibroblasts. The obtained transgene-free riPS cells exhibit the typical characteristics of pluripotent stem cells; moreover, they are amenable to subsequent genetic modification by homologous recombination. Although they can contribute significantly to chimeric formation, no germline transmission has been achieved. Although this partial success in achieving competency is encouraging, it suggests that more efforts are still needed to derive ground-state riPS cells. Stem Cells Translational Medicine 2017;6:340-351. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  1. Identification and quantitation of morphological cell types in electrophoretically separated human embryonic kidney cell cultures

    NASA Technical Reports Server (NTRS)

    Williams, K. B.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    Four major cell types were identified by phase microscopy in early passage human embryonic kidney cell cultures. They are small and large epithelioid, domed, and fenestrated cells. Fibroblasts are also present in some explants. The percent of each cell type changes with passage number as any given culture grows. As a general rule, the fraction of small epithelioid cells increases, while the fraction of fenestrated cells, always small, decreases further. When fibroblasts are present, they always increase in percentage of the total cell population. Electrophoretic separation of early passage cells showed that the domed cells have the highest electrophoretic mobility, fibroblasts have an intermediate high mobility, small epithelioid cells have a low mobility, broadly distributed, and fenestrated cells have the lowest mobility. All cell types were broadly distributed among electrophoretic subfractions, which were never pure but only enriched with respect to a given cell type.

  2. Identification and quantitation of morphological cell types in electrophoretically separated human embryonic kidney cell cultures

    NASA Technical Reports Server (NTRS)

    Williams, K. B.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    Four major cell types were identified by phase microscopy in early passage human embryonic kidney cell cultures. They are small and large epithelioid, domed, and fenestrated cells. Fibroblasts are also present in some explants. The percent of each cell type changes with passage number as any given culture grows. As a general rule, the fraction of small epithelioid cells increases, while the fraction of fenestrated cells, always small, decreases further. When fibroblasts are present, they always increase in percentage of the total cell population. Electrophoretic separation of early passage cells showed that the domed cells have the highest electrophoretic mobility, fibroblasts have an intermediate high mobility, small epithelioid cells have a low mobility, broadly distributed, and fenestrated cells have the lowest mobility. All cell types were broadly distributed among electrophoretic subfractions, which were never pure but only enriched with respect to a given cell type.

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

    NASA Astrophysics Data System (ADS)

    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.

  4. The dynamin-related GTPase Drp1 is required for embryonic and brain development in mice.

    PubMed

    Wakabayashi, Junko; Zhang, Zhongyan; Wakabayashi, Nobunao; Tamura, Yasushi; Fukaya, Masahiro; Kensler, Thomas W; Iijima, Miho; Sesaki, Hiromi

    2009-09-21

    The dynamin-related guanosine triphosphatase Drp1 mediates the division of mitochondria and peroxisomes. To understand the in vivo function of Drp1, complete and tissue-specific mouse knockouts of Drp1 were generated. Drp1-null mice die by embryonic day 11.5. This embryonic lethality is not likely caused by gross energy deprivation, as Drp1-null cells showed normal intracellular adenosine triphosphate levels. In support of the role of Drp1 in organelle division, mitochondria formed extensive networks, and peroxisomes were elongated in Drp1-null embryonic fibroblasts. Brain-specific Drp1 ablation caused developmental defects of the cerebellum in which Purkinje cells contained few giant mitochondria instead of the many short tubular mitochondria observed in control cells. In addition, Drp1-null embryos failed to undergo developmentally regulated apoptosis during neural tube formation in vivo. However, Drp1-null embryonic fibroblasts have normal responses to apoptotic stimuli in vitro, suggesting that the apoptotic function of Drp1 depends on physiological cues. These findings clearly demonstrate the physiological importance of Drp1-mediated organelle division in mice.

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

    PubMed

    Denissova, Natalia G; Tereshchenko, Irina V; Cui, Eric; Stambrook, Peter J; Shao, Changshun; Tischfield, Jay A

    2011-10-01

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

  6. PINOCYTOSIS IN FIBROBLASTS

    PubMed Central

    Steinman, Ralph M.; Silver, Jonathan M.; Cohn, Zanvil A.

    1974-01-01

    Horseradish peroxidase (HRP) was used as a marker to determine the rate of ongoing pinocytosis in several fibroblast cell lines. The enzyme was interiorized in the fluid phase without evidence of adsorption to the cell surface. Cytochemical reaction product was not found on the cell surface and was visualized only within intracellular vesicles and granules. Uptake was directly proportional to the administered concentration of HRP and to the duration of exposure. The rate of HRP uptake was 0.0032–0.0035% of the administered load per 106 cells per hour for all cells studied with one exception: L cells, after reaching confluence, progressively increased their pinocytic activity two- to fourfold. After uptake of HRP, L cells inactivated HRP with a half-life of 6–8 h. Certain metabolic requirements of pinocytosis were then studied in detail in L cells. Raising the environmental temperature increased pinocytosis over a range of 2–38°C. The Q10 was 2.7 and the activation energy, 17.6 kcal/mol. Studies on the levels of cellular ATP in the presence of various metabolic inhibitors (fluoride, 2-desoxyglycose, azide, and cyanide) showed that L cells synthesized ATP by both glycolytic and respiratory pathways. A combination of a glycolytic and a respiratory inhibitor was needed to depress cellular ATP levels as well as pinocytic activity to 10–20% of control values, whereas drugs administered individually had only partial effects. In spite of the availability of an accurate quantitative assay for fluid and solute uptake, the function of pinocytosis in tissue culture cells remains unknown. PMID:4140194

  7. Rheology of embryonic avian blood.

    PubMed

    Al-Roubaie, Sarah; Jahnsen, Espen D; Mohammed, Masud; Henderson-Toth, Caitlin; Jones, Elizabeth A V

    2011-12-01

    Shear stress, a mechanical force created by blood flow, is known to affect the developing cardiovascular system. Shear stress is a function of both shear rate and viscosity. While established techniques for measuring shear rate in embryos have been developed, the viscosity of embryonic blood has never been known but always assumed to be like adult blood. Blood is a non-Newtonian fluid, where the relationship between shear rate and shear stress is nonlinear. In this work, we analyzed the non-Newtonian behavior of embryonic chicken blood using a microviscometer and present the apparent viscosity at different hematocrits, different shear rates, and at different stages during development from 4 days (Hamburger-Hamilton stage 22) to 8 days (about Hamburger-Hamilton stage 34) of incubation. We chose the chicken embryo since it has become a common animal model for studying hemodynamics in the developing cardiovascular system. We found that the hematocrit increases with the stage of development. The viscosity of embryonic avian blood in all developmental stages studied was shear rate dependent and behaved in a non-Newtonian manner similar to that of adult blood. The range of shear rates and hematocrits at which non-Newtonian behavior was observed is, however, outside the physiological range for the larger vessels of the embryo. Under low shear stress conditions, the spherical nucleated blood cells that make up embryonic blood formed into small aggregates of cells. We found that the apparent blood viscosity decreases at a given hematocrit during embryonic development, not due to changes in protein composition of the plasma but possibly due to the changes in cellular composition of embryonic blood. This decrease in apparent viscosity was only visible at high hematocrit. At physiological values of hematocrit, embryonic blood viscosity did not change significantly with the stage of development.

  8. Disseminated Cerebrospinal Embryonal Tumor in the Adult

    PubMed Central

    Armocida, Daniele; Caporlingua, Federico; Lapadula, Gennaro; Elefante, Grazia Maria; Antonelli, Manila; Salvati, Maurizio

    2016-01-01

    Introduction. According to the 2016 World Health Organization classification of Tumors of the Central Nervous System, the term Primitive Neuroectodermal Tumor has been replaced by the term Embryonal Tumor (ET). We present a case of disseminated cerebrospinal ET presenting in an adult patient. Illustrative Case. A 49-year-old male presenting with low back pain, dysuria, and hypoesthesia of the lower extremities referred to our emergency department. Brain and whole spine contrast-enhanced MRI documented a diffusively disseminated heterogeneous neoplasm with intradural extra- and intramedullary involvement of the cervicothoracic tract and cauda equina. A primary biopsy of the lumbosacral localization was performed through L5 bilateral laminectomy. Histologic diagnosis was Embryonal Tumor Not Otherwise Specified. The patient underwent chemotherapy with postoperative adjuvant alternating Vincristine-Doxorubicin-Ifosfamide (VAI) and Ifosfamide-Etoposide (IE). Discussion. Spinal ETs are exceedingly rare especially when presenting in the adult patient. Neurosurgical and oncologic management is still unclear. When feasible, surgical removal should always be performed to obtain a histologic diagnosis. Postoperative adjuvant therapy might entail both chemo- and radiotherapy; however a consensus on this matter is still lacking. PMID:27818821

  9. Differential Thy-1 expression by splenic fibroblasts defines functionally distinct subsets.

    PubMed

    Borrello, M A; Phipps, R P

    1996-11-01

    Fibroblasts have an important structural role in the spleen, as they provide a scaffold of extracellular matrix in which cells of the immune system reside. Aside from their vague recognition as "stromal" or "reticular" components of the spleen, these cells have not been characterized. In this study, normal fibroblast lines from mouse [B6D2(F1)] spleen were established. The fibroblast phenotype of these lines was confirmed by their morphology, expression of vimentin, as well as their lack of epithelial and endothelial cell markers, their failure to display the hematopoietic marker CD45, and their inability to phagocytize. Interestingly, 50-65% of the splenic fibroblasts expressed the Thy-1 antigen, while a subpopulation of Thy-1-negative fibroblasts existed. FACS on the basis of Thy-1, as well as limiting dilution cloning, yielded stable lines and clones of Thy-1+ and Thy-1- splenic fibroblasts. Phenotypic characterization revealed that both subsets synthesized collagen and expressed class I MHC, ICAM-1, VCAM-1, and CD44 constitutively. However, intriguing differences existed between the fibroblast subpopulations. Thy-1+ splenic fibroblasts produced significantly greater levels of IL-6 than did their Thy-1- counterparts. After treatment with IFN-gamma (150 U/ml, 72 hr), Thy-1-, but not Thy-1+, splenic fibroblasts expressed class II MHC and presented antigen to an I-A(b)-restricted T cell line. This suggests that the Thy-1- fibroblasts may present antigen to T lymphocytes in vivo under inflammatory conditions. Thus, splenic fibroblasts are a heterogeneous and dynamic cell type poised in an immunologically relevant location to interact with bone marrow-derived cells under normal and fibrotic conditions.

  10. Tumor-associated fibroblasts predominantly come from local and not circulating precursors

    PubMed Central

    Arina, Ainhoa; Idel, Christian; Hyjek, Elizabeth M.; Alegre, Maria-Luisa; Wang, Ying; Bindokas, Vytautas P.; Weichselbaum, Ralph R.; Schreiber, Hans

    2016-01-01

    Fibroblasts are common cell types in cancer stroma and lay down collagen required for survival and growth of cancer cells. Although some cancer therapy strategies target tumor fibroblasts, their origin remains controversial. Multiple publications suggest circulating mesenchymal precursors as a source of tumor-associated fibroblasts. However, we show by three independent approaches that tumor fibroblasts derive primarily from local, sessile precursors. First, transplantable tumors developing in a mouse expressing green fluorescent reporter protein (EGFP) under control of the type I collagen (Col-I) promoter (COL-EGFP) had green stroma, whereas we could not find COL-EGFP+ cells in tumors developing in the parabiotic partner lacking the fluorescent reporter. Lack of incorporation of COL-EGFP+ cells from the circulation into tumors was confirmed in parabiotic pairs of COL-EGFP mice and transgenic mice developing autochthonous intestinal adenomas. Second, transplantable tumors developing in chimeric mice reconstituted with bone marrow cells from COL-EGFP mice very rarely showed stromal fibroblasts expressing EGFP. Finally, cancer cells injected under full-thickness COL-EGFP skin grafts transplanted in nonreporter mice developed into tumors containing green stromal cells. Using multicolor in vivo confocal microscopy, we found that Col-I–expressing fibroblasts constituted approximately one-third of the stromal mass and formed a continuous sheet wrapping the tumor vessels. In summary, tumors form their fibroblastic stroma predominantly from precursors present in the local tumor microenvironment, whereas the contribution of bone marrow-derived circulating precursors is rare. PMID:27317748

  11. Mechanotransduction in Embryonic Vascular Development

    PubMed Central

    Roman, Beth L.; Pekkan, Kerem

    2015-01-01

    A plethora of biochemical signals provides spatial and temporal cues that carefully orchestrate the complex process of vertebrate embryonic development. The embryonic vasculature develops not only in the context of these biochemical cues, but also in the context of the biomechanical forces imparted by blood flow. In the mature vasculature, different blood flow regimes induce distinct genetic programs, and significant progress has been made toward understanding how these forces are perceived by endothelial cells and transduced into biochemical signals. However, it cannot be assumed that paradigms that govern the mature vasculature are pertinent to the developing embryonic vasculature. The embryonic vasculature can respond to the mechanical forces of blood flow, and these responses are critical in vascular remodeling, certain aspects of sprouting angiogenesis, and maintenance of arterial-venous identity. Here, we review data regarding mechanistic aspects of endothelial cell mechanotransduction, with a focus on the response to shear stress, and elaborate upon the multifarious effects of shear stress on the embryonic vasculature. In addition, we discuss emerging predictive vascular growth models and highlight the prospect of combining signaling pathway information with computational modeling. We assert that correlation of precise measurements of hemodynamic parameters with effects on endothelial cell gene expression and cell behavior is required for fully understanding how blood flow-induced loading governs normal vascular development and shapes congenital cardiovascular abnormalities. PMID:22744845

  12. Embryonic death and the creation of human embryonic stem cells.

    PubMed

    Landry, Donald W; Zucker, Howard A

    2004-11-01

    The creation of human embryonic stem cells through the destruction of a human embryo pits the value of a potential therapeutic tool against that of an early human life. This contest of values has resulted in a polarized debate that neglects areas of common interest and perspective. We suggest that a common ground for pursuing research on human embryonic stem cells can be found by reconsidering the death of the human embryo and by applying to this research the ethical norms of essential organ donation.

  13. [Effects of dexamethasone and vitamin B12 on expression of fibroblast growth factor 10 and fibroblast growth factor receptor 2b during early palatogenesis].

    PubMed

    He, Wei; Lu, Sheng-jun; Li, Cheng-hao; Zhou, Jing-lin; Meng, Tian; Zheng, Qian; Shi, Bing

    2010-10-01

    To observe the alteration of fibroblast growth factor 10 (Fgf10) and fibroblast growth factor receptor 2 (Fgfr2b) signal in mouse embryonic palate after dexamethasone and vitamin B12 exposure. Dams were divided teratogenetic group, antagomistic group and control group and were respectively injected dexamethasone, dexamethasone and vitamin B12, and normal sodium. Dams were killed and fetus was collected at embryo 12.5 and 13.5 day. The expression of Fgf10 and Fgfr2b and mesenchymal cells proliferation of mouse embryonic by western blotting and BrdU assay were checked. Fgf10 and Fgfr2b expression was down-regulated and mesenchymal cells proliferation was inhibited significantly after dexamethasone exposure. After vitamin B12 treatment, Fgf10 and Fgfr2b expression did not restore, but cells proliferation was recovered. Dexamethasone and vitamin B12 affected the expression of Fgf10 and Fgfr2b of mouse embryonic palate and mesenchyme cells proliferation, but the change was disaccord.

  14. Cardiac Fibrosis: The Fibroblast Awakens

    PubMed Central

    Travers, Joshua G.; Kamal, Fadia A.; Robbins, Jeffrey; Yutzey, Katherine E.; Blaxall, Burns C.

    2016-01-01

    Myocardial fibrosis is a significant global health problem associated with nearly all forms of heart disease. Cardiac fibroblasts comprise an essential cell type in the heart that is responsible for the homeostasis of the extracellular matrix; however upon injury, these cells transform to a myofibroblast phenotype and contribute to cardiac fibrosis. This remodeling involves pathological changes that include chamber dilation, cardiomyocyte hypertrophy and apoptosis, and ultimately leads to the progression to heart failure. Despite the critical importance of fibrosis in cardiovascular disease, our limited understanding of this cell population impedes the development of potential therapies that effectively target this cell type and its pathological contribution to disease progression. This review summarizes current knowledge regarding the origins and roles of fibroblasts, mediators and signaling pathways known to influence fibroblast function after myocardial injury, as well as novel therapeutic strategies under investigation to attenuate cardiac fibrosis. PMID:26987915

  15. Contested embryonic culture in Japan--public discussion, and human embryonic stem cell research in an aging welfare society.

    PubMed

    Sleeboom-Faulkner, Margaret

    2010-01-01

    This article explores the reasons for the lack of a broad discussion on bioethical regulation of human embryonic stem cell research (hESR) in Japan and asks why scientists experience difficulties accessing resources for hESR despite the acclaimed indifference of dominant Japanese culture to embryo research. The article shows how various social actors express their views on the embryo and oocyte donation in terms of dominant Japanese culture, foiled against what is regarded as Western culture. Second, it shows how the lack of concern with hESR should be understood in the context of public health policies and communications and bioethics decision making in Japan. Finally, it interprets the meaning of the embryo in the context of Japan as an aging modern welfare society, explaining how policymakers have come to emphasize the urgency of infertility problems over issues around abortion and embryonic life.

  16. PTBP1 Is Required for Embryonic Development before Gastrulation

    PubMed Central

    Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A. Francis; Solimena, Michele

    2011-01-01

    Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures. PMID:21423341

  17. PTBP1 is required for embryonic development before gastrulation.

    PubMed

    Suckale, Jakob; Wendling, Olivia; Masjkur, Jimmy; Jäger, Melanie; Münster, Carla; Anastassiadis, Konstantinos; Stewart, A Francis; Solimena, Michele

    2011-02-17

    Polypyrimidine-tract binding protein 1 (PTBP1) is an important cellular regulator of messenger RNAs influencing the alternative splicing profile of a cell as well as its mRNA stability, location and translation. In addition, it is diverted by some viruses to facilitate their replication. Here, we used a novel PTBP1 knockout mouse to analyse the tissue expression pattern of PTBP1 as well as the effect of its complete removal during development. We found evidence of strong PTBP1 expression in embryonic stem cells and throughout embryonic development, especially in the developing brain and spinal cord, the olfactory and auditory systems, the heart, the liver, the kidney, the brown fat and cartilage primordia. This widespread distribution points towards a role of PTBP1 during embryonic development. Homozygous offspring, identified by PCR and immunofluorescence, were able to implant but were arrested or retarded in growth. At day 7.5 of embryonic development (E7.5) the null mutants were about 5x smaller than the control littermates and the gap in body size widened with time. At mid-gestation, all homozygous embryos were resorbed/degraded. No homozygous mice were genotyped at E12 and the age of weaning. Embryos lacking PTBP1 did not display differentiation into the 3 germ layers and cavitation of the epiblast, which are hallmarks of gastrulation. In addition, homozygous mutants displayed malformed ectoplacental cones and yolk sacs, both early supportive structure of the embryo proper. We conclude that PTBP1 is not required for the earliest isovolumetric divisions and differentiation steps of the zygote up to the formation of the blastocyst. However, further post-implantation development requires PTBP1 and stalls in homozygous null animals with a phenotype of dramatically reduced size and aberration in embryonic and extra-embryonic structures.

  18. Targeting Mll1 H3K4 methyltransferase activity to guide cardiac lineage specific reprogramming of fibroblasts.

    PubMed

    Liu, Liu; Lei, Ienglam; Karatas, Hacer; Li, Yangbing; Wang, Li; Gnatovskiy, Leonid; Dou, Yali; Wang, Shaomeng; Qian, Li; Wang, Zhong

    2016-01-01

    Generation of induced cardiomyocytes (iCMs) directly from fibroblasts offers a great opportunity for cardiac disease modeling and cardiac regeneration. A major challenge of iCM generation is the low conversion rate. To address this issue, we attempted to identify small molecules that could potentiate the reprogramming ability towards cardiac fate by removing inhibitory roadblocks. Using mouse embryonic fibroblasts as the starting cell source, we first screened 47 cardiac development related epigenetic and transcription factors, and identified an unexpected role of H3K4 methyltransferase Mll1 and related factor Men1 in inhibiting iCM reprogramming. We then applied small molecules (MM408 and MI503) of Mll1 pathway inhibitors and observed an improved efficiency in converting embryonic fibroblasts and cardiac fibroblasts into functional cardiomyocyte-like cells. We further observed that these inhibitors directly suppressed the expression of Mll1 target gene Ebf1 involved in adipocyte differentiation. Consequently, Mll1 inhibition significantly decreased the formation of adipocytes during iCM induction. Therefore, Mll1 inhibitors likely increased iCM efficiency by suppressing alternative lineage gene expression. Our studies show that targeting Mll1 dependent H3K4 methyltransferase activity provides specificity in the process of cardiac reprogramming. These findings shed new light on the molecular mechanisms underlying cardiac conversion of fibroblasts and provide novel targets and small molecules to improve iCM reprogramming for clinical applications.

  19. Lefty-1 alleviates TGF-β1-induced fibroblast-myofibroblast transdifferentiation in NRK-49F cells.

    PubMed

    Zhang, Lijun; Zhang, Jie; Xu, Changgeng; Zhou, Xiangjun; Wang, Wei; Zheng, Renping; Hu, Wei; Wu, Pin

    2015-01-01

    Fibroblast activation and proliferation are important for fibroblast-myofibroblast transdifferentiation, a crucial process in the pathological changes that define renal interstitial fibrosis. The left-right determination factor (Lefty) is an important cytokine of the transforming growth factor (TGF)-β family, with two variants, Lefty-1 and Lefty-2, in mice. Lefty has diverse functions, such as the regulation of embryonic development, the inhibition of TGF-β1 signaling, and the suppression of tumor activity. However, whether Lefty-1 influences fibroblast activation and proliferation, and consequently prevents fibroblast-myofibroblast transdifferentiation, remains unclear. This study aimed to investigate whether Lefty-1 can attenuate TGF-β1-induced fibroblast-myofibroblast transdifferentiation in normal rat kidney interstitial fibroblast cells (NRK-49F), as well as the mechanisms underlying any effects. Results showed that the typical fibroblast cell morphology of NRK-49F cells was altered after TGF-β1 treatment and that Lefty-1 significantly prevented this change in a dose-dependent manner. Further analyses demonstrated decreased proliferating cell nuclear antigen, cyclin D1, collagen I(A1), alpha-smooth muscle actin, and fibronectin expression. Lefty-1 further induced remarkable reductions in TGF-β1-induced Smad3 and mitogen-activated protein kinase-10/c-Jun N-terminal kinase (JNK-3) signaling, and enhanced expression of the antifibrotic factor bone morphogenetic protein (BMP)-5. However, without TGF-β1, Lefty-1 had no effect on Smad3, JNK-3, and BMP-5 activation and fibroblast-myofibroblast transdifferentiation. Taken together, these findings indicate that Lefty-1 can alleviate TGF-β1-mediated activation and the proliferation of fibroblasts. Furthermore, Lefty-1 may prevent fibroblast-myofibroblast transdifferentiation in part via modulations of Smad3, JNK-3, and BMP-5 activities in the TGF-β/BMP signaling pathway.

  20. Derivation of Porcine Embryonic Stem-Like Cells from In Vitro-Produced Blastocyst-Stage Embryos

    PubMed Central

    Hou, Dao-Rong; Jin, Yong; Nie, Xiao-Wei; Zhang, Man-Ling; Ta, Na; Zhao, Li-Hua; Yang, Ning; Chen, Yuan; Wu, Zhao-Qiang; Jiang, Hai-Bin; Li, Yan-Ru; Sun, Qing-Yuan; Dai, Yi-Fan; Li, Rong-Feng

    2016-01-01

    Efficient isolation of embryonic stem (ES) cells from pre-implantation porcine embryos has remained a challenge. Here, we describe the derivation of porcine embryonic stem-like cells (pESLCs) by seeding the isolated inner cell mass (ICM) from in vitro-produced porcine blastocyst into α-MEM with basic fibroblast growth factor (bFGF). The pESL cells kept the normal karyotype and displayed flatten clones, similar in phenotype to human embryonic stem cells (hES cells) and rodent epiblast stem cells. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers such as OCT4, NANOG, SOX2, SSEA-4, TRA-1-60, and TRA-1-81 as determined by both immunofluorescence and RT-PCR. Additionally, these cells formed embryoid body (EB), teratomas and also differentiated into 3 germ layers in vitro and in vivo. Microarray analysis showed the expression of the pluripotency markers, PODXL, REX1, SOX2, KLF5 and NR6A1, was significantly higher compared with porcine embryonic fibroblasts (PEF), but expression of OCT4, TBX3, REX1, LIN28A and DPPA5, was lower compared to the whole blastocysts or ICM of blastocyst. Our results showed that porcine embryonic stem-like cells can be established from in vitro-produced blastocyst-stage embryos, which promote porcine naive ES cells to be established. PMID:27173828

  1. When Lack of Evidence Is Evidence of Lack.

    PubMed

    Pickering, Neil

    2015-12-01

    In their recent article "A Gentle Ethical Defence of Homeopathy," Levy, Gadd, Kerridge, and Komesaroff use the claim that "lack of evidence is not equivalent to evidence of lack" as a component of their ethical defence of homeopathy. In response, this article argues that they cannot use this claim to shore up their ethical arguments. This is because it is false.

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

    PubMed

    Questa, María; Romorini, Leonardo; Blüguermann, Carolina; Solari, Claudia María; Neiman, Gabriel; Luzzani, Carlos; Scassa, María Élida; Sevlever, Gustavo Emilio; Guberman, Alejandra Sonia; Miriuka, Santiago Gabriel

    2016-03-01

    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. Copyright © 2015 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

  3. Initial specification of the epibranchial placode in zebrafish embryos depends on the fibroblast growth factor signal.

    PubMed

    Nikaido, Masataka; Doi, Kazunao; Shimizu, Takashi; Hibi, Masahiko; Kikuchi, Yutaka; Yamasu, Kyo

    2007-02-01

    In vertebrates, cranial sensory ganglia are mainly derived from ectodermal placodes, which are focal thickenings at characteristic positions in the embryonic head. Here, we provide the first description of the early development of the epibranchial placode in zebrafish embryos using sox3 as a molecular marker. By the one-somite stage, we saw a pair of single sox3-expressing domains appear lateral to the future hindbrain. The sox3 domain, which is referred to here as the early lateral placode, is segregated during the early phase of segmentation to form a pax2a-positive medial area and a pax2a-negative lateral area. The medial area subsequently developed to form the otic placode, while the lateral area was further segregated along the anteroposterior axis, giving rise to four sox3-positive subdomains by 26 hr postfertilization. Given their spatial relationship with the expression of the markers for the epibranchial ganglion, as well as their positions and temporal changes, we propose that these four domains correspond to the facial, glossopharyngeal, vagal, and posterior lateral line placodes in an anterior-to-posterior order. The expression of sox3 in the early lateral placode was absent in mutants lacking functional fgf8, while implantation of fibroblast growth factor (FGF) beads restored the sox3 expression. Using SU5402, which inhibits the FGF signal, we were able to demonstrate that formation of both the early lateral domains and later epibranchial placodes depends on the FGF signal operating at the beginning of somitogenesis. Together, these data provide evidence for the essential role of FGF signals in the development of the epibranchial placodes.

  4. Effect of UVC radiation on mouse fibroblasts deficient for FAS-associated protein with death domain.

    PubMed

    Begović, Lidija; Antunovic, Maja; Matic, Igor; Furcic, Ivana; Baricevic, Ana; Vojvoda Parcina, Valerija; Peharec Štefanić, Petra; Nagy, Biserka; Marijanovic, Inga

    2016-08-01

    Ultraviolet (UV) radiation-induced apoptosis enabled us to study the mechanism of DNA damage and to investigate how cells avoid consequences of damaged DNA. Cells with extensive DNA damage activate extrinsic and intrinsic pathways of apoptosis. The extrinsic pathway is coupled to a FAS-associated protein with death domain (FADD), an adaptor protein molecule necessary for mediating apoptotic signals through the cell. Viability and apoptosis of wild-type and FADD-deficient mouse embryonic fibroblasts were investigated 1, 3, 24 and 48 h after exposure to three doses (50, 75 and 300 J/m(2)) of UVC radiation. Morphological changes were observed using DNA binding dyes (Hoechst and propidium iodide) while biochemical changes were monitored using immunodetection of the poly (ADP-ribose) polymerase (PARP) protein cleavage and caspase-3 activity assay. Results showed that the difference in cell death response between wild-type and FADD-deficient cells depended on dose and incubation time after exposure to UVC radiation. FADD-deficient cells are more sensitive to UVC radiation. Even though FADD-deficient cells lack an adapter protein of apoptotic extrinsic pathway, higher doses of UVC triggered their apoptotic response, while wild-type cells die mainly due to necrosis. A different pattern of caspase 3 activity and PARP cleavage was observed 24 h after radiation between two cell lines confirming higher apoptotic response in FADD-deficient cells. Wild-type cells can execute apoptosis via both, the mitochondrial and the receptor-mediated pathway whereas FADD-deficient cells can only activate the intrinsic pathway. There is a difference in UVC radiation response between two cell lines indicating the role of FADD in the selection of cell death modality.

  5. Absence of K-Ras Reduces Proliferation and Migration But Increases Extracellular Matrix Synthesis in Fibroblasts.

    PubMed

    Muñoz-Félix, José M; Fuentes-Calvo, Isabel; Cuesta, Cristina; Eleno, Nélida; Crespo, Piero; López-Novoa, José M; Martínez-Salgado, Carlos

    2016-10-01

    The involvement of Ras-GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H- and N-Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K-Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K-Ras knockout (KO) mouse embryonic fibroblasts (K-ras(-/-) ) stimulated with transforming growth factor-β1 (TGF-β1) exhibited reduced proliferation and impaired mobility than wild-type fibroblasts. Moreover, an increase on ECM production was observed in K-Ras KO fibroblasts in basal conditions. The absence of K-Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF-β1-induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K-ras but reduced migration and ECM proteins expression only in wild-type fibroblasts, while the PI3K-AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K-Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K-Ras may be a crucial mediator in TGF-β1-mediated effects in this cell type. J. Cell. Physiol. 231: 2224-2235, 2016. © 2016 Wiley Periodicals, Inc.

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

    PubMed

    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.

  7. Emergence of embryonic pattern through contact inhibition of locomotion.

    PubMed

    Davis, John R; Huang, Chieh-Yin; Zanet, Jennifer; Harrison, Sam; Rosten, Edward; Cox, Susan; Soong, Daniel Y; Dunn, Graham A; Stramer, Brian M

    2012-12-01

    The pioneering cell biologist Michael Abercrombie first described the process of contact inhibition of locomotion more than 50 years ago when migrating fibroblasts were observed to rapidly change direction and migrate away upon collision. Since then, we have gleaned little understanding of how contact inhibition is regulated and only lately observed its occurrence in vivo. We recently revealed that Drosophila macrophages (haemocytes) require contact inhibition for their uniform embryonic dispersal. Here, to investigate the role that contact inhibition plays in the patterning of haemocyte movements, we have mathematically analysed and simulated their contact repulsion dynamics. Our data reveal that the final pattern of haemocyte distribution, and the details and timing of its formation, can be explained by contact inhibition dynamics within the geometry of the Drosophila embryo. This has implications for morphogenesis in general as it suggests that patterns can emerge, irrespective of external cues, when cells interact through simple rules of contact repulsion.

  8. [Fibroblast growth factors and their effects in pancreas organogenesis].

    PubMed

    Gnatenko, D A; Kopantzev, E P; Sverdlov, E D

    2017-05-01

    Fibroblast growth factors (FGF) - growth factors that regulate many important biological processes, including proliferation and differentiation of embryonic cells during organogenesis. In this review, we will summarize current information about the involvement of FGFs in the pancreas organogenesis. Pancreas organogenesis is a complex process, which involves constant signaling from mesenchymal tissue. This orchestrates the activation of various regulator genes at specific stages, determining the specification of progenitor cells. Alterations in FGF/FGFR signaling pathway during this process lead to incorrect activation of the master genes, which leads to different pathologies during pancreas development. Understanding the full picture about role of FGF factors in pancreas development will make it possible to more accurately understand their role in other pathologies of this organ, including carcinogenesis.

  9. Derivation of Transgene-Free Rat Induced Pluripotent Stem Cells Approximating the Quality of Embryonic Stem Cells.

    PubMed

    Li, Shuping; Lan, He; Men, Hongsheng; Wu, Yuanyuan; Li, Ning; Capecchi, Mario R; Bryda, Elizabeth C; Wu, Sen

    2016-09-13

    : Although a variety of reprogramming strategies have been reported to create transgene-free induced pluripotent stem (iPS) cells from differentiated cell sources, a fundamental question still remains: Can we generate safe iPS cells that have the full spectrum of features of corresponding embryonic stem (ES) cells? Studies in transgene-free mouse iPS cells have indicated a positive answer to this question. However, the reality is that no other species have a derived transgene-free iPS cell line that can truly mimic ES cell quality. Specifically, critical data for chimera formation and germline transmission are generally lacking. To date, the rat is the only species, other than the mouse, that has commonly recognized authentic ES cells that can be used for direct comparison with measure features of iPS cells. To help find the underlying reasons of the current inability to derive germline-competent ES/iPS cells in nonrodent animals, we first used optimized culture conditions to isolate and establish rat ES cell lines and demonstrated they are fully competent for chimeric formation and germline transmission. We then used episomal vectors bearing eight reprogramming genes to improve rat iPS (riPS) cell generation from Sprague-Dawley rat embryonic fibroblasts. The obtained transgene-free riPS cells exhibit the typical characteristics of pluripotent stem cells; moreover, they are amenable to subsequent genetic modification by homologous recombination. Although they can contribute significantly to chimeric formation, no germline transmission has been achieved. Although this partial success in achieving competency is encouraging, it suggests that more efforts are still needed to derive ground-state riPS cells. To date, no species other than the mouse have derived transgene-free induced pluripotent stem (iPS) cells that can truly mimic ES cell quality. In the current study, episomal vectors were used to obtain rat transgene-free iPS cells, which contributed to chimeric

  10. Novel anti-adipogenic activity produced by human fibroblasts

    PubMed Central

    Lehmann, Geniece M.; Woeller, Collynn F.; Pollock, Stephen J.; O'Loughlin, Charles W.; Gupta, Shikha; Feldon, Steven E.

    2010-01-01

    Fatty tissue is generally found in distinct “depots” distributed throughout the human body. Adipocytes from each of the various depots differ in their metabolic capacities and their responses to environmental stimuli. Although a general understanding of the factors responsible for adipogenic transformation has been achieved, much is not understood about the mechanisms of adipose tissue deposition and the phenotypes of the adipocytes found within each depot. A clue to the factors regulating fat deposition may come from studies of adipogenesis using primary human orbital fibroblasts from patients with thyroid eye disease, a condition in which intense inflammation leads to expansion of orbital adipose tissue via differentiation of fibroblasts to adipocytes. We have previously demonstrated that adipogenesis of orbital fibroblasts is negatively correlated with cellular expression of the Thy-1 surface marker. In this study, we developed a novel imaging flow cytometric approach for the assessment of adipogenesis to test the hypothetical dependence of adipogenic potential on lack of Thy-1 expression. Using this technique, we learned that Thy-1-positive fibroblasts are, in fact, capable of differentiating into adipocytes but are less likely to do so because they secrete a paracrine anti-adipogenic factor. It is possible that such a factor plays an important role in the prevention of excess fat deposition in the normal orbit and may even be exploited as a therapy for the treatment of obesity, a major worldwide health concern. PMID:20554910

  11. Peroxisomal organization in normal and cerebrohepatorenal (Zellweger) syndrome fibroblasts.

    PubMed Central

    Santos, M J; Ojeda, J M; Garrido, J; Leighton, F

    1985-01-01

    The reported absence of morphologically detectable peroxisomes in liver and kidney tissue cells from patients affected by the autosomic recessive, inherited metabolic disease known as cerebrohepatorenal, or Zellweger, syndrome was studied in fibroblasts, assuming it to be a generalized defect. Normal cultured fibroblasts were shown to contain peroxisomes according to morphological, biochemical, and subcellular fractionation criteria: particle-bound catalase and fatty acyl-CoA oxidase copurify in subcellular fractionation by differential centrifugation or isopycnic equilibrium in continuous density gradients and peroxidase-positive organelles of approximately equal to 0.1 micron in diameter are detected in the cytoplasm. In Zellweger cultured fibroblasts, these peroxisomal enzymes are present; however, they behave as cytosolic enzymes in the different subcellular fractionation procedures employed and peroxisomes are not detected cytochemically. These findings support the hypothesis that the lack of peroxisomes in this genetic disease is the consequence of a defect in the assembly of the peroxisomal constituents. Furthermore, the value of fibroblasts for subcellular analysis of peroxisomal defects is illustrated. Images PMID:2995971

  12. Pericellular versican regulates the fibroblast-myofibroblast transition: a role for ADAMTS5 protease-mediated proteolysis.

    PubMed

    Hattori, Noriko; Carrino, David A; Lauer, Mark E; Vasanji, Amit; Wylie, James D; Nelson, Courtney M; Apte, Suneel S

    2011-09-30

    The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5(-/-) mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5(-/-) cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcan(hdf/+)) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5(-/-) cells resulted from increased PCM versican content, we generated Adamts5(-/-);Vcan(hdf/+) mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5(-/-) mice. In Adamts5(-/-) fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates.

  13. Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins

    PubMed Central

    Janmaat, C. J.; de Rooij, K. E; Locher, H; de Groot, S. C.; de Groot, J. C. M. J.; Frijns, J. H. M.; Huisman, M. A.

    2015-01-01

    In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality. PMID:26678612

  14. Synthetic peptide TEKKRRETVEREKE derived from ezrin induces differentiation of NIH/3T3 fibroblasts.

    PubMed

    Chulkina, Marina; Negmadjanov, Ulugbek; Lebedeva, Ekaterina; Pichugin, Aleksey; Mazurov, Dmitriy; Ataullakhanov, Ravshan; Holmuhamedov, Ekhson

    2017-09-15

    Synthetic 14 AA peptide (Gepon) derived from the hinge region of ezrin, a protein that links cell surface molecules to intracellular actin filaments, accelerates and facilitates wound and ulcer healing in clinical applications. However, the molecular mechanisms underlying this phenomenon and involved in enhanced healing of wounds with Gepon are not yet understood. The purpose of current study was to investigate intracellular signaling pathways involved in the effect of this peptide on wild type and genetically modified (CD44 KO) NIH/3T3 embryonic mouse fibroblasts. Gepon treatment of NIH/3T3 cells resulted in morphological and biochemical changes, characteristic of differentiated fibroblasts. While treatment of NIH/3T3 cells with TGF-β1 triggered the activation of both canonical and non-canonical signaling pathways, exposure of fibroblasts to Gepon activated only the ERK1/2 dependent pathway without modulating SMAD dependent signaling pathway. Knocking out hyaluronic acid CD44 receptor did not change Gepon or TGF-β1 dependent activation of intracellular signaling pathways and assembling of α-SMA-positive filaments. Gepon dependent differentiation of NIH/3T3 fibroblasts is based on activation of ERK1/2 kinase, non-canonical intracellular signaling pathway. Our data suggest that the treatment of fibroblasts with Gepon triggers activation of the non-canonical (SMAD independent) intracellular signaling pathway that involves ERK1/2kinase phosphorylation. Activation of the MAPK signaling pathway and the increase in formation of α-SMA containing stress filaments induced by Gepon were independent on presence of CD44 receptor in NIH/3T3 fibroblasts. Thus, our observation designates the significance and sufficiency of MAPK pathway mediated activation of fibroblasts with Gepon for healing of erosion, ulcers and wounds. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

    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.

  16. S100A13-C2A binary complex structure-a key component in the acidic fibroblast growth factor for the non-classical pathway

    SciTech Connect

    Mohan, Sepuru K.; Rani, Sandhya G.; Kumar, Sriramoju M.; Yu Chin

    2009-03-13

    Fibroblast growth factors (FGFs) are key regulators of cell proliferation, differentiation, tumor-induced angiogenesis and migration. FGFs are essential for early embryonic development, organ formation and angiogenesis. They play important roles in tumor formation, inflammation, wound healing and restenosis. The biological effects of FGFs are mediated through the activation of the four transmembrane phosphotyrosine kinase receptors (FGFRs) in the presence of heparin sulfate proteoglycans (HSPGs) and therefore require the release of FGFs into the extracellular space. However, FGF-1 lacks the signal peptide required for the releasing of these proteins through the classical endoplasmic reticulum (ER)-Golgi secretary pathway. Maciag et al. demonstrated that FGF-1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex [M. Landriscina, R. Soldi, C. Bagala, I. Micucci, S. Bellum, F. Tarantini, I. Prudovsky, T. Maciag, S100A13 participates in the release of fibroblast growth factor 1 in response to heat shock in vitro, J. Biol. Chem. 276 (2001) 22544-22552; C.M. Carreira, T.M. LaVallee, F. Tarantini, A. Jackson, J.T. Lathrop, B. Hampton, W.H. Burgess, T. Maciag, S100A13 is involved in the regulation of fibroblast growth factor-1 and p40 synaptotagmin-1 release in vitro, J. Biol. Chem. 273 (1998) 22224-22231; T.M. LaValle, F. Tarantini, S. Gamble, C.M. Carreira, A. Jackson, T. Maciag, Synaptotagmin-1 is required for fibroblast growth factor-1 release, J. Biol. Chem. 273 (1998) 22217-22223; C. Bagala, V. Kolev, A. Mandinova, R. Soldi, C. Mouta, I. Graziani, I, Prudovsky, T. Maciag, The alternative translation of synaptotagmin 1 mediates the non-classical release of FGF1, Biochem. Biophys. Res. Commun. 310 (2003) 1041-1047]. The protein constituents of this complex include FGF-1, S100A13 (a Ca{sup 2+}-binding protein), and the p40 form of synaptotagmin 1 (Syt1). To understand the molecular events in the FGF-1 releasing

  17. Role of fibroblast growth factor receptor signaling in kidney development.

    PubMed

    Bates, Carlton M

    2007-03-01

    Fibroblast growth factor receptors (Fgfrs) are expressed in the ureteric bud and metanephric mesenchyme of the developing kidney. Furthermore, in vitro and in vivo studies have shown that exogenous fibroblast growth factors (Fgfs) increase growth and maturation of the metanephric mesenchyme and ureteric bud. Deletion of fgf7, fgf10, and fgfr2IIIb (the receptor isoform that binds Fgf7 and Fgf10) in mice lead to smaller kidneys with fewer collecting ducts and nephrons. Overexpression of a dominant negative receptor isoform in transgenic mice has revealed more striking defects including renal aplasia or severe dysplasia. Moreover, deletion of many fgf ligands and receptors in mice results in early embryonic lethality, making it difficult to determine their roles in kidney development. Recently, conditional targeting approaches revealed that deletion of fgf8 from the metanephric mesenchyme interrupts nephron formation. Furthermore, deletion of fgfr2 from the ureteric bud resulted in both ureteric bud branching and stromal mesenchymal patterning defects. Deletion of both fgfr1 and fgfr2 in the metanephric mesenchyme resulted in renal aplasia, characterized by defects in metanephric mesenchyme formation and initial ureteric bud elongation and branching. Thus, Fgfr signaling is critical for growth and patterning of all renal lineages at early and later stages of kidney development.

  18. Role of fibroblast growth factors in organ regeneration and repair.

    PubMed

    El Agha, Elie; Kosanovic, Djuro; Schermuly, Ralph T; Bellusci, Saverio

    2016-05-01

    In its broad sense, regeneration refers to the renewal of lost cells, tissues or organs as part of the normal life cycle (skin, hair, endometrium etc.) or as part of an adaptive mechanism that organisms have developed throughout evolution. For example, worms, starfish and amphibians have developed remarkable regenerative capabilities allowing them to voluntarily shed body parts, in a process called autotomy, only to replace the lost parts afterwards. The bizarre myth of the fireproof homicidal salamander that can survive fire and poison apple trees has persisted until the 20th century. Salamanders possess one of the most robust regenerative machineries in vertebrates and attempting to draw lessons from limb regeneration in these animals and extrapolate the knowledge to mammals is a never-ending endeavor. Fibroblast growth factors are potent morphogens and mitogens that are highly conserved among the animal kingdom. These growth factors play key roles in organogenesis during embryonic development as well as homeostatic balance during postnatal life. In this review, we provide a summary about the current knowledge regarding the involvement of fibroblast growth factor signaling in organ regeneration and repair. We also shed light on the use of these growth factors in previous and current clinical trials in a wide array of human diseases.

  19. Angiogenesis is repressed by ethanol exposure during chick embryonic development.

    PubMed

    Wang, Guang; Zhong, Shan; Zhang, Shi-yao; Ma, Zheng-lai; Chen, Jian-long; Lu, Wen-hui; Cheng, Xin; Chuai, Manli; Lee, Kenneth Ka Ho; Lu, Da-xiang; Yang, Xuesong

    2016-05-01

    It is now known that excess alcohol consumption during pregnancy can cause fetal alcohol syndrome to develop. However, it is not known whether excess ethanol exposure could directly affect angiogenesis in the embryo or angiogenesis being indirectly affected because of ethanol-induced fetal alcohol syndrome. Using the chick yolk sac membrane (YSM) model, we demonstrated that ethanol exposure dramatically inhibited angiogenesis in the YSM of 9-day-old chick embryos, in a dose-dependent manner. Likewise, the anti-angiogenesis effect of ethanol could be seen in the developing vessel plexus (at the same extra-embryonic regions) during earlier stages of embryo development. The anti-angiogenic effect of ethanol was found associated with excess reactive oxygen species (ROS) production; as glutathione peroxidase activity increased while superoxide dismutase 1 and 2 activities decreased in the YSMs. We further validated this observation by exposing chick embryos to 2,2'-azobis-amidinopropane dihydrochloride (a ROS inducer) and obtained a similar anti-angiogenesis effect as ethanol treatment. Semiquantitative reverse transcription-polymerase chain reaction analysis of the experimental YSMs revealed that expression of angiogenesis-related genes, vascular endothelial growth factor and its receptor, fibroblast growth factor 2 and hypoxia-inducible factor, were all repressed following ethanol and 2,2'-azobis-amidinopropane dihydrochloride treatment. In summary, our results suggest that excess ethanol exposure inhibits embryonic angiogenesis through promoting superfluous ROS production during embryo development. Copyright © 2015 John Wiley & Sons, Ltd.

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

    PubMed

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    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.

  1. EDA-Containing Fibronectin Increases Proliferation of Embryonic Stem Cells

    PubMed Central

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F.; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    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. PMID:24244705

  2. GABA uptake in embryonic palate mesenchymal cells of two mouse strains.

    PubMed

    Wee, E L; Zimmerman, E F

    1985-12-01

    To obtain further evidence that the inhibitory neurotransmitter GABA functions in palate development, the presence of an active GABA uptake mechanism was sought using primary cultures of embryonic palate mesenchymal cells. Uptake was compared from cells of two inbred mouse strains in which the SWV strain shows greater sensitivity than the AJ strain to effects of GABA on palate morphogenesis and of diazepam in producing cleft palate. Palate cells were capable of accumulating [3H]GABA by saturable uptake mechanisms characteristic of a high and low affinity active transport as indicated by temperature, Na+ ion and carrier dependence as well as Km and Vmax values that were comparable to other biological systems. The Vmax of the high-affinity uptake system from cells of the SWV strain was 1.8 fold higher than that of the AJ. GABA uptake was also observed in fibroblasts from various sources including embryonic mouse limb cells, human skin fibroblasts and 3T3 cells. When active GABA uptake was measured in skin fibroblasts from the mouse SWV and AJ strains, the rate of uptake from SWV cells under high affinity conditions was also 1.8 fold greater than in AJ cells. Thus active GABA uptake appears to be genetically regulated in non-neural cells which may contribute to differential responses to GABA.

  3. Rhodamine-based sensor for real-time imaging of mitochondrial ATP in living fibroblasts.

    PubMed

    de la Fuente-Herreruela, Diego; Gónzalez-Charro, Vicente; Almendro-Vedia, Víctor G; Morán, María; Martín, Miguel Ángel; Lillo, M Pilar; Natale, Paolo; López-Montero, Iván

    2017-09-22

    Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL(+) into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL(+) to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  4. Fibroblast growth factor 10 represses premature cell differentiation during establishment of the intestinal progenitor niche.

    PubMed

    Nyeng, Pia; Bjerke, Maureen Ann; Norgaard, Gitte Anker; Qu, Xiaoling; Kobberup, Sune; Jensen, Jan

    2011-01-01

    Spatio-temporal regulation of the balance between cell renewal and cell differentiation is of vital importance for embryonic development and adult homeostasis. Fibroblast growth factor signaling relayed from the mesenchyme to the epithelium is necessary for progenitor maintenance during organogenesis of most endoderm-derived organs, but it is still ambiguous whether the signal is exclusively mitogenic. Furthermore, the downstream mechanisms are largely unknown. In order to elucidate these questions we performed a complementary analysis of fibroblast growth factor 10 (Fgf10), gain-of-function and loss-of-function in the embryonic mouse duodenum, where the progenitor niche is clearly defined and differentiation proceeds in a spatially organized manner. In agreement with a role in progenitor maintenance, FGF10 is expressed in the duodenal mesenchyme during early development while the cognate receptor FGFR2b is expressed in the epithelial progenitor niche. Fgf10 gain-of-function in the epithelium leads to spatial expansion of the progenitor niche and repression of cell differentiation, while loss-of-function results in premature cell differentiation and subsequent epithelial hypoplasia. We conclude that FGF10 mediated mesenchymal-to-epithelial signaling maintains the progenitor niche in the embryonic duodenum primarily by repressing cell differentiation, rather than through mitogenic signaling. Furthermore, we demonstrate that FGF10-signaling targets include ETS-family transcription factors, which have previously been shown to regulate epithelial maturation and tumor progression.

  5. FSP1+ fibroblast subpopulation is essential for the maintenance and regeneration of medullary thymic epithelial cells

    PubMed Central

    Sun, Lina; Sun, Chenming; Liang, Zhanfeng; Li, Hongran; Chen, Lin; Luo, Haiying; Zhang, Hongmei; Ding, Pengbo; Sun, Xiaoning; Qin, Zhihai; Zhao, Yong

    2015-01-01

    Thymic epithelial cells (TECs) form a 3-dimentional network supporting thymocyte development and maturation. Besides epithelium and thymocytes, heterogeneous fibroblasts are essential components in maintaining thymic microenvironments. However, thymic fibroblast characteristics, development and function remain to be determined. We herein found that thymic non-hematopoietic CD45-FSP1+ cells represent a unique Fibroblast specific protein 1 (FSP1)—fibroblast-derived cell subset. Deletion of these cells in FSP1-TK transgenic mice caused thymus atrophy due to the loss of TECs, especially mature medullary TECs (MHCIIhigh, CD80+ and Aire+). In a cyclophosphamide-induced thymus injury and regeneration model, lack of non-hematopoietic CD45-FSP1+ fibroblast subpopulation significantly delayed thymus regeneration. In fact, thymic FSP1+ fibroblasts released more IL-6, FGF7 and FSP1 in the culture medium than their FSP1- counterparts. Further experiments showed that the FSP1 protein could directly enhance the proliferation and maturation of TECs in the in vitro culture systems. FSP1 knockout mice had significantly smaller thymus size and less TECs than their control. Collectively, our studies reveal that thymic CD45-FSP1+ cells are a subpopulation of fibroblasts, which is crucial for the maintenance and regeneration of TECs especially medullary TECs through providing IL-6, FGF7 and FSP1. PMID:26445893

  6. Apoptosis-Like Cell Death Induction and Aberrant Fibroblast Properties in Human Incisional Hernia Fascia

    PubMed Central

    Diaz, Ramon; Quiles, Maria T.; Guillem-Marti, Jordi; Lopez-Cano, Manuel; Huguet, Pere; Ramon-y-Cajal, Santiago; Reventos, Jaume; Armengol, Manel; Arbos, Maria A.

    2011-01-01

    Incisional hernia often occurs following laparotomy and can be a source of serious problems. Although there is evidence that a biological cause may underlie its development, the mechanistic link between the local tissue microenvironment and tissue rupture is lacking. In this study, we used matched tissue-based and in vitro primary cell culture systems to examine the possible involvement of fascia fibroblasts in incisional hernia pathogenesis. Fascia biopsies were collected at surgery from incisional hernia patients and non-incisional hernia controls. Tissue samples were analyzed by histology and immunoblotting methods. Fascia primary fibroblast cultures were assessed at morphological, ultrastructural, and functional levels. We document tissue and fibroblast loss coupled to caspase-3 activation and induction of apoptosis-like cell-death mechanisms in incisional hernia fascia. Alterations in cytoskeleton organization and solubility were also observed. Incisional hernia fibroblasts showed a consistent phenotype throughout early passages in vitro, which was characterized by significantly enhanced cell proliferation and migration, reduced adhesion, and altered cytoskeleton properties, as compared to non-incisional hernia fibroblasts. Moreover, incisional hernia fibroblasts displayed morphological and ultrastructural alterations compatible with autophagic processes or lysosomal dysfunction, together with enhanced sensitivity to proapoptotic challenges. Overall, these data suggest an ongoing complex interplay of cell death induction, aberrant fibroblast function, and tissue loss in incisional hernia fascia, which may significantly contribute to altered matrix maintenance and tissue rupture in vivo. PMID:21641387

  7. The role of fibroblast Tiam1 in tumor cell invasion and metastasis.

    PubMed

    Xu, K; Rajagopal, S; Klebba, I; Dong, S; Ji, Y; Liu, J; Kuperwasser, C; Garlick, J A; Naber, S P; Buchsbaum, R J

    2010-12-16

    The co-evolution of tumors and their microenvironment involves bidirectional communication between tumor cells and tumor-associated stroma. Various cell types are present in tumor-associated stroma, of which fibroblasts are the most abundant. The Rac exchange factor Tiam1 is implicated in multiple signaling pathways in epithelial tumor cells and lack of Tiam1 in tumor cells retards tumor growth in Tiam1 knockout mouse models. Conversely, tumors arising in Tiam1 knockout mice have increased invasiveness. We have investigated the role of Tiam1 in tumor-associated fibroblasts as a modulator of tumor cell invasion and metastasis, using retroviral delivery of short hairpin RNA to suppress Tiam1 levels in three different experimental models. In spheroid co-culture of mammary epithelial cells and fibroblasts, Tiam1 silencing in fibroblasts led to increased epithelial cell outgrowth into matrix. In tissue-engineered human skin, Tiam1 silencing in dermal fibroblasts led to increased invasiveness of epidermal keratinocytes with pre-malignant features. In a model of human breast cancer in mice, co-implantation of mammary fibroblasts inhibited tumor invasion and metastasis, which was reversed by Tiam1 silencing in co-injected fibroblasts. These results suggest that stromal Tiam1 may have a role in modulating the effects of the tumor microenvironment on malignant cell invasion and metastasis. This suggests a set of pathways for further investigation, with implications for future therapeutic targets.

  8. Embryonic markers of cone differentiation

    PubMed Central

    Rodgers, Helen M.; Belcastro, Marycharmain; Sokolov, Maxim

    2016-01-01

    Purpose Photoreceptor cells are born in two distinct phases of vertebrate retinogenesis. In the mouse retina, cones are born primarily during embryogenesis, while rod formation occurs later in embryogenesis and early postnatal ages. Despite this dichotomy in photoreceptor birthdates, the visual pigments and phototransduction machinery are not reactive to visual stimulus in either type of photoreceptor cell until the second postnatal week. Several markers of early cone formation have been identified, including Otx2, Crx, Blimp1, NeuroD, Trβ2, Rorβ, and Rxrγ, and all are thought to be involved in cellular determination. However, little is known about the expression of proteins involved in cone visual transduction during early retinogenesis. Therefore, we sought to characterize visual transduction proteins that are expressed specifically in photoreceptors during mouse embryogenesis. Methods Eye tissue was collected from control and phosducin-null mice at embryonic and early postnatal ages. Immunohistochemistry and quantitative reverse transcriptase-PCR (qPCR) were used to measure the spatial and temporal expression patterns of phosducin (Pdc) and cone transducin γ (Gngt2) proteins and transcripts in the embryonic and early postnatal mouse retina. Results We identified the embryonic expression of phosducin (Pdc) and cone transducin γ (Gngt2) that coincides temporally and spatially with the earliest stages of cone histogenesis. Using immunohistochemistry, the phosducin protein was first detected in the retina at embryonic day (E)12.5, and cone transducin γ was observed at E13.5. The phosducin and cone transducin γ proteins were seen only in the outer neuroblastic layer, consistent with their expression in photoreceptors. At the embryonic ages, phosducin was coexpressed with Rxrγ, a known cone marker, and with Otx2, a marker of photoreceptors. Pdc and Gngt2 mRNAs were detected as early as E10.5 with qPCR, although at low levels. Conclusions Visual transduction

  9. Fibroblast growth factor receptor levels decrease during chick embryogenesis

    PubMed Central

    1990-01-01

    Two putative receptors for fibroblast growth factor (FGF) of approximately 150 and 200 kD were identified in membrane preparations from chick embryos. Specific binding (femtomoles/milligram) of 125I- aFGF to whole chick embryonic membranes was relatively constant from day 2 to 7, then decreased fivefold between days 7 and 13. Day-19 chick embryos retained 125I-aFGF binding at low levels to brain, eye, and liver tissues but not to skeletal muscle or cardiac tissues. The 200-kD FGF receptor began to decline between day 4.5 and 7 and was barely detectable by day 9, whereas the 150-kD FGF receptor began to decline by day 7 but was still detectable in day-9 embryonic membranes. It is not known whether the two FGF-binding proteins represent altered forms of one polypeptide, but it is clear that their levels undergo differential changes during development. Because endogenous chick FGF may remain bound to FGF receptor in membrane preparations, membranes were treated with acidic (pH 4.0) buffers to release bound FGF; such treatment did not affect 125I-aFGF binding and moderately increased the number of binding sites in day-7 and -19 embryos. Consequently, the observed loss of high affinity 125I-aFGF binding sites and FGF-binding polypeptides most likely represents a loss of FGF receptor protein. These experiments provide in vivo evidence to support the hypothesis that regulation of FGF receptor levels may function as a mechanism for controlling FGF-dependent processes during embryonic development. PMID:2153684

  10. Embryonic Heart Progenitors and Cardiogenesis

    PubMed Central

    Brade, Thomas; Pane, Luna S.; Moretti, Alessandra; Chien, Kenneth R.; Laugwitz, Karl-Ludwig

    2013-01-01

    The mammalian heart is a highly specialized organ, comprised of many different cell types arising from distinct embryonic progenitor populations during cardiogenesis. Three precursor populations have been identified to contribute to different myocytic and nonmyocytic cell lineages of the heart: cardiogenic mesoderm cells (CMC), the proepicardium (PE), and cardiac neural crest cells (CNCCs). This review will focus on molecular cues necessary for proper induction, expansion, and lineage-specific differentiation of these progenitor populations during cardiac development in vivo. Moreover, we will briefly discuss how the knowledge gained on embryonic heart progenitor biology can be used to develop novel therapeutic strategies for the management of congenital heart disease as well as for improvement of cardiac function in ischemic heart disease. PMID:24086063

  11. Embryonic development during chronic acceleration

    NASA Technical Reports Server (NTRS)

    Smith, A. H.; Abbott, U. K.

    1982-01-01

    Experiments carried out on chicken eggs indicate that the embryo is affected during very early development, especially over the first four days, and during hatching. In the first four days, the brain develops as well as the anlage for all other organs. In addition, the heart commences to function and the extraembryonic membranes that compartmentalize the egg contents form. The latter require an appreciable extension and folding of tissue which may be disrupted by the mechanical load. Observations of embryonic abnormalities that occur during chronic acceleration suggest an inhibition of development of the axial skeleton, which is rarely seen otherwise, a general retardation of embryonic growth, and circulatory problems. The final stages of development (after 18 days) involve the uptake of fluids, the transition to aerial respiration, and the reorientation of the embryo into a normal hatching position. At 4 G mortality is very high during this period, with a majority of embryos failing to reorient into the normal hatching position.

  12. Embryonic development during chronic acceleration

    NASA Technical Reports Server (NTRS)

    Smith, A. H.; Abbott, U. K.

    1982-01-01

    Experiments carried out on chicken eggs indicate that the embryo is affected during very early development, especially over the first four days, and during hatching. In the first four days, the brain develops as well as the anlage for all other organs. In addition, the heart commences to function and the extraembryonic membranes that compartmentalize the egg contents form. The latter require an appreciable extension and folding of tissue which may be disrupted by the mechanical load. Observations of embryonic abnormalities that occur during chronic acceleration suggest an inhibition of development of the axial skeleton, which is rarely seen otherwise, a general retardation of embryonic growth, and circulatory problems. The final stages of development (after 18 days) involve the uptake of fluids, the transition to aerial respiration, and the reorientation of the embryo into a normal hatching position. At 4 G mortality is very high during this period, with a majority of embryos failing to reorient into the normal hatching position.

  13. Electroporation of Embryonic Kidney Explants

    NASA Astrophysics Data System (ADS)

    Haddad, Nicholas; Houle, Daniel; Gupta, Indra R.

    Metanephric kidney development in the mouse begins at embryonic day (E) 10.5, when the ureteric bud (UB), an outgrowth of the epithelial nephric duct, invades the neighboring metanephric mesenchyme (MM). The ureteric bud then undergoes a series of branching events to form the collecting duct network of the adult kidney (Fig. 19.1). As each ureteric bud tip forms, the adjacent undifferentiated mesenchyme is induced to epithelialize and form a nephron, the functional unit of the adult kidney that filters waste. Rodent embryonic kidneys can be dissected and cultured as explants such that branching morphogenesis and nephrogenesis can be observed ex vivo (Rothenpieler and Dressler, 1993; Vega et al., 1996; Piscione et al., 1997; Gupta et al., 2003).

  14. Deep and superficial keloid fibroblasts contribute differentially to tissue phenotype in a novel in vivo model of keloid scar.

    PubMed

    Supp, Dorothy M; Hahn, Jennifer M; Glaser, Kathryn; McFarland, Kevin L; Boyce, Steven T

    2012-06-01

    Keloids are thick fibrous scars that are refractory to treatment and unique to humans. The lack of keloid animal models has hampered development of effective therapies. The authors' goal was to develop an animal model of keloids using grafted engineered skin substitutes composed of keloid-derived cells. To demonstrate the model's utility, differences between deep and superficial keloid fibroblasts were investigated. Engineered skin substitutes were prepared using six combinations of cells: 1, normal keratinocytes and normal fibroblasts; 2, normal keratinocytes and deep keloid fibroblasts; 3, normal keratinocytes and superficial keloid fibroblasts; 4, keloid keratinocytes and normal fibroblasts; 5, keloid keratinocytes and deep keloid fibroblasts; and 6, keloid keratinocytes and superficial keloid fibroblasts. Engineered skin substitutes stably grafted to athymic mice were evaluated for wound area, thickness, and gene expression. Deep keloid fibroblasts displayed elevated expression of type 1 collagen alpha 1 (COL1A1), transforming growth factor β-1, periostin, plasminogen activator inhibitor 2, and inhibin beta A compared with superficial keloid fibroblasts and normal fibroblasts. After grafting, engineered skin substitutes in group 5 were significantly thicker than controls and had increased COL1A1 expression. Engineered skin substitutes in group 6 showed significantly increased area. Histologic analysis revealed abnormal collagen organization in engineered skin substitutes containing deep keloid fibroblasts or superficial keloid fibroblasts. Aspects of the phenotypes of engineered skin substitutes prepared with keloid cells are analogous to thickening and spreading of human keloid scars. Therefore, use of keloid engineered skin substitutes is a valuable new tool for the study of keloid scarring.

  15. Absence of Rybp Compromises Neural Differentiation of Embryonic Stem Cells

    PubMed Central

    Kovacs, Gergo; Szabo, Viktoria; Pirity, Melinda K.

    2016-01-01

    Rybp (Ring1 and Yy1 Binding Protein) is a transcriptional regulator and member of the noncanonical polycomb repressive complex 1 with essential role in early embryonic development. We have previously described that alteration of Rybp dosage in mouse models induced striking neural tube defects (NTDs), exencephaly, and disorganized neurocortex. In this study we further investigated the role of Rybp in neural differentiation by utilising wild type (rybp +/+) and rybp null mutant (rybp −/−) embryonic stem cells (ESCs) and tried to uncover underlying molecular events that are responsible for the observed phenotypic changes. We found that rybp null mutant ESCs formed less matured neurons, astrocytes, and oligodendrocytes from existing progenitors than wild type cells. Furthermore, lack of rybp coincided with altered gene expression of key neural markers including Pax6 and Plagl1 pinpointing a possible transcriptional circuit among these genes. PMID:26788067

  16. Adaptive response to l-serine deficiency is mediated by p38 MAPK activation via 1-deoxysphinganine in normal fibroblasts.

    PubMed

    Sayano, Tomoko; Kawano, Yuki; Kusada, Wataru; Arimoto, Yashiho; Esaki, Kayoko; Hamano, Momoko; Udono, Miyako; Katakura, Yoshinori; Ogawa, Takuya; Kato, Hisanori; Hirabayashi, Yoshio; Furuya, Shigeki

    2016-04-01

    Reduced availability of l-serine limits cell proliferation and leads to an adaptation to l-serine-deficient environment, the underlying molecular mechanism of which remain largely unexplored. Genetic ablation of 3-phosphoglycerate dehydrogenase (Phgdh), which catalyzes the first step of de novo l-serine synthesis, led to diminished cell proliferation and the activation of p38 MAPK and stress-activated protein kinase/Jun amino-terminal kinase in mouse embryonic fibroblasts under l-serine depletion. The resultant l-serine deficiency induced cyclin-dependent kinase inhibitor 1a (Cdkn1a; p21) expression, which was mediated by p38 MAPK. Survival of the Phgdh-deficient mouse embryonic fibroblasts was markedly reduced by p38 MAPK inhibition under l-serine depletion, whereas p38 MAPK could be activated by 1-deoxysphinganine, an atypical alanine-derived sphingoid base that was found to accumulate in l-serine-depleted mouse embryonic fibroblasts. These observations provide persuasive evidence that when the external l-serine supply is limited, l-serine synthesized de novo in proliferating cells serves as a metabolic gatekeeper to maintain cell survival and the functions necessary for executing cell cycle progression. Gene Expression Omnibus, accession number GSE55687.

  17. Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

    PubMed

    Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

    2015-02-25

    Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process.

  18. Cellular Dysfunction in the Diabetic Fibroblast

    PubMed Central

    Lerman, Oren Z.; Galiano, Robert D.; Armour, Mary; Levine, Jamie P.; Gurtner, Geoffrey C.

    2003-01-01

    Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O2), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 ± 1.3 pg/ml versus 34.8 ± 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show

  19. Removal of maternal retinoic acid by embryonic CYP26 is required for correct Nodal expression during early embryonic patterning

    PubMed Central

    Uehara, Masayuki; Yashiro, Kenta; Takaoka, Katsuyoshi; Yamamoto, Masamichi; Hamada, Hiroshi

    2009-01-01

    The abundance of retinoic acid (RA) is determined by the balance between its synthesis by retinaldehyde dehydrogenase (RALDH) and its degradation by CYP26. In particular, the dynamic expression of three CYP26 genes controls the regional level of RA within the body. Pregastrulation mouse embryos express CYP26 but not RALDH. We now show that mice lacking all three CYP26 genes manifest duplication of the body axis as a result of expansion of the Nodal expression domain throughout the epiblast. Mouse Nodal was found to contain an RA-responsive element in intron 1 that is highly conserved among mammals. In the absence of CYP26, maternally derived RA activates Nodal expression in the entire epiblast of pregastrulation embryos via this element. These observations suggest that maternal RA must be removed by embryonic CYP26 for correct Nodal expression during embryonic patterning. PMID:19605690

  20. Effect of in vitro adaptation of Marek's disease virus on pock induction on the chorioallantoic membrane of embryonated chicken eggs.

    PubMed Central

    Sharma, J M; Coulson, B D; Young, E

    1976-01-01

    Cell-associated preparations of several isolates of Marek's disease virus produced more pocks on the chorioallantoic membrane of embryonated chicken eggs than plaques in duck embryo fibroblasts, thus indicating that lesion response in eggs was more sensitive than cytopathic response in duck embryo fibroblasts for assaying low-passage Marek's disease virus. Adaptation of the virus to cell cultures by serial passages, however, substantially reduced its pock response so that the titer ratio (plaque-forming units in duck embryo fibroblasts/pock-forming units in eggs) of cell culture-adapted Marek's disease virus was 1 or higher. The decreased pock response could not be attributed to selection of preexisting virus variant(s) with low affinity for chorioallantoic membrane because cloned Marek's disease virus had a good pock response at low cell culture passage levels, but this response decreased as the virus was attenuated by serial cell culture passage. PMID:175018

  1. Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells with Sleeping Beauty Transposon-Based Stable Gene Delivery.

    PubMed

    Sebe, Attila; Ivics, Zoltán

    2016-01-01

    Human induced pluripotent stem (iPS) cells are a source of patient-specific pluripotent stem cells and resemble human embryonic stem (ES) cells in gene expression profiles, morphology, pluripotency, and in vitro differentiation potential. iPS cells are applied in disease modeling, drug screenings, toxicology screenings, and autologous cell therapy. In this protocol, we describe how to derive human iPS cells from fibroblasts by Sleeping Beauty (SB) transposon-mediated gene transfer of reprogramming factors. First, the components of the non-viral Sleeping Beauty transposon system, namely a transposon vector encoding reprogramming transcription factors and a helper plasmid expressing the SB transposase, are electroporated into human fibroblasts. The reprogramming cassette undergoes transposition from the transfected plasmids into the fibroblast genome, thereby resulting in stable delivery of the reprogramming factors. Reprogramming by using this protocol takes ~4 weeks, after which the iPS cells are isolated and clonally propagated.

  2. Direct reprogramming of fibroblasts into skeletal muscle progenitor cells by transcription factors enriched in undifferentiated subpopulation of satellite cells.

    PubMed

    Ito, Naoki; Kii, Isao; Shimizu, Noriaki; Tanaka, Hirotoshi; Shin'ichi, Takeda

    2017-08-14

    Satellite cells comprise a functionally heterogeneous population of stem cells in skeletal muscle. Separation of an undifferentiated subpopulation and elucidation of its molecular background are necessary to identify the reprogramming factors to induce skeletal muscle progenitor cells. In this study, we found that intracellular esterase activity distinguishes a subpopulation of cultured satellite cells with high stemness using esterase-sensitive cell staining reagent, calcein-AM. Gene expression analysis of this subpopulation revealed that defined combinations of transcription factors (Pax3, Mef2b, and Pitx1 or Pax7, Mef2b, and Pitx1 in embryonic fibroblasts, and Pax7, Mef2b and MyoD in adult fibroblasts) reprogrammed fibroblasts into skeletal muscle progenitor cells. These reprogrammed cells formed Dystrophin-positive mature muscle fibers when transplanted into a mouse model of Duchenne muscular dystrophy. These results highlight the new marker for heterogenous population of cultured satellite cells, potential therapeutic approaches and cell sources for degenerative muscle diseases.

  3. Embryonic mammary signature subsets are activated in Brca1-/- and basal-like breast cancers

    PubMed Central

    2013-01-01

    Introduction Cancer is often suggested to result from development gone awry. Links between normal embryonic development and cancer biology have been postulated, but no defined genetic basis has been established. We recently published the first transcriptomic analysis of embryonic mammary cell populations. Embryonic mammary epithelial cells are an immature progenitor cell population, lacking differentiation markers, which is reflected in their very distinct genetic profiles when compared with those of their postnatal descendents. Methods We defined an embryonic mammary epithelial signature that incorporates the most highly expressed genes from embryonic mammary epithelium when compared with the postnatal mammary epithelial cells. We looked for activation of the embryonic mammary epithelial signature in mouse mammary tumors that formed in mice in which Brca1 had been conditionally deleted from the mammary epithelium and in human breast cancers to determine whether any genetic links exist between embryonic mammary cells and breast cancers. Results Small subsets of the embryonic mammary epithelial signature were consistently activated in mouse Brca1-/- tumors and human basal-like breast cancers, which encoded predominantly transcriptional regulators, cell-cycle, and actin cytoskeleton components. Other embryonic gene subsets were found activated in non-basal-like tumor subtypes and repressed in basal-like tumors, including regulators of neuronal differentiation, transcription, and cell biosynthesis. Several embryonic genes showed significant upregulation in estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and/or grade 3 breast cancers. Among them, the transcription factor, SOX11, a progenitor cell and lineage regulator of nonmammary cell types, is found highly expressed in some Brca1-/- mammary tumors. By using RNA interference to silence SOX11 expression in breast cancer cells, we found evidence that SOX11 regulates breast cancer cell

  4. Immunological studies of the embryonic muscle cell surface. Antiserum to the prefusion myoblast

    PubMed Central

    1979-01-01

    Xenogeneic antisera raised in rabbits have been used to detect compositional changes at the cell surfaces of differentiating embryonic chick skeletal muscle. In this report, we present the serological characterization of antiserum (Anti-M-24) against muscle tissue and developmental stage-specific cell surface antigens of the prefusion myoblast. Cells from primary cultures of 12-d-old embryonic chick hindlimb muscle were injected into rabbits, and the resulting antisera were selectively absorbed to obtain immunological specificity. Cytotoxicity and immunohistochemical assays were used to test this antiserum. Absorption with embryonic or adult chick heart, brain, retina, liver, erythrocytes, or skeletal muscle fibroblasts failed to remove all reactivity of Anti-M-24 for myogenic cells at all stages of development. After absorption with embryonic myotubes, however, Anti-M- 24 no longer reacted with differentiated myofibers, but did react with prefusion myoblasts. The myoblast surface antigens detected with Anti-M- 24 are components of the muscle cell membrane: (a) these macromolecules are free to diffuse laterally within the myoblast membrane; (b) Anti-M- 24, in the presence of complement, induced lysis of the muscle cell membrane; and (c) intact monolayers of viable myoblasts completely absorbed reactivity of Anti-M-24 for myoblasts. These antigens are not loosely adsorbed culture medium components or an artifact of tissue culture because: (a) absorption of Anti-M-24 with homogenized embryonic muscle removed all antibodies to cultured myoblasts; (b) Anti-M-24 reacted with myoblast surfaces in vivo; and (c) absorption of Anti-M-24 with culture media did not affect the titer of this antiserum for myoblasts. We conclude that myogenic cells at all stages of development possess externally exposed antigens which are undetected on other embryonic and adult chick tissues. In addition, myoblasts exhibit surface antigenic determinants that are either masked, absent, or present

  5. [Species and tissue differences of reparative DNA synthesis in embryonic cell cultures treated with carcinogens].

    PubMed

    Budunova, I V; Belitskiĭ, G A

    1982-01-01

    DNA repair synthesis (RS) was studied in embryonic cell cultures exposed to different carcinogenic factors: UV-light, N-methyl-N-nitro-N-nitrosoguanidine, 4-nitroquinoline-1-oxide, aflatoxin BI and 7,12-dimethylbenz(a)anthracene. DNA RS level was shown to be higher in human liver cells than in murine ones. Tissue-dependent differences in DNA RS of cells damaged by carcinogens were found, too. RS-activity was higher in human, mouse and rat fibroblast cultures than in liver cultures of the same species. RS level in human kidney cultures was similar to that in human fibroblasts. The said differences should be taken into account in the evaluation of the results of testing of chemical agents for carcinogenicity, using their ability to cause DNA repair synthesis.

  6. Fibronectin is not Present in the Focal Adhesions Formed between Normal Cultured Fibroblasts and Their Substrata

    NASA Astrophysics Data System (ADS)

    Chen, Wen-Tien; Singer, S. J.

    1980-12-01

    Fibronectin is an extracellular matrix protein that has been implicated in the spreading and adhesion of cultured fibroblasts to their substrata. In this paper, double immunoelectron microscopic labeling experiments for fibronectin and for concanavalin A-binding proteins on the cell surface were carried out on ultrathin frozen sections of cultures of embryonic chicken heart fibroblasts. On cross sections through the focal adhesions of the cell to the substratum there was substantial labeling for concanavalin A-binding proteins but no detectable labeling for fibronectin, whereas both the binding proteins and fibronectin were extensively labeled elsewhere on the cell surface and substratum. These results demonstrate that fibronectin is not present within the sites of focal adhesions. Therefore, the functions of fibronectin in cell spreading and adhesion are not directly mediated through its binding at focal adhesion sites. An alternative model is presented which can account for such fibronectin functions.

  7. Mutations in fibroblast growth factor receptors: Phenotypic consequences during eukaryotic development

    SciTech Connect

    Park, W.J.; Bellus, G.A.; Jabs, E.W.

    1995-10-01

    Recently, a tremendous amount of excitement and interest has been generated by the rapid succession of discoveries in the human fibroblast growth factor receptor (FGFR) field. In less than a year, mutations in three FGFRs (FGFR1-FGFR3) have been associated with three skeletal dysplasias and four craniosynostotic syndromes. FGFRs are members of the receptor tyrosine kinase family that bind fibroblast growth factors (FGFs). The FGF family consists of structurally related polypeptides that play a key role in numerous aspects of embryogenesis, growth, and homeostasis. FGFs have a potent growth stimulatory and/or differentiation-inducing effect on cells such as those derived from the early-embryonic mesoderm or ectoderm. In addition to mitogenesis and differentiation, FGFs also stimulate chemotaxis, cell survival, and angiogenesis. FGFs mediate cellular responses on binding to and activation of FGFRs. 45 refs., 2 figs., 1 tab.

  8. BMPs functionally replace Klf4 and support efficient reprogramming of mouse fibroblasts by Oct4 alone

    PubMed Central

    Chen, Jiekai; Liu, Jing; Yang, Jiaqi; Chen, You; Chen, Jing; Ni, Su; Song, Hong; Zeng, Lingwen; Ding, Ke; Pei, Duanqing

    2011-01-01

    Generation of induced pluripotent stem cells by defined factors has become a useful model to investigate the mechanism of reprogramming and cell fate determination. However, the precise mechanism of factor-based reprogramming remains unclear. Here, we show that Klf4 mainly acts at the initial phase of reprogramming to initiate mesenchymal-to-epithelial transition and can be functionally replaced by bone morphogenetic proteins (BMPs). BMPs boosted the efficiency of Oct4/Sox2-mediated reprogramming of mouse embryonic fibroblasts (MEFs) to ∼1%. BMPs also promoted single-factor Oct4-based reprogramming of MEFs and tail tibial fibroblasts. Our studies clarify the contribution of Klf4 in reprogramming and establish Oct4 as a singular setter of pluripotency in differentiated cells. PMID:21135873

  9. Question of bone marrow stromal fibroblast traffic

    SciTech Connect

    Maloney, M.A.; Lamela, R.A.; Patt, H.M.

    1985-01-01

    Bone marrow stromal fibroblasts (CFU-F) normally do not exchange bone marrow sites in vivo. Restitution of the CFU-F after radiation damage is primarily recovery by the local fibroblasts from potentially lethal damage. Migration of stromal fibroblasts from shielded sites to an irradiated site makes a minimal contribution, if any, to CFU-F recovery. Determination of the relative contribution of donor stromal cells in bone marrow transplants by karyotyping the proliferating bone marrow stromal cells in vitro may not reflect the relative distribution of fibroblasts in the marrow. If there is residual damage to the host stromal fibroblasts from treatment before transplantation, these cells may not be able to proliferate in vitro. Therefore, an occasional transplanted fibroblast may contribute most of the metaphase figures scored for karyotype.

  10. Maintenance of human embryonic stem cells in animal serum- and feeder layer-free culture conditions.

    PubMed

    Amit, Michal; Itskovitz-Eldor, Joseph

    2006-01-01

    The availability of human embryonic stem cells (hESCs) reflects their outstanding potential for research areas such as human developmental biology, teratology, and cell-based therapies. To allow their continuous growth as undifferentiated cells, isolation and culturing were traditionally conducted on mouse embryonic fibroblast feeder layers, using medium supplemented with fetal bovine serum. However, these conditions allow possible exposure of the cells to animal pathogens. Because both research and future clinical application require an animal-free and well-defined culture system for hESCs, these conventional conditions would prevent the use of hESCs in human therapy. This chapter describes optional culture conditions based on either animal-free or feeder-free culture methods for hESCs.

  11. Small RNA-directed epigenetic programming of embryonic stem cell cardiac differentiation.

    PubMed

    Ghanbarian, Hossein; Wagner, Nicole; Michiels, Jean-François; Cuzin, François; Wagner, Kay-Dietrich; Rassoulzadegan, Minoo

    2017-02-06

    Microinjection of small noncoding RNAs in one-cell embryos was reported in several instances to result in transcriptional activation of target genes. To determine the molecular mechanisms involved and to explore whether such epigenetic regulations could play a role in early development, we used a cell culture system as close as possible to the embryonic state. We report efficient cardiac differentiation of embryonic stem (ES) cells induced by small non-coding RNAs with sequences of Cdk9, a key player in cardiomyocyte differentiation. Transfer of oligoribonucleotides representing parts of the Cdk9 mRNA into ES and mouse embryo fibroblast cultures resulted in upregulation of transcription. Dependency on Argonaute proteins and endogenous antisense transcripts indicated that the inducer oligoribonucleotides were processed by the RNAi machinery. Upregulation of Cdk9 expression resulted in increased efficiency of cardiac differentiation suggesting a potential tool for stem cell-based regenerative medicine.

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

    SciTech Connect

    Yamano, Noriko; Kimura, Tohru; Watanabe-Kushima, Shoko; Shinohara, Takashi; Nakano, Toru

    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.

  13. Small RNA-directed epigenetic programming of embryonic stem cell cardiac differentiation

    PubMed Central

    Ghanbarian, Hossein; Wagner, Nicole; Michiels, Jean-François; Cuzin, François; Wagner, Kay-Dietrich; Rassoulzadegan, Minoo

    2017-01-01

    Microinjection of small noncoding RNAs in one-cell embryos was reported in several instances to result in transcriptional activation of target genes. To determine the molecular mechanisms involved and to explore whether such epigenetic regulations could play a role in early development, we used a cell culture system as close as possible to the embryonic state. We report efficient cardiac differentiation of embryonic stem (ES) cells induced by small non-coding RNAs with sequences of Cdk9, a key player in cardiomyocyte differentiation. Transfer of oligoribonucleotides representing parts of the Cdk9 mRNA into ES and mouse embryo fibroblast cultures resulted in upregulation of transcription. Dependency on Argonaute proteins and endogenous antisense transcripts indicated that the inducer oligoribonucleotides were processed by the RNAi machinery. Upregulation of Cdk9 expression resulted in increased efficiency of cardiac differentiation suggesting a potential tool for stem cell-based regenerative medicine. PMID:28165496

  14. Diphenylhydantoin affects glycosaminoglycans and collagen production by human fibroblasts from cleft palate patients.

    PubMed

    Bosi, G; Evangelisti, R; Valeno, V; Carinci, F; Pezzetti, F; Calastrini, C; Bodo, M; Carinci, P

    1998-08-01

    During embryonic development, the proper production of extracellular matrix molecules mediates morphogenetic processes involved in palatogenesis. In the present study, we investigated whether any differences exist in glycosaminoglycan (GAG) and collagen synthesis between palate fibroblasts from infants, with or without cleft palate, in two age ranges. Subsequently, the effects of diphenylhydantoin (PHT), a teratogen known to induce cleft palate in human and mammalian newborns, on extracellular matrix (ECM) production were studied. We found that cleft palate fibroblasts (CPFs) synthesize greater amounts of GAG and collagen than normal fibroblasts (NFs). CPFs produced less cellular hyaluronic acid (HA) and more sulphated GAG. HA was the principal GAG species in the medium, and its percentage was lower in one- to three-year-old CPFs. Cleft palate fibroblasts produced more extracellular chondroitin 4- and 6-sulphate (CS) and dermatan sulphate (DS). Associated with a higher production of sulphated GAG, we observed a higher synthesis of type III and type I collagen with a normal ratio of alpha2(I) to alpha1(I) chains. PHT treatment of NFs reduced collagen and GAG synthesis, with a marked effect on sulphated GAG. The drug changed collagen synthesis, whereas it did not affect GAG production in CPFs whose phenotype may already be impaired. These findings indicate that, in CPFs, modifications in the pattern of ECM components, which are most likely responsible for the anomalous development, persist in infants. In addition, NFs and CPFs with a different phenotype respond differently to PHT treatment.

  15. Ceramide inhibits CCN2 expression in fibroblasts

    PubMed Central

    Kennedy, Laura; Parapuram, Sunil; Greenspoon, Jill

    2008-01-01

    Connective tissue growth factor (CTGF, CCN2) is induced in response to TGFβ in fibroblasts. In this report, we show that C2 ceramide reduced the ability of TGFβ to induce CCN2 protein, mRNA and promoter activity in fibroblasts. C2 ceramide reduced the ability of TGFβ to induce the generic Smad responsive promoter/reporter construct SBE-luciferase. These results suggest that C2 ceramide reduces the action of TGFβ in fibroblasts via Smad antagonism. PMID:18649016

  16. Cell motility and local viscoelasticity of fibroblasts.

    PubMed

    Park, S; Koch, D; Cardenas, R; Käs, J; Shih, C K

    2005-12-01

    Viscoelastic changes of the lamellipodial actin cytoskeleton are a fundamental element of cell motility. Thus, the correlation between the local viscoelastic properties of the lamellipodium (including the transitional region to the cell body) and the speed of lamellipodial extension is studied for normal and malignantly transformed fibroblasts. Using our atomic force microscopy-based microrheology technique, we found different mechanical properties between the lamellipodia of malignantly transformed fibroblasts (H-ras transformed and SV-T2 fibroblasts) and normal fibroblasts (BALB 3T3 fibroblasts). The average elastic constants, K, in the leading edge of SV-T2 fibroblasts (0.48 +/- 0.51 kPa) and of H-ras transformed fibroblasts (0.42 +/- 0.35 kPa) are significantly lower than that of BALB 3T3 fibroblasts (1.01 +/- 0.40 kPa). The analysis of time-lapse phase contrast images shows that the decrease in the elastic constant, K, for malignantly transformed fibroblasts is correlated with the enhanced motility of the lamellipodium. The measured mean speeds are 6.1 +/- 4.5 microm/h for BALB 3T3 fibroblasts, 13.1 +/- 5.2 microm/h for SV-T2 fibroblasts, and 26.2 +/- 11.5 microm/h for H-ras fibroblasts. Furthermore, the elastic constant, K, increases toward the cell body in many instances which coincide with an increase in actin filament density toward the cell body. The correlation between the enhanced motility and the decrease in viscoelastic moduli supports the Elastic Brownian Ratchet model for driving lamellipodia extension.

  17. Cell Motility and Local Viscoelasticity of Fibroblasts

    PubMed Central

    Park, S.; Koch, D.; Cardenas, R.; Käs, J.; Shih, C. K.

    2005-01-01

    Viscoelastic changes of the lamellipodial actin cytoskeleton are a fundamental element of cell motility. Thus, the correlation between the local viscoelastic properties of the lamellipodium (including the transitional region to the cell body) and the speed of lamellipodial extension is studied for normal and malignantly transformed fibroblasts. Using our atomic force microscopy-based microrheology technique, we found different mechanical properties between the lamellipodia of malignantly transformed fibroblasts (H-ras transformed and SV-T2 fibroblasts) and normal fibroblasts (BALB 3T3 fibroblasts). The average elastic constants, K, in the leading edge of SV-T2 fibroblasts (0.48 ± 0.51 kPa) and of H-ras transformed fibroblasts (0.42 ± 0.35 kPa) are significantly lower than that of BALB 3T3 fibroblasts (1.01 ± 0.40 kPa). The analysis of time-lapse phase contrast images shows that the decrease in the elastic constant, K, for malignantly transformed fibroblasts is correlated with the enhanced motility of the lamellipodium. The measured mean speeds are 6.1 ± 4.5 μ m/h for BALB 3T3 fibroblasts, 13.1 ± 5.2 μm/h for SV-T2 fibroblasts, and 26.2 ± 11.5 μm/h for H-ras fibroblasts. Furthermore, the elastic constant, K, increases toward the cell body in many instances which coincide with an increase in actin filament density toward the cell body. The correlation between the enhanced motility and the decrease in viscoelastic moduli supports the Elastic Brownian Ratchet model for driving lamellipodia extension. PMID:16199496

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

    PubMed Central

    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

  19. Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

    PubMed

    Chang, Chan-Jung; Mitra, Koyel; Koya, Mariko; Velho, Michelle; Desprat, Romain; Lenz, Jack; Bouhassira, Eric E

    2011-01-01

    We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

  20. The validated embryonic stem cell test to predict embryotoxicity in vitro.

    PubMed

    Seiler, Andrea E M; Spielmann, Horst

    2011-06-16

    In the embryonic stem cell test (EST), differentiation of mouse embryonic stem cells (mESCs) is used as a model to assess embryotoxicity in vitro. The test was successfully validated by the European Center for the Validation of Alternative Methods (ECVAM) and models fundamental mechanisms in embryotoxicity, such as cytotoxicity and differentiation. In addition, differences in sensitivity between differentiated (adult) and embryonic cells are also taken into consideration. To predict the embryotoxic potential of a test substance, three endpoints are assessed: the inhibition of differentiation into beating cardiomyocytes, the cytotoxic effects on stem cells and the cytotoxic effects on 3T3 fibroblasts. A special feature of the EST is that it is solely based on permanent cell lines so that primary embryonic cells and tissues from pregnant animals are not needed. In this protocol, we describe the ECVAM-validated method, in which the morphological assessment of contracting cardiomyocytes is used as an endpoint for differentiation, and the molecular-based FACS-EST method, in which highly predictive protein markers specific for developing heart tissue were selected. With these methods, the embryotoxic potency of a compound can be assessed in vitro within 10 or 7 d, respectively.

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

    PubMed

    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

  2. Zebularine and scriptaid significantly improve epigenetic reprogramming of yak fibroblasts and cloning efficiency.

    PubMed

    Xiong, Xianrong; Lan, Daoliang; Li, Jian; Zhong, Jincheng; Zi, Xiangdong; Ma, Li; Wang, Yong

    2013-08-01

    Abnormal epigenetic reprogramming of the donor nucleus after somatic cell nuclear transfer (SCNT) is thought to be the main cause of low cloning efficiency. Following SCNT, the donor nucleus often fails to express early embryonic genes and establish a normal embryonic pattern of chromatin modification. Therefore, in this study, we have attempted to improve epigenetic reprogramming of the donor nucleus and cloned embryos with Zebularine and Scriptaid. Yak fibroblasts were treated with 20 μM Zebularine alone or 20 μM Zebularine plus 0.5 μM Scriptaid for 24 h, whereas yak cloned embryos were treated exclusively with 0.5 μM Scriptaid for 12 h. There was no effect on cellular viability and proliferation after drug treatment. The treatment of fibroblasts with Zebularine or Zebularine plus Scriptaid increased histone acetylation of histone 3 lysine 9 (H3K9), but decreased the level of DNA methylation of Oct-4 and Sox-2 promoter regions. When donor cells were used after Zebularine plus Scriptaid treatment to reconstruct cloned embryos and then treated with Scriptaid, the developmental competence and cryosurvival of embryos were improved significantly. In addition, the relative expression of Oct-4 and Sox-2 were increased significantly. The expression levels of Dnmt-1 and Hdac-1 were significantly decreased when fibroblasts and cloned embryos were treated with Zebularine or Scriptaid. This work provides functional evidence that treatment with Zebularine and Scriptaid modifies the epigenetic status of yak fibroblasts, subsequently enhancing in vitro developmental potential and the quality of yak cloned embryos.

  3. Culture and Manipulation of Embryonic Cells

    PubMed Central

    Edgar, Lois G.; Goldstein, Bob

    2012-01-01

    The direct manipulation of embryonic cells is an important tool for addressing key questions in cell and developmental biology. C. elegans is relatively unique among genetic model systems in being amenable to manipulation of embryonic cells. Embryonic cell manipulation has allowed the identification of cell interactions by direct means, and it has been an important technique for dissecting mechanisms by which cell fates are specified, cell divisions are oriented, and morphogenesis is accomplished. Here, we present detailed methods for isolating, manipulating and culturing embryonic cells of C. elegans. PMID:22226523

  4. Gastrointestinal Fibroblasts Have Specialized, Diverse Transcriptional Phenotypes: A Comprehensive Gene Expression Analysis of Human Fibroblasts

    PubMed Central

    Ishii, Genichiro; Aoyagi, Kazuhiko; Sasaki, Hiroki; Ochiai, Atsushi

    2015-01-01

    Background Fibroblasts are the principal stromal cells that exist in whole organs and play vital roles in many biological processes. Although the functional diversity of fibroblasts has been estimated, a comprehensive analysis of fibroblasts from the whole body has not been performed and their transcriptional diversity has not been sufficiently explored. The aim of this study was to elucidate the transcriptional diversity of human fibroblasts within the whole body. Methods Global gene expression analysis was performed on 63 human primary fibroblasts from 13 organs. Of these, 32 fibroblasts from gastrointestinal organs (gastrointestinal fibroblasts: GIFs) were obtained from a pair of 2 anatomical sites: the submucosal layer (submucosal fibroblasts: SMFs) and the subperitoneal layer (subperitoneal fibroblasts: SPFs). Using hierarchical clustering analysis, we elucidated identifiable subgroups of fibroblasts and analyzed the transcriptional character of each subgroup. Results In unsupervised clustering, 2 major clusters that separate GIFs and non-GIFs were observed. Organ- and anatomical site-dependent clusters within GIFs were also observed. The signature genes that discriminated GIFs from non-GIFs, SMFs from SPFs, and the fibroblasts of one organ from another organ consisted of genes associated with transcriptional regulation, signaling ligands, and extracellular matrix remodeling. Conclusions GIFs are characteristic fibroblasts with specific gene expressions from transcriptional regulation, signaling ligands, and extracellular matrix remodeling related genes. In addition, the anatomical site- and organ-dependent diversity of GIFs was also discovered. These features of GIFs contribute to their specific physiological function and homeostatic maintenance, and create a functional diversity of the gastrointestinal tract. PMID:26046848

  5. The influence of microscale topography on fibroblast attachment and motility.

    PubMed

    Berry, Catherine C; Campbell, Gordon; Spadiccino, Antonio; Robertson, Mary; Curtis, Adam S G

    2004-11-01

    The ability of a cell to attach and migrate on a substrate or scaffold is important in the field of tissue engineering and biomaterials, and is thus extensively studied. When considering tissue-engineering applications, a highly porous scaffold is required to guide cell growth and proliferation in three dimensions. However existing scaffolds are less than ideal for actual applications, not only as they lack mechanical strength due to pore size and have regular distribution, but also they do not ensure cell attachment, in-growth and organisation. In this study, microfabrication technology was used to create regular arrays of pits on a two-dimensional quartz surface (7, 15 and 25 microm diameter, 20 and 40 microm spacing). The patterned surface thus exhibited spatially separated mechanical edges akin to the basic structural element of a three-dimensional network, and was used as a model system for studying the effects of substrate microgeometry on fibroblast attachment and motility. Results clearly showed that fibroblast interaction with the pit edges depended on both diameter, and therefore angle of circumference, and inter pit spacing, with the largest diameter permitting cells to enter the pits. Interestingly, the highest cell proliferation rates were recorded on the smaller pits. Such information may provide details on possible pore sizes for use in synthetic tissue engineering scaffolds that aim to support fibroblast in-growth and subsequent proliferation.

  6. Ectoderm-mesoderm crosstalk in the embryonic limb: The role of fibroblast growth factor signaling.

    PubMed

    Mariani, Francesca V; Fernandez-Teran, Marian; Ros, Maria A

    2017-04-01

    In this commentary we focus on the function of FGFs during limb development and morphogenesis. Our goal is to understand, interpret and, when possible, reconcile the interesting findings and conflicting results that remain unexplained. For example, the cell death pattern observed after surgical removal of the AER versus genetic removal of the AER-Fgfs is strikingly different and the field is at an impasse with regard to an explanation. We also discuss the idea that AER function may involve signaling components in addition to the AER-FGFs and that signaling from the non-AER ectoderm may also have a significant contribution. We hope that a re-evaluation of current studies and a discussion of outstanding questions will motivate new experiments, especially considering the availability of new technologies, that will fuel further progress toward understanding the intricate ectoderm-to-mesoderm crosstalk during limb development. Developmental Dynamics 246:208-216, 2017. © 2016 Wiley Periodicals, Inc.

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

    SciTech Connect

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

    2016-05-13

    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.

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

    PubMed

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

    2016-05-13

    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. Copyright © 2016. Published by Elsevier Inc.

  9. PED/PEA-15 controls fibroblast motility and wound closure by ERK1/2-dependent mechanisms.

    PubMed

    Buonomo, Roberta; Giacco, Ferdinando; Vasaturo, Angela; Caserta, Sergio; Guido, Stefano; Pagliara, Valentina; Garbi, Corrado; Mansueto, Gelsomina; Cassese, Angela; Perruolo, Giuseppe; Oriente, Francesco; Miele, Claudia; Beguinot, Francesco; Formisano, Pietro

    2012-05-01

    Cell migration is dependent on the control of signaling events that play significant roles in creating contractile force and in contributing to wound closure. We evaluated wound closure in fibroblasts from mice overexpressing (TgPED) or lacking ped/pea-15 (KO), a gene overexpressed in patients with type 2 diabetes. Cultured skin fibroblasts isolated from TgPED mice showed a significant reduction in the ability to recolonize wounded area during scratch assay, compared to control fibroblasts. This difference was observed both in the absence and in the presence of mytomicin C, an inhibitor of mitosis. In time-lapse experiments, TgPED fibroblasts displayed about twofold lower velocity and diffusion coefficient, as compared to controls. These changes were accompanied by reduced spreading and decreased formation of stress fibers and focal adhesion plaques. At the molecular level, TgPED fibroblasts displayed decreased RhoA activation and increased abundance of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). Inhibition of ERK1/2 activity by PD98059 restored RhoA activation, cytoskeleton organization and cell motility, and almost completely rescued wound closure of TgPED fibroblasts. Interestingly, skin fibroblasts isolated from KO mice displayed an increased wound closure ability. In vivo, healing of dorsal wounds was delayed in TgPED and accelerated in KO mice. Thus, PED/PEA-15 may affect fibroblast motility by a mechanism, at least in part, mediated by ERK1/2.

  10. Embryonic stem cell-specific signature in cervical cancer.

    PubMed

    Organista-Nava, Jorge; Gómez-Gómez, Yazmín; Gariglio, Patricio

    2014-03-01

    The wide range of invasive and noninvasive lesion phenotypes associated with high-risk human papillomavirus (HR-HPV) infection in cervical cancer (CC) indicates that not only the virus but also specific cervical epithelial cells in the transformation zone (TZ), such as stem cells (SCs), play an important part in the development of cervical neoplasia. In this review, we focused in an expression signature that is specific to embryonic SCs and to poorly differentiated cervical malignant tumors and we hypothesize that this expression signature may play an important role to promote cell growth, survival, colony formation, lack of adhesion, as well as cell invasion and migration in CC.

  11. Pulse propagation by a capacitive mechanism drives embryonic blood flow.

    PubMed

    Anton, Halina; Harlepp, Sebastien; Ramspacher, Caroline; Wu, Dave; Monduc, Fabien; Bhat, Sandeep; Liebling, Michael; Paoletti, Camille; Charvin, Gilles; Freund, Jonathan B; Vermot, Julien

    2013-11-01

    Pulsatile flow is a universal feature of the blood circulatory system in vertebrates and can lead to diseases when abnormal. In the embryo, blood flow forces stimulate vessel remodeling and stem cell proliferation. At these early stages, when vessels lack muscle cells, the heart is valveless and the Reynolds number (Re) is low, few details are available regarding the mechanisms controlling pulses propagation in the developing vascular network. Making use of the recent advances in optical-tweezing flow probing approaches, fast imaging and elastic-network viscous flow modeling, we investigated the blood-flow mechanics in the zebrafish main artery and show how it modifies the heart pumping input to the network. The movement of blood cells in the embryonic artery suggests that elasticity of the network is an essential factor mediating the flow. Based on these observations, we propose a model for embryonic blood flow where arteries act like a capacitor in a way that reduces heart effort. These results demonstrate that biomechanics is key in controlling early flow propagation and argue that intravascular elasticity has a role in determining embryonic vascular function.

  12. Effects of embryonic cyclosporine exposures on brain development and behavior.

    PubMed

    Clift, Danielle E; Thorn, Robert J; Passarelli, Emily A; Kapoor, Mrinal; LoPiccolo, Mary K; Richendrfer, Holly A; Colwill, Ruth M; Creton, Robbert

    2015-04-01

    Cyclosporine, a calcineurin inhibitor, is successfully used as an immunosuppressant in transplant medicine. However, the use of this pharmaceutical during pregnancy is concerning since calcineurin is thought to play a role in neural development. The risk for human brain development is difficult to evaluate because of a lack of basic information on the sensitive developmental times and the potentially pleiotropic effects on brain development and behavior. In the present study, we use zebrafish as a model system to examine the effects of embryonic cyclosporine exposures. Early embryonic exposures reduced the size of the eyes and brain. Late embryonic exposures did not affect the size of the eyes or brain, but did lead to substantial behavioral defects at the larval stages. The cyclosporine-exposed larvae displayed a reduced avoidance response to visual stimuli, low swim speeds, increased resting, an increase in thigmotaxis, and changes in the average distance between larvae. Similar results were obtained with the calcineurin inhibitor FK506, suggesting that most, but not all, effects on brain development and behavior are mediated by calcineurin inhibition. Overall, the results show that cyclosporine can induce either structural or functional brain defects, depending on the exposure window. The observed functional brain defects highlight the importance of quantitative behavioral assays when evaluating the risk of developmental exposures.

  13. Effects of embryonic cyclosporine exposures on brain development and behavior

    PubMed Central

    Clift, Danielle E.; Thorn, Robert J.; Passarelli, Emily A.; Kapoor, Mrinal; LoPiccolo, Mary K.; Richendrfer, Holly A.; Colwill, Ruth M.; Creton, Robbert

    2015-01-01

    Cyclosporine, a calcineurin inhibitor, is successfully used as an immunosuppressant in transplant medicine. However, the use of this pharmaceutical during pregnancy is concerning, since calcineurin is thought to play a role in neural development. The risk for human brain development is difficult to evaluate, because of a lack of basic information on the sensitive developmental times and the potentially pleiotropic effects on brain development and behavior. In the present study, we use zebrafish as a model system to examine the effects of embryonic cyclosporine exposures. Early embryonic exposures reduced the size of the eyes and brain. Late embryonic exposures did not affect the size of the eyes or brain, but did lead to substantial behavioral defects at the larval stages. The cyclosporine-exposed larvae displayed a reduced avoidance response to visual stimuli, low swim speeds, increased resting, an increase in thigmotaxis, and changes in the average distance between larvae. Similar results were obtained with the calcineurin inhibitor FK506, suggesting that most, but not all, effects on brain development and behavior are mediated by calcineurin inhibition. Overall, the results show that cyclosporine can induce either structural or functional brain defects, depending on the exposure window. The observed functional brain defects highlight the importance of quantitative behavioral assays when evaluating the risk of developmental exposures. PMID:25591474

  14. Discoidin domain receptor 2 regulates the adhesion of fibroblasts to 3D collagen matrices.

    PubMed

    Kim, Daehwan; You, Eunae; Min, Na Young; Lee, Kwang-Ho; Kim, Hyoung Kyu; Rhee, Sangmyung

    2013-05-01

    The collagen matrix constitutes the primary extracellular matrix (ECM) portion of mammalian connective tissues in which the interaction of the cell and the surrounding collagen fibers has a significant impact on cell and tissue physiology, including morphogenesis, development and motility. Discoidin domain receptors (DDR1 and DDR2) have been identified as the receptor tyrosine kinases that are activated upon collagen binding. However, there is a lack of evidence regarding the effect of DDRs on the mechanical interaction between fibroblasts and ECM. In this study, we demonstrated that one of the major phosphotyrosine proteins in human fibroblasts during 3D collagen matrix polymerization is DDR2. Treatment of fibroblasts in 3D collagen matrices with platelet-derived growth factor (PDFG) has been shown to increase DDR2 phosphorylation. Silencing of DDR2 with siRNA in fibroblasts significantly reduced the number of dendritic extensions regardless of whether cells were cultured in the collagen or fibronectin 3D matrices. Decreasing dendritic extensions in DDR2-silenced cells has also been shown to decrease the ability of fibroblast entanglement to collagen fibrils in 3D collagen matrices. Finally, we also showed that the silencing of DDR2 decreased the cell migration in 3D nested collagen matrices but had no effect on 3D floating matrix contraction. Collectively, these results suggest that DDR2 functioning is required for the membrane dynamics to control the mechanical attachment of fibroblasts to the 3D collagen matrices in an integrin-independent manner.

  15. Autophagy in cancer associated fibroblasts promotes tumor cell survival

    PubMed Central

    Martinez-Outschoorn, Ubaldo E; Trimmer, Casey; Lin, Zhao; Whitaker-Menezes, Diana; Chiavarina, Barbara; Zhou, Jie; Wang, Chengwang; Pavlides, Stephanos; Martinez-Cantarin, Maria P; Capozza, Franco; Witkiewicz, Agnieszka K; Flomenberg, Neal; Howell, Anthony; Pestell, Richard G; Caro, Jaime

    2010-01-01

    -associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (−/−) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the “autophagic tumor stroma model of cancer metabolism”, and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a “lethal” tumor microenvironment. PMID:20855962

  16. Vertebrate Embryonic Cleavage Pattern Determination.

    PubMed

    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.

  17. The enigma of embryonic diapause.

    PubMed

    Renfree, Marilyn B; Fenelon, Jane C

    2017-09-15

    Embryonic diapause - a period of embryonic suspension at the blastocyst stage - is a fascinating phenomenon that occurs in over 130 species of mammals, ranging from bears and badgers to mice and marsupials. It might even occur in humans. During diapause, there is minimal cell division and greatly reduced metabolism, and development is put on hold. Yet there are no ill effects for the pregnancy when it eventually continues. Multiple factors can induce diapause, including seasonal supplies of food, temperature, photoperiod and lactation. The successful reactivation and continuation of pregnancy then requires a viable embryo, a receptive uterus and effective molecular communication between the two. But how do the blastocysts survive and remain viable during this period of time, which can be up to a year in some cases? And what are the signals that bring it out of suspended animation? Here, we provide an overview of the process of diapause and address these questions, focussing on recent molecular data. © 2017. Published by The Company of Biologists Ltd.

  18. Interferon Regulatory Factor 4 Contributes to Transformation of v-Rel-Expressing Fibroblasts

    PubMed Central

    Hrdličková, Radmila; Nehyba, Jiří; Bose, Henry R.

    2001-01-01

    The avian homologue of the interferon regulatory factor 4 (IRF-4) and a novel splice variant lacking exon 6, IRF-4ΔE6, were isolated and characterized. Chicken IRF-4 is expressed in lymphoid organs, less in small intestine, and lungs. IRF-4ΔE6 mRNA, though less abundant than full-length IRF-4, was detected in lymphoid tissues, with the highest levels observed in thymic cells. IRF-4 is highly expressed in v-Rel-transformed lymphocytes, and the expression of IRF-4 is increased in v-Rel- and c-Rel-transformed fibroblasts relative to control cells. The expression of IRF-4 from retrovirus vectors morphologically transformed primary fibroblasts, increased their saturation density, proliferation, and life span, and promoted their growth in soft agar. IRF-4 and v-Rel cooperated synergistically to transform fibroblasts. The expression of IRF-4 antisense RNA eliminated formation of soft agar colonies by v-Rel and reduced the proliferation of v-Rel-transformed cells. v-Rel-transformed fibroblasts produced interferon 1 (IFN1), which inhibits fibroblast proliferation. Infection of fibroblasts with retroviruses expressing v-Rel resulted in an increase in the mRNA levels of IFN1, the IFN receptor, STAT1, JAK1, and 2′,5′-oligo(A) synthetase. The exogenous expression of IRF-4 in v-Rel-transformed fibroblasts decreased the production of IFN1 and suppressed the expression of several genes in the IFN transduction pathway. These results suggest that induction of IRF-4 expression by v-Rel likely facilitates transformation of fibroblasts by decreasing the induction of this antiproliferative pathway. PMID:11533227

  19. Immunolocalization of fibroblast growth factor receptors 1 and 2 in mouse palate development.

    PubMed

    Lee, S; Crisera, C A; Erfani, S; Maldonado, T S; Lee, J J; Alkasab, S L; Longaker, M T

    2001-06-01

    Recent evidence has implicated mutations of fibroblast growth factor receptors (FGF-R) in the pathogenesis of craniosynostotic syndromes. Cleft palate can be a component of such syndromes. The expression of FGF-R1 and FGF-R2 has been delineated in normally developing cranium, where they seem to regulate cellular differentiation and proliferation, respectively. The specific role of fibroblast growth factor signaling in mammalian palate development is unclear. The authors investigated the patterns of expression of FGF-R1 and FGF-R2 throughout mouse palatal development in the embryo. Time-dated CD-1 mouse heads (n = 135) were harvested at embryonic ages 12.5, 13.5, 14.5, 15.5, and 16.5 days (term gestation = 19.5 days), fixed in paraformaldehyde, embedded in paraffin, and sectioned. In addition, paired palatal shelves (n = 30) were isolated by means of microdissection from embryonic day--13.5 embryos, grown on Millipore filters in serum-free medium in vitro for 24, 48, 72, or 96 hours and processed for histological analysis. Immunohistochemical analysis for FGF-R1 and FGF-R2 was performed on the in vivo and in vitro specimens. FGF-R1 and FGF-R2 were found to be specifically expressed in the epithelium of the developing palatal shelves from the time of their outgrowth from the maxillary processes through completion of fusion in vivo and in vitro. Expression of both receptors was particularly strong during the phases of medial epithelial-medial epithelial contact between the individual shelves, through the formation of the medial epithelial seam, to the ultimate dissolution of the seam. Such a pattern of expression seems to implicate fibroblast growth factor signaling in the regulation of the critical phase of fusion of the bilateral shelves. The expression of both FGF-R1 and FGF-R2 in the lateral palatal mesenchyme, where such secondary structures as tooth primordia and bone begin to appear, also suggests a role for fibroblast growth factor signaling in the induction

  20. Murine Dermal Fibroblast Isolation by FACS.

    PubMed

    Walmsley, Graham G; Maan, Zeshaan N; Hu, Michael S; Atashroo, David A; Whittam, Alexander J; Duscher, Dominik; Tevlin, Ruth; Marecic, Owen; Lorenz, H Peter; Gurtner, Geoffrey C; Longaker, Michael T

    2016-01-07

    Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin(-)) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

  1. Mice Lacking Hbp1 Function Are Viable and Fertile

    PubMed Central

    Wilhelm, Dagmar; Jans, David A.; Bowles, Josephine; Koopman, Peter

    2017-01-01

    Fetal germ cell development is tightly regulated by the somatic cell environment, and is characterised by cell cycle states that differ between XY and XX gonads. In the testis, gonocytes enter G1/G0 arrest from 12.5 days post coitum (dpc) in mice and maintain cell cycle arrest until after birth. Failure to correctly maintain G1/G0 arrest can result in loss of germ cells or, conversely, germ cell tumours. High mobility group box containing transcription factor 1 (HBP1) is a transcription factor that was previously identified in fetal male germ cells at the time of embryonic cell cycle arrest. In somatic cells, HBP1 is classified as a tumour suppressor protein, known to regulate proliferation and senescence. We therefore investigated the possible role of HBP1 in the initiation and maintenance of fetal germ cell G1/G0 arrest using the mouse model. We identified two splice variants of Hbp1, both of which are expressed in XY and XX fetal gonads, but only one of which is localised to the nucleus in in vitro assays. To investigate Hbp1 loss of function, we used embryonic stem (ES) cells carrying a Genetrap mutation for Hbp1 to generate mice lacking Hbp1 function. We found that Hbp1-genetrap mouse mutant germ cells proliferated correctly throughout development, and adult males were viable and fertile. Multiple Hbp1-LacZ reporter mouse lines were generated, unexpectedly revealing Hbp1 embryonic expression in hair follicles, eye and limbs. Lastly, in a model of defective germ cell G1/G0 arrest, the Rb1-knockout model, we found no evidence for Hbp1 mis-regulation, suggesting that the reported RB1-HBP1 interaction is not critical in the germline, despite co-expression. PMID:28107452

  2. The Effects of Fibroblast Co-Culture and Activin A on in vitro Growth of Mouse Preantral Follicles

    PubMed Central

    Karimpour Malekshah, Abbasali; Heidari, Mahmoud; Parivar, Kazem; Azami, Nasrin Sadat

    2014-01-01

    Background: This study was conducted to evaluate fibroblast co-culture and Activin A on in vitro maturation and fertilization of mouse preantral follicles. Methods: The ovaries from 12-14-day-old mice were dissected, and 120-150 μm preantral follicles were cultured individually in α-MEM as based medium for 12 days. A total number of 456 follicles were cultured in four conditions: (i) base medium as control group (n = 113), (ii) base medium supplemented with 30 ng/ml Activin A (n = 115), (iii) base medium co-cultured with mouse embryonic fibroblast (n = 113), and (iv) base medium supplemented with 30 ng/ml Activin A and co-cultured with fibroblast (n = 115). Rate of growth, survivability, antrum formation, ovulation, embryonic development and steroid production were evaluated. Analysis of Variance and Duncan test were applied for analyzing. Results: Both co-culture and co-culture + Activin A groups showed significant difference (P<0.05) in growth (on days 4, 6, and 8 of culture period) and survival rates. However, there was no significant difference in antrum formation, ovulation rate, and embryonic development of ovulated oocytes. There were significant differences (P<0.05) in the estradiol production on days 8, 10, and 12 between co-culture + Activin A and the control group. Progesterone production also was significant (P<0.05) in co-culture + Activin A group on days 6, 8, 10, and 12 compared to control group. Conclusion: Fibroblast co-culture and Activin A promoted growth and survivability of preantral follicles. However, simultaneous use of them was more efficient. PMID:24375163

  3. Role of human pulp fibroblasts in angiogenesis.

    PubMed

    Tran-Hung, L; Mathieu, S; About, I

    2006-09-01

    After pulp amputation, complete pulp healing requires not only reparative dentin production but also fibroblast proliferation, nerve fiber growth, and neoangiogenesis. This study was designed to investigate the role of pulp fibroblasts in angiogenesis. Human pulp fibroblasts from third molars co-cultured with human umbilical vein endothelial cells induced the organization of endothelial cells and the formation of tubular structures corresponding to capillaries in vivo. The direct contact between both cells was not necessary to induce angiogenesis, and the observed effect was due to soluble factors. This was confirmed with neutralizing antibodies against FGF-2 and VEGF, which decreased the angiogenic effects of these soluble factors. Immunohistochemistry showed that both FGF-2 and VEGF were expressed in human dental pulp fibroblasts, and this expression increased after injury. These results suggest that the pulp fibroblasts secrete angiogenic factors, which are necessary for complete pulp healing, particularly at the pulp injury site.

  4. Embryonic Mechanical and Soluble Cues Regulate Tendon Progenitor Cell Gene Expression as a Function of Developmental Stage and Anatomical Origin

    PubMed Central

    Brown, Jeffrey P; Finley, Violet G; Kuo, Catherine K

    2014-01-01

    Stem cell-based engineering strategies for tendons have yet to yield a normal functional tissue, due in part to a need for tenogenic factors. Additionally, the ability to evaluate differentiation has been challenged by a lack of markers for differentiation. We propose to inform tendon regeneration with developmental cues involved in normal tissue formation and with phenotypic markers that are characteristic of differentiating tendon progenitor cells (TPCs). Mechanical forces, fibroblast growth factor (FGF)-4 and transforming growth factor (TGF)-β2 are implicated in embryonic tendon development, yet the isolated effects of these factors on differentiating TPCs are unknown. Additionally, developmental mechanisms vary between limb and axial tendons, suggesting the respective cell types are programmed to respond uniquely to exogenous factors. To characterize developmental cues and benchmarks for differentiation toward limb vs. axial phenotypes, we dynamically loaded and treated TPCs with growth factors and assessed gene expression profiles as a function of developmental stage and anatomical origin. Based on scleraxis expression, TGFβ2 was tenogenic for TPCs at all stages, while loading was for late-stage cells only, and FGF4 had no effect despite regulation of other genes. When factors were combined, TGF 2 continued to be tenogenic, while FGF4 appeared anti-tenogenic. Various treatments elicited distinct responses by axial vs. limb TPCs of specific stages. These results identified tenogenic factors, suggest tendon engineering strategies should be customized for tissues by anatomical origin, and provide stage-specific gene expression profiles of limb and axial TPCs as benchmarks with which to monitor tenogenic differentiation of stem cells. PMID:24231248

  5. Assessment of differentiation aspects by the morphological classification of embryoid bodies derived from human embryonic stem cells.

    PubMed

    Kim, Jung Mo; Moon, Sung-Hwan; Lee, Sung Geum; Cho, Youn Jeong; Hong, Ki Sung; Lee, Jae Ho; Lee, Hey Jin; Chung, Hyung-Min

    2011-11-01

    In general, the formation of embryoid bodies (EBs) is a commonly known method for initial induction of human embryonic stem cells (hESCs) into their derivatives in vitro. Despite the ability of EBs to mimic developmental processing, the specification and classifications of EBs are not yet well known. Because EBs show various differentiation potentials depending on the size and morphology of the aggregated cells, specification is difficult to attain. Here, we sought to classify the differentiation potentials of EBs by morphologies to enable one to control the differentiation of specific lineages from hESCs with high efficiency. To induce the differentiation of EB formation, we established floating cultures of undifferentiated hESCs in Petri dishes with hESC medium lacking basic fibroblast growth factor. Cells first aggregated into balls; ∼10 days after suspension culture, some different types of EB morphology were present, which we classified as cystic-, bright cavity-, and dark cavity-type EBs. Next, we analyzed the characteristics of each type of EB for its capacity to differentiate into the 3 germ layers via multiplex polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry. Our results indicated that most cells within the cystic EBs were composed of endoderm lineage populations, and both of the cavity EB types were well organized with 3 germ-layer cells. However, the differentiation capacity of the bright cavity EBs was faster than that of the dark cavity EBs. Thus, the bright cavity EBs in this study, which showed equal differentiation tendencies compared with other types of EBs, may serve as the standard for in vitro engineering of EBs. These results indicate that the classification of EB morphologies allows the estimation of the differentiation status of the EBs and may allow the delineation of subsets of conditions necessary for EBs to differentiate into specific cell types.

  6. Application of a novel population of multipotent stem cells derived from skin fibroblasts as donor cells in bovine SCNT.

    PubMed

    Pan, Shaohui; Chen, Wuju; Liu, Xu; Xiao, Jiajia; Wang, Yanqin; Liu, Jun; Du, Yue; Wang, Yongsheng; Zhang, Yong

    2015-01-01

    Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4(+) cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4(+) cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4(+) cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4(+) cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4(-) cells. Moreover, blastocysts derived from SSEA-4(+) cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4(-) derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4(+) cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle.

  7. Application of a Novel Population of Multipotent Stem Cells Derived from Skin Fibroblasts as Donor Cells in Bovine SCNT

    PubMed Central

    Pan, Shaohui; Chen, Wuju; Liu, Xu; Xiao, Jiajia; Wang, Yanqin; Liu, Jun; Du, Yue; Wang, Yongsheng; Zhang, Yong

    2015-01-01

    Undifferentiated stem cells are better donor cells for somatic cell nuclear transfer (SCNT), resulting in more offspring than more differentiated cells. While various stem cell populations have been confirmed to exist in the skin, progress has been restricted due to the lack of a suitable marker for their prospective isolation. To address this fundamental issue, a marker is required that could unambiguously prove the differentiation state of the donor cells. We therefore utilized magnetic activated cell sorting (MACS) to separate a homogeneous population of small SSEA-4+ cells from a heterogeneous population of bovine embryonic skin fibroblasts (BEF). SSEA-4+ cells were 8-10 μm in diameter and positive for alkaline phosphatase (AP). The percentage of SSEA-4+ cells within the cultured BEF population was low (2-3%). Immunocytochemistry and PCR analyses revealed that SSEA-4+ cells expressed pluripotency-related markers, and could differentiate into cells comprising all three germ layers in vitro. They remained undifferentiated over 20 passages in suspension culture. In addition, cloned embryos derived from SSEA-4 cells showed significant differences in cleavage rate and blastocyst development when compared with those from BEF and SSEA-4− cells. Moreover, blastocysts derived from SSEA-4+ cells showed a higher total cell number and lower apoptotic index as compared to BEF and SSEA-4– derived cells. It is well known that nuclei from pluripotent stem cells yield a higher cloning efficiency than those from adult somatic cells, however, pluripotent stem cells are relatively difficult to obtain from bovine. The SSEA-4+ cells described in the current study provide an attractive candidate for SCNT and a promising platform for the generation of transgenic cattle. PMID:25602959

  8. Transactivation of Latent Marek's Disease Herpesvirus Genes in QT35, a Quail Fibroblast Cell Line, by Herpesvirus of Turkeys

    PubMed Central

    Yamaguchi, T.; Kaplan, S. L.; Wakenell, P.; Schat, K. A.

    2000-01-01

    The QT35 cell line was established from a methylcholanthrene-induced tumor in Japanese quail (Coturnix coturnix japonica) (C. Moscovici, M. G. Moscovici, H. Jimenez, M. M. Lai, M. J. Hayman, and P. K. Vogt, Cell 11:95–103, 1977). Two independently maintained sublines of QT35 were found to be positive for Marek's disease virus (MDV)-like genes by Southern blotting and PCR assays. Sequence analysis of fragments of the ICP4, ICP22, ICP27, VP16, meq, pp14, pp38, open reading frame (ORF) L1, and glycoprotein B (gB) genes showed a strong homology with the corresponding fragments of MDV genes. Subsequently, a serotype 1 MDV-like herpesvirus, tentatively name QMDV, was rescued from QT35 cells in chicken kidney cell (CKC) cultures established from 6- to 9-day-old chicks inoculated at 8 days of embryonation with QT35 cells. Transmission electron microscopy failed to show herpesvirus particles in QT35 cells, but typical intranuclear herpesvirus particles were detected in CKCs. Reverse transcription-PCR analysis showed that the following QMDV transcripts were present in QT35 cells: sense and antisense meq, ORF L1, ICP4, and latency-associated transcripts, which are antisense to ICP4. A transcript of approximately 4.5 kb was detected by Northern blotting using total RNA from QT35 cells. Inoculation of QT35 cells with herpesvirus of turkeys (HVT)-infected chicken embryo fibroblasts (CEF) but not with uninfected CEF resulted in the activation of ICP22, ICP27, VP16, pp38, and gB. In addition, the level of ICP4 mRNA was increased compared to that in QT35 cells. The activation by HVT resulted in the production of pp38 protein. It was not possible to detect if the other activated genes were translated due to the lack of serotype 1-specific monoclonal antibodies. PMID:11024146

  9. Emodin ameliorates bleomycin-induced pulmonary fibrosis in rats by suppressing epithelial-mesenchymal transition and fibroblast activation

    PubMed Central

    Guan, Ruijuan; Wang, Xia; Zhao, Xiaomei; Song, Nana; Zhu, Jimin; Wang, Jijiang; Wang, Jin; Xia, Chunmei; Chen, Yonghua; Zhu, Danian; Shen, Linlin

    2016-01-01

    Aberrant activation of TGF-β1 is frequently encountered and promotes epithelial-mesenchymal transition (EMT) and fibroblast activation in pulmonary fibrosis. The present study investigated whether emodin mediates its effect via suppressing TGF-β1-induced EMT and fibroblast activation in bleomycin (BLM)-induced pulmonary fibrosis in rats. Here, we found that emodin induced apoptosis and inhibited cellular proliferation, migration and differentiation in TGF-β1-stimulated human embryonic lung fibroblasts (HELFs). Emodin suppressed TGF-β1-induced EMT in a dose- and time-dependent manner in alveolar epithelial A549 cells. Emodin also inhibited TGF-β1-induced Smad2, Smad3 and Erk1/2 activation, suggesting that Smad2/3 and Erk1/2 inactivation mediated the emodin-induced effects on TGF-β1-induced EMT. Additionally, we provided in vivo evidence suggesting that emodin apparently alleviated BLM-induced pulmonary fibrosis and improved pulmonary function by inhibiting TGF-β1 signaling and subsequently repressing EMT, fibroblast activation and extracellular matrix (ECM) deposition. Taken together, our data suggest that emodin mediates its effects mainly via inhibition of EMT and fibroblast activation and thus has a potential for the treatment of pulmonary fibrosis. PMID:27774992

  10. Infrared inhibition of embryonic hearts

    NASA Astrophysics Data System (ADS)

    Wang, Yves T.; Rollins, Andrew M.; Jenkins, Michael W.

    2016-06-01

    Infrared control is a new technique that uses pulsed infrared lasers to thermally alter electrical activity. Originally developed for nerves, we have applied this technology to embryonic hearts using a quail model, previously demonstrating infrared stimulation and, here, infrared inhibition. Infrared inhibition enables repeatable and reversible block, stopping cardiac contractions for several seconds. Normal beating resumes after the laser is turned off. The block can be spatially specific, affecting propagation on the ventricle or initiation on the atrium. Optical mapping showed that the block affects action potentials and not just calcium or contraction. Increased resting intracellular calcium was observed after a 30-s exposure to the inhibition laser, which likely resulted in reduced mechanical function. Further optimization of the laser illumination should reduce potential damage. Stopping cardiac contractions by disrupting electrical activity with infrared inhibition has the potential to be a powerful tool for studying the developing heart.

  11. Mechanisms of embryonic stomach development.

    PubMed

    McCracken, Kyle W; Wells, James M

    2017-06-01

    The stomach is a digestive organ that has important roles in human physiology and pathophysiology. The developmental origin of the stomach is the embryonic foregut, which also gives rise a number of other structures. There are several signaling pathways and transcription factors that are known to regulate stomach development at different stages, including foregut patterning, stomach specification, and gastric regionalization. These developmental events have important implications in later homeostasis and disease in the adult stomach. Here we will review the literature that has shaped our current understanding of the molecular mechanisms that coordinate gastric organogenesis. Further we will discuss how developmental paradigms have guided recent efforts to differentiate stomach tissue from pluripotent stem cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. The Polymerase Activity of Mammalian DNA Pol ζ Is Specifically Required for Cell and Embryonic Viability

    PubMed Central

    Lange, Sabine S.; Tomida, Junya; Boulware, Karen S.; Bhetawal, Sarita; Wood, Richard D.

    2016-01-01

    DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/- Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/- Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents. PMID:26727495

  13. Altered Cellular Homeostasis in Murine MPS I Fibroblasts: Evidence of Cell-Specific Physiopathology.

    PubMed

    Viana, Gustavo Monteiro; do Nascimento, Cinthia Castro; Paredes-Gamero, Edgar Julian; D'Almeida, Vânia

    2017-02-21

    Mucopolysaccharidosis type I (MPS I), a rare autosomal recessive disease, is caused by a deficiency of the lysosomal enzyme alfa-L-iduronidase. Impaired enzyme activity promotes glycosaminoglycans accumulation in several tissues and organs, leading to complex multisystemic complications. Several studies using animal models indicated different intracellular pathways involving MPS I physiopathology; however, the exact mechanisms underlying this syndrome are still not understood. Previous results from our group showed alterations in ionic homeostasis and cell viability of splenocytes and macrophages in Idua-/- mice. In the present study, we found altered intracellular ionic homeostasis in a different cell type (fibroblasts) from the same murine model. Idua-/- fibroblasts from 3-month-old mice presented higher cytoplasmatic and endoplasmic reticulum Ca(2+) concentration, lower levels of mitochondrial Ca(2+) and mitochondrial membrane potential and higher cytoplasmatic pH when compared to Idua+/+ animals. Also, Idua-/- fibroblasts were more resistant to the apoptotic induction with staurosporine, indicating a possible resistance to apoptotic induction in those cells. In addition, despite the intracellular ionic imbalance, no significant alterations were found in apoptosis and autophagy in Idua-/- fibroblasts, which implies that the ionic alterations did not activate those pathways. The investigation of mechanisms underlying the cellular physiopathology of lysosomal diseases is crucial for a better understanding about the progression of these diseases. Since splenocytes, macrophages, and fibroblasts have different embryonic origins and distinct structural and functional features, potentially altered signaling pathways found in a cell-specific manner in an alfa-L-iduronidase-deficient environment provide additional understanding of the clinical multisystemic presentation of this disease and provide new basis for improved therapeutic approaches.

  14. Expression of fibulin-6 in failing hearts and its role for cardiac fibroblast migration.

    PubMed

    Chowdhury, Arpita; Herzog, Christine; Hasselbach, Lisa; Khouzani, Houra Loghmani; Zhang, Jinli; Hammerschmidt, Matthias; Rudat, Carsten; Kispert, Andreas; Gaestel, Matthias; Menon, Manoj B; Tudorache, Igor; Hilfiker-Kleiner, Denise; Mühlfeld, Christian; Schmitto, Jan Dieter; Müller, Martin; Theilmeier, Gregor

    2014-09-01

    The cardiac extracellular matrix (ECM) undergoes a dynamic transition following myocardial infarction. Fibulin-6 is expressed in cell junctions particularly in tissues subjected to significant mechanical stress. Fibulin-6 deficiency results in defective cell migration in nematodes and early embryonic lethality in mice. The role of fibulin-6 in healthy and failing myocardium is unknown. We have examined the expression and distribution pattern of fibulin-6 during myocardial remodelling (MR) and detailed its effect on the migratory function of cardiac fibroblasts (CFs) in response to TGF-β1. In healthy murine myocardium, fibulin-6 expression is largely confined to larger coronary arteries. It is induced during the early and the late phase of remodelling after infarction in murine hearts predominantly in the scar-muscle junction. Similar results are obtained in human ischaemic cardiomyopathy. Fibulin-6 is mostly expressed in close vicinity to vimentin-positive cells and is also abundantly expressed in vitro in cultured neonatal CF. TGF-β1 does not induce smooth muscle actin in fibroblasts deficient of fibulin-6, which also compromised their migration. Cells that had migrated expressed more fibulin-6 compared with stationary cells. Plated on fibulin-6-depleted matrix, stress fibre induction in fibroblast in response to TGF-β1 was impaired. In ex vivo explant cultures from post-infarct myocardium, the number of emigrating fibroblasts was also significantly reduced by fibulin-6 siRNA knockdown. Fibulin-6, a fibroblast-released ECM protein, may play an important role during MR by imparting an effect on CF migration in close and complementary interplay with TGF-β1 signalling. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

  15. Microporous Dermal-Mimetic Electrospun Scaffolds Pre-Seeded with Fibroblasts Promote Tissue Regeneration in Full-Thickness Skin Wounds

    PubMed Central

    Bonvallet, Paul P.; Schultz, Matthew J.; Mitchell, Elizabeth H.; Bain, Jennifer L.; Culpepper, Bonnie K.; Thomas, Steven J.; Bellis, Susan L.

    2015-01-01

    Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone) electrospun scaffold (70:30 col/PCL) containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM), and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344) rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14%) over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold). Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration. Collectively these

  16. Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse Embryonic Stem Cells

    PubMed Central

    Aiba, Kazuhiro; Nedorezov, Timur; Piao, Yulan; Nishiyama, Akira; Matoba, Ryo; Sharova, Lioudmila V.; Sharov, Alexei A.; Yamanaka, Shinya; Niwa, Hitoshi; Ko, Minoru S. H.

    2009-01-01

    Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three ‘cell lineage trajectories’, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. PMID:19112179

  17. Analysis of essential pathways for self-renewal in common marmoset embryonic stem cells.

    PubMed

    Nii, Takenobu; Marumoto, Tomotoshi; Kawano, Hirotaka; Yamaguchi, Saori; Liao, Jiyuan; Okada, Michiyo; Sasaki, Erika; Miura, Yoshie; Tani, Kenzaburo

    2014-01-01

    Common marmoset (CM) is widely recognized as a useful non-human primate for disease modeling and preclinical studies. Thus, embryonic stem cells (ESCs) derived from CM have potential as an appropriate cell source to test human regenerative medicine using human ESCs. CM ESCs have been established by us and other groups, and can be cultured in vitro. However, the growth factors and downstream pathways for self-renewal of CM ESCs are largely unknown. In this study, we found that basic fibroblast growth factor (bFGF) rather than leukemia inhibitory factor (LIF) promoted CM ESC self-renewal via the activation of phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT) pathway on mouse embryonic fibroblast (MEF) feeders. Moreover, bFGF and transforming growth factor β (TGFβ) signaling pathways cooperatively maintained the undifferentiated state of CM ESCs under feeder-free condition. Our findings may improve the culture techniques of CM ESCs and facilitate their use as a preclinical experimental resource for human regenerative medicine.

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

    PubMed Central

    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

  19. Establishment of an exogenous LIF-free culture system for mouse embryonic stem cells.

    PubMed

    Feng, Shumei; Mo, Lijuan; Wu, Rongrong; Chen, Xiaopan; Zhang, Ming

    2009-09-01

    Mouse embryonic stem cells (mESCs) have played a key role in the newly emerging fields of stem cell research. The traditional derivation and culture of mESCs have been based on the use of mouse embryonic fibroblasts (MEFs) treated with exogenous leukemia inhibitory factor (LIF). However, the rapid senescence of MEFs, coupled with the high cost of LIF, has significantly hampered the widespread use of mESCs in stem cell research. Thus, we present a novel exogenous LIF-free culture system for general mESCs applications, comprising fibroblast-like cells derived from the rabbit spleen (RSFs). We demonstrated that mESCs cultured on RSFs (mESCs-RSFs) maintained all mESC features after prolonged LIF-free culture, including alkaline phosphatase, cell surface markers (SSEA-1), molecular markers (OCT-4, NANOG, TERT, REX-1), karyotype, and pluripotency. The high expression level of both LIF and WNT3A in the RSFs may account for their ability to maintain mESCs without exogenous LIF. Moreover, this exogenous LIF-free culture system was verified to be of microbiological quality through analysis with electron transmission microscopy.

  20. Characterization and rescue of telomeric abnormalities in ICF syndrome type I fibroblasts

    PubMed Central

    Yehezkel, Shiran; Shaked, Rony; Sagie, Shira; Berkovitz, Ron; Shachar-Bener, Hofit; Segev, Yardena; Selig, Sara

    2013-01-01

    Mutations in the human DNA methyltransferase 3B (DNMT3B) gene lead to ICF (immunodeficiency, centromeric region instability, and facial anomalies) syndrome type I. We have previously described a telomere-related phenotype in cells from these patients, involving severe hypomethylation of subtelomeric regions, abnormally short telomeres and high levels of telomeric-repeat-containing RNA (TERRA). Here we demonstrate that ICF-patient fibroblasts carry abnormally short telomeres at a low population doubling (PD) and enter senescence prematurely. Accordingly, we attempted to rescue the senescence phenotype by ectopic expression of human telomerase, which led to elongated telomeres with hypomethylated subtelomeres. The senescence phenotype was overcome under these conditions, thus dissociating subtelomeric-DNA hypomethylation per se from the senescence phenotype. In addition, we examined whether the subtelomeric methylation could be restored by expression of a normal copy of full length DNMT3B1 in ICF fibroblasts. Ectopic expression of DNMT3B1 failed to rescue the abnormal hypomethylation at subtelomeres. However, partial rescue of subtelomeric-hypomethylation was achieved by co-expression of DNMT3B1 together with DNA methyltransferase 3-like (DNMT3L), encoding a protein that functions as a stimulator of DNMT3A and DNMT3B. DNMT3B1 and DNMT3L are predominantly expressed during early embryonic development, suggesting that de novo subtelomeric DNA methylation during crucial stages of human embryonic development may be necessary for setting and maintaining normal telomere length. PMID:23450006

  1. SOX17 Regulates Conversion of Human Fibroblasts Into Endothelial Cells and Erythroblasts by Dedifferentiation Into CD34(+) Progenitor Cells.

    PubMed

    Zhang, Lianghui; Jambusaria, Ankit; Hong, Zhigang; Marsboom, Glenn; Toth, Peter T; Herbert, Brittney-Shea; Malik, Asrar B; Rehman, Jalees

    2017-06-20

    The mechanisms underlying the dedifferentiation and lineage conversion of adult human fibroblasts into functional endothelial cells have not yet been fully defined. Furthermore, it is not known whether fibroblast dedifferentiation recapitulates the generation of multipotent progenitors during embryonic development, which give rise to endothelial and hematopoietic cell lineages. Here we established the role of the developmental transcription factor SOX17 in regulating the bilineage conversion of fibroblasts by the generation of intermediate progenitors. CD34(+) progenitors were generated after the dedifferentiation of human adult dermal fibroblasts by overexpression of pluripotency transcription factors. Sorted CD34(+) cells were transdifferentiated into induced endothelial cells and induced erythroblasts using lineage-specific growth factors. The therapeutic potential of the generated cells was assessed in an experimental model of myocardial infarction. Induced endothelial cells expressed specific endothelial cell surface markers and also exhibited the capacity for cell proliferation and neovascularization. Induced erythroblasts expressed erythroid surface markers and formed erythroid colonies. Endothelial lineage conversion was dependent on the upregulation of the developmental transcription factor SOX17, whereas suppression of SOX17 instead directed the cells toward an erythroid fate. Implantation of these human bipotential CD34(+) progenitors into nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice resulted in the formation of microvessels derived from human fibroblasts perfused with mouse and human erythrocytes. Endothelial cells generated from human fibroblasts also showed upregulation of telomerase. Cell implantation markedly improved vascularity and cardiac function after myocardial infarction without any evidence of teratoma formation. Dedifferentiation of fibroblasts to intermediate CD34(+) progenitors gives rise to endothelial cells and

  2. Embryonic stem cell potency fluctuates with endogenous retrovirus activity.

    PubMed

    Macfarlan, Todd S; Gifford, Wesley D; Driscoll, Shawn; Lettieri, Karen; Rowe, Helen M; Bonanomi, Dario; Firth, Amy; Singer, Oded; Trono, Didier; Pfaff, Samuel L

    2012-07-05

    Embryonic stem (ES) cells are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass, which lacks the ability to produce all extraembryonic tissues. Here we identify a rare transient cell population within mouse ES and induced pluripotent stem (iPS) cell cultures that expresses high levels of transcripts found in two-cell (2C) embryos in which the blastomeres are totipotent. We genetically tagged these 2C-like ES cells and show that they lack the inner cell mass pluripotency proteins Oct4 (also known as Pou5f1), Sox2 and Nanog, and have acquired the ability to contribute to both embryonic and extraembryonic tissues. We show that nearly all ES cells cycle in and out of this privileged state, which is partially controlled by histone-modifying enzymes. Transcriptome sequencing and bioinformatic analyses showed that many 2C transcripts are initiated from long terminal repeats derived from endogenous retroviruses, suggesting this foreign sequence has helped to drive cell-fate regulation in placental mammals.

  3. Transcriptional control of cardiac fibroblast plasticity.

    PubMed

    Lighthouse, Janet K; Small, Eric M

    2016-02-01

    Cardiac fibroblasts help maintain the normal architecture of the healthy heart and are responsible for scar formation and the healing response to pathological insults. Various genetic, biomechanical, or humoral factors stimulate fibroblasts to become contractile smooth muscle-like cells called myofibroblasts that secrete large amounts of extracellular matrix. Unfortunately, unchecked myofibroblast activation in heart disease leads to pathological fibrosis, which is a major risk factor for the development of cardiac arrhythmias and heart failure. A better understanding of the molecular mechanisms that control fibroblast plasticity and myofibroblast activation is essential to develop novel strategies to specifically target pathological cardiac fibrosis without disrupting the adaptive healing response. This review highlights the major transcriptional mediators of fibroblast origin and function in development and disease. The contribution of the fetal epicardial gene program will be discussed in the context of fibroblast origin in development and following injury, primarily focusing on Tcf21 and C/EBP. We will also highlight the major transcriptional regulatory axes that control fibroblast plasticity in the adult heart, including transforming growth factor β (TGFβ)/Smad signaling, the Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF) axis, and Calcineurin/transient receptor potential channel (TRP)/nuclear factor of activated T-Cell (NFAT) signaling. Finally, we will discuss recent strategies to divert the fibroblast transcriptional program in an effort to promote cardiomyocyte regeneration. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling".

  4. Fibroblast migration in fibrin gel matrices.

    PubMed Central

    Brown, L. F.; Lanir, N.; McDonagh, J.; Tognazzi, K.; Dvorak, A. M.; Dvorak, H. F.

    1993-01-01

    In healing wounds and many solid tumors, locally increased microvascular permeability results in extravasation of fibrinogen and its extravascular coagulation to form a fibrin gel, with concomitant covalent cross-linking of fibrin by factor XIIIa. Subsequently, inflammatory cells, fibroblasts, and endothelial cells migrate into the gel and organize it into granulation tissue and later into mature collagenous connective tissue. To gain insight into some of the cell migration events associated with these processes, we developed a quantitative in vitro assay that permits the study of fibroblast migration in fibrin gels. Early passage human or rat fibroblasts were allowed to attach to tissue culture dishes and then were overlaid with a thin layer of fibrinogen that was clotted with thrombin. Fibroblasts began to migrate upwards into the fibrin within 24 hours and their numbers and the distance migrated were quantified over several days. The extent of fibroblast migration was affected importantly by the nature of the fibrin clot. Fibroblasts migrated optimally into gels prepared from fibrinogen at concentrations of -3 mg/ml; ie, near normal plasma fibrinogen levels. Migration was greatly enhanced by extensive cross-linking of the fibrin alpha-chains by factor XIIIa, as occurs when clotting takes place in vivo. When fibrinogen was clotted in Dulbecco's modified Eagle's medium, gamma-chains were cross-linked, but alpha-chain cross-linking was strikingly inhibited, and fibroblasts migrated poorly. Gels prepared from factor XIII-depleted fibrinogen exhibited neither alpha-nor gamma-chain cross-linking and did not support fibroblast migration. Further purification of fibrinogen by anion exchange high pressure liquid chromatography depleted fibrinogen of fibronectin, plasminogen, and other impurities; this purified fibrinogen clotted to form fibrin gels that supported reproducible fibroblast migration. Images Figure 1 Figure 2 Figure 4 Figure 6 PMID:8424460

  5. 28. Embryonic and adult stem cell therapy.

    PubMed

    Henningson, Carl T; Stanislaus, Marisha A; Gewirtz, Alan M

    2003-02-01

    Stem cells are characterized by the ability to remain undifferentiated and to self-renew. Embryonic stem cells derived from blastocysts are pluripotent (able to differentiate into many cell types). Adult stem cells, which were traditionally thought to be monopotent multipotent, or tissue restricted, have recently also been shown to have pluripotent properties. Adult bone marrow stem cells have been shown to be capable of differentiating into skeletal muscle, brain microglia and astroglia, and hepatocytes. Stem cell lines derived from both embryonic stem and embryonic germ cells (from the embryonic gonadal ridge) are pluripotent and capable of self-renewal for long periods. Therefore embryonic stem and germ cells have been widely investigated for their potential to cure diseases by repairing or replacing damaged cells and tissues. Studies in animal models have shown that transplantation of fetal, embryonic stem, or embryonic germ cells may be able to treat some chronic diseases. In this review, we highlight recent developments in the use of stem cells as therapeutic agents for three such diseases: Diabetes, Parkinson disease, and congestive heart failure. We also discuss the potential use of stem cells as gene therapy delivery cells and the scientific and ethical issues that arise with the use of human stem cells.

  6. Differentiation of nonhuman primate embryonic stem cells along neural lineages

    PubMed Central

    Kuai, Xiao Ling; Gagliardi, Christine; Flaat, Mette; Bunnell, Bruce A.

    2009-01-01

    The differentiation of embryonic stem cells (ESCs) into neurons and glial cells represents a promising cell-based therapy for neurodegenerative diseases. Because the rhesus macaque is physiologically and phylogenetically similar to humans, it is a clinically relevant animal model for ESC research. In this study, the pluripotency and neural differentiation potential of a rhesus monkey ESC line (ORMES6) was investigated. ORMES6 was derived from an in vitro produced blastocyst, which is the same way human ESCs have been derived. ORMES6 stably expressed the embryonic transcription factors POU5F1 (Oct4), Sox2 and NANOG. Stage-specific embryonic antigen 4 (SSEA 4) and the glycoproteins TRA-1-60 and TRA-1-81 were also expressed. The embryoid bodies (EBs) formed from ORMES6 ESCs spontaneously gave rise to cells of three germ layers. After exposure to basic fibroblast growth factor (bFGF) for 14–16 days, columnar rosette cells formed in the EB outgrowths. Sox2, microtubule-associated protein (MAP2), β-tublinIII and glial fibrillary acidic protein (GFAP) genes and Nestin, FoxD3, Pax6 and β-tublinIII antigens were expressed in the rosette cells. Oct4 and NANOG expression were remarkably down-regulated in these cells. After removal of bFGF from the medium, the rosette cells differentiated along neural lineages. The differentiated cells expressed MAP2, β-tublinIII, Neuro D and GFAP genes. Most differentiated cells expressed early neuron-specific antigen β-tublinIII (73±4.7%) and some expressed intermediate neuron antigen MAP2 (18±7.2%). However, some differentiated cells expressed the glial cell antigens A2B5 (7.17%±1.2%), GFAP (4.93±1.9%), S100 (7±3.5%) and O4 (0.2±70.2%). The rosette cells were transplanted into the striatum of immune-deficient NIHIII mice. The cells persisted for approximately 2 weeks and expressed Ki67, NeuN, MAP2 and GFAP. These results demonstrate that the rhesus monkey ESC line ORMES6 retains the pluripotent characteristics of ESCs and can be

  7. Wound healing and the role of fibroblasts.

    PubMed

    Bainbridge, P

    2013-08-01

    Fibroblasts are critical in supporting normal wound healing, involved in key processes such as breaking down the fibrin clot, creating new extra cellular matrix (ECM) and collagen structures to support the other cells associated with effective wound healing, as well as contracting the wound. This article explores and summarises the research evidence on the role of fibroblasts, their origins and activation, and how they navigate the wound bed, as well as how their activity leads to wound contraction. This article also explores the local conditions at the wound site, which activate, regulate and ultimately reduce the fibroblast activity as the skin's integrity returns on healing.

  8. Fibroblast heterogeneity in the cancer wound

    PubMed Central

    Öhlund, Daniel; Elyada, Ela

    2014-01-01

    Fibroblasts regulate the structure and function of healthy tissues, participate transiently in tissue repair after acute inflammation, and assume an aberrant stimulatory role during chronic inflammatory states including cancer. Such cancer-associated fibroblasts (CAFs) modulate the tumor microenvironment and influence the behavior of neoplastic cells in either a tumor-promoting or tumor-inhibiting manner. These pleiotropic functions highlight the inherent plasticity of fibroblasts and may provide new avenues to understand and therapeutically intervene in malignancies. We discuss the emerging themes of CAF biology in the context of tumorigenesis and therapy. PMID:25071162

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

    SciTech Connect

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

    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. Functional melanocytes are readily reprogrammable from multilineage-differentiating stress-enduring (muse) cells, distinct stem cells in human fibroblasts.

    PubMed

    Tsuchiyama, Kenichiro; Wakao, Shohei; Kuroda, Yasumasa; Ogura, Fumitaka; Nojima, Makoto; Sawaya, Natsue; Yamasaki, Kenshi; Aiba, Setsuya; Dezawa, Mari

    2013-10-01

    The induction of melanocytes from easily accessible stem cells has attracted attention for the treatment of melanocyte dysfunctions. We found that multilineage-differentiating stress-enduring (Muse) cells, a distinct stem cell type among human dermal fibroblasts, can be readily reprogrammed into functional melanocytes, whereas the remainder of the fibroblasts do not contribute to melanocyte differentiation. Muse cells can be isolated as cells positive for stage-specific embryonic antigen-3, a marker for undifferentiated human embryonic stem cells, and differentiate into cells representative of all three germ layers from a single cell, while also being nontumorigenic. The use of certain combinations of factors induces Muse cells to express melanocyte markers such as tyrosinase and microphthalmia-associated transcription factor and to show positivity for the 3,4-dihydroxy-L-phenylalanine reaction. When Muse cell-derived melanocytes were incorporated into three-dimensional (3D) cultured skin models, they localized themselves in the basal layer of the epidermis and produced melanin in the same manner as authentic melanocytes. They also maintained their melanin production even after the 3D cultured skin was transplanted to immunodeficient mice. This technique may be applicable to the efficient production of melanocytes from accessible human fibroblasts by using Muse cells, thereby contributing to autologous transplantation for melanocyte dysfunctions, such as vitiligo.

  11. Early Neurobehavioral Development of Mice Lacking Endogenous PACAP.

    PubMed

    Farkas, Jozsef; Sandor, Balazs; Tamas, Andrea; Kiss, Peter; Hashimoto, Hitoshi; Nagy, Andras D; Fulop, Balazs D; Juhasz, Tamas; Manavalan, Sridharan; Reglodi, Dora

    2017-04-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide. In addition to its diverse physiological roles, PACAP has important functions in the embryonic development of various tissues, and it is also considered as a trophic factor during development and in the case of neuronal injuries. Data suggest that the development of the nervous system is severely affected by the lack of endogenous PACAP. Short-term neurofunctional outcome correlates with long-term functional deficits; however, the early neurobehavioral development of PACAP-deficient mice has not yet been evaluated. Therefore, the aim of the present study was to describe the postnatal development of physical signs and neurological reflexes in mice partially or completely lacking PACAP. We examined developmental hallmarks during the first 3 weeks of the postnatal period, during which period most neurological reflexes and motor coordination show most intensive development, and we describe the neurobehavioral development using a complex battery of tests. In the present study, we found that PACAP-deficient mice had slower weight gain throughout the observation period. Interestingly, mice partially lacking PACAP weighed significantly less than homozygous mice. There was no difference between male and female mice during the first 3 weeks. Some other signs were also more severely affected in the heterozygous mice than in the homozygous mice, such as air righting, grasp, and gait initiation reflexes. Interestingly, incisor teeth erupted earlier in mice lacking PACAP. Motor coordination, shown by the number of foot-faults on an elevated grid, was also less developed in PACAP-deficient mice. In summary, our results show that mice lacking endogenous PACAP have slower weight gain during the first weeks of development and slower neurobehavioral development regarding a few developmental hallmarks.

  12. Super-telomeres in transformed human fibroblasts.

    PubMed

    Chiodi, Ilaria; Belgiovine, Cristina; Zongaro, Samantha; Ricotti, Roberta; Horard, Beatrice; Lossani, Andrea; Focher, Federico; Gilson, Eric; Giulotto, Elena; Mondello, Chiara

    2013-08-01

    Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to the deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 rose with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis.

  13. The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy.

    PubMed

    Mohamed, Tamer M A; Abou-Leisa, Riham; Stafford, Nicholas; Maqsood, Arfa; Zi, Min; Prehar, Sukhpal; Baudoin-Stanley, Florence; Wang, Xin; Neyses, Ludwig; Cartwright, Elizabeth J; Oceandy, Delvac

    2016-03-29

    The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition.

  14. Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts.

    PubMed

    Small, M B; Hubbard, K; Pardinas, J R; Marcus, A M; Dhanaraj, S N; Sethi, K A

    1996-09-01

    Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization.

  15. The plasma membrane calcium ATPase 4 signalling in cardiac fibroblasts mediates cardiomyocyte hypertrophy

    PubMed Central

    Mohamed, Tamer M. A.; Abou-Leisa, Riham; Stafford, Nicholas; Maqsood, Arfa; Zi, Min; Prehar, Sukhpal; Baudoin-Stanley, Florence; Wang, Xin; Neyses, Ludwig; Cartwright, Elizabeth J.; Oceandy, Delvac

    2016-01-01

    The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition. PMID:27020607

  16. Differences in ( sup 14 C)glycerol utilization in normal and familial hypercholesterolemic fibroblasts

    SciTech Connect

    Shireman, R.B.; Durieux, J. )

    1991-01-01

    It is known that cultured fibroblasts from familial hypercholesterolemia (FH) patients lack the normal cell receptor for low density lipoprotein (LDL) and that the absence of receptor-mediated transport of LDL cholesterol into these cells results in increased cellular synthesis of cholesterol. After 20 h perincubation in lipid-free medium, cultured FH fibroblasts incorporated significantly greater amounts of ({sup 14}C)glycerol into cellular lipids than did normal fibroblasts. Relative to the control medium which contained only bovine serum albumin (BSA), preincubation with 5% fetal bovine serum or 50 micrograms LDL/ml decreased ({sup 14}C)glycerol incorporation by both cell types. FH cells utilized more ({sup 14}C)glycerol for phospholipid synthesis and less for triglyceride synthesis than normal cells. This study indicates that LDL may be important in the transport of glycerides, as well as cholesterol, to cells.

  17. Role of microglia in embryonic neurogenesis

    PubMed Central

    Tong, Chih Kong

    2016-01-01

    Microglia begin colonizing the developing brain as early as embryonic day 9, prior to the emergence of neurons and other glia. Their ontogeny is also distinct from other central nervous system cells, as they derive from yolk sac hematopoietic progenitors and not neural progenitors. In this review, we feature these unique characteristics of microglia and assess the spatiotemporal similarities between microglia colonization of the central nervous system and embryonic neurogenesis. We also infer to existing evidence for microglia function from embryonic through to postnatal neurodevelopment to postulate roles for microglia in neurogenesis. PMID:27555616

  18. Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay

    PubMed Central

    2013-01-01

    Background Carbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents. Several studies have identified the capacity of CNT to stimulate the proliferation of fibroblasts. We developed and validated experimentally here a simple and rapid in vitro assay to evaluate the capacity of a nanomaterial to exert a direct pro-fibrotic effect on fibroblasts. Methods The activity of several multi-wall (MW)CNT samples (NM400, the crushed form of NM400 named NM400c, NM402 and MWCNTg 2400) and asbestos (crocidolite) was investigated in vitro and in vivo. The proliferative response to MWCNT was assessed on mouse primary lung fibroblasts, human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB-3T3) and mouse lung fibroblasts (MLg) by using different assays (cell counting, WST-1 assay and propidium iodide PI staining) and dispersion media (fetal bovine serum, FBS and bovine serum albumin, BSA). C57BL/6 mice were pharyngeally aspirated with the same materials and lung fibrosis was assessed after 2 months by histopathology, quantification of total collagen lung content and pro-fibrotic cytokines in broncho-alveolar lavage fluid (BALF). Results MWCNT (NM400 and NM402) directly stimulated fibroblast proliferation in vitro in a dose-dependent manner and induced lung fibrosis in vivo. NM400 stimulated the proliferation of all tested fibroblast types, independently of FBS- or BSA- dispersion. Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays. Crocidolite also stimulated fibroblast proliferation and induced pulmonary fibrosis, although to a lesser extent than NM400 and NM402. In contrast, shorter CNT (NM400c and MWCNTg 2400) did not induce any fibroblast proliferation or collagen accumulation in vivo, supporting the idea that CNT structure is an important parameter for inducing lung fibrosis. Conclusions In this study, an optimized proliferation assay using BSA as a dispersant, MLg cells as targets

  19. Selective MicroRNA-Offset RNA Expression in Human Embryonic Stem Cells

    PubMed Central

    Juhila, Juuso; Holm, Frida; Weltner, Jere; Trokovic, Ras; Mikkola, Milla; Toivonen, Sanna; Balboa, Diego; Lampela, Riina; Icay, Katherine; Tuuri, Timo; Otonkoski, Timo; Wong, Garry; Hovatta, Outi

    2015-01-01

    Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs) and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs. PMID:25822230

  20. Depressed adrenomedullin in the embryonic transforming growth factor-beta1 null mouse becomes elevated postnatally.

    PubMed

    Bodegas, Elena; Martínez, Alfredo; Ozbun, Laurent L; Garayoa, Mercedes; Letterio, John J; Montuenga, Luis M; Jakowlew, Sonia B

    2004-02-01

    Transforming growth factor-beta (TGF-beta) and adrenomedullin are multifunctional regulatory proteins which are expressed in developing embryonic and adult tissues. Because of their colocalization, TGF-beta1 and adrenomedullin may be able to coordinately act to influence development and differentiation. In order to learn more about the biology of adrenomedullin in the absence of the effects of TGF-beta1 in vivo, we examined adrenomedullin in the TGF-beta1 null mouse. A generally lower amount of adrenomedullin was detected by immunohistochemical staining analysis in multiple tissues from embryonic TGF-beta1 null mice compared to wildtype animals, including the heart, lung, brain, liver, and kidney, among others. In contrast, immunohistochemical staining for adrenomedullin was more intense in tissues of the postnatal TGF-beta1 null mouse compared to the wildtype mouse. These observations were confirmed by quantitative real time RT-PCR for adrenomedullin in both embryos and postnatal animals, as well as in cultured mouse embryo fibroblasts from TGF-beta1 null and wildtype mice. In addition, when cultured mouse embryo fibroblasts were treated with a neutralizing monoclonal antibody against TGF-beta1, the levels of adrenomedullin expression were statistically reduced compared to untreated cells. Our data show that expression of adrenomedullin is reduced in tissues of the developing embryonic TGF-beta1 null mouse compared to the wildtype mouse, but increases during postnatal development in TGF-beta1 null mice. The elevated expression of adrenomedullin which occurs postnatally in the TGF-beta1 null mouse may be a cause or a consequence of the multifocal wasting syndrome which is characteristic of postnatal TGF-beta1 null mice.

  1. Aggregating embryonic but not somatic nuclear transfer embryos increases cloning efficiency in cattle.

    PubMed

    Misica-Turner, Pavla M; Oback, Fleur C; Eichenlaub, Michael; Wells, David N; Oback, Björn

    2007-02-01

    Our objectives were to compare the cellular and molecular effects of aggregating bovine embryonic vs. somatic cell nuclear transfer (ECNT vs. SCNT) embryos and to determine whether aggregation can improve cattle cloning efficiency. We reconstructed cloned embryos from: 1) morula-derived blastomeres, 2) six adult male ear skin fibroblast lines, 3) one fetal female lung fibroblast line (BFF), and 4) two transgenic clonal strains derived from BFF. Embryos were cultured either singularly (1X) or as aggregates of three (3X). In vitro-fertilized (IVF) 1X and 3X embryos served as controls. After aggregation, the in vitro development of ECNT but not that of SCNT or IVF embryos was strongly compromised. The inner cell mass (ICM), total cell (TC) numbers, and ICM:TC ratios significantly increased for all the aggregates. The relative concentration of the key embryonic transcript POU5F1 (or OCT4) did not correlate with these increases, remaining unchanged in the ECNT and IVF aggregates and decreasing significantly in the SCNT aggregates. Overall, the IVF and 3X ECNT but not the 1X ECNT embryos had significantly higher relative POU5F1 levels than the SCNT embryos. High POU5F1 levels correlated with high in vivo survival, while no such correlation was noted for the ICM:TC ratios. Development to weaning was more than doubled in the ECNT aggregates (10/51 or 20% vs. 7/85 or 8% for 3X vs. 1X, respectively; P < 0.05). In contrast, the SCNT and IVF controls showed no improvement in survival. These data reveal striking biological differences between embryonic and somatic clones in response to aggregation.

  2. Hyperforin inhibits cell proliferation and differentiation in mouse embryonic stem cells.

    PubMed

    Nakamura, K; Aizawa, K; Yamauchi, J; Tanoue, A

    2013-10-01

    Hyperforin, a phloroglucinol derivative of St. John's Wort, has been identified as the major molecule responsible for this plant's products anti-depressant effects. It can be expected that exposure to St. John's Wort during pregnancy occurs with some frequency although embryotoxic or teratogenic effects of St. John's Wort and hyperforin have not yet been experimentally examined in detail. In this study, to determine any embryotoxic effects of hyperforin, we have attempted to determine whether hyperforin affects growth and survival processes of employing mouse embryonic stem (mES) cells (representing embryonic tissue) and fibroblasts (representing adult tissues). We used a modified embryonic stem cell test, which has been validated as an in vitro developmental toxicity protocol, mES cells, to assess embryotoxic potential of chemicals under investigation. We have identified that high concentrations of hyperforin inhibited mouse ES cell population growth and induced apoptosis in fibroblasts. Under our cell culture conditions, ES cells mainly differentiated into cardiomyocytes, although various other cell types were also produced. In this condition, hyperforin affected ES cell differentiation into cardiomyocytes in a dose-dependent manner. Analysis of tissue-specific marker expression also revealed that hyperforin at high concentrations partially inhibited ES cell differentiation into mesodermal and endodermal lineages. Hyperforin is currently used in the clinic as a safe and effective antidepressant. Our data indicate that at typical dosages it has only a low risk of embryotoxicity; ingestion of large amounts of hyperforin by pregnant women, however, may pose embryotoxic and teratogenic risks. © 2013 John Wiley & Sons Ltd.

  3. Assembly of embryonic and extra-embryonic stem cells to mimic embryogenesis in vitro.

    PubMed

    Harrison, Sarah Ellys; Sozen, Berna; Christodoulou, Neophytos; Kyprianou, Christos; Zernicka-Goetz, Magdalena

    2017-03-02

    Mammalian embryogenesis requires intricate interactions between embryonic and extra-embryonic tissues to orchestrate and coordinate morphogenesis with changes in developmental potential. Here, we combine mouse embryonic stem cells (ESCs) and extra-embryonic trophoblast stem cells (TSCs) in a 3D-scaffold to generate structures whose morphogenesis is remarkably similar to natural embryos. By using genetically-modified stem cells and specific inhibitors, we show embryogenesis of ESC- and TSC-derived embryos, ETS-embryos, depends on crosstalk involving Nodal signaling. When ETS-embryos develop, they spontaneously initiate expression of mesoderm and primordial germ cell markers asymmetrically on the embryonic and extra-embryonic border, in response to Wnt and BMP signaling. Our study demonstrates the ability of distinct stem cell types to self-assemble in vitro to generate embryos whose morphogenesis, architecture, and constituent cell-types resemble natural embryos.

  4. Isolation (from a basal cell carcinoma) of a functionally distinct fibroblast-like cell type that overexpresses Ptch.

    PubMed

    Dicker, Anthony J; Serewko, Magdalena M; Russell, Terry; Rothnagel, Joseph A; Strutton, Geoff M; Dahler, Alison L; Saunders, Nicholas A

    2002-05-01

    In this study we report on the isolation and characterization of a nonepithelial, nontumorigenic cell type (BCC1) derived from a basal cell carcinoma from a patient. The BCC1 cells share many characteristics with dermal fibroblasts, such as the expression of vimentin, lack of expression of cytokeratins, and insensitivity to agents that cause growth inhibition and differentiation of epithelial cells; however, significant differences between BCC1 cells and fibroblasts also exist. For example, BCC1 cells are stimulated to undergo DNA synthesis in response to interferon-gamma, whereas dermal fibroblasts are not. More over, BCC1 cells overexpress the basal cell carcinoma-specific genes ptch and ptch2. These data indicate that basal cell carcinomas are associated with a functionally distinct population of fibroblast-like cells that overexpress known tumor-specific markers (ptch and ptch2).

  5. Retinoic acid induces TGFbeta-dependent autocrine fibroblast growth.

    PubMed

    Fadloun, A; Kobi, D; Delacroix, L; Dembélé, D; Michel, I; Lardenois, A; Tisserand, J; Losson, R; Mengus, G; Davidson, I

    2008-01-17

    To evaluate the role of murine TFIID subunit TAF4 in activation of cellular genes by all-trans retinoic acid (T-RA), we have characterized the T-RA response of taf4(lox/-) and taf4(-/-) embryonic fibroblasts. T-RA regulates almost 1000 genes in taf4(lox/-) cells, but less than 300 in taf4(-/-) cells showing that TAF4 is required for T-RA regulation of most, but not all cellular genes. We further show that T-RA-treated taf4(lox/-) cells exhibit transforming growth factor (TGF)beta-dependent autocrine growth and identify a set of genes regulated by loss of TAF4 and by T-RA corresponding to key mediators of the TGFbeta signalling pathway. T-RA rapidly and potently induces expression of connective tissue growth factor (CTGF) via a conserved DR2 type response element in its proximal promoter leading to serum-free autocrine growth. These results highlight the role of TAF4 as a cofactor in the cellular response to T-RA and identify the genetic programme of a novel cross talk between the T-RA and TGFbeta pathways that leads to deregulated cell growth.

  6. Interactions between fibroblast growth factors and Notch regulate neuronal differentiation.

    PubMed

    Faux, C H; Turnley, A M; Epa, R; Cappai, R; Bartlett, P F

    2001-08-01

    The differentiation of precursor cells into neurons has been shown to be influenced by both the Notch signaling pathway and growth factor stimulation. In this study, the regulation of neuronal differentiation by these mechanisms was examined in the embryonic day 10 neuroepithelial precursor (NEP) population. By downregulating Notch1 expression and by the addition of a Delta1 fusion protein (Delta Fc), it was shown that signaling via the Notch pathway inhibited neuron differentiation in the NEP cells, in vitro. The expression of two of the Notch receptor homologs, Notch1 and Notch3, and the ligand Delta1 in these NEP cells was found to be influenced by a number of different growth factors, indicating a potential interaction between growth factors and Notch signaling. Interestingly, none of the growth factors examined promoted neuron differentiation; however, the fibroblast growth factors (FGFs) 1 and 2 potently inhibited differentiation. FGF1 and FGF2 upregulated the expression of Notch and decreased expression of Delta1 in the NEP cells. In addition, the inhibitory response of the cells to the FGFs could be overcome by downregulating Notch1 expression and by disrupting Notch cleavage and signaling by the ablation of the Presenilin1 gene. These results indicate that FGF1 and FGF2 act via the Notch pathway, either directly or indirectly, to inhibit differentiation. Thus, signaling through the Notch receptor may be a common regulator of neuronal differentiation within the developing forebrain.

  7. Fibroblast growth factors, old kids on the new block.

    PubMed

    Li, Xiaokun; Wang, Cong; Xiao, Jian; McKeehan, Wallace L; Wang, Fen

    2016-05-01

    The fibroblast growth factors (FGFs) are a family of cell intrinsic regulatory peptides that control a broad spectrum of cellular activities. The family includes canonic FGFs that elicit their activities by activating the FGF receptor (FGFR) tyrosine kinase and non-canonic members that elicit their activities intracellularly and via FGFR-independent mechanisms. The FGF signaling axis is highly complex due to the existence of multiple isoforms of both ligands and receptors, as well as cofactors that include the chemically heterogeneous heparan sulfate (HS) cofactors, and in the case of endocrine FGFs, the Klotho coreceptors. Resident FGF signaling controls embryonic development, maintains tissue homeostasis, promotes wound healing and tissue regeneration, and regulates functions of multiple organs. However, ectopic or aberrant FGF signaling is a culprit for various diseases, including congenital birth defects, metabolic disorder, and cancer. The molecular mechanisms by which the specificity of FGF signaling is achieved remain incompletely understood. Since its application as a druggable target has been gradually recognized by pharmaceutical companies and translational researchers, understanding the determinants of FGF signaling specificity has become even more important in order to get into the position to selectively suppress a particular pathway without affecting others to minimize side effects.

  8. Lactic Acid Bacteria Convert Human Fibroblasts to Multipotent Cells

    PubMed Central

    Ohta, Kunimasa; Kawano, Rie; Ito, Naofumi

    2012-01-01

    The human gastrointestinal tract is colonized by a vast community of symbionts and commensals. Lactic acid bacteria (LAB) form a group of related, low-GC-content, gram-positive bacteria that are considered to offer a number of probiotic benefits to general health. While the role of LAB in gastrointestinal microecology has been the subject of extensive study, little is known about how commensal prokaryotic organisms directly influence eukaryotic cells. Here, we demonstrate the generation of multipotential cells from adult human dermal fibroblast cells by incorporating LAB. LAB-incorporated cell clusters are similar to embryoid bodies derived from embryonic stem cells and can differentiate into endodermal, mesodermal, and ectodermal cells in vivo and in vitro. LAB-incorporated cell clusters express a set of genes associated with multipotency, and microarray analysis indicates a remarkable increase of NANOG, a multipotency marker, and a notable decrease in HOX gene expression in LAB-incorporated cells. During the cell culture, the LAB-incorporated cell clusters stop cell division and start to express early senescence markers without cell death. Thus, LAB-incorporated cell clusters have potentially wide-ranging implications for cell generation, reprogramming, and cell-based therapy. PMID:23300571

  9. Propagation of human embryonic and induced pluripotent stem cells in an indirect co-culture system

    PubMed Central

    Abraham, Sheena; Sheridan, Steven D.; Laurent, Louise C.; Albert, Kelsey; Stubban, Christopher; Ulitsky, Igor; Miller, Bradley; Loring, Jeanne F.; Rao, Raj R.

    2010-01-01

    We have developed and validated a microporous poly(ethylene terephthalate) membrane-based indirect co-culture system for human pluripotent stem cell (hPSC) propagation, which allows real-time conditioning of the culture medium with human fibroblasts while maintaining the complete separation of the two cell types. The propagation and pluripotent characteristics of a human embryonic stem cell (hESC) line and a human induced pluripotent stem cell (hiPSC) line were studied in prolonged culture in this system. We report that hPSCs cultured on membranes by indirect co-culture with fibroblasts were indistinguishable by multiple criteria from hPSCs cultured directly on a fibroblast feeder layer. Thus this co-culture system is a significant advance in hPSC culture methods, providing a facile stem cell expansion system with continuous medium conditioning while preventing mixing of hPSCs and feeder cells. This membrane culture method will enable testing of novel feeder cells and differentiation studies using co-culture with other cell types, and will simplify stepwise changes in culture conditions for staged differentiation protocols. PMID:20117095

  10. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells.

    PubMed

    Stojkovic, Petra; Lako, Majlinda; Stewart, Rebecca; Przyborski, Stefan; Armstrong, Lyle; Evans, Jerome; Murdoch, Alison; Strachan, Tom; Stojkovic, Miodrag

    2005-03-01

    Human embryonic stem cells (hESCs) have great potential as a source of cells for therapeutic uses, but their culture requires the support of mouse or human cells, either directly as a feeder cell layer or indirectly as a source of conditioned medium in feeder-free culture systems. Unfortunately, the risks of cross-transfer of pathogens from xenogeneic or allogeneic feeders or cell by-products limit their medical applications. In addition, not all human feeders support the growth of hESCs equally well, and ethical concerns have been raised regarding the derivation of feeder cells from aborted human fetuses. We report here the culture of hESCs on a novel feeder cell system, comprising fibroblast-like cells derived from the spontaneous differentiation of hESCs. Isogenicity of the hESCs and hESC-derived fibroblasts was confirmed by micro satellite analysis. The nature of the hESC-derived fibroblasts was identified by the expression of specific markers. This feeder system permits continuous growth of undifferentiated and pluripotent hESCs, as demonstrated by the expression of specific hESC markers, by the formation of teratomas after injection of hESCs into severely combined immunodeficient mice, and by in vitro differentiation of hESCs into differentiated cells of ectodermal, endodermal, and mesodermal origin. Feeder cells derived from hESCs offers a potentially more secure autogeneic and genotypically homogenous system for the growth of undifferentiated hESCs.

  11. Aging adult porcine fibroblasts can support nuclear transfer and transcription factor-mediated reprogramming.

    PubMed

    Li, Xia; Zhang, Pengfei; Jiang, Shaoshuai; Ding, Biao; Zuo, Xiaoyuan; Li, Yunsheng; Cao, Zubing; Zhang, Yunhai

    2017-10-03

    Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs) technology are two classical reprogramming methods. Donor cell types can affect the reprogramming results in the above two methods. We here used porcine embryonic fibroblasts (PEFs) and adult porcine ear skin fibroblasts (APEFs) and adipose-derived stem cells (ADSCs) as donor cells for SCNT and source cells for iPSCs to study their in vitro developmental capability and colony-formation efficiency, respectively. For SCNT, fusion and cleavage rate has no significant difference among PEFs, ADSCs and APEFs. The rate and total cell number of blastocysts in the APEF group were significant lower than that in PEFs and ADSCs. For transcription factor-mediated reprogramming, the reprogramming efficiency of ADSCs were significantly higher than PEFs and APEFs and there is no significant difference between PEFs and APEFs. Furthermore, PEFs, APEFs and ADSCs can be used to generate iPSCs. Fianlly, somatic cloned pigs could still be successfully generated from APEFs, suggesting terminally differentiated aging adult somatic cells could be reprogrammed into a totipotent state. Considering the easy availability of animal tissue and the costs of establishing cell lines, aging porcine ear fibroblasts can support nuclear transfer-mediated and transcription factor-based reprogramming. © 2017 Japanese Society of Animal Science.

  12. Stromal Fibroblasts in Tertiary Lymphoid Structures: A Novel Target in Chronic Inflammation.

    PubMed

    Barone, Francesca; Gardner, David H; Nayar, Saba; Steinthal, Nathalie; Buckley, Christopher D; Luther, Sanjiv A

    2016-01-01

    Tertiary lymphoid structures (TLS) are organized aggregates of lymphocytes, myeloid, and stromal cells that provide ectopic hubs for acquired immune responses. TLS share phenotypical and functional features with secondary lymphoid organs (SLO); however, they require persistent inflammatory signals to arise and are often observed at target sites of autoimmune disease, chronic infection, cancer, and organ transplantation. Over the past 10 years, important progress has been made in our understanding of the role of stromal fibroblasts in SLO development, organization, and function. A complex and stereotyped series of events regulate fibroblast differentiation from embryonic life in SLOs to lymphoid organ architecture observed in adults. In contrast, TLS-associated fibroblasts differentiate from postnatal, locally activated mesenchyme, predominantly in settings of inflammation and persistent antigen presentation. Therefore, there are critical differences in the cellular and molecular requirements that regulate SLO versus TLS development that ultimately impact on stromal and hematopoietic cell function. These differences may contribute to the pathogenic nature of TLS in the context of chronic inflammation and malignant transformation and offer a window of opportunity for therapeutic interventions in TLS associated pathologies.

  13. Stromal Fibroblasts in Tertiary Lymphoid Structures: A Novel Target in Chronic Inflammation

    PubMed Central

    Barone, Francesca; Gardner, David H.; Nayar, Saba; Steinthal, Nathalie; Buckley, Christopher D.; Luther, Sanjiv A.

    2016-01-01

    Tertiary lymphoid structures (TLS) are organized aggregates of lymphocytes, myeloid, and stromal cells that provide ectopic hubs for acquired immune responses. TLS share phenotypical and functional features with secondary lymphoid organs (SLO); however, they require persistent inflammatory signals to arise and are often observed at target sites of autoimmune disease, chronic infection, cancer, and organ transplantation. Over the past 10 years, important progress has been made in our understanding of the role of stromal fibroblasts in SLO development, organization, and function. A complex and stereotyped series of events regulate fibroblast differentiation from embryonic life in SLOs to lymphoid organ architecture observed in adults. In contrast, TLS-associated fibroblasts differentiate from postnatal, locally activated mesenchyme, predominantly in settings of inflammation and persistent antigen presentation. Therefore, there are critical differences in the cellular and molecular requirements that regulate SLO versus TLS development that ultimately impact on stromal and hematopoietic cell function. These differences may contribute to the pathogenic nature of TLS in the context of chronic inflammation and malignant transformation and offer a window of opportunity for therapeutic interventions in TLS associated pathologies. PMID:27877173

  14. Exposure of cardiac fibroblasts to the herbicide nitrofen causes altered interactions with the extracellular matrix.

    PubMed

    Borck, A; Massey, E; Loftis, M J; Carver, W

    2004-02-01

    A large proportion of congenital heart defects result from dysmorphogenesis of valvuloseptal precursors, the endocardial cushions. Intrinsic to formation and maturation of these tissues are developmental changes in cell-cell and cell-extracellular matrix interactions. Interactions between cells and the extracellular matrix play critical roles in modulating cellular processes including proliferation, migration, differentiation and even survival. While significant progress is being made in the elucidation of the cellular events involved in valvuloseptal development, little is known regarding how environmental factors may affect this process. Embryonic exposure to the herbicide nitrofen has been shown to result in congenital heart defects associated with altered endocardial cushion formation or maturation. The present studies were performed to begin to address the cellular mechanisms of these nitrofen-induced effects. Heart fibroblasts were isolated and treated with varying doses of nitrofen in vitro. Experiments were performed to determine the effects of this herbicide on important cellular processes including migration, proliferation and apoptosis. These studies illustrated a dose-dependent decrease in collagen gel contraction and proliferation in response to nitrofen. Assays were also performed to determine the effects of nitrofen on fibroblast gene expression. Increased expression of collagen type I and specific integrins were seen following nitrofen exposure. These studies illustrate that nitrofen has direct effects on cardiac fibroblast proliferation and extracellular matrix remodeling, cellular events important in valvuloseptal development.

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

    PubMed

    Salmenperä, Pertteli; Karhemo, Piia-Riitta; Räsänen, Kati; Laakkonen, Pirjo; Vaheri, Antti

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. FGF signalling controls anterior extraembryonic and embryonic fate in the beetle Tribolium.

    PubMed

    Sharma, Rahul; Beermann, Anke; Schröder, Reinhard

    2013-09-01

    Fibroblast growth factor (FGF) signalling plays a key role in early embryonic development and cell migration in vertebrates and in invertebrates. To gain novel insights into FGF signalling in an arthropod, we characterized the fgf1b ortholog in the beetle Tribolium that is not represented in the Drosophila genome. We found that FGF1b dependent signalling organizes the anterior to posterior axis of the early embryo. The loss of Tc-fgf1b function in Tribolium by RNA interference resulted in the reduction of the anteriormost extraembryonic fate, in an anterior shift of embryonic fate and in the loss or malformation of anterior embryonic structures. Without intact extraembryonic membranes the serosa and the amnion, Tc-fgf1b(RNAi) embryos did not undergo morphogenetic movements and remained posteriorly localized throughout embryogenesis. Only weakly affected embryos developed into a cuticle that show dorsally curved bodies with head defects and a dorsal opening. Except for the posterior dorsal amnion, the overall topology of the dorsal-ventral axis seemed unaffected. Moreover, FGF signalling was not required for the onset of mesoderm formation but for fine-tuning this tissue during later development. We also show that in affected embryos the dorsal epidermis was expanded and expressed Tc-dpp at a higher level. We conclude that in the Tribolium blastoderm embryo, FGF1-signalling organizes patterning along the AP-axis and also balances the expression level of Dpp in the dorsal epidermis, a tissue critically involved in dorsal closure.

  17. An Integrated Transcriptome Atlas of Embryonic Hair Follicle Progenitors, Their Niche, and the Developing Skin.

    PubMed

    Sennett, Rachel; Wang, Zichen; Rezza, Amélie; Grisanti, Laura; Roitershtein, Nataly; Sicchio, Cristina; Mok, Ka Wai; Heitman, Nicholas J; Clavel, Carlos; Ma'ayan, Avi; Rendl, Michael

    2015-09-14

    Defining the unique molecular features of progenitors and their niche requires a genome-wide, whole-tissue approach with cellular resolution. Here, we co-isolate embryonic hair follicle (HF) placode and dermal condensate cells, precursors of adult HF stem cells and the dermal papilla/sheath niche, along with lineage-related keratinocytes and fibroblasts, Schwann cells, melanocytes, and a population inclusive of all remaining skin cells. With next-generation RNA sequencing, we define gene expression patterns in the context of the entire embryonic skin, and through transcriptome cross-comparisons, we uncover hundreds of enriched genes in cell-type-specific signatures. Axon guidance signaling and many other pathway genes are enriched in multiple signatures, implicating these factors in driving the large-scale cellular rearrangements necessary for HF formation. Finally, we share all data in an interactive, searchable companion website. Our study provides an overarching view of signaling within the entire embryonic skin and captures a molecular snapshot of HF progenitors and their niche. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Neurotrophic factors improve motoneuron survival and function of muscle reinnervated by embryonic neurons.

    PubMed

    Grumbles, Robert M; Sesodia, Sanjay; Wood, Patrick M; Thomas, Christine K

    2009-07-01

    Motoneuron death can occur over several spinal levels with disease or trauma, resulting in muscle denervation. We tested whether cotransplantation of embryonic neurons with 1 or more neurotrophic factors into peripheral nerve improved axon regeneration, muscle fiber area, reinnervation, and function to a greater degree than cell transplantation alone. Sciatic nerves of adult Fischer rats were cut to denervate muscles; 1 week later, embryonic ventral spinal cord cells (days 14-15) were transplanted into the tibial nerve stump as the only source of neurons for muscle reinnervation. Factors that promote motoneuron survival (cardiotrophin 1; fibroblast growth factor 2; glial cell line-derived neurotrophic factor; insulin-like growth factor 1; leukemia inhibitory factor; and hepatocyte growth factor) were added to the transplant individually or in combinations. Inclusion of a single factor with the cells resulted in comparable myelinated axon counts, muscle fiber areas, and evoked electromyographic activity to cells alone 10 weeks after transplantation. Only cell transplantation with glial cell line-derived neurotrophic factor, hepatocyte growth factor, and insulin-like growth factor 1 significantly increased motoneuron survival, myelinated axon counts, muscle reinnervation, and evoked electromyographic activity compared with cells alone. Thus, immediate application of a specific combination of factors to dissociated embryonic neurons improves survival of motoneurons and the long-term function of reinnervated muscle.

  19. Identification and characterization of secondary neural tube-derived embryonic neural stem cells in vitro.

    PubMed

    Shaker, Mohammed R; Kim, Joo Yeon; Kim, Hyun; Sun, Woong

    2015-05-15

    Secondary neurulation is an embryonic progress that gives rise to the secondary neural tube, the precursor of the lower spinal cord region. The secondary neural tube is derived from aggregated Sox2-expressing neural cells at the dorsal region of the tail bud, which eventually forms rosette or tube-like structures to give rise to neural tissues in the tail bud. We addressed whether the embryonic tail contains neural stem cells (NSCs), namely secondary NSCs (sNSCs), with the potential for self-renewal in vitro. Using in vitro neurosphere assays, neurospheres readily formed at the rosette and neural-tube levels, but less frequently at the tail bud tip level. Furthermore, we identified that sNSC-generated neurospheres were significantly smaller in size compared with cortical neurospheres. Interestingly, various cell cycle analyses revealed that this difference was not due to a reduction in the proliferation rate of NSCs, but rather the neuronal commitment of sNSCs, as sNSC-derived neurospheres contain more committed neuronal progenitor cells, even in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). These results suggest that the higher tendency for sNSCs to spontaneously differentiate into progenitor cells may explain the limited expansion of the secondary neural tube during embryonic development.

  20. Human embryonic stem cells: mother of all cell and tissue types.

    PubMed

    Trounson, Alan

    2002-01-01

    Pluripotential embryonic stem (ES) cells have been derived very efficiently from spare human embryos produced by IVF and grown in culture to the nascent blastocyst stage. The inner cell mass (ICM) is isolated by immunosurgery and grown on selected embryonic fibroblast monolayer cultures. ICM cells lose their memory for axis during formation of ES cell colonies and are then unable to integrate tissue formation with a body plan. ES cells form teratomas in vivo with cells and tissues representative of the three major embryonic lineages (ectoderm, mesoderm, endoderm). The ES cells are continuously renewable and can be directed to differentiate into early progenitors of neural stem cells (Noggin cells) and from there into mature neurons and glia (astrocytes and oligodendrocytes). The neural stem cells formed from human ES cells repopulate the brains of newborn mice when injected into the lateral cerebral ventricles, forming astrocytes dominantly in the parenchyma. The human neural cells can be observed migrating from the subventricular areas along the rostral migratory stream. Human neurons can be found in the olfactory bulb. Human ES cells can also be directed into cardiomyocytes when co-cultured with visceral endoderm-like cells (END-2). These observations provide further scope to explore stem cell therapies, gene therapies and drug discovery. For compatible transplantation, ES may need to be derived with a range of HLA types or by nuclear transplantation or stem cell fusion.

  1. Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar–ductal metaplasia

    PubMed Central

    Liu, Xin; Pitarresi, Jason R.; Cuitiño, Maria C.; Kladney, Raleigh D.; Woelke, Sarah A.; Sizemore, Gina M.; Nayak, Sunayana G.; Egriboz, Onur; Schweickert, Patrick G.; Yu, Lianbo; Trela, Stefan; Schilling, Daniel J.; Halloran, Shannon K.; Li, Maokun; Dutta, Shourik; Fernandez, Soledad A.; Rosol, Thomas J.; Lesinski, Gregory B.; Shakya, Reena; Ludwig, Thomas; Konieczny, Stephen F.; Leone, Gustavo; Wu, Jinghai; Ostrowski, Michael C.

    2016-01-01

    The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a KrasG12D mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo. Additionally, Smo-deleted fibroblasts stimulated the growth of KrasG12D/Tp53R172H pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating KrasG12D-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts. PMID:27633013

  2. Abnormal iron metabolism in fibroblasts from a patient with the neurodegenerative disease hereditary ferritinopathy

    PubMed Central

    2010-01-01

    Background Nucleotide duplications in exon 4 of the ferritin light polypeptide (FTL) gene cause the autosomal dominant neurodegenerative disease neuroferritinopathy or hereditary ferritinopathy (HF). Pathologic examination of patients with HF has shown abnormal ferritin and iron accumulation in neurons and glia in the central nervous system (CNS) as well as in cells of other organ systems, including skin fibroblasts. To gain some understanding on the molecular basis of HF, we characterized iron metabolism in primary cultures of human skin fibroblasts from an individual with the FTL c.497_498dupTC mutation. Results Compared to normal controls, HF fibroblasts showed abnormal iron metabolism consisting of increased levels of ferritin polypeptides, divalent metal transporter 1, basal iron content and reactive oxygen species, and decreased levels of transferrin receptor-1 and IRE-IRP binding activity. Conclusions Our data indicates that HF fibroblasts replicate the abnormal iron metabolism observed in the CNS of patients with HF. We propose that HF fibroblasts are a unique cellular model in which to study the role of abnormal iron metabolism in the pathogenesis of HF without artifacts derived from over-expression or lack of endogenous translational regulatory elements. PMID:21067605

  3. Proliferation of human fibroblasts in vitro after exposure to orbital implants.

    PubMed

    Mawn, L A; Jordan, D R; Gilberg, S

    2001-08-01

    Porous orbital implants allow fibrovascular ingrowth and integration with the extraocular muscles. The available implants have different structural characteristics, which may influence orbital response. We studied the proliferation of orbital fibroblasts in vitro after exposure to four different orbital implants. Four orbital implant biomaterials were studied: hydroxyapatite (Bio-Eye), synthetic hydroxyapatite, porous polyethylene (Medpor) (pore sizes 150 microm and 400 microm) and aluminium oxide (Bioceramic implant). Human fibroblasts obtained from orbital fat at the time of elective blepharoplasty were cultured and then exposed to the individual implants. Cell growth was assessed with immunocytochemical analysis using bromodeoxyuridine, a thymidine analogue. After DNA denaturation, the cells were washed, incubated with secondary antibody and vis