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  1. 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

  2. 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.

  3. 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

  4. [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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. CP27 affects viability, proliferation, attachment and gene expression in embryonic fibroblasts.

    PubMed

    Luan, X; Diekwisch, T G H

    2002-08-01

    CP27 is a gene that has been cloned from an E11 early embryonic library and has been suggested to mediate early organogenesis (Diekwisch et al., 1999, Gene 235, 19). We have hypothesized that CP27 exhibits its effects on organogenesis by affecting individual cell function. Based on the CP27 expression pattern we have selected the CP27 expressing embryonic fibroblast cell line BALB/c 3T3 to determine the effects of CP27 on cell function. CP27 loss of function strategies were performed by adding 5, 12.5 or 25 micro g/ml anti-CP27 antibody to cultured BALB/c 3T3 cells and comparing the results to controls in which identical concentrations of rabbit serum were added to the culture medium. Other controls included an antibody against another extracellular matrix protein amelogenin (negative control) and anti-CP27 antibodies directed against other areas of the CP27 molecule (positive control). Following cell culture, cell viability, apoptosis, cell proliferation, cell shape, cellular attachment and fibronectin matrix production were assayed using MTT colourimetric assay, BrdU staining, morphometry, immunostaining and western blot analysis. Block of CP27 function using an antibody strategy resulted in the following significant changes: (i) reduced viability, (ii) increased number of apoptotic cells, (iii) reduced proliferation, (iv) alterations in cell shape, (v) loss of attachment, and (vi) reduction in fibronectin matrix production. There was also a redistribution in fibronectin matrix organization demonstrated by immunohistochemistry. We conclude that CP27 plays an important role in the maintance of normal cell function and that CP27 block leads to significant changes in cellular behaviour.

  15. 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.

  16. 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.

  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. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  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. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

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

    PubMed

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

    2012-01-01

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

  12. 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

  13. 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

  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. 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

  16. Heparan sulfation-dependent fibroblast growth factor signaling maintains embryonic stem cells primed for differentiation in a heterogeneous state.

    PubMed

    Lanner, Fredrik; Lee, Kian Leong; Sohl, Marcus; Holmborn, Katarina; Yang, Henry; Wilbertz, Johannes; Poellinger, Lorenz; Rossant, Janet; Farnebo, Filip

    2010-02-01

    Embryonic stem (ES) cells continuously decide whether to maintain pluripotency or differentiate. While exogenous leukemia inhibitory factor and BMP4 perpetuate a pluripotent state, less is known about the factors initiating differentiation. We show that heparan sulfate (HS) proteoglycans are critical coreceptors for signals inducing ES cell differentiation. Genetic targeting of NDST1 and NDST2, two enzymes required for N-sulfation of proteoglycans, blocked differentiation. This phenotype was rescued by HS presented in trans or by soluble heparin. NaClO(3) (-), which reduces sulfation of proteoglycans, potently blocked differentiation of wild-type cells. Mechanistically, N-sulfation was identified to be critical for functional autocrine fibroblast growth factor 4 (FGF4) signaling. Microarray analysis identified the pluripotency maintaining transcription factors Nanog, KLF2/4/8, Tbx3, and Tcf3 to be negatively regulated, whereas markers of differentiation such as Gbx2, Dnmt3b, FGF5, and Brachyury were induced by sulfation-dependent FGF receptor (FGFR) signaling. We show that several of these genes are heterogeneously expressed in ES cells, and that targeting of heparan sulfation or FGFR-signaling facilitated a homogenous Nanog/KLF4/Tbx3 positive ES cell state. This finding suggests that the recently discovered heterogeneous state of ES cells is regulated by HS-dependent FGFR signaling. Similarly, culturing blastocysts with NaClO(3) (-) eliminated GATA6-positive primitive endoderm progenitors generating a homogenous Nanog-positive inner cell mass. Functionally, reduction of sulfation robustly improved de novo ES cell derivation efficiency. We conclude that N-sulfated HS is required for FGF4 signaling to maintain ES cells primed for differentiation in a heterogeneous state. Inhibiting this pathway facilitates a more naïve ground state.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  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. 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.

  10. 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.

  11. 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.

  12. 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.

  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. 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.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. In Vitro Study of Mutagenesis Induced by Crocidolite-Exposed Alveolar Macrophages NR8383 in Cocultured Big Blue Rat2 Embryonic Fibroblasts.

    PubMed

    Guichard, Yves; Gaté, Laurent; Darne, Christian; Bottin, Marie-Claire; Langlais, Cristina; Micillino, Jean-Claude; Goutet, Michèle; Julien, Schmit; Stéphane, Binet

    2010-01-01

    Asbestos-induced mutagenicity in the lung may involve reactive oxygen/nitrogen species (ROS/RNS) released by alveolar macrophages. With the aim of proposing an alternative in vitro mutagenesis test, a coculture system of rat alveolar macrophages (NR8383) and transgenic Big Blue Rat2 embryonic fibroblasts was developed and tested with a crocidolite sample. Crocidolite exposure induced no detectable increase in ROS production from NR8383, contrasting with the oxidative burst that occurred following a brief exposure (1 hour) to zymosan, a known macrophage activator. In separated cocultures, crocidolite and zymosan induced different changes in the gene expressions involved in cellular inflammation in NR8383 and Big Blue. In particular, both particles induced up-regulation of iNOS expression in Big Blue, suggesting the formation of potentially genotoxic nitrogen species. However, crocidolite exposure in separated or mixed cocultures induced no mutagenic effects whereas an increase in Big Blue mutants was detected after exposure to zymosan in mixed cocultures. NR8383 activation by crocidolite is probably insufficient to induce in vitro mutagenic events. The mutagenesis assay based on the coculture of NR8383 and Big Blue cannot be used as an alternative in vitro method to assess the mutagenic properties of asbestos fibres.

  5. Poly(2-hydroxyethyl methacrylate)-b-poly(L-Lysine) cationic hybrid materials for non-viral gene delivery in NIH 3T3 mouse embryonic fibroblasts.

    PubMed

    Johnson, Renjith P; Uthaman, Saji; John, Johnson V; Heo, Min Seon; Park, In Kyu; Suh, Hongsuk; Kim, Il

    2014-09-01

    In order to develop efficient and nontoxic gene delivery vectors, a series of biocompatible block copolymers, poly[(2-hydroxyethyl methacrylate)40 -block-(L-lysine)n ] (n = 40, 80, 120, 150), are prepared by combining an atom transfer radical polymerization of 2-hydroxyethyl methacrylate with a ring-opening polymerization of N(ϵ) -(carbobenzoxy)-L-lysine N-carboxyanhydride. The block copolymers are successfully condensed with plasmid DNA (pDNA) into nanosized (<200 nm) polyplexes. As a representative sample, p(HEMA)40 -b-p(lys)150 is utilized to confirm the effective cellular and nuclear uptake of pDNA. The polymer/pDNA polyplexes exhibit very low cytotoxicity and enhanced transfection activity by being easily taken up into mouse embryonic fibroblast cell line (NIH 3T3). Thus, the chimeric block copolymers provide a means for developing versatile nonviral gene vectors harboring the ideal requirements of low cytotoxicity, good stability, and high transfection efficiency for gene therapy.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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…

  3. 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

  4. 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.

  5. 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).

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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...

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. [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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. 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

  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. 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.

  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. 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.

  12. 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

  13. 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.

  14. 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

  15. [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.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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.

  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. 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.

  8. 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.

  9. 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.

  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. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

    PubMed

    Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

    2015-09-01

    Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  9. 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

  10. 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.

  11. Copper accumulation and compartmentalization in mouse fibroblast lacking metallothionein and copper chaperone, Atox1

    SciTech Connect

    Miyayama, Takamitsu; Suzuki, Kazuo T.; Ogra, Yasumitsu

    2009-06-01

    Copper (Cu) is the active center of some enzymes because of its redox-active property, although that property could have harmful effects. Because of this, cells have strict regulation/detoxification systems for this metal. In this study, multi-disciplinary approaches, such as speciation and elemental imaging of Cu, were applied to reveal the detoxification mechanisms for Cu in cells bearing a defect in Cu-regulating genes. Although Cu concentration in metallothionein (MT)-knockout cells was increased by the knockdown of the Cu chaperone, Atox1, the concentrations of the Cu influx pump, Ctr1, and another Cu chaperone, Ccs, were paradoxically increased; namely, the cells responded to the Cu deficiency despite the fact that cellular Cu concentration was actually increased. Cu imaging showed that the elevated Cu was compartmentalized in cytoplasmic vesicles. Together, the results point to the novel roles of MT and cytoplasmic vesicles in the detoxification of Cu in mammalian cells.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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).

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  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. 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

  16. 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.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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

  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. 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".

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  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. 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

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  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. 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.

  15. 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.

  16. Smooth muscle differentiation in scleroderma fibroblastic cells.

    PubMed Central

    Sappino, A. P.; Masouyé, I.; Saurat, J. H.; Gabbiani, G.

    1990-01-01

    Using antibodies to alpha-smooth muscle actin and desmin on paraffin-embedded formalin-fixed tissue sections, the authors demonstrate that fibroblastic cells of localized and systemic scleroderma lesions express features of smooth muscle differentiation. Eleven of eleven skin specimens of systemic sclerosis patients and two of four skin specimens of localized scleroderma displayed the presence of fibroblasts expressing alpha-smooth muscle actin, a cell population that predominated in areas of prominent collagen deposition. A similar fibroblastic phenotype was found in the esophagus, the liver, and the lung specimens obtained from four patients who died of progressive systemic sclerosis. Immunostaining for desmin, performed on adjacent tissue sections, demonstrated that a minority of these fibroblastic cells present in skin and visceral lesions contained this protein. The authors' observations indicate that scleroderma fibroblasts are phenotypically related to the stromal cells previously identified in hypertrophic scars, fibromatoses, and desmoplasia; they might provide novel criteria for the characterization of scleroderma lesions and help to identify the factors responsible for phenotypic modulations in fibroblastic cells. Images Figure 1 Figure 2 Figure 3 PMID:1698026

  17. 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

  18. 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

  19. 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.

  20. 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

  1. Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

    PubMed Central

    Lin, Audrey; Hokugo, Akishige; Choi, Jae; Nishimura, Ichiro

    2010-01-01

    Wounds created in the oral cavity heal rapidly and leave minimal scarring. We have examined a role of a previously isolated cDNA from oral wounds encoding wound inducible transcript-3.0 (wit3.0), also known as fibroblast growth factor receptor 1 oncogene partner 2 (FGFR1OP2). FGFR1OP2/wit3.0 was highly expressed in oral wound fibroblasts without noticeable up-regulation of α-smooth muscle actin. In silico analyses, denaturing and nondenaturing gel Western blot, and immunocytology together demonstrated that FGFR1OP2/wit3.0 were able to dimerize and oligomerize through coiled-coil structures and appeared to associate with cytoskeleton networks in oral wound fibroblasts. Overexpression of FGFR1OP2/wit3.0 increased the floating collagen gel contraction of naïve oral fibroblasts to the level of oral wound fibroblasts, which was in turn attenuated by small-interfering RNA knockdown. The FGFR1OP2/wit3.0 synthesis did not affect the expression of collagen I as well as procontractile peptides such as α-smooth muscle actin, and transforming growth factor-β1 had no effect on FGFR1OP2/wit3.0 expression. Fibroblastic cells derived from embryonic stem cells carrying FGFR1OP2/wit3.0 (+/−) mutation showed significant retardation in cell migration. Thus, we postulate that FGFR1OP2/wit3.0 may regulate cell motility and stimulate wound closure. FGFR1OP2/wit3.0 was not up-regulated during skin wound healing; however, when treated with FGFR1OP2/wit3.0 -expression vector, the skin wound closure was significantly accelerated, resulting in the limited granulation tissue formation. Our data suggest that FGFR1OP2/wit3.0 may possess a therapeutic potential for wound management. PMID:19959814

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Role of fibroblast growth factor receptor signaling in kidney development.

    PubMed

    Bates, Carlton M

    2011-09-01

    Fibroblast growth factor receptors (Fgfrs) are expressed throughout the developing kidney. Several early studies have shown that exogenous fibroblast growth factors (Fgfs) affect growth and maturation of the metanephric mesenchyme (MM) and ureteric bud (UB). Transgenic mice that over-express a dominant negative receptor isoform develop renal aplasia/severe dysplasia, confirming the importance of Fgfrs in renal development. Furthermore, global deletion of Fgf7, Fgf10, and Fgfr2IIIb (isoform that binds Fgf7 and Fgf10) in mice leads to small kidneys with fewer collecting ducts and nephrons. Deletion of Fgfrl1, a receptor lacking intracellular signaling domains, causes severe renal dysgenesis. Conditional targeting of Fgf8 from the MM interrupts nephron formation. Deletion of Fgfr2 from the UB results in severe ureteric branching and stromal mesenchymal defects, although loss of Frs2α (major signaling adapter for Fgfrs) in the UB causes only mild renal hypoplasia. Deletion of both Fgfr1 and Fgfr2 in the MM results in renal aplasia with defects in MM formation and initial UB elongation and branching. Loss of Fgfr2 in the MM leads to many renal and urinary tract anomalies as well as vesicoureteral reflux. Thus, Fgfr signaling is critical for patterning of virtually all renal lineages at early and later stages of development.

  7. LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts.

    PubMed

    Zhang, Hui; Sweezey, Neil B; Kaplan, Feige

    2015-02-15

    Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1(+/-) mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5(LGL1KD)). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5(LGL1KD) suppressed cell growth and increased apoptosis of annexin V(+) staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5(LGL1KD) cells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development.

  8. Repeated Nrf2 stimulation using sulforaphane protects fibroblasts from ionizing radiation

    SciTech Connect

    Mathew, Sherin T.; Bergström, Petra; Hammarsten, Ola

    2014-05-01

    Most of the cytotoxicity induced by ionizing radiation is mediated by radical-induced DNA double-strand breaks. Cellular protection from free radicals can be stimulated several fold by sulforaphane-mediated activation of the transcription factor Nrf2 that regulates more than 50 genes involved in the detoxification of reactive substances and radicals. Here, we report that repeated sulforaphane treatment increases radioresistance in primary human skin fibroblasts. Cells were either treated with sulforaphane for four hours once or with four-hour treatments repeatedly for three consecutive days prior to radiation exposure. Fibroblasts exposed to repeated-sulforaphane treatment showed a more pronounced dose-dependent induction of Nrf2-regulated mRNA and reduced amount of radiation-induced free radicals compared with cells treated once with sulforaphane. In addition, radiation- induced DNA double-strand breaks measured by gamma-H2AX foci were attenuated following repeated sulforaphane treatment. As a result, cellular protection from ionizing radiation measured by the 5-ethynyl-2′-deoxyuridine (EdU) assay was increased, specifically in cells exposed to repeated sulforaphane treatment. Sulforaphane treatment was unable to protect Nrf2 knockout mouse embryonic fibroblasts, indicating that the sulforaphane-induced radioprotection was Nrf2-dependent. Moreover, radioprotection by repeated sulforaphane treatment was dose-dependent with an optimal effect at 10 uM, whereas both lower and higher concentrations resulted in lower levels of radioprotection. Our data indicate that the Nrf2 system can be trained to provide further protection from radical damage. - Highlights: • Repeated treatment with sulforaphane protects fibroblasts from ionizing radiation • Repeated sulforaphane treatment attenuates radiation induced ROS and DNA damage • Sulforaphane mediated protection is Nrf2 dependent.

  9. Medaka haploid embryonic stem cells.

    PubMed

    Hong, Yunhan

    2010-01-01

    The appearance of diploidy, the presence of two genomes or chromosome sets, is a fundamental hallmark of eukaryotic evolution and bisexual reproduction, because diploidy offers the basis for the bisexual life cycle, allowing for oscillation between diploid and haploid phases. Meiosis produces haploid gametes. At fertilization, male and female gametes fuse to restore diploidy in a zygote, which develops into a new life. At sex maturation, diploid cells enter into meiosis, culminating in the production of haploid gametes. Therefore, diploidy ensures pluripotency, cell proliferation, and functions, whereas haploidy is restricted only to the post-meiotic gamete phase of germline development and represents the end point of cell growth. Diploidy is advantageous for evolution. Haploidy is ideal for genetic analyses, because any recessive mutations of essential genes will show a clear phenotype in the absence of a second gene copy. Recently, my laboratory succeeded in the generation of medaka haploid embryonic stem (ES) cells capable of whole animal production. Therefore, haploidy in a vertebrate is able to support stable cell culture and pluripotency. This finding anticipates the possibility to generate haploid ES cells in other vertebrate species such as zebrafish. These medaka haploid ES cells elegantly combine haploidy and pluripotency, offering a unique yeast-like system for in vitro genetic analyses of molecular, cellular, and developmental events in various cell lineages. This chapter is aimed to describe the strategy of haploid ES cell derivation and their characteristics, and illustrate the perspectives of haploid ES cells for infertility treatment, genetic screens, and analyses.

  10. Embryonic development in Zungaro jahu.

    PubMed

    Marques, Camila; Faustino, Francine; Bertolucci, Bruno; Paes, Maria do Carmo Faria; Silva, Regiane Cristina da; Nakaghi, Laura Satiko Okada

    2017-02-01

    The aim of this study was to characterize the embryonic development of Zungaro jahu, a fresh water teleostei commonly known as 'jaú'. Samples were collected at pre-determined times from oocyte release to larval hatching and analysed under light microscopy, transmission electron microscopy and scanning electron microscopy. At the first collection times, the oocytes and eggs were spherical and yellowish, with an evident micropyle. Embryo development took place at 29.4 ± 1.5°C and was divided into seven stages: zygote, cleavage, morula, blastula, gastrula, organogenesis, and hatching. The differentiation of the animal and vegetative poles occured during the zygote stage, at 10 min post-fertilization (mpf), leading to the development of the egg cell at 15 mpf. From 20 to 75 mpf, successive cleavages resulted in the formation of 2, 4, 8, 16, 32 and 64 blastomeres. The morula stage was observed between 90 and 105 mpf, and the blastula and gastrula stage at 120 and 180 mpf; respectively. The end of the gastrula stage was characterized by the presence of the yolk plug at 360 mpf. Organogenesis followed, with differentiation of the cephalic and caudal regions, elongation of the embryo by the cephalo-caudal axis, and somitogenesis. Hatching occurred at 780 mpf, with mean larval total length of 3.79 ± 0.11 mm.

  11. Embryonal rhabdomyosarcoma: A rare oral tumor

    PubMed Central

    Datta, Sila; Ray, Jay Gopal; Deb, Tushar; Patsa, Santanu

    2016-01-01

    Rhabdomyosarcoma is the malignant neoplasm of striated muscle and a relatively uncommon tumor of the oral cavity. Embryonal variety is the most common subtype, observed in children below 10 years of age but occasionally seen in adolescents and young adults. The present report describes a case of embryonal rhabdomyosarcoma in the left posterior buccal mucosa, with extension in the adjacent alveolus, soft palate, oropharynx and nasopharynx of a 17-year-old female. PMID:27721622

  12. Design and characteristics of cytotoxic fibroblast growth factor 1 conjugate for fibroblast growth factor receptor-targeted cancer therapy

    PubMed Central

    Szlachcic, Anna; Zakrzewska, Malgorzata; Lobocki, Michal; Jakimowicz, Piotr; Otlewski, Jacek

    2016-01-01

    Fibroblast growth factor receptors (FGFRs) are attractive candidate cancer therapy targets as they are overexpressed in multiple types of tumors, such as breast, prostate, bladder, and lung cancer. In this study, a natural ligand of FGFR, an engineered variant of fibroblast growth factor 1 (FGF1V), was conjugated to a potent cytotoxic drug, monomethyl auristatin E (MMAE), and used as a targeting agent for cancer cells overexpressing FGFRs, similar to antibodies in antibody–drug conjugates. The FGF1V–valine–citrulline–MMAE conjugate showed a favorable stability profile, bound FGFRs on the cell surface specifically, and efficiently released the drug (MMAE) upon cleavage by the lysosomal protease cathepsin B. Importantly, the conjugate showed a prominent cytotoxic effect toward cell lines expressing FGFR. FGF1V–vcMMAE was highly cytotoxic at concentrations even an order of magnitude lower than those found for free MMAE. This effect was FGFR-specific as cells lacking FGFR did not show any increased mortality. PMID:27563235

  13. Characterization of cholesterol homeostasis in sphingosine-1-phosphate lyase-deficient fibroblasts reveals a Niemann-Pick disease type C-like phenotype with enhanced lysosomal Ca2+ storage

    PubMed Central

    Vienken, Hans; Mabrouki, Nathalie; Grabau, Katja; Claas, Ralf Frederik; Rudowski, Agnes; Schömel, Nina; Pfeilschifter, Josef; Lütjohann, Dieter; van Echten-Deckert, Gerhild; Meyer zu Heringdorf, Dagmar

    2017-01-01

    Sphingosine-1-phosphate (S1P) lyase irreversibly cleaves S1P, thereby catalysing the ultimate step of sphingolipid degradation. We show here that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1−/−-MEFs), in which S1P and sphingosine accumulate, have features of Niemann-Pick disease type C (NPC) cells. In the presence of serum, overall cholesterol content was elevated in Sgpl1−/−-MEFs, due to upregulation of the LDL receptor and enhanced cholesterol uptake. Despite this, activation of sterol regulatory element-binding protein-2 was increased in Sgpl1−/−-MEFs, indicating a local lack of cholesterol at the ER. Indeed, free cholesterol was retained in NPC1-containing vesicles, which is a hallmark of NPC. Furthermore, upregulation of amyloid precursor protein in Sgpl1−/−-MEFs was mimicked by an NPC1 inhibitor in Sgpl1+/+-MEFs and reduced by overexpression of NPC1. Lysosomal pH was not altered by S1P lyase deficiency, similar to NPC. Interestingly, lysosomal Ca2+ content and bafilomycin A1-induced [Ca2+]i increases were enhanced in Sgpl1−/−-MEFs, contrary to NPC. These results show that both a primary defect in cholesterol trafficking and S1P lyase deficiency cause overlapping phenotypic alterations, and challenge the present view on the role of sphingosine in lysosomal Ca2+ homeostasis. PMID:28262793

  14. Cubilin, a high affinity receptor for fibroblast growth factor 8, is required for cell survival in the developing vertebrate head.

    PubMed

    Cases, Olivier; Perea-Gomez, Aitana; Aguiar, Diego P; Nykjaer, Anders; Amsellem, Sabine; Chandellier, Jacqueline; Umbhauer, Muriel; Cereghini, Silvia; Madsen, Mette; Collignon, Jérôme; Verroust, Pierre; Riou, Jean-François; Creuzet, Sophie E; Kozyraki, Renata

    2013-06-07

    Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity.

  15. Cubilin, a High Affinity Receptor for Fibroblast Growth Factor 8, Is Required for Cell Survival in the Developing Vertebrate Head*

    PubMed Central

    Cases, Olivier; Perea-Gomez, Aitana; Aguiar, Diego P.; Nykjaer, Anders; Amsellem, Sabine; Chandellier, Jacqueline; Umbhauer, Muriel; Cereghini, Silvia; Madsen, Mette; Collignon, Jérôme; Verroust, Pierre; Riou, Jean-François; Creuzet, Sophie E.; Kozyraki, Renata

    2013-01-01

    Cubilin (Cubn) is a multiligand endocytic receptor critical for the intestinal absorption of vitamin B12 and renal protein reabsorption. During mouse development, Cubn is expressed in both embryonic and extra-embryonic tissues, and Cubn gene inactivation results in early embryo lethality most likely due to the impairment of the function of extra-embryonic Cubn. Here, we focus on the developmental role of Cubn expressed in the embryonic head. We report that Cubn is a novel, interspecies-conserved Fgf receptor. Epiblast-specific inactivation of Cubn in the mouse embryo as well as Cubn silencing in the anterior head of frog or the cephalic neural crest of chick embryos show that Cubn is required during early somite stages to convey survival signals in the developing vertebrate head. Surface plasmon resonance analysis reveals that fibroblast growth factor 8 (Fgf8), a key mediator of cell survival, migration, proliferation, and patterning in the developing head, is a high affinity ligand for Cubn. Cell uptake studies show that binding to Cubn is necessary for the phosphorylation of the Fgf signaling mediators MAPK and Smad1. Although Cubn may not form stable ternary complexes with Fgf receptors (FgfRs), it acts together with and/or is necessary for optimal FgfR activity. We propose that plasma membrane binding of Fgf8, and most likely of the Fgf8 family members Fgf17 and Fgf18, to Cubn improves Fgf ligand endocytosis and availability to FgfRs, thus modulating Fgf signaling activity. PMID:23592779

  16. Cancer-associated fibroblasts in digestive tumors

    PubMed Central

    Huang, Lei; Xu, A-Man; Liu, Sha; Liu, Wei; Li, Tuan-Jie

    2014-01-01

    The significant influence of tumor stroma on malignant cells has been extensively investigated in this era of targeted therapy. The tumor microenvironment, as a dynamic system, is orchestrated by various cells including tumor vascular composing cells, inflammatory cells and fibroblasts. As a major and important component in tumor stroma, increasing evidence has shown that spindle-shaped cancer-associated fibroblasts (CAFs) are a significant modifier of cancer evolution, and promote tumorigenesis, tumor invasion and metastasis by stimulating angiogenesis, malignant cell survival, epithelial-mesenchymal transition (EMT) and proliferation via direct cell-to-cell contact or secretion of soluble factors in most digestive solid tumors. CAFs are thought to be activated, characterized by the expression of α-smooth muscle actin, fibroblast activated protein, fibroblast specific protein, vimentin, fibronectin, etc. They are hypothesized to originate from normal or aged fibroblasts, bone marrow-derived mesenchymal cells, or vascular endothelial cells. EMT may also be an important process generating CAFs, and most probably, CAFs may originate from multiple cells. A close link exists between EMT, tumor stem cells, and chemo-resistance of tumor cells, which is largely orchestrated by CAFs. CAFs significantly induce immunosuppression, and may be a prognostic marker in various malignancies. Targeted therapy toward CAFs has displayed promising anticancer efficacy, which further reinforces the necessity to explore the relationship between CAFs and their hosts. PMID:25548479

  17. Laminin-511 expression is associated with the functionality of feeder cells in human embryonic stem cell culture.

    PubMed

    Hongisto, Heidi; Vuoristo, Sanna; Mikhailova, Alexandra; Suuronen, Riitta; Virtanen, Ismo; Otonkoski, Timo; Skottman, Heli

    2012-01-01

    Fibroblast feeder cells play an important role in supporting the derivation and long term culture of undifferentiated, pluripotent human embryonic stem cells (hESCs). The feeder cells secrete various growth factors and extracellular matrix (ECM) proteins into extracellular milieu. However, the roles of the feeder cell-secreted factors are largely unclear. Animal feeder cells and use of animal serum also make current feeder cell culture conditions unsuitable for derivation of clinical grade hESCs. We established xeno-free feeder cell lines using human serum (HS) and studied their function in hESC culture. While human foreskin fibroblast (hFF) feeder cells were clearly hESC supportive, none of the established xeno-free human dermal fibroblast (hDF) feeder cells were able to maintain undifferentiated hESC growth. The two fibroblast types were compared for their ECM protein synthesis, integrin receptor expression profiles and key growth factor secretion. We show that hESC supportive feeder cells produce laminin-511 and express laminin-binding integrins α3ß1, α6ß1 and α7ß1. These results indicate specific laminin isoforms and integrins in maintenance of hESC pluripotency in feeder-dependent cultures. In addition, several genes with a known or possible role for hESC pluripotency were differentially expressed in distinct feeder cells.

  18. Acute loss of Cited2 impairs Nanog expression and decreases self-renewal of mouse embryonic stem cells.

    PubMed

    Kranc, Kamil R; Oliveira, Daniel V; Armesilla-Diaz, Alejandro; Pacheco-Leyva, Ivette; Catarina Matias, Ana; Luisa Escapa, Ana; Subramani, Chithra; Wheadon, Helen; Trindade, Marlene; Nichols, Jennifer; Kaji, Keisuke; Enver, Tariq; Bragança, José

    2015-03-01

    Identifying novel players of the pluripotency gene regulatory network centered on Oct4, Sox2, and Nanog as well as delineating the interactions within the complex network is key to understanding self-renewal and early cell fate commitment of embryonic stem cells (ESC). While overexpression of the transcriptional regulator Cited2 sustains ESC pluripotency, its role in ESC functions remains unclear. Here, we show that Cited2 is important for proliferation, survival, and self-renewal of mouse ESC. We position Cited2 within the pluripotency gene regulatory network by defining Nanog, Tbx3, and Klf4 as its direct targets. We also demonstrate that the defects caused by Cited2 depletion are, at least in part, rescued by Nanog constitutive expression. Finally, we demonstrate that Cited2 is required for and enhances reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells.

  19. Promotion of human early embryonic development and blastocyst outgrowth in vitro using autocrine/paracrine growth factors.

    PubMed

    Kawamura, Kazuhiro; Chen, Yuan; Shu, Yimin; Cheng, Yuan; Qiao, Jie; Behr, Barry; Pera, Renee A Reijo; Hsueh, Aaron J W

    2012-01-01

    Studies using animal models demonstrated the importance of autocrine/paracrine factors secreted by preimplantation embryos and reproductive tracts for embryonic development and implantation. Although in vitro fertilization-embryo transfer (IVF-ET) is an established procedure, there is no evidence that present culture conditions are optimal for human early embryonic development. In this study, key polypeptide ligands known to be important for early embryonic development in animal models were tested for their ability to improve human early embryo development and blastocyst outgrowth in vitro. We confirmed the expression of key ligand/receptor pairs in cleavage embryos derived from discarded human tri-pronuclear zygotes and in human endometrium. Combined treatment with key embryonic growth factors (brain-derived neurotrophic factor, colony-stimulating factor, epidermal growth factor, granulocyte macrophage colony-stimulating factor, insulin-like growth factor-1, glial cell-line derived neurotrophic factor, and artemin) in serum-free media promoted >2.5-fold the development of tri-pronuclear zygotes to blastocysts. For normally fertilized embryos, day 3 surplus embryos cultured individually with the key growth factors showed >3-fold increases in the development of 6-8 cell stage embryos to blastocysts and >7-fold increase in the proportion of high quality blastocysts based on Gardner's criteria. Growth factor treatment also led to a 2-fold promotion of blastocyst outgrowth in vitro when day 7 surplus hatching blastocysts were used. When failed-to-be-fertilized oocytes were used to perform somatic cell nuclear transfer (SCNT) using fibroblasts as donor karyoplasts, inclusion of growth factors increased the progression of reconstructed SCNT embryos to >4-cell stage embryos. Growth factor supplementation of serum-free cultures could promote optimal early embryonic development and implantation in IVF-ET and SCNT procedures. This approach is valuable for infertility

  20. Pro-inflammatory phenotype of COPD fibroblasts not compatible with repair in COPD lung.

    PubMed

    Zhang, Jing; Wu, Lian; Qu, Jie-ming; Bai, Chun-xue; Merrilees, Mervyn J; Black, Peter N

    2012-07-01

    Chronic obstructive pulmonary disease (COPD) is characterized by loss of elastic fibres from small airways and alveolar walls, with the decrease in elastin increasing with disease severity. It is unclear why there is a lack of repair of elastic fibres. We have examined fibroblasts cultured from lung tissue from subjects with or without COPD to determine if the secretory profile explains lack of tissue repair. In this study, fibroblasts were cultured from lung parenchyma of patients with mild COPD [Global initiative for chronic Obstructive Lung Disease (GOLD) 1, n= 5], moderate to severe COPD (GOLD 2-3, n= 12) and controls (non-COPD, n= 5). Measurements were made of proliferation, senescence-associated β-galactosidase-1, mRNA expression of IL-6, IL-8, MMP-1, tropoelastin and versican, and protein levels for IL-6, IL-8, PGE(2,) tropoelastin, insoluble elastin, and versican. GOLD 2-3 fibroblasts proliferated more slowly (P < 0.01), had higher levels of senescence-associated β-galactosidase-1 (P < 0.001) than controls and showed significant increases in mRNA and/or protein for IL-6 (P < 0.05), IL-8 (P < 0.01), MMP-1 (P < 0.05), PGE(2) (P < 0.05), versican (P < 0.05) and tropoelastin (P < 0.05). mRNA expression and/or protein levels of tropoelastin (P < 0.01), versican (P < 0.05), IL-6 (P < 0.05) and IL-8 (P < 0.05) were negatively correlated with FEV1% of predicted. Insoluble elastin was not increased. In summary, fibroblasts from moderate to severe COPD subjects display a secretory phenotype with up-regulation of inflammatory molecules including the matrix proteoglycan versican, and increased soluble, but not insoluble, elastin. Versican inhibits assembly of tropoelastin into insoluble elastin and we conclude that the pro-inflammatory phenotype of COPD fibroblasts is not compatible with repair of elastic fibres.

  1. Generation of KCL017 research grade human embryonic stem cell line carrying a mutation in VHL gene

    PubMed Central

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL017 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting splicing site of the VHL gene encoding von Hippel–Lindau tumor suppressor E3 ubiquitin protein ligase (676 + 3 A > T). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345980

  2. Generation of KCL016 research grade human embryonic stem cell line carrying a mutation in VHL gene.

    PubMed

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

    2016-01-01

    The KCL016 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting splicing site of the VHL gene encoding von Hippel-Lindau tumor suppressor E3 ubiquitin protein ligase (676+3A>T). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  3. Generation of KCL016 research grade human embryonic stem cell line carrying a mutation in VHL gene

    PubMed Central

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

    2016-01-01

    The KCL016 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting splicing site of the VHL gene encoding von Hippel–Lindau tumor suppressor E3 ubiquitin protein ligase (676 + 3A > T). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345783

  4. Generation of KCL017 research grade human embryonic stem cell line carrying a mutation in VHL gene.

    PubMed

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-03-01

    The KCL017 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation affecting splicing site of the VHL gene encoding von Hippel-Lindau tumor suppressor E3 ubiquitin protein ligase (676+3A>T). The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays.

  5. Generation of KCL024 research grade human embryonic stem cell line carrying a mutation in NF1 gene

    PubMed Central

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL024 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neurofibromin (c.3739–3742 ∆ TTTG). Mutations in this gene have been linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345975

  6. Generation of KCL025 research grade human embryonic stem cell line carrying a mutation in NF1 gene

    PubMed Central

    Hewitson, Heema; Wood, Victoria; Kadeva, Neli; Cornwell, Glenda; Codognotto, Stefano; Stephenson, Emma; Ilic, Dusko

    2016-01-01

    The KCL025 human embryonic stem cell line was derived from an embryo donated for research that carried an autosomal dominant mutation in the NF1 gene encoding neurofibromin (c.3739–3742 ΔTTTG). Mutations in this gene have been linked to neurofibromatosis type 1, juvenile myelomonocytic leukemia and Watson syndrome. The ICM was isolated using laser microsurgery and plated on γ-irradiated human foreskin fibroblasts. Both the derivation and cell line propagation were performed in an animal product-free environment. Pluripotent state and differentiation potential were confirmed by in vitro assays. PMID:27345978

  7. Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

    SciTech Connect

    Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

    1982-02-01

    The metabolism of (1,2,6,7-3H)testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC.

  8. Divergent fibroblast growth factor signaling pathways in lung fibroblast subsets: where do we go from here?

    PubMed

    Ruiz-Camp, Jordi; Morty, Rory E

    2015-10-15

    Lung fibroblasts play a key role in postnatal lung development, namely, the formation of the alveolar gas exchange units, through the process of secondary septation. Although evidence initially highlighted roles for fibroblasts in the production and remodeling of the lung extracellular matrix, more recent studies have described the presence of different fibroblast subsets in the developing lung. These subsets include myofibroblasts and lipofibroblasts and their precursors. These cells are believed to play different roles in alveologenesis and are localized to different regions of the developing septa. The precise roles played by these different fibroblast subsets remain unclear. Understanding the signaling pathways that control the discrete functions of these fibroblast subsets would help to clarify the roles and the regulation of lung fibroblasts during lung development. Here, we critically evaluate a recent report that described divergent fibroblast growth factor (FGF) signaling pathways in two different subsets of lung fibroblasts that express different levels of green fluorescent protein (GFP) driven by the platelet-derived growth factor receptor-α promoter. The GFP expression was used as a surrogate for lipofibroblasts (GFP(low)) and myofibroblasts (GFP(high)). It was suggested that Fgf10/Fgf1 and Fgf18/Fgfr3 autocrine pathways may be operative in GFP(low) and GFP(high) cells, respectively, and that these pathways might regulate the proliferation and migration of different fibroblast subsets during alveologenesis. These observations lay important groundwork for the further exploration of FGF function during normal lung development, as well as in aberrant lung development associated with bronchopulmonary dysplasia.

  9. Trisomy 8 restricted to cultured fibroblasts.

    PubMed Central

    Niss, R; Passarge, E

    1976-01-01

    In the course of re-examing cultured fibroblasts stored in liquid nitrogen from a patient with developmental retardation, solitary left kidney, and Wilms tumour, a cell line trisomic for chromosome 8 was found. Trisomy 8 was restricted to fibroblasts in the first 22 subcultures and was absent in later passages as well as in lymphocytes. A familial pericentric inversion of chromosome 2 was observed in three generations including the propositus but was though to be unrelated to the clinical problem. Multiple spontaneous chromosomal rearrangements were seen in several late subcultures. Images PMID:180293

  10. [Morphological fibroblastic changes in cytomegalovirus infection].

    PubMed

    Parkhomenko, Iu V; Solnyshkova, T G; Tishkivich, O A; Shakhgil'dian, V I; Nikonova, E A

    2006-01-01

    Cytomegalovirus (CMV) infection is widely spread among population. While immunocompetent patients suffer rarely from this virus, it can lead to a lethal outcome in immunocompromised patients. An electron microscopic study has detected fibroblastic morphological changes of a definite cytodestructive character. The nuclei of some fibroblasts have chromatine condensation. A clear zone arising due to vacuolization near this inclusion may reflect nuclear rearrangement leading to further CMV metamorphosis of the cell. This metamorphosis is characteristic of the changes developing in the cells of different parenchymatous organs.

  11. Distinct phenotype and therapeutic potential of gingival fibroblasts.

    PubMed

    Häkkinen, Lari; Larjava, Hannu; Fournier, Benjamin P J

    2014-09-01

    Gingiva of the oral mucosa provides a practical source to isolate fibroblasts for therapeutic purposes because the tissue is easily accessible, tissue discards are common during routine clinical procedures and wound healing after biopsy is fast and results in complete wound regeneration with very little morbidity or scarring. In addition, gingival fibroblasts have unique traits, including neural crest origin, distinct gene expression and synthetic properties and potent immunomodulatory functions. These characteristics may provide advantages for certain therapeutic approaches over other more commonly used cells, including skin fibroblasts, both in intraoral and extra-oral sites. However, identity and phenotype of gingival fibroblasts, like other fibroblasts, are still not completely understood. Gingival fibroblasts are phenotypically heterogeneous, and these…fibroblast subpopulations may play different roles in tissue maintenance, regeneration and pathologies. The purpose of this review is to summarize what is currently known about gingival fibroblasts, their distinct potential for tissue regeneration and their potential therapeutic uses in the future.

  12. Electromechanical integration of cardiomyocytes derived from human embryonic stem cells.

    PubMed

    Kehat, Izhak; Khimovich, Leonid; Caspi, Oren; Gepstein, Amira; Shofti, Rona; Arbel, Gil; Huber, Irit; Satin, Jonathan; Itskovitz-Eldor, Joseph; Gepstein, Lior

    2004-10-01

    Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.

  13. The neuro-steroid, 5-androstene 3β,17α diol; induces endoplasmic reticulum stress and autophagy through PERK/eIF2α signaling in malignant glioma cells and transformed fibroblasts.

    PubMed

    Jia, Wentao; Loria, Roger M; Park, Margaret A; Yacoub, Adly; Dent, Paul; Graf, Martin R

    2010-12-01

    In this study, we identified a mechanism by which the neuro-steroid, 5-androstene 3β,17α diol (17α-AED) induces autophagy in human malignant glioma cells and transformed fibroblasts. 17α-AED treatment induced endoplasmic reticulum (ER) stress, identified by the partial activation of an unfolded protein response in T98G, U87MG, U251MG, LN-18, LN-229 and LN-Z308 glioma cell lines. In this regard, there were increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose-regulated protein of 78kDa transcripts but no splicing of X-box-binding protein 1 mRNA or processing of activating transcription factor-6 in glioma cells treated with the neuro-steroid. 17α-AED induced eukaryotic translational initiation factor 2α (eIF2α) phosphorylation in glioma cells which correlated with microtubule-associated protein-light chain 3 (LC3) conversion from LC3-I to -II. In transformed murine embryonic fibroblasts (MEFs) that are deficient of eIF2α function or T98G glioma cells transfected with a dominant-negative eIF2α construct, 17α-AED induced LC3 conversion was significantly reduced as compared to control cells. Neuro-steroid treatment caused the activation of the eIF2α kinase, protein kinase-like ER kinase (PERK) but not other eIF2α kinases in glioma cells. Moreover, eIF2α phosphorylation and LC3 conversion, in response to 17α-AED treatment, was blocked in MEFs that lacked PERK activity. T98G cells transfected with a dominant-negative PERK construct exhibited an attenuated response to neuro-steroid treatment in terms of decreases in: eIF2α activation; CHOP expression; the incidence of autophagy; and cytotoxicity. These results demonstrate that ER stress is linked to 17α-AED induced autophagy by PERK/eIF2α signaling in human malignant glioma cells and transformed fibroblasts.

  14. Stromal fibroblasts facilitate cancer cell invasion by a novel invadopodia-independent matrix degradation process

    PubMed Central

    Krueger, Eugene W.; Chen, Jing; Qiang, Li; McNiven, Mark A.

    2015-01-01

    Metastatic invasion of tumors into peripheral tissues is known to rely upon protease-mediated degradation of the surrounding stroma. This remodeling process utilizes complex, actin-based, specializations of the plasma membrane termed invadopodia that act both to sequester and release matrix metalloproteinases. Here we report that cells of mesenchymal origin, including tumor-associated fibroblasts, degrade substantial amounts of surrounding matrix by a mechanism independent of conventional invadopodia. These degradative sites lack the punctate shape of conventional invadopodia to spread along the cell base and are reticular and/or fibrous in character. In marked contrast to invadopodia, this degradation does not require the action of Src kinase, Cdc42, or Dyn2. Rather, inhibition of Dyn2 causes a dramatic upregulation of stromal matrix degradation. Further, expression and activity of matrix metalloproteinases are differentially regulated between tumor cells and stromal fibroblasts. This matrix remodeling by fibroblasts increases the invasive capacity of tumor cells, thereby illustrating how the tumor microenvironment can contribute to metastasis. These findings provide evidence for a novel matrix remodeling process conducted by stromal fibroblasts that is substantially more effective than conventional invadopodia, distinct in structural organization, and regulated by disparate molecular mechanisms. PMID:25982272

  15. ANT2-defective fibroblasts exhibit normal mitochondrial bioenergetics

    PubMed Central

    Prabhu, Dolly; Goldstein, Amy C.; El-Khoury, Riyad; Rak, Malgorzata; Edmunds, Lia; Rustin, Pierre; Vockley, Jerry; Schiff, Manuel

    2015-01-01

    Adenine nucleotide translocase 2 (ANT2) transports glycolytic ATP across the inner mitochondrial membrane. Patients with ANT2 deletion were recently reported. We aimed at characterizing mitochondrial functions in ANT2-defective fibroblasts. In spite of ANT2 expression in fibroblasts, we observed no difference between ANT2-defective and control fibroblasts for mitochondrial respiration, respiratory chain activities, mitochondrial membrane potential and intracellular ATP levels. This indicates that ANT2 insufficiency does not alter fibroblast basal mitochondrial bioenergetics. PMID:26000237

  16. Polar Bodies – more a lack of understanding than a lack of respect

    PubMed Central

    Schmerler, Samuel; Wessel, Gary

    2011-01-01

    Polar bodies are as diverse as the organisms that produce them. Although in many animals these cells often die following meiotic maturation of the oocyte, in other organisms they are an essential and diverse part of embryonic development. Here we highlight some of this diversity and summarize the evolutionary basis for their utility. PMID:21268179

  17. Histone H1 Depletion Impairs Embryonic Stem Cell Differentiation

    PubMed Central

    Cao, Kaixiang; Krauth, Beth; Ho, Po-Yi; Medrzycki, Magdalena; Berhe, Dawit T.; Pan, Chenyi; McDevitt, Todd C.; Fan, Yuhong

    2012-01-01

    Pluripotent embryonic stem cells (ESCs) are known to possess a relatively open chromatin structure; yet, despite efforts to characterize the chromatin signatures of ESCs, the role of chromatin compaction in stem cell fate and function remains elusive. Linker histone H1 is important for higher-order chromatin folding and is essential for mammalian embryogenesis. To investigate the role of H1 and chromatin compaction in stem cell pluripotency and differentiation, we examine the differentiation of embryonic stem cells that are depleted of multiple H1 subtypes. H1c/H1d/H1e triple null ESCs are more resistant to spontaneous differentiation in adherent monolayer culture upon removal of leukemia inhibitory factor. Similarly, the majority of the triple-H1 null embryoid bodies (EBs) lack morphological structures representing the three germ layers and retain gene expression signatures characteristic of undifferentiated ESCs. Furthermore, upon neural differentiation of EBs, triple-H1 null cell cultures are deficient in neurite outgrowth and lack efficient activation of neural markers. Finally, we discover that triple-H1 null embryos and EBs fail to fully repress the expression of the pluripotency genes in comparison with wild-type controls and that H1 depletion impairs DNA methylation and changes of histone marks at promoter regions necessary for efficiently silencing pluripotency gene Oct4 during stem cell differentiation and embryogenesis. In summary, we demonstrate that H1 plays a critical role in pluripotent stem cell differentiation, and our results suggest that H1 and chromatin compaction may mediate pluripotent stem cell differentiation through epigenetic repression of the pluripotency genes. PMID:22589736

  18. Plasma treated polyethylene grafted with adhesive molecules for enhanced adhesion and growth of fibroblasts.

    PubMed

    Rimpelová, Silvie; Kasálková, Nikola Slepičková; Slepička, Petr; Lemerová, Helena; Švorčík, Václav; Ruml, Tomáš

    2013-04-01

    The cell-material interface plays a crucial role in the interaction of cells with synthetic materials for biomedical use. The application of plasma for tailoring polymer surfaces is of abiding interest and holds a great promise in biomedicine. In this paper, we describe polyethylene (PE) surface tuning by Ar plasma irradiating and subsequent grafting of the chemically active PE surface with adhesive proteins or motives to support cell attachment. These simple modifications resulted in changed polymer surface hydrophilicity, roughness and morphology, which we thoroughly characterized. The effect of our modifications on adhesion and growth was tested in vitro using mouse embryonic fibroblasts (NIH 3T3 cell line). We demonstrate that the plasma treatment of PE had a positive effect on the adhesion, spreading, homogeneity of distribution and moderately on proliferation activity of NIH 3T3 cells. This effect was even more pronounced on PE coated with biomolecules.

  19. Fibroblast growth factors: from molecular evolution to roles in development, metabolism and disease.

    PubMed

    Itoh, Nobuyuki; Ornitz, David M

    2011-02-01

    Fibroblast growth factors (FGFs) are a family of structurally related polypeptides that are essential for embryonic development and that function postnatally as homoeostatic factors, in the response to injury, in the regulation of electrical excitability of cells and as hormones that regulate metabolism. In humans, FGF signalling is involved in developmental, neoplastic, metabolic and neurological diseases. Fgfs have been identified in metazoans but not in unicellular organisms. In vertebrates, FGFs can be classified as having intracrine, paracrine and endocrine functions. Paracrine and endocrine FGFs act via cell-surface FGF receptors (FGFRs); while, intracrine FGFs act independent of FGFRs. The evolutionary history of the Fgf family indicates that an intracrine Fgf is the likely ancestor of the Fgf family. During metazoan evolution, the Fgf family expanded in two phases, after the separation of protostomes and deuterostomes and in the evolution of early vertebrates. These expansions enabled FGFs to acquire diverse actions and functions.

  20. Transcription factor–mediated reprogramming of fibroblasts to expandable, myelinogenic oligodendrocyte progenitor cells

    PubMed Central

    Najm, Fadi J.; Lager, Angela M.; Zaremba, Anita; Wyatt, Krysta; Caprariello, Andrew V.; Factor, Daniel C.; Karl, Robert T.; Maeda, Tadao; Miller, Robert H.; Tesar, Paul J.

    2013-01-01

    Cell-based therapies for myelin disorders, such as multiple sclerosis and leukodystrophies, require technologies to generate functional oligodendrocyte progenitor cells. Here we describe direct conversion of mouse embryonic and lung fibroblasts to “induced” oligodendrocyte progenitor cells (iOPCs) using sets of either eight or three defined transcription factors. iOPCs exhibit a bipolar morphology and global gene expression profile consistent with bona fide OPCs. They can be expanded in vitro for at least five passages while retaining the ability to differentiate into multiprocessed oligodendrocytes. When transplanted to hypomyelinated mice, iOPCs are capable of ensheathing host axons and generating compact myelin. Lineage conversion of somatic cells to expandable iOPCs provides a strategy to study the molecular control of oligodendrocyte lineage identity and may facilitate neurological disease modeling and autologous remyelinating therapies. PMID:23584611

  1. Role of MEF feeder cells in direct reprogramming of mousetail-tip fibroblasts.

    PubMed

    Chen, Mengfei; Sun, Xuerong; Jiang, Ruzhang; Shen, Wenjuan; Zhong, Xiufeng; Liu, Bingqian; Qi, Ying; Huang, Bing; Xiang, Andy Peng; Ge, Jian

    2009-12-01

    Pluripotent stem cells can be induced from somatic cells by the transcription factors Oct3/4, Sox2, c-Myc and Klf4 when co-cultured with mouse embryonic fibroblast (MEF) feeder cells. To date, the role of the feeder cells in the reprogramming process remains unclear. In this study, using a comparative analysis, we demonstrated that MEF feeder cells did not accelerate reprogramming or increase the frequency of induced pluripotent stem (iPS) cell colonies. However, feeder conditions did improve the growth of primary iPS colonies and were necessary for passaging the primary colonies after reprogramming was achieved. We further developed a feeder-free culture system for supporting iPS growth and sustaining pluripotency by adding bFGF and activin A (bFA) to the medium. These data will facilitate the generation of human iPS cells without animal feeders for regenerative medicine.

  2. The influence of Pyk2 on the mechanical properties in fibroblasts

    SciTech Connect

    Klemm, Anna H.; Kienle, Sandra; Rheinlaender, Johannes; Schaeffer, Tilman E.; Goldmann, Wolfgang H.

    2010-03-19

    The cell surface receptor integrin is involved in signaling mechanical stresses via the focal adhesion complex (FAC) into the cell. Within FAC, the focal adhesion kinase (FAK) and Pyk2 are believed to act as important scaffolding proteins. Based on the knowledge that many signal transducing molecules are transiently immobilized within FAC connecting the cytoskeleton with integrins, we applied magnetic tweezer and atomic force microscopic measurements to determine the influence of FAK and Pyk2 in cells mechanically. Using mouse embryonic fibroblasts (MEF; FAK{sup +/+}, FAK{sup -/-}, and siRNA-Pyk2 treated FAK{sup -/-} cells) provided a unique opportunity to describe the function of FAK and Pyk2 in more detail and to define their influence on FAC and actin distribution.

  3. Exogenous fibroblast growth factor 8 rescues development of mouse diastemal vestigial tooth ex vivo.

    PubMed

    Li, Lu; Yuan, Guohua; Liu, Chao; Zhang, Lu; Zhang, Yanding; Chen, YiPing; Chen, Zhi

    2011-06-01

    Regression of vestigial tooth buds results in the formation of the toothless diastema, a unique feature of the mouse dentition. Revitalization of the diastemal vestigial tooth bud provides an excellent model for studying tooth regeneration and replacement. It has been previously shown that suppression of fibroblast growth factor (FGF) signaling in the diastema results in vestigial tooth bud regression. In this study, we report that application of exogenous FGF8 to the mouse embryonic diastemal region rescues diastemal tooth development. However, this rescue of diastemal tooth development occurs only in an isolated diastemal regions and not in the mandibular quadrant, which includes the incisor and molar germs. FGF8 promotes cell proliferation and inhibits apoptosis in diastemal tooth epithelium, and revitalizes the tooth developmental program, as evidenced by the expression of genes critical for normal tooth development. Our results also support the idea that the adjacent tooth germs contribute to the suppression of diastemal vestigial tooth buds by means of multiple signals.

  4. Use of fibroblast growth factor 2 for expansion of chondrocytes and tissue engineering

    NASA Technical Reports Server (NTRS)

    Martin, Ivan (Inventor); Freed, Lisa E. (Inventor); Langer, Robert (Inventor); Vunjak-Novakovic, Gordana (Inventor)

    2003-01-01

    The present invention provides an improved method for expanding cells for use in tissue engineering. In particular the method provides specific biochemical factors to supplement cell culture medium during the expansion process in order to reproduce events occurring during embryonic development with the goal of regenerating tissue equivalents that resemble natural tissues both structurally and functionally. These specific biochemical factors improve proliferation of the cells and are capable of de-differentiation mature cells isolated from tissue so that the differentiation potential of the cells is preserved. The bioactive molecules also maintain the responsiveness of the cells to other bioactive molecules. Specifically, the invention provides methods for expanding chondrocytes in the presence of fibroblast growth factor 2 for use in regeneration of cartilage tissue.

  5. Gaining myocytes or losing fibroblasts: Challenges in cardiac fibroblast reprogramming for infarct repair.

    PubMed

    Nagalingam, Raghu S; Safi, Hamza A; Czubryt, Michael P

    2016-04-01

    Unlike most somatic tissues, the heart possesses a very limited inherent ability to repair itself following damage. Attempts to therapeutically salvage the myocardium after infarction, either by sparing surviving myocytes or by injection of exogenous cells of varied provenance, have met with limited success. Cardiac fibroblasts are numerous, resistant to hypoxia, and amenable to phenotype reprogramming to cardiomyocytes - a potential panacea to an intractable problem. However, the long-term effects of mass conversion of fibroblasts are as-yet unknown. Since fibroblasts play key roles in normal cardiac function, treating these cells as a ready source of replacements for myocytes may have the effect of swapping one problem for another. This review briefly examines the roles of cardiac fibroblasts, recaps the strides made so far in their reprogramming to cardiomyocytes both in vitro and in vivo, and discusses the potential ramifications of large-scale cellular identity swapping. While such therapy offers great promise, the potential repercussions require consideration and careful study.

  6. Embryonic requirements for ErbB signaling in neural crest development and adult pigment pattern formation

    PubMed Central

    Budi, Erine H.; Patterson, Larissa B.; Parichy, David M.

    2009-01-01

    SUMMARY Vertebrate pigment cells are derived from neural crest cells and are a useful system for studying neural crest-derived traits during post-embryonic development. In zebrafish, neural crest-derived melanophores differentiate during embryogenesis to produce stripes in the early larva. Dramatic changes to the pigment pattern occur subsequently during the larva-to-adult transformation, or metamorphosis. At this time, embryonic melanophores are replaced by newly differentiating metamorphic melanophores that form the adult stripes. Mutants with normal embryonic/early larval pigment patterns but defective adult patterns identify factors required uniquely to establish, maintain, or recruit the latent precursors to metamorphic melanophores. We show that one such mutant, picasso, lacks most metamorphic melanophores and results from mutations in the ErbB gene erbb3b, encoding an EGFR-like receptor tyrosine kinase. To identify critical periods for ErbB activities, we treated fish with pharmacological ErbB inhibitors and also knocked-down erbb3b by morpholino injection. These analyses reveal an embryonic critical period for ErbB signaling in promoting later pigment pattern metamorphosis, despite the normal patterning of embryonic/early larval melanophores. We further demonstrate a peak requirement during neural crest migration that correlates with early defects in neural crest pathfinding and peripheral ganglion formation. Finally, we show that erbb3b activities are both autonomous and non-autonomous to the metamorphic melanophore lineage. These data identify a very early, embryonic, requirement for erbb3b in the development of much later metamorphic melanophores, and suggest complex modes by which ErbB signals promote adult pigment pattern development. PMID:18508863

  7. Lack of Set Theory Relevant Prerequisite Knowledge

    ERIC Educational Resources Information Center

    Dogan-Dunlap, Hamide

    2006-01-01

    Many students struggle with college mathematics topics due to a lack of mastery of prerequisite knowledge. Set theory language is one such prerequisite for linear algebra courses. Many students' mistakes on linear algebra questions reveal a lack of mastery of set theory knowledge. This paper reports the findings of a qualitative analysis of a…

  8. Scaffolding for Three-Dimensional Embryonic Vasculogenesis

    NASA Astrophysics Data System (ADS)

    Kraehenbuehl, Thomas P.; Aday, Sezin; Ferreira, Lino S.

    Biomaterial scaffolds have great potential to support efficient vascular differentiation of embryonic stem cells. Vascular cell fate-specific biochemical and biophysical cues have been identified and incorporated into three-dimensional (3D) biomaterials to efficiently direct embryonic vasculogenesis. The resulting vascular-like tissue can be used for regenerative medicine applications, further elucidation of biophysical and biochemical cues governing vasculogenesis, and drug discovery. In this chapter, we give an overview on the following: (1) developmental cues for directed differentiation of human embryonic stem cells (hESCs) into vascular cells, (2) 3D vascular differentiation in embryoid bodies (EBs), (3) preparation of 3D scaffolds for the vascular differentiation of hESCs, and (4) the most significant studies combining scaffolding and hESCs for development of vascular-like tissue.

  9. Looking older: Fibroblast Collapse and Therapeutic Implications

    PubMed Central

    Fisher, Gary J.; Varani, James; Voorhees, John J.

    2010-01-01

    Background Skin appearance is a primary indicator of age. During the last decade, substantial progress has been made towards understanding underlying mechanisms of human skin aging. This understanding provides the basis for current use and new development of anti-aging treatments. Objective To present state of the art knowledge pertaining to mechanisms involved in skin aging, with specific focus on the dermal collagen matrix. Results A major feature of aged skin is fragmentation of the dermal collagen matrix. Fragmentation results from actions of specific enzymes (matrix metalloproteinases), and impairs the structural integrity of the dermis. Fibroblasts that produce and organize the collagen matrix cannot attach to fragmented collagen. Loss of attachment prevents fibroblasts from receiving mechanical information from their support and they collapse. Stretch is critical for normal balanced production of collagen and collagen-degrading enzymes. In aged skin, collapsed fibroblasts produce low levels of collagen and high levels of collagen–degrading enzymes. This imbalance advances the aging process, in a self-perpetuating, never-ending deleterious cycle. Clinically-proven anti-aging treatments such as topical retinoic acid, CO2 laser resurfacing, and intradermal injection of cross-linked hyaluronic acid stimulate production of new undamaged collagen. Attachment of fibroblasts to this new collagen allows stretch, which in turn balances collagen production/degradation and thereby slows the aging process. Conclusion Collagen fragmentation is responsible for loss of structural integrity and impairment of fibroblast function in aged human skin. Treatments that stimulate production of new, non-fragmented collagen should provide substantial improvement to the appearance and health of aged skin. PMID:18490597

  10. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration.

    PubMed

    Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

    2017-03-28

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy.

  11. Rho/Rock cross-talks with transforming growth factor-β/Smad pathway participates in lung fibroblast-myofibroblast differentiation.

    PubMed

    Ji, Hong; Tang, Haiying; Lin, Hongli; Mao, Jingwei; Gao, Lili; Liu, Jia; Wu, Taihua

    2014-11-01

    The differentiation of fibroblasts, which are promoted by transforming growth factor-β (TGF-β)/Smad, is involved in the process of pulmonary fibrosis. The Rho/Rho-associated coiled-coil-forming protein kinase (Rock) pathway may regulate the fibroblast differentiation and myofibroblast expression of α-smooth muscle actin (α-SMA), however, the mechanism is not clear. The aim of the present study was to evaluate the role of Rho/Rock and TGF-β/Smad in TGF-β1-induced lung fibroblasts differentiation. Human embryonic lung fibroblasts were stimulated by TGF-β1, Y-27632 (inhibitor of Rho/Rock signaling) and staurosporine (inhibitor of TGF-β/Smad signaling). The α-SMA expression, cell cycle progression, content of the extracellular matrix (ECM) in cell culture supernatants and the expression of RhoA, RhoC, Rock1 and Smad2 were detected. The results demonstrated that α-SMA-positive cells significantly increased following TGF-β1 stimulation. Rho/Rock and TGF-β/Smad inhibitors suppressed TGF-β1-induced lung fibroblast differentiation. The inhibitors increased G0/G1 and decreased S and G2/M percentages. The concentrations of the ECM proteins in the supernatant were significantly increased by TGF-β1 stimulation, whereas they were decreased by inhibitor stimulation. RhoA, RhoC, Rock1, Smad2 and tissue inhibitor of metalloproteinase-1 were upregulated by TGF-β1 stimulation. The Rho/Rock inhibitor downregulated Smad2 expression and the TGF-β/Smad inhibitor downregulated RhoA, RhoC and Rock1 expression. Therefore, the Rho/Rock pathway and Smad signaling were involved in the process of lung fibroblasts transformation, induced by TGF-β1, to myofibroblasts. The two pathways may undergo cross-talk in the lung fibroblasts differentiation in vitro.

  12. Stepwise proteolytic activation of type I procollagen to collagen within the secretory pathway of tendon fibroblasts in situ.

    PubMed

    Canty-Laird, Elizabeth G; Lu, Yinhui; Kadler, Karl E

    2012-01-15

    Proteolytic cleavage of procollagen I to collagen I is essential for the formation of collagen fibrils in the extracellular matrix of vertebrate tissues. Procollagen is cleaved by the procollagen N- and C-proteinases, which remove the respective N- and C-propeptides from procollagen. Procollagen processing is initiated within the secretory pathway in tendon fibroblasts, which are adept in assembling an ordered extracellular matrix of collagen fibrils in vivo. It was thought that intracellular processing was restricted to the TGN (trans-Golgi network). In the present study, brefeldin A treatment of tendon explant cultures showed that N-proteinase activity is present in the resulting fused ER (endoplasmic reticulum)-Golgi compartment, but that C-proteinase activity is restricted to the TGN in embryonic chick tendon fibroblasts. In late embryonic and postnatal rat tail and postnatal mouse tail tendon, C-proteinase activity was detected in TGN and pre-TGN compartments. Preventing activation of the procollagen N- and C-proteinases with the furin inhibitor Dec-RVKR-CMK (decanoyl-Arg-Val-Lys-Arg-chloromethylketone) indicated that only a fraction of intracellular procollagen cleavage was mediated by newly activated proteinases. In conclusion, the N-propeptides are removed earlier in the secretory pathway than the C-propeptides. The removal of the C-propeptides in post-Golgi compartments most probably indicates preparation of collagen molecules for fibril formation at the cell-matrix interface.

  13. Embryonic stem cell patents and human dignity.

    PubMed

    Resnik, David B

    2007-09-01

    This article examines the assertion that human embryonic stem cells patents are immoral because they violate human dignity. After analyzing the concept of human dignity and its role in bioethics debates, this article argues that patents on human embryos or totipotent embryonic stem cells violate human dignity, but that patents on pluripotent or multipotent stem cells do not. Since patents on pluripotent or multipotent stem cells may still threaten human dignity by encouraging people to treat embryos as property, patent agencies should carefully monitor and control these patents to ensure that patents are not inadvertently awarded on embryos or totipotent stem cells.

  14. Embryonic Stem Cell Patents and Human Dignity

    PubMed Central

    Resnik, David B.

    2009-01-01

    This article examines the assertion that human embryonic stem cells patents are immoral because they violate human dignity. After analyzing the concept of human dignity and its role in bioethics debates, this article argues that patents on human embryos or totipotent embryonic stem cells violate human dignity, but that patents on pluripotent or multipotent stem cells do not. Since patents on pluripotent or multipotent stem cells may still threaten human dignity by encouraging people to treat embryos as property, patent agencies should carefully monitor and control these patents to ensure that patents are not inadvertently awarded on embryos or totipotent stem cells. PMID:17922198

  15. Inner Ear Hair Cell-Like Cells from Human Embryonic Stem Cells

    PubMed Central

    Ronaghi, Mohammad; Nasr, Marjan; Ealy, Megan; Durruthy-Durruthy, Robert; Waldhaus, Joerg; Diaz, Giovanni H.; Joubert, Lydia-Marie; Oshima, Kazuo

    2014-01-01

    In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation. PMID:24512547

  16. Emerin organizes actin flow for nuclear movement and centrosome orientation in migrating fibroblasts.

    PubMed

    Chang, Wakam; Folker, Eric S; Worman, Howard J; Gundersen, Gregg G

    2013-12-01

    In migrating fibroblasts, rearward movement of the nucleus orients the centrosome toward the leading edge. Nuclear movement results from coupling rearward-moving, dorsal actin cables to the nucleus by linear arrays of nesprin-2G and SUN2, termed transmembrane actin-associated nuclear (TAN) lines. A-type lamins anchor TAN lines, prompting us to test whether emerin, a nuclear membrane protein that interacts with lamins and TAN line proteins, contributes to nuclear movement. In fibroblasts depleted of emerin, nuclei moved nondirectionally or completely failed to move. Consistent with these nuclear movement defects, dorsal actin cable flow was nondirectional in cells lacking emerin. TAN lines formed normally in cells lacking emerin and were coordinated with the erratic nuclear movements, although in 20% of the cases, TAN lines slipped over immobile nuclei. Myosin II drives actin flow, and depletion of myosin IIB, but not myosin IIA, showed similar nondirectional nuclear movement and actin flow as in emerin-depleted cells. Myosin IIB specifically coimmunoprecipitated with emerin, and emerin depletion prevented myosin IIB localization near nuclei. These results show that emerin functions with myosin IIB to polarize actin flow and nuclear movement in fibroblasts, suggesting a novel function for the nuclear envelope in organizing directional actin flow and cytoplasmic polarity.

  17. Functional genetic targeting of embryonic kidney progenitor cells ex vivo.

    PubMed

    Junttila, Sanna; Saarela, Ulla; Halt, Kimmo; Manninen, Aki; Pärssinen, Heikki; Lecca, M Rita; Brändli, André W; Sims-Lucas, Sunder; Skovorodkin, Ilya; Vainio, Seppo J

    2015-05-01

    The embryonic mammalian metanephric mesenchyme (MM) is a unique tissue because it is competent to generate the nephrons in response to Wnt signaling. An ex vivo culture in which the MM is separated from the ureteric bud (UB), the natural inducer, can be used as a classic tubule induction model for studying nephrogenesis. However, technological restrictions currently prevent using this model to study the molecular genetic details before or during tubule induction. Using nephron segment-specific markers, we now show that tubule induction in the MM ex vivo also leads to the assembly of highly segmented nephrons. This induction capacity was reconstituted when MM tissue was dissociated into a cell suspension and then reaggregated (drMM) in the presence of human recombinant bone morphogenetic protein 7/human recombinant fibroblast growth factor 2 for 24 hours before induction. Growth factor-treated drMM also recovered the capacity for organogenesis when recombined with the UB. Cell tracking and time-lapse imaging of chimeric drMM cultures indicated that the nephron is not derived from a single progenitor cell. Furthermore, viral vector-mediated transduction of green fluorescent protein was much more efficient in dissociated MM cells than in intact mesenchyme, and the nephrogenic competence of transduced drMM progenitor cells was preserved. Moreover, drMM cells transduced with viral vectors mediating Lhx1 knockdown were excluded from the nephric tubules, whereas cells transduced with control vectors were incorporated. In summary, these techniques allow reproducible cellular and molecular examinations of the mechanisms behind nephrogenesis and kidney organogenesis in an ex vivo organ culture/organoid setting.

  18. Embryonic stem cells: testing the germ-cell theory.

    PubMed

    Hochedlinger, Konrad

    2011-10-25

    The exact cellular origin of embryonic stem cells remains elusive. Now a new study provides compelling evidence that embryonic stem cells, established under conventional culture conditions, originate from a transient germ-cell state.

  19. Effects of Feeder Cells on Dopaminergic Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Zhao, Zhenqiang; Ma, Yanlin; Chen, Zhibin; Liu, Qian; Li, Qi; Kong, Deyan; Yuan, Kunxiong; Hu, Lan; Wang, Tan; Chen, Xiaowu; Peng, Yanan; Jiang, Weimin; Yu, Yanhong; Liu, Xinfeng

    2016-01-01

    Mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (HFFs) are used for the culture of human embryonic stem cells (hESCs). MEFs and HFFs differed in their capacity to support the proliferation and pluripotency of hESCs and could affect cardiac differentiation potential of hESCs. The aim of this study was to evaluate the effect of MEFs and HFFs feeders on dopaminergic differentiation of hESCs lines. To minimize the impact of culture condition variation, two hESCs lines were cultured on mixed feeder cells (MFCs, MEFs: HFFs = 1:1) and HFFs feeder, respectively, and then were differentiated into dopaminergic (DA) neurons under the identical protocol. Dopaminergic differentiation was evaluated by immunocytochemistry, quantitative fluorescent real-time PCR, transmission and scanning electron microscopy, and patch clamp. Our results demonstrated that these hESCs-derived neurons were genuine and functional DA neurons. However, compared to hESCs line on MFCs feeder, hESCs line on HFFs feeder had a higher proportion of tyrosine hydroxylase (TH) positive cells and expressed higher levels of FOXA2, PITX3, NURR1, and TH genes. In addition, the values of threshold intensity and threshold membrane potential of DA neurons from hESCs line on HFFs feeder were lower than those of DA neurons from hESCs line on the MFCs feeder. In conclusion, HFFs feeder not only facilitated the differentiation of hESCs cells into dopaminergic neurons, but also induced hESCs-derived DA neurons to express higher electrophysiological excitability. Therefore, feeder cells could affect not only dopaminergic differentiation potential of different hESCs lines, but also electrophysiological properties of hESCs-derived DA neurons. PMID:28066186

  20. Mast cell differentiation depends on T cells and granule synthesis on fibroblasts.

    PubMed Central

    Davidson, S; Mansour, A; Gallily, R; Smolarski, M; Rofolovitch, M; Ginsburg, H

    1983-01-01

    Mast cell differentiation was generated in the following three experimental situations: (i) infection of mice with Schistosoma Mansoni or with Nippostrongylus brasiliensis and growth of the lymph node cells in the presence of the corresponding helminth antigen; (ii) immunization with horse serum and growth of blood and lymph node cells in the presence of the horse serum; (iii) exposure of T-cell-depleted suspensions of lymph node cells from unimmunized mice to T-cell factor (TCF) released into medium of the young cultures of (i) and (ii). This differentiation was also obtained when lymph node cells from athymic nude mice were exposed to TCF. The cell suspensions were plated on X-irradiated fibroblast monolayers prepared from embryonic mouse skin. Screening of the suspensions before plating on the fibroblasts in culture revealed no young forms of mast cells, and none were present in culture of nude mice lymph node cells maintained without TCF. Primordial appearance of metachromatic granules generally in the golgi zone was first seen in many 'large lymphoid cells' as early as 18 hr after plating. This was followed by increase in the cytoplasm volume, increase in granule number and mitosis, ending at 10-18 days with homogeneous populations of mature mast cells. When the mesenteric lymph node cells from mice infected with the helminths were grown in the absence of fibroblasts but in the presence of the antigen, homogeneous populations of cells with extended cytoplasm, filled with unstained vacuoles developed during days 7-13. These cells did not contain histamine (or at most 0.2 microgram per 10(6) vacuolated cells). When these cells were plated on fibroblast monolayers clear granule formation in all the vacuoles was seen 2 days later. It increased progressively in size and staining intensity, until the vacuoles transformed into typical mast cell granules. By the fourth day the vacuolated cells attained the typical mast cell morphology and the histamine content greatly

  1. A model of electrical conduction in cardiac tissue including fibroblasts.

    PubMed

    Sachse, Frank B; Moreno, A P; Seemann, G; Abildskov, J A

    2009-05-01

    Fibroblasts are abundant in cardiac tissue. Experimental studies suggested that fibroblasts are electrically coupled to myocytes and this coupling can impact cardiac electrophysiology. In this work, we present a novel approach for mathematical modeling of electrical conduction in cardiac tissue composed of myocytes, fibroblasts, and the extracellular space. The model is an extension of established cardiac bidomain models, which include a description of intra-myocyte and extracellular conductivities, currents and potentials in addition to transmembrane voltages of myocytes. Our extension added a description of fibroblasts, which are electrically coupled with each other and with myocytes. We applied the extended model in exemplary computational simulations of plane waves and conduction in a thin tissue slice assuming an isotropic conductivity of the intra-fibroblast domain. In simulations of plane waves, increased myocyte-fibroblast coupling and fibroblast-myocyte ratio reduced peak voltage and maximal upstroke velocity of myocytes as well as amplitudes and maximal downstroke velocity of extracellular potentials. Simulations with the thin tissue slice showed that inter-fibroblast coupling affected rather transversal than longitudinal conduction velocity. Our results suggest that fibroblast coupling becomes relevant for small intra-myocyte and/or large intra-fibroblast conductivity. In summary, the study demonstrated the feasibility of the extended bidomain model and supports the hypothesis that fibroblasts contribute to cardiac electrophysiology in various manners.

  2. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq

    PubMed Central

    Treutlein, Barbara; Lee, Qian Yi; Camp, J. Gray; Mall, Moritz; Koh, Winston; Shariati, Seyed Ali Mohammad; Sim, Sopheak; Neff, Norma F.; Skotheim, Jan M.; Wernig, Marius; Quake, Stephen R.

    2016-01-01

    Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states1–3. However, the intermediates through which individual cells progress are largely undefined. Here we used single-cell RNA-seq4–7 at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts (MEFs) to induced neuronal (iN) cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts suggesting the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation. PMID:27281220

  3. Proliferative response patterns of human fibroblasts after photoinjury with 4,5',8-trimethylpsoralen

    SciTech Connect

    Cohen, S.R.; Carter, D.M.; Gala, M.

    1981-01-01

    The extent of growth suppression and recovery following exposure to 4,5',8-trimethylpsoralen plus uv-A irradiation was studied in 3 diploid human fibroblast strains. Inhibition of cellular proliferation was dose-dependent within the concentration range of TMP that was tested, using a constant level of uv-A. The population generation times for all cell strains were progressively lengthened under these conditions while maximal cell densities were reduced. At 2 to 4 x 10(-7) M TMP in the presence of uv-A, there was a triphasic pattern of growth which consisted of proliferative activity during the first 24 to 36 h, followed by complete growth inhibition for variable periods of time and a recovery period of log phase proliferation that was not as vigorous as measured for untreated cells. There were also declines in the percentage of cells labeled with 3H-Tdr at various times after TMP-uv-A treatment. These measurements were essentially identical for the three fibroblast strains evaluated. In that the cells employed for these investigations were derived from embryonic pulmonary tissue, neonatal foreskin and the buttock skin of an adult male, it seems unlikely that donor age and tissue source were important variables in determining growth response patterns after TMP-uv-A exposure. Because proliferative recovery was attenuated after this photochemical injury, researchers conclude that the biologic effect(s) of TMP-uv-A extend beyond the known period of psoralen-DNA cross-link removal.

  4. Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq.

    PubMed

    Treutlein, Barbara; Lee, Qian Yi; Camp, J Gray; Mall, Moritz; Koh, Winston; Shariati, Seyed Ali Mohammad; Sim, Sopheak; Neff, Norma F; Skotheim, Jan M; Wernig, Marius; Quake, Stephen R

    2016-06-16

    Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

  5. The many roads to Rome: induction of neural precursor cells from fibroblasts.

    PubMed

    Lujan, Ernesto; Wernig, Marius

    2012-10-01

    The experimental induction of specific cell fates in related or unrelated lineages has fascinated developmental biologists for decades. The evaluation of altered cell fates in response to ectopic expression during embryonic development has been a standard assay for interrogating gene function. However, until recently examples of cell lineage conversions were limited to closely related and primitive cell types. The induction of pluripotency in fibroblasts prominently highlighted that combinations of transcription factors can be extremely powerful and are much more effective than single genes. On the basis of this conclusion we previously identified transcription factor combinations that directly induce functional neuronal cells from mesodermal and endodermal cells. This work has evoked numerous additional studies demonstrating direct lineage conversion into neural and other lineages. Here, we review the generation of neural progenitor cells from fibroblasts, which is the newest addition to the arena of induced cell types. Surprisingly, two fundamentally different approaches have been taken to induce this cell type, one direct approach and another that involves the intermediate generation of a partially reprogrammed pluripotent state.

  6. Deficiency in ubiquitin-like protein Ubl4A impairs migration of fibroblasts and macrophages.

    PubMed

    Zhao, Yu; Zhang, Huaiyuan; Affonso, Carlos; Bonomo, Raiza; Mañas, Adriana; Xiang, Jialing

    2017-01-29

    Ubiquitin-like protein Ubl4A is a small, multi-functional protein with no ubiquitination activity. We have previously demonstrated that Ubl4A directly interacts with actin-related protein 2/3 complex (Arp2/3) and promotes Arp2/3-dependent actin branching, thereby accelerating plasma membrane translocation of protein kinase Akt upon insulin stimulation. Here, we show that Ubl4A is critical for plasma membrane protrusion and cell migration. Ubl4A, F-actin and Arp2/3 are co-localized at the cell leading edges during wound closure. Knockout of Ubl4A significantly reduces actin-mediated membrane protrusion and delays wound healing by primary mouse embryonic fibroblasts. Consistently, the ability of fibroblasts to migrate out of corneal tissue ex vivo is also impaired in Ubl4A-deficient mice. Furthermore, cell motility, but not phagocytosis, is significantly decreased in Ubl4A-deficient macrophages compared with wild-type controls. These results imply an important role for Ubl4A in cell migration-associated pathophysiological processes.

  7. Regulation of proliferation and histone acetylation in embryonic neural precursors by CREB/CREM signaling

    PubMed Central

    Parlato, Rosanna; Mandl, Claudia; Hölzl-Wenig, Gabriele; Liss, Birgit; Tucker, Kerry L; Ciccolini, Francesca

    2014-01-01

    The transcription factor CREB (cAMP-response element binding protein) regulates differentiation, migration, survival and activity-dependent gene expression in the developing and mature nervous system. However, its specific role in the proliferation of embryonic neural progenitors is still not completely understood. Here we investigated how CREB regulates proliferation of mouse embryonic neural progenitors by a conditional mutant lacking Creb gene in neural progenitors. In parallel, we explored possible compensatory effects by the genetic ablation of another member of the same gene family, the cAMP-responsive element modulator (Crem). We show that CREB loss differentially impaired the proliferation, clonogenic potential and self-renewal of precursors derived from the ganglionic eminence (GE), in comparison to those derived from the cortex. This phenotype was associated with a specific reduction of histone acetylation in the GE of CREB mutant mice, and this reduction was rescued in vivo by inhibition of histone deacetylation. These observations indicate that the impaired proliferation could be caused by a reduced acetyltransferase activity in Creb conditional knock-out mice. These findings support a crucial role of CREB in controlling embryonic neurogenesis and propose a novel mechanism by which CREB regulates embryonic neural development. PMID:27504469

  8. The embryonic origin of the ampullate silk glands of the spider Cupiennius salei.

    PubMed

    Hilbrant, Maarten; Damen, Wim G M

    2015-05-01

    Silk production in spiders is considered a key innovation, and to have been vital for the diversification of the clade. The evolutionary origin of the organs involved in spider silk production, however, and in particular of the silk glands, is poorly understood. Homologies have been proposed between these and other glands found in arachnids, but lacking knowledge of the embryonic development of spider silk glands hampers an evaluation of hypotheses. This study focuses on the embryonic origin of the largest silk glands of the spider Cupiennius salei, the major and minor ampullate glands. We show how the ampullate glands originate from ectodermal invaginations on the embryonic spinneret limb buds, in relation to morphogenesis of these buds. Moreover, we visualize the subsequent growth of the ampullate glands in sections of the early postembryonic stages. The invaginations are shown to correlate with expression of the proneural gene CsASH2, which is remarkable since it has been proposed that spider silk glands and their nozzles originate from sensory bristles. Hence, by confirming the ectodermal origin of spider silk glands, and by describing the (post-)embryonic morphogenesis of the ampullate glands, this work provides a starting point for further investigating into the genetic program that underlies their development.

  9. Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions

    PubMed Central

    Jung, Juwon; Baek, Jin Ah; Seol, Hye Won; Choi, Young Min

    2016-01-01

    Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies. PMID:27294211

  10. Hoxc13 mutant mice lack external hair.

    PubMed

    Godwin, A R; Capecchi, M R

    1998-01-01

    Hox genes are usually expressed temporally and spatially in a colinear manner with respect to their positions in the Hox complex. Consistent with the expected pattern for a paralogous group 13 member, early embryonic Hoxc13 expression is found in the nails and tail. Hoxc13 is also expressed in vibrissae, in the filiform papillae of the tongue, and in hair follicles throughout the body; a pattern that apparently violates spatial colinearity. Mice carrying mutant alleles of Hoxc13 have been generated by gene targeting. Homozygotes have defects in every region in which gene expression is seen. The most striking defect is brittle hair resulting in alopecia (hairless mice). One explanation for this novel role is that Hoxc13 has been recruited for a function common to hair, nail, and filiform papilla development.

  11. Ontogeny of basic fibroblast growth factor binding sites in mouse ocular tissues

    SciTech Connect

    Fayein, N.A.; Courtois, Y.; Jeanny, J.C. )

    1990-05-01

    Basic fibroblast growth factor (bFGF) binding to ocular tissues has been studied by autoradiographical and biochemical approaches directly performed on sections during mouse embryonic and postnatal development. Frozen sections of embryos (9 to 18 days), newborns, and adults (1 day to 6 months) were incubated with iodinated bFGF. One specific FGF binding site (KD = 2.5 nM) is colocalized with heparan sulfate proteoglycans of the basement membranes and is heparitinase sensitive. It first appears at Day 9 around the neural tube, the optic vesicles, and below the head ectoderm and by Day 14 of embryonic development is found in all basement membranes of the eye. At Day 16, very intensely labeled patches appear, corresponding to mast cells which have been characterized by metachromatic staining of their heparin-rich granulations with toluidine blue. In addition to the latter binding, we have also observed a general diffuse distribution of silver grains on all tissues and preferentially in the ecto- and neuroectodermic tissues. From Days 17-18, there is heterogeneous labeling inside the retina, localized in the pigmented epithelium and in three different layers colocalized with the inner and outer plexiform layers and with the inner segments of the photoreceptors. This binding is heparitinase resistant but N-glycanase sensitive and may represent a second specific binding site corresponding to cellular FGF receptors (KD = 280 pM). Both types of binding patterns observed suggest a significant role for bFGF in eye development and physiology.

  12. Generation of LIF-independent induced pluripotent stem cells from canine fetal fibroblasts.

    PubMed

    Gonçalves, N J N; Bressan, F F; Roballo, K C S; Meirelles, F V; Xavier, P L P; Fukumasu, H; Williams, C; Breen, M; Koh, S; Sper, R; Piedrahita, J; Ambrósio, C E

    2017-04-01

    Takahashi and Yamanaka established the first technique in which transcription factors related to pluripotency are incorporated into the genome of somatic cells to enable reprogramming of these cells. The expression of these transcription factors enables a differentiated somatic cell to reverse its phenotype to an embryonic state, generating induced pluripotent stem cells (iPSCs). iPSCs from canine fetal fibroblasts were produced through lentiviral polycistronic human and mouse vectors (hOSKM/mOSKM), aiming to obtain pluripotent stem cells with similar features to embryonic stem cells (ESC) in this animal model. The cell lines obtained in this study were independent of LIF or any other supplemental inhibitors, resistant to enzymatic procedure (TrypLE Express Enzyme), and dependent on bFGF. Clonal lines were obtained from slightly different protocols with maximum reprogramming efficiency of 0.001%. All colonies were positive for alkaline phosphatase, embryoid body formation, and spontaneous differentiation and expressed high levels of endogenous OCT4 and SOX2. Canine iPSCs developed tumors at 120 days post-injection in vivo. Preliminary chromosomal evaluations were performed by FISH hybridization, revealing no chromosomal abnormality. To the best of our knowledge, this report is the first to describe the ability to reprogram canine somatic cells via lentiviral vectors without supplementation and with resistance to enzymatic action, thereby demonstrating the pluripotency of these cell lines.

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

    SciTech Connect

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

    2010-02-01

    The conventional paradigm in radiation biology has been that DNA is the primary target for energy deposition following exposure to ionizing radiation. However, studies focusing on the non-target effects of radiation, i.e. effects occurring in cells not directly exposed to radiation, imply that the target of exposure is larger than what has traditionally been assumed and could have significant implications for radiation health risks. We have conducted an extensive study of the low-LET bystander effect including multiple cell lines and endpoints and various radiation sources and exposure scenarios. In no instance do we see evidence of a low-LET induced bystander effect. However, direct comparison for alpha particle exposure showed a statistically significant media transfer bystander effect for high-LET but not for low-LET radiation. From our results it is evident that there are many confounding factors mitigating bystander responses as reported in the literature and for the cell lines we studied that there is a LET dependence for the observed responses. Our observations reflect the inherent variability in biological systems and the difficulties in extrapolating from in vitro models to radiation risks in humans.

  14. Report: EPA Travel Program Lacks Necessary Controls

    EPA Pesticide Factsheets

    Report #10-P-0078, March 9, 2010. The EPA travel program, which comprises EPA policies and GovTrip, lacks necessary control procedures to assure all travel authorizations were necessary and in the best interest of the government.

  15. Two factor-based reprogramming of rodent and human fibroblasts into Schwann cells

    PubMed Central

    Mazzara, Pietro Giuseppe; Massimino, Luca; Pellegatta, Marta; Ronchi, Giulia; Ricca, Alessandra; Iannielli, Angelo; Giannelli, Serena Gea; Cursi, Marco; Cancellieri, Cinzia; Sessa, Alessandro; Del Carro, Ubaldo; Quattrini, Angelo; Geuna, Stefano; Gritti, Angela; Taveggia, Carla; Broccoli, Vania

    2017-01-01

    Schwann cells (SCs) generate the myelin wrapping of peripheral nerve axons and are promising candidates for cell therapy. However, to date a renewable source of SCs is lacking. In this study, we show the conversion of skin fibroblasts into induced Schwann cells (iSCs) by driving the expression of two transcription factors, Sox10 and Egr2. iSCs resembled primary SCs in global gene expression profiling and PNS identity. In vitro, iSCs wrapped axons generating compact myelin sheaths with regular nodal structures. Conversely, iSCs from Twitcher mice showed a severe loss in their myelinogenic potential, demonstrating that iSCs can be an attractive system for in vitro modelling of PNS diseases. The same two factors were sufficient to convert human fibroblasts into iSCs as defined by distinctive molecular and functional traits. Generating iSCs through direct conversion of somatic cells offers opportunities for in vitro disease modelling and regenerative therapies. PMID:28169300

  16. Fibroblasts induce heparin synthesis in chondroitin sulfate E containing human bone marrow-derived mast cells

    SciTech Connect

    Gilead, L.; Bibi, O.; Razin, E. )

    1990-09-15

    Human bone marrow-derived mast cells (hBMMCs), differentiated in vitro in suspension culture and under the influence of human peripheral blood mononuclear cells conditioned medium (hCM), were tested for their response to recombinant human interleukin-3 (rhIL-3) and for their behavior in different microenvironments. The hBMMCs were incubated in the presence of rhIL-3 and the changes in their proliferation rate were determined. Recombinant hIL-3 induced a more than sixfold increase in 3H-thymidine uptake into the hBMMC DNA in a dose-dependent manner. Human CM used as a control for proliferation response induced a more than eightfold maximal proliferation rate increase. Rabbit anti-rhIL-3 completely inhibited hBMMC 3H-thymidine uptake induced by rhIL-3 and decreased the hCM-induced proliferation by approximately 50%. These hBMMCs were cocultured with four different mytomicin C-treated cell monolayers and assayed for phenotypic changes. After only 2 days in coculture with either embryonic mouse skin-derived fibroblasts (MESFs) or human skin-derived fibroblasts (HSFs), a marked increase in granule number and density was noted on staining with toluidine blue. Mast cells that initially stained alcian blue+/safranin- at day 0 of coculture became alcian blue+/safranin+ during the coculture period. Human BMMC proteoglycan synthesis shifted from approximately 85% chondroitin sulfate E to approximately 60% heparin within 14 to 19 days of coculture with the MESF monolayer and to approximately 50% heparin within 19 days of coculture with the HSF monolayer. None of the above-mentioned changes were noted in cocultures of hBMMCs with 3T3 cell line fibroblast monolayers or in cocultures with bovine vascular endothelium (BVE) cell monolayers.

  17. Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion.

    PubMed

    Mazur, Anna; Holthoff, Emily; Vadali, Shanthi; Kelly, Thomas; Post, Steven R

    2016-01-01

    Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages.

  18. TGF-β1 Induces Polypyrimidine Tract-Binding Protein to Alter Fibroblasts Proliferation and Fibronectin Deposition in Keloid

    PubMed Central

    Jiao, Hu; Dong, Ping; Yan, Li; Yang, Zhigang; Lv, Xiaoyan; Li, Qiuchen; Zong, Xianlei; Fan, Jincai; Fu, Xin; Liu, Xia; Xiao, Ran

    2016-01-01

    Human dermal fibrotic disease keloid has been a clinical challenge because of its tumour-like growth and the lack of effective therapy. Dysregulated alternative splicing events have been demonstrated in tumours and fibrosis. In the current study, for the first time, it was demonstrated that the splicing regulator polypyrimidine tract-binding protein (PTB), which plays a pivotal role in tumour proliferation, invasion and metastasis, is overexpressed in keloid tissues and fibroblasts. Additionally, TGF-β1 upregulated the expressions of PTB and its upstream regulator, C-MYC, in keloid fibroblasts. Furthermore, we suppressed PTB using siRNA in keloid fibroblasts and in a keloid xenograft nude mouse model. PTB knockdown significantly slowed the proliferation of keloid fibroblasts and accelerated the regression of transplanted keloid tissues, which was accompanied by a shift in the alternative splicing of USP5 and RTN4. Moreover, when PTB was suppressed, there was a reduction in excessive deposition of FN1 and COL3A1 in transplanted keloid tissues. However, only FN1 was downregulated in keloid fibroblasts that were cultured in media supplemented with TGF-β1. Our study provides evidence for the role of PTB in keloid pathophysiology and offers a novel therapeutic target for keloids. Most importantly, the role TGF-β1 regulation of PTB may provide new insights into the mechanisms underlying inflammatory cytokine-induced fibrosis. PMID:27897224

  19. Myxoinflammatory fibroblastic sarcoma of the face.

    PubMed

    Gómez Martín, Cristina; Ortega, María I; Aramburu, José A; Fernández-Cañamaque, José L

    2012-08-01

    Myxoinflammatory fibroblastic sarcoma (MIFS) is a rare low-grade tumor of modified fibroblasts, with tendency to local recurrence. This unusual entity typically presents as a slow-growing painless mass in the distal extremities of middle-aged subjects. A 48-year-old woman presented to our clinic with a painless subcutaneous mass in the right temporal region. Excisional biopsy made the rare diagnosis of MIFS. Histologic examination showed the unique features that characterize this lesion: a myxoid component with a superimposed inflammatory infiltrate and the presence of distinctive, large, and bizarre Reed-Stemberg-like cells. A second wide tumor bed resection was performed, achieving clear margins. No adjuvant therapy was administered, and the patient is free of disease at 18 months postoperatively. To the best of our knowledge, this is the first reported case of MIFS presenting in the face. This adds another possibility for differential diagnoses of soft tissue tumors of the face.

  20. Mechanoregulation of gene expression in fibroblasts

    PubMed Central

    Wang, James H.-C.; Thampatty, Bhavani P.; Lin, Jeen-Shang; Im, Hee-Jeong

    2010-01-01

    Mechanical loads placed on connective tissues alter gene expression in fibroblasts through mechanotransduction mechanisms by which cells convert mechanical signals into cellular biological events, such as gene expression of extracellular matrix components (e.g., collagen). This mechanical regulation of ECM gene expression affords maintenance of connective tissue homeostasis. However, mechanical loads can also interfere with homeostatic cellular gene expression and consequently cause the pathogenesis of connective tissue diseases such as tendinopathy and osteoarthritis. Therefore, the regulation of gene expression by mechanical loads is closely related to connective tissue physiology and pathology. This article reviews the effects of various mechanical loading conditions on gene regulation in fibroblasts and discusses several mechanotransduction mechanisms. Future research directions in mechanoregulation of gene expression are also suggested. PMID:17331678

  1. Three-dimensional epithelial tissues generated from human embryonic stem cells.

    PubMed

    Hewitt, Kyle J; Shamis, Yulia; Carlson, Mark W; Aberdam, Edith; Aberdam, Daniel; Garlick, Jonathan A

    2009-11-01

    The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

  2. Reciprocal regulatory interactions between the Notch and Ras signaling pathways in the Drosophila embryonic mesoderm.

    PubMed

    Carmena, Ana; Buff, Eugene; Halfon, Marc S; Gisselbrecht, Stephen; Jiménez, Fernando; Baylies, Mary K; Michelson, Alan M

    2002-04-15

    Convergent intercellular signals must be precisely integrated in order to elicit specific biological responses. During specification of muscle and cardiac progenitors from clusters of equivalent cells in the Drosophila embryonic mesoderm, the Ras/MAPK pathway--activated by both epidermal and fibroblast growth factor receptors--functions as an inductive cellular determination signal, while lateral inhibition mediated by Notch antagonizes this activity. A critical balance between these signals must be achieved to enable one cell of an equivalence group to segregate as a progenitor while its neighbors assume a nonprogenitor identity. We have investigated whether these opposing signals directly interact with each other, and we have examined how they are integrated by the responding cells to specify their unique fates. Our findings reveal that Ras and Notch do not function independently; rather, we have uncovered several modes of cross-talk between these pathways. Ras induces Notch, its ligand Delta, and the epidermal growth factor receptor antagonist, Argos. We show that Delta and Argos then synergize to nonautonomously block a positive autoregulatory feedback loop that amplifies a fate-inducing Ras signal. This feedback loop is characterized by Ras-mediated upregulation of proximal components of both the epidermal and fibroblast growth factor receptor pathways. In turn, Notch activation in nonprogenitors induces its own expression and simultaneously suppresses both Delta and Argos levels, thereby reinforcing a unidirectional inhibitory response. These reciprocal interactions combine to generate the signal thresholds that are essential for proper specification of progenitors and nonprogenitors from groups of initially equivalent cells.

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

    PubMed

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

    2014-02-01

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

  4. Inhibition of fibroblast proliferation by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Shenker, B J; Kushner, M E; Tsai, C C

    1982-01-01

    We have examined soluble sonic extracts of Actinobacillus actinomycetemcomitans for their ability to alter human and murine fibroblast proliferation. We found that extracts of all A. actinomycetemcomitans strains examined (both leukotoxic and nonleukotoxic) caused a dose-dependent inhibition of both murine and human fibroblast proliferation as assessed by DNA synthesis ([3H]thymidine incorporation). Addition of sonic extract simultaneously with [3H]thymidine had no effect on incorporation, indicating that suppression was not due to the presence of excessive amounts of cold thymidine. Inhibition of DNA synthesis was also paralleled by decreased RNA synthesis ([3H]uridine incorporation) and by a decrease in cell growth as assessed by direct cell counts; there was no effect on cell viability. The suppressive factor(s) is heat labile; preliminary purification and characterization studies indicate that it is a distinct and separate moiety from other A. actinomycetemcomitans mediators previously reported, including leukotoxin, immune suppressive factor, and endotoxin. Although it is not clear how A. actinomycetemcomitans acts to cause disease, we propose that one aspect of the pathogenicity of this organism rests in its ability to inhibit fibroblast growth, which in turn could contribute to the collagen loss associated with certain forms of periodontal disease, in particular juvenile periodontitis. PMID:7152684

  5. Fibroblasts as architects of cancer pathogenesis.

    PubMed

    Marsh, Timothy; Pietras, Kristian; McAllister, Sandra S

    2013-07-01

    Studies of epithelial cancers (i.e., carcinomas) traditionally focused on transformation of the epithelium (i.e., the cancer cells) and how aberrant signaling within the cancer cells modulates the surrounding tissue of origin. In more recent decades, the normal cells, blood vessels, molecules, and extracellular components that surround the tumor cells, collectively known as the "tumor microenvironment" or "stroma", have received increasing attention and are now thought to be key regulators of tumor initiation and progression. Of particular relevance to the work reviewed herein are the fibroblasts, which make up the major cell type within the microenvironment of most carcinomas. Due to their inherent heterogeneity, plasticity, and function, it is perhaps not surprising that fibroblasts are ideal modulators of normal and cancerous epithelium; however, these aspects also present challenges if we are to interrupt their tumor-supportive functions. Here, we review the current body of knowledge and the many questions that still remain about the special entity known as the cancer-associated fibroblast. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.

  6. Three key variables involved in feeder preparation for the maintenance of human embryonic stem cells.

    PubMed

    Zhou, Di; Liu, Tiancheng; Zhou, Xiaoying; Lu, Guangxiu

    2009-07-01

    Although the development of a feeder-free culture system for future applications of human embryonic stem cells (hESCs), at present the regular culture system uses mitotically inactivated mouse embryonic fibroblasts (mEFs) as feeder cells for maintaining undifferentiated hESCs. Mitomycin C (MMC) is used to inactivate mEFs, but this causes DNA damage, and it is unclear whether MMC remains in the culture system after several washes. Three variables have been evaluated with respect to feeder preparation and MMC involvement, including mEF exposure to MMC, density of feeder cells, and different wash steps during the preparation of feeder cells. These variables are critical to the subsequent planting of hESCs because remnants of MMC would be unsafe with respect to long-term culture of hESCs The novel data here evaluates the remnant amounts of MMC in a hESCs culture system using HPLC/MS/MS. The ultimate objective of this study is the control of MMC within a safe range.

  7. Effects and possible mechanisms of simulated-microgravity on zebrafish embryonic cell

    NASA Astrophysics Data System (ADS)

    Hang, Xiaoming; Sun, Yeqing; Wu, Di; Li, Yixiao; Wang, Ruonan

    2016-07-01

    Cellular level studies are helpful for revealing the underlying mechanisms of microgravity effects on living organisms. Many cell types, ranging from bacteria to mammalian cells, are sensitive to the microgravity environment. In this study, zebrafish embryonic cells (ZF4) were exposed to simulated-microgravity (SMG) for different times to investigate the effects and possible mechanisms of microgravity on fibroblasts. A significant arrest in G2/M phase was detected in ZF4 cells after 24 or 48 hour of SMG exposure, respectively. The mRNA levels of G2/M phase regulators cyclinB1 and cdc2 were significantly decreased, while wee1 was significantly increased. Additionally, CEP135, a core centrosome protein throughout the cell cycle, seems to play a key role in modulating this effect. Quantitative analysis showed that cep135 expression was significantly increased, while CEP135 protein expression level was significantly decreased two times after SMG. Further investigation demonstrated the transfection of dre-miR-22a, a miRNA for targeting cep135, also induced G2/M arrest in ZF4 cells. These results suggest that SMG induced G2/M arrest in ZF4 cells may due to the regulation of dre-miR-22a and its target cep135. Key Words: Simulated-microgravity; zebrafish embryonic cell; G2/M arrest; molecular mechanism

  8. RPLP1, a crucial ribosomal protein for embryonic development of the nervous system.

    PubMed

    Perucho, Laura; Artero-Castro, Ana; Guerrero, Sergi; Ramón y Cajal, Santiago; LLeonart, Matilde E; Wang, Zhao-Qi

    2014-01-01

    Ribosomal proteins are pivotal to development and tissue homeostasis. RP Large P1 (Rplp1) overexpression is associated with tumorigenesis. However, the physiological function of Rplp1 in mammalian development remains unknown. In this study, we disrupted Rplp1 in the mouse germline and central nervous system (Rplp1CNSΔ). Rplp1 heterozygosity caused body size reductions, male infertility, systemic abnormalities in various tissues and a high frequency of early postnatal death. Rplp1CNSΔ newborn mice exhibited perinatal lethality and brain atrophy with size reductions of the neocortex, midbrain and ganglionic eminence. The Rplp1 knockout neocortex exhibited progenitor cell proliferation arrest and apoptosis due to the dysregulation of key cell cycle and apoptosis regulators (cyclin A, cyclin E, p21CIP1, p27KIP1, p53). Similarly, Rplp1 deletion in pMEFs led to proliferation arrest and premature senescence. Importantly, Rplp1 deletion in primary mouse embryonic fibroblasts did not alter global protein synthesis, but did change the expression patterns of specific protein subsets involved in protein folding and the unfolded protein response, cell death, protein transport and signal transduction, among others. Altogether, we demonstrated that the translation "fine-tuning" exerted by Rplp1 is essential for embryonic and brain development and for proper cell proliferation.

  9. Expression of liver fatty acid binding protein alters growth and differentiation of embryonic stem cells.

    PubMed

    Schroeder, F; Atshaves, B P; Starodub, O; Boedeker, A L; Smith, R R; Roths, J B; Foxworth, W B; Kier, A B

    2001-03-01

    Although expression of liver fatty acid binding protein (L-FABP) modulates cell growth, it is not known if L-FABP also alters cell morphology and differentiation. Therefore, pluripotent embryonic stem cells were transfected with cDNA encoding L-FABP and a series of clones expressing increasing levels of L-FABP were isolated. Untransfected ES cells, as well as ES cells transfected only with empty vector, spontaneously differentiated from rounded adipocyte-like to fibroblast-like morphology, concomitant with marked reduction in expression of stage-specific embryonic antigen (SSEA-1). These changes in morphology and expression of SSEA-1 were greatest in ES cell clones expressing L-FABP above a threshold level. Immunofluorescence confocal microscopy revealed that L-FABP was primarily localized in a diffuse-cytosolic pattern along with a lesser degree of punctate L-FABP expression in the nucleus. Nuclear localization of L-FABP was preferentially increased in clones expressing higher levels of L-FABP. In summary, L-FABP expression altered ES cell morphology and expression of SSEA-1. Taken together with the fact that L-FABP was detected in the nucleus, these data suggested that L-FABP may play a more direct, heretofore unknown, role in regulating ES cell differentiation by acting in the nucleus as well as cytoplasm.

  10. Beta-amyloid Toxicity and Reversal in Embryonic Rat Septal Neurons

    PubMed Central

    Jarvis, Karen; Assis-Nascimento, Poincyane; Mudd, Laura M.; Montague, Jeremy R.

    2009-01-01

    Alzheimer’s disease is characterized mainly by loss of neurons from the septal nucleus. In this study, neurons from the septal nucleus of the embryonic day 16 (E16) rat were grown in culture with a plane of astrocytes from the embryonic rat and in a defined medium in the absence of serum. Neurons were treated with beta-amyloid (Aβ: 0.1, 1, and 10 µM) on day in vitro (DIV) 1 and DIV 4 and fluorescent microscopy was used to measure survival and apoptosis following exposure of the treated cells on DIV 7. Reversal of neurotoxicity was studied using the potentially neuroprotective agents nerve growth factor (NGF, 100 ng/ml), basic fibroblast growth factor (bFGF, 5 ng/ml), insulin-like growth factors (IGF1 and IGF2, 10 ng/ml), and estrogen (10 nM), administered on DIV 4 and DIV 5, that is, subsequent to the Aβ (10 µM)-induced neurotoxicity. Aβ caused a significant decrease in survival at 10 µM, and a significant increase in apoptosis at 0.1 and 10 µM. IGF1, IGF2 and bFGF all caused a reversal of the Aβ-induced neurotoxic effect on survival while NGF and estrogen did not under these experimental conditions. PMID:17709203

  11. Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells.

    PubMed

    Min, Irene M; Waterfall, Joshua J; Core, Leighton J; Munroe, Robert J; Schimenti, John; Lis, John T

    2011-04-01

    Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, "bivalent" ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.

  12. Expression pattern of embryonic stem cell markers in DFAT cells and ADSCs.

    PubMed

    Gao, Qian; Zhao, Lili; Song, Ziyi; Yang, Gongshe

    2012-05-01

    Mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability under the condition of ceiling method, named dedifferentiated fat cells (DFAT cells). These cells exhibit multilineage potential as adipose tissue-derived stromal cells (ADSCs). However, the stem molecular signature of DFAT cells and the difference distinct from ADSCs are still not sure. To study the molecular signature of DFAT cells better, highly purified mature adipocytes were obtained from rats and the purity was more than 98%, and about 98.6% were monocytes. These mature adipocytes dedifferentiated into fibroblast-like cells spontaneously by the ceiling culture method, these cells proliferated rapidly in vitro, grew in the same direction and formed vertex, and expressed extensively embryonic stem cell markers such as Oct4, Sox2, c-Myc, and Nanog, surface antigen SSEA-1, CD105, and CD31, moreover, these cells possessed ALP and telomerase activity. The expression level was Oct4 1.3%, Sox2 1.3%, c-Myc 1.2%, Nanog 1.2%, CD105 0.6%, CD31 0.6% and SSEA-1 0.4%, respectively, which was lower than that in ADSCs, but the purity of DFAT cells was much higher than that of ADSCs. In conclusion, DFAT cells is a highly purified stem cell population, and expressed some embryonic stem cell markers like ADSCs, which seems to be a good candidate source of adult stem cells for the future cell replacement therapy.

  13. Transcriptional repression of p27 is essential for murine embryonic development

    PubMed Central

    Teratake, Youichi; Kuga, Chisa; Hasegawa, Yuta; Sato, Yoshiharu; Kitahashi, Masayasu; Fujimura, Lisa; Watanabe-Takano, Haruko; Sakamoto, Akemi; Arima, Masafumi; Tokuhisa, Takeshi; Hatano, Masahiko

    2016-01-01

    The Nczf gene has been identified as one of Ncx target genes and encodes a novel KRAB zinc-finger protein, which functions as a sequence specific transcriptional repressor. In order to elucidate Nczf functions, we generated Nczf knockout (Nczf−/−) mice. Nczf−/− mice died around embryonic day 8.5 (E8.5) with small body size and impairment of axial rotation. Histopathological analysis revealed that the cell number decreased and pyknotic cells were occasionally observed. We examined the expression of cell cycle related genes in Nczf−/− mice. p27 expression was increased in E8.0 Nczf−/− mice compared to that of wild type mice. Nczf knockdown by siRNA resulted in increased expression of p27 in mouse embryonic fibroblasts (MEFs). Furthermore, p27 promoter luciferase reporter gene analysis confirmed the regulation of p27 mRNA expression by Nczf. Nczf−/−; p27−/− double knockout mice survived until E11.5 and the defect of axial rotation was restored. These data suggest that p27 repression by Nczf is essential in the developing embryo. PMID:27196371

  14. The culture of human embryonic stem cells in microchannel perfusion bioreactors

    NASA Astrophysics Data System (ADS)

    Korin, Natanel; Bransky, Avishay; Dinnar, Uri; Levenberg, Shulamit

    2007-12-01

    The culture of human Embryonic Stem (ES) cells in microchannel bioreactors can be highly beneficial for ES cell biology studies and ES tissue engineering applications. In the present study we examine the use of Human Foreskin Fibroblasts (HFF) cells as feeder cells for human ES culture in a microchannel perfusion bioreactor. PDMS microchannels (depth:130 micron) were fabricated using conventional soft-lithography techniques. The channels were sterilized, coated with a human fibronectin solution and seeded with cells. Following a period of static incubation, culture medium was perfused through the channels at various flow rates and cell growth was monitored throughout the culture process. Mass transport and fluid mechanics models were used to evaluate the culture conditions (shear stress, oxygen levels within the micro-bioreactor as a function of the medium flow rate. The conditions for successful long-term culture (>7 days) of HFF under flow were established. Experiments with human embryonic stem cells cultured in microchannels show that the conditions essential to co-culture human ES cell on HFF cells under perfusion differ from the conditions necessary for HFF cell culture. Human ES cells were found to be highly sensitive to flow and culture conditions and did not grow under flow rates which were suitable for HFF long-term culture. Successful culture of undifferentiated human ES cell colonies in a perfusion micro-bioreactor is a basic step towards utilizing microfluidic techniques to explore stem cell biology.

  15. Comparison of electronic cigarette refill fluid cytotoxicity using embryonic and adult models.

    PubMed

    Bahl, Vasundhra; Lin, Sabrina; Xu, Nicole; Davis, Barbara; Wang, Yu-huan; Talbot, Prue

    2012-12-01

    Electronic cigarettes (EC) and refill fluids are distributed with little information on their pre- and postnatal health effects. This study compares the cytotoxicity of EC refill fluids using embryonic and adult cells and examines the chemical characteristics of refill fluids using HPLC. Refill solutions were tested on human embryonic stem cells (hESC), mouse neural stem cells (mNSC), and human pulmonary fibroblasts (hPF) using the MTT assay, and NOAELs and IC(50)s were determined from dose-response curves. Spectral analysis was performed when products of the same flavor had different MTT outcomes. hESC and mNSC were generally more sensitive to refill solutions than hPF. All products from one company were cytotoxic to hESC and mNSC, but non-cytotoxic to hPF. Cytotoxicity was not due to nicotine, but was correlated with the number and concentration of chemicals used to flavor fluids. Additional studies are needed to fully assess the prenatal effect of refill fluids.

  16. Generation of induced pluripotent stem cells with high efficiency from human embryonic renal cortical cells

    PubMed Central

    Yao, Ling; Chen, Ruifang; Wang, Pu; Zhang, Qi; Tang, Hailiang; Sun, Huaping

    2016-01-01

    Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) emerges as a prospective therapeutic angle in regenerative medicine and a tool for drug screening. Although increasing numbers of iPSCs from different sources have been generated, there has been limited progress in yield of iPSC. Here, we show that four Yamanaka factors Oct4, Sox2, Klf4 and c-Myc can convert human embryonic renal cortical cells (hERCCs) to pluripotent stem cells with a roughly 40-fold higher reprogramming efficiency compared with that of adult human dermal fibroblasts. These iPSCs show pluripotency in vitro and in vivo, as evidenced by expression of pluripotency associated genes, differentiation into three embryonic germ layers by teratoma tests, as well as neuronal fate specification by embryoid body formation. Moreover, the four exogenous genes are effectively silenced in these iPSCs. This study highlights the use of hERCCs to generate highly functional human iPSCs which may aid the study of genetic kidney diseases and accelerate the development of cell-based regenerative therapy. PMID:27904699

  17. The docking protein FRS2alpha is an essential component of multiple fibroblast growth factor responses during early mouse development.

    PubMed

    Gotoh, N; Manova, K; Tanaka, S; Murohashi, M; Hadari, Y; Lee, A; Hamada, Y; Hiroe, T; Ito, M; Kurihara, T; Nakazato, H; Shibuya, M; Lax, I; Lacy, E; Schlessinger, J

    2005-05-01

    The docking protein FRS2alpha is a major mediator of fibroblast growth factor (FGF) signaling. However, the physiological role of FRS2alpha in vivo remains unknown. In this report, we show that Frs2alpha-null mouse embryos have a defect in anterior-posterior (A-P) axis formation and are developmentally retarded, resulting in embryonic lethality by embryonic day 8. We demonstrate that FRS2alpha is essential for the maintenance of self-renewing trophoblast stem (TS) cells in response to FGF4 in the extraembryonic ectoderm (ExE) that gives rise to tissues of the placenta. By analyzing chimeric embryos, we found that FRS2alpha also plays a role in cell movement through the primitive streak during gastrulation. In addition, experiments are presented demonstrating that Bmp4 expression in TS cells is controlled by mitogen-activated protein kinase-dependent FGF4 stimulation. Moreover, both the expression of Bmp4 in ExE and activation of Smad1/5 in epiblasts are reduced in Frs2alpha-null embryos. These experiments underscore the critical role of FRS2alpha in mediating multiple processes during embryonic development and reveal a potential new link between FGF and Bmp4 signaling pathways in early embryogenesis.

  18. Alteration of Skin Properties with Autologous Dermal Fibroblasts

    PubMed Central

    Thangapazham, Rajesh L.; Darling, Thomas N.; Meyerle, Jon

    2014-01-01

    Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue. They are primarily responsible for synthesizing collagen and glycosaminoglycans; components of extracellular matrix supporting the structural integrity of the skin. Dermal fibroblasts play a pivotal role in cutaneous wound healing and skin repair. Preclinical studies suggest wider applications of dermal fibroblasts ranging from skin based indications to non-skin tissue regeneration in tendon repair. One clinical application for autologous dermal fibroblasts has been approved by the Food and Drug Administration (FDA) while others are in preclinical development or various stages of regulatory approval. In this context, we outline the role of fibroblasts in wound healing and discuss recent advances and the current development pipeline for cellular therapies using autologous dermal fibroblasts. The microanatomic and phenotypic differences of fibroblasts occupying particular locations within the skin are reviewed, emphasizing the therapeutic relevance of attributes exhibited by subpopulations of fibroblasts. Special focus is provided to fibroblast characteristics that define regional differences in skin, including the thick and hairless skin of the palms and soles as compared to hair-bearing skin. This regional specificity and functional identity of fibroblasts provides another platform for developing regional skin applications such as the induction of hair follicles in bald scalp or alteration of the phenotype of stump skin in amputees to better support their prosthetic devices. PMID:24828202

  19. Alteration of skin properties with autologous dermal fibroblasts.

    PubMed

    Thangapazham, Rajesh L; Darling, Thomas N; Meyerle, Jon

    2014-05-13

    Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue. They are primarily responsible for synthesizing collagen and glycosaminoglycans; components of extracellular matrix supporting the structural integrity of the skin. Dermal fibroblasts play a pivotal role in cutaneous wound healing and skin repair. Preclinical studies suggest wider applications of dermal fibroblasts ranging from skin based indications to non-skin tissue regeneration in tendon repair. One clinical application for autologous dermal fibroblasts has been approved by the Food and Drug Administration (FDA) while others are in preclinical development or various stages of regulatory approval. In this context, we outline the role of fibroblasts in wound healing and discuss recent advances and the current development pipeline for cellular therapies using autologous dermal fibroblasts. The microanatomic and phenotypic differences of fibroblasts occupying particular locations within the skin are reviewed, emphasizing the therapeutic relevance of attributes exhibited by subpopulations of fibroblasts. Special focus is provided to fibroblast characteristics that define regional differences in skin, including the thick and hairless skin of the palms and soles as compared to hair-bearing skin. This regional specificity and functional identity of fibroblasts provides another platform for developing regional skin applications such as the induction of hair follicles in bald scalp or alteration of the phenotype of stump skin in amputees to better support their prosthetic devices.

  20. Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model

    PubMed Central

    Geng, Xuehui; Shuda, Yoko; Ostrowski, Stephen M.; Lukianov, Stefan; Jenkins, Frank J.; Honda, Kord; Maricich, Stephen M.; Moore, Patrick S.; Chang, Yuan

    2015-01-01

    Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting. PMID:26544690

  1. The infarcted cardiac microenvironment cannot selectively promote embryonic stem cell differentiation into cardiomyocytes.

    PubMed

    Chen, You-Ren; Li, Yang; Chen, Li; Yang, Xin-Chun; Su, Pi-Xiong; Cai, Jun

    2011-01-01

    Postinfarct congestive heart failure is one of the leading causes of morbidity and mortality in industrialized countries. It is controversial whether embryonic stem cells are feasible sources for in situ cardiac regeneration in infarcted hearts. In order to investigate whether the infarcted cardiac microenvironment could selectively promote embryonic stem cell differentiation into cardiomyocytes, we assessed the cardiac differentiation potential of mouse embryonic stem cells (mESCs) injected into normal (n=16) or acutely infarcted rat hearts (n=18). We found that the transplanted 4',6-diamidino-2-phenylindole (DAPI)-labeled mESCs were able to survive and form stable intracardiac grafts both in normal and infarcted hearts, along with macrophages found specifically in the engraftment area. Two to four weeks after mESC transplantation, we found that more DAPI-positive mESCs differentiated into cardiomyocytes, marked by cardiac troponin T (cTnT), in normal than those in infarcted hearts (2.67±0.79% vs. 1.06±0.52%, P<.01). However, the discrepancy between the percentage of DAPI-positive cells that express cTnT in normal and that in infarcted hearts was diminished after 4 weeks (1.17±0.98% vs. 1.07±1.02%, P>.05), when the transverse striation began to present in the mESCs-derived cardiomyocytes. In addition, mESCs differentiated into vimentin-positive cardiac fibroblasts in normal and infracted hearts. Our results indicated that transplanted mESCs cannot only survive but differentiate into cardiomyocytes in infarcted rat hearts. However, the infarcted cardiac microenvironment cannot selectively promote mESCs differentiation into cardiomyocytes.

  2. Histone demethylase JMJD5 is essential for embryonic development

    SciTech Connect

    Oh, Sangphil; Janknecht, Ralf

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Histone demethylase JMJD5 is essential for embryogenesis. Black-Right-Pointing-Pointer Transcription of tumor suppressor p53 is upregulated in JMJD5 knockout embryos. Black-Right-Pointing-Pointer JMJD5 may antagonize p53-dependent growth inhibition and apoptosis. Black-Right-Pointing-Pointer JMJD5 is overexpressed in leukemias and breast cancer. -- Abstract: Histone lysine methylation is pivotal in regulating chromatin structure and thus profoundly affects the transcriptome. JMJD5 (jumonji C domain-containing 5) is a histone demethylase that specifically removes methyl moieties from dimethylated lysine 36 on histone H3 and exerts a pro-proliferative effect on breast cancer cells. Here, we generated JMJD5 knockout mice in order to study the physiological significance of this enzyme. Whereas heterozygous knockout mice displayed no overt phenotype, homozygous JMJD5 knockouts died around day 10 of embryonal development. JMJD5{sup -/-} embryos showed delayed development already at E8.5 and were actively resorbed at E10.5. While strong JMJD5 expression was observed only in the yolk sac at E8.5, JMJD5 was robustly expressed in E10.5 embryos at several sites, including the heart and eye. Lack of JMJD5 resulted in transcriptional upregulation of the tumor suppressor p53. Concurrently, the cell cycle inhibitor p21 and the pro-apoptotic molecule Noxa, both of which are prominent p53 target genes, became strongly upregulated in JMJD5{sup -/-} embryos. Collectively, our data indicate that JMJD5 is essential during embryonal development and a repressor of p53 expression. The latter suggests that JMJD5 has oncogenic activity and accordingly JMJD5 is upregulated in leukemias and breast cancer.

  3. The enforced expression of c-Myc in pig fibroblasts triggers mesenchymal-epithelial transition (MET) via F-actin reorganization and RhoA/Rock pathway inactivation.

    PubMed

    Shi, Jun-Wen; Liu, Wei; Zhang, Ting-Ting; Wang, Sheng-Chun; Lin, Xiao-Lin; Li, Jing; Jia, Jun-Shuang; Sheng, Hong-Fen; Yao, Zhi-Fang; Zhao, Wen-Tao; Zhao, Zun-Lan; Xie, Rao-Ying; Yang, Sheng; Gao, Fei; Fan, Quan-Rong; Zhang, Meng-Ya; Yue, Min; Yuan, Jin; Gu, Wei-Wang; Yao, Kai-Tai; Xiao, Dong

    2013-04-01

    In previous studies from other labs it has been well demonstrated that the ectopic expression of c-Myc in mammary epithelial cells can induce epithelial-mesenchymal transition (EMT), whereas in our pilot experiment, epithelial-like morphological changes were unexpectedly observed in c-Myc-expressing pig fibroblasts [i.e., porcine embryonic fibroblasts (PEFs) and porcine dermal fibroblasts (PDFs)] and pig mesenchymal stem cells, suggesting that the same c-Myc gene is entitled to trigger EMT in epithelial cells and mesenchymal-epithelial transition (MET) in fibroblasts. This prompted us to characterize the existence of a MET in c-Myc-expressing PEFs and PDFs at the molecular level. qRT-PCR, immunofluorescence and western blot analysis illustrated that epithelial-like morphological changes were accompanied by the increased expression of epithelial markers [such as cell adhesion proteins (E-cadherin, α-catenin and Bves), tight junction protein occludin and cytokeratins (Krt8 and Krt18)], the reduced expression of mesenchymal markers [vimentin, fibronectin 1 (FN1), snail1, collagen family of proteins (COL1A1, COL5A2) and matrix metalloproteinase (MMP) family (MMP12 and MMP14)] and the decreased cell motility and increased cell adhesion in c-Myc-expressing PEFs and PDFs. Furthermore, the ectopic expression of c-Myc in pig fibroblasts disrupted the stress fiber network, suppressed the formation of filopodia and lamellipodia, and resulted in RhoA/Rock pathway inactivation, which finally participates in epithelial-like morphological conversion. Taken together, these findings demonstrate, for the first time, that the enforced expression of c-Myc in fibroblasts can trigger MET, to which cytoskeleton depolymerization and RhoA/Rock pathway inactivation contribute.

  4. Kid's Green Movement Lacks Basis in Reality.

    ERIC Educational Resources Information Center

    Chemecology, 1992

    1992-01-01

    Discussed is the idea that kids are highly politically correct environmentalists on the surface, but underneath they seem to be missing the connection with nature. The author attributes this ignorance of all things natural to things such as kid's preference for video games, television, and lack of access and time. The importance of parental…

  5. Lack of Communications: The Most Common Deficiency.

    ERIC Educational Resources Information Center

    Allen, Thomas R., Jr.

    1979-01-01

    A survey with employers and teacher-coordinators of cooperative education programs showed that young employees' most common deficiencies are in communication skills, both written and oral. Poor handwriting was the leading complaint, followed by misspelling, ignorance of grammar and rhetoric, poor customer relations, and lack of comprehension and…

  6. Chicken alpha-globin switching depends on autonomous silencing of the embryonic pi globin gene by epigenetics mechanisms.

    PubMed

    Rincón-Arano, Héctor; Guerrero, Georgina; Valdes-Quezada, Christian; Recillas-Targa, Félix

    2009-10-15

    Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional regulation. Here we determined the patterns of chicken alpha-globin gene expression during development and erythroid differentiation. Previously published data suggested that the promoter regions of alpha-globin genes contain the complete information for proper developmental regulation. However, our data show a preferential trans-activation of the embryonic alpha-globin gene independent of the developmental or differentiation stage. We also found that DNA methylation and histone deacetylation play key roles in silencing the expression of the embryonic pi gene in definitive erythrocytes. However, drug-mediated reactivation of the embryonic gene during definitive erythropoiesis dramatically impaired the expression of the adult genes, suggesting gene competition or interference for enhancer elements. Our results also support a model in which the lack of open chromatin marks and localized recruitment of chicken MeCP2 contribute to autonomous gene silencing of the embryonic alpha-globin gene in a developmentally specific manner. We propose that epigenetic mechanisms are necessary for in vivo chicken alpha-globin gene switching through differential gene silencing of the embryonic alpha-globin gene in order to allow proper activation of adult alpha-globin genes.

  7. OCT guided microinjections for mouse embryonic research

    NASA Astrophysics Data System (ADS)

    Larin, Kirill V.; Syed, Saba H.; Coughlin, Andrew J.; Wang, Shang; West, Jennifer L.; Dickinson, Mary E.; Larina, Irina V.

    2013-02-01

    Optical coherence tomography (OCT) is gaining popularity as live imaging tool for embryonic research in animal models. Recently we have demonstrated that OCT can be used for live imaging of cultured early mouse embryos (E7.5-E10) as well as later stage mouse embryos in utero (E12.5 to the end of gestation). Targeted delivery of signaling molecules, drugs, and cells is a powerful approach to study normal and abnormal development, and image guidance is highly important for such manipulations. Here we demonstrate that OCT can be used to guide microinjections of gold nanoshell suspensions in live mouse embryos. This approach can potentially be used for variety of applications such as guided injections of contrast agents, signaling molecules, pharmacological agents, cell transplantation and extraction, as well as other image-guided micromanipulations. Our studies also reveal novel potential for gold nanoshells in embryonic research.

  8. Mechanically patterning the embryonic airway epithelium.

    PubMed

    Varner, Victor D; Gleghorn, Jason P; Miller, Erin; Radisky, Derek C; Nelson, Celeste M

    2015-07-28

    Collections of cells must be patterned spatially during embryonic development to generate the intricate architectures of mature tissues. In several cases, including the formation of the branched airways of the lung, reciprocal signaling between an epithelium and its surrounding mesenchyme helps generate these spatial patterns. Several molecular signals are thought to interact via reaction-diffusion kinetics to create distinct biochemical patterns, which act as molecular precursors to actual, physical patterns of biological structure and function. Here, however, we show that purely physical mechanisms can drive spatial patterning within embryonic epithelia. Specifically, we find that a growth-induced physical instability defines the relative locations of branches within the developing murine airway epithelium in the absence of mesenchyme. The dominant wavelength of this instability determines the branching pattern and is controlled by epithelial growth rates. These data suggest that physical mechanisms can create the biological patterns that underlie tissue morphogenesis in the embryo.

  9. Mechanically patterning the embryonic airway epithelium

    PubMed Central

    Varner, Victor D.; Gleghorn, Jason P.; Miller, Erin; Radisky, Derek C.; Nelson, Celeste M.

    2015-01-01

    Collections of cells must be patterned spatially during embryonic development to generate the intricate architectures of mature tissues. In several cases, including the formation of the branched airways of the lung, reciprocal signaling between an epithelium and its surrounding mesenchyme helps generate these spatial patterns. Several molecular signals are thought to interact via reaction-diffusion kinetics to create distinct biochemical patterns, which act as molecular precursors to actual, physical patterns of biological structure and function. Here, however, we show that purely physical mechanisms can drive spatial patterning within embryonic epithelia. Specifically, we find that a growth-induced physical instability defines the relative locations of branches within the developing murine airway epithelium in the absence of mesenchyme. The dominant wavelength of this instability determines the branching pattern and is controlled by epithelial growth rates. These data suggest that physical mechanisms can create the biological patterns that underlie tissue morphogenesis in the embryo. PMID:26170292

  10. Fibrogenic Lung Injury Induces Non-Cell-Autonomous Fibroblast Invasion.

    PubMed

    Ahluwalia, Neil; Grasberger, Paula E; Mugo, Brian M; Feghali-Bostwick, Carol; Pardo, Annie; Selman, Moisés; Lagares, David; Tager, Andrew M

    2016-06-01

    Pathologic accumulation of fibroblasts in pulmonary fibrosis appears to depend on their invasion through basement membranes and extracellular matrices. Fibroblasts from the fibrotic lungs of patients with idiopathic pulmonary fibrosis (IPF) have been demonstrated to acquire a phenotype characterized by increased cell-autonomous invasion. Here, we investigated whether fibroblast invasion is further stimulated by soluble mediators induced by lung injury. We found that bronchoalveolar lavage fluids from bleomycin-challenged mice or patients with IPF contain mediators that dramatically increase the matrix invasion of primary lung fibroblasts. Further characterization of this non-cell-autonomous fibroblast invasion suggested that the mediators driving this process are produced locally after lung injury and are preferentially produced by fibrogenic (e.g., bleomycin-induced) rather than nonfibrogenic (e.g., LPS-induced) lung injury. Comparison of invasion and migration induced by a series of fibroblast-active mediators indicated that these two forms of fibroblast movement are directed by distinct sets of stimuli. Finally, knockdown of multiple different membrane receptors, including platelet-derived growth factor receptor-β, lysophosphatidic acid 1, epidermal growth factor receptor, and fibroblast growth factor receptor 2, mitigated the non-cell-autonomous fibroblast invasion induced by bronchoalveolar lavage from bleomycin-injured mice, suggesting that multiple different mediators drive fibroblast invasion in pulmonary fibrosis. The magnitude of this mediator-driven fibroblast invasion suggests that its inhibition could be a novel therapeutic strategy for pulmonary fibrosis. Further elaboration of the molecular mechanisms that drive non-cell-autonomous fibroblast invasion consequently may provide a rich set of novel drug targets for the treatment of IPF and other fibrotic lung diseases.

  11. A trade-off between embryonic development rate and immune function of avian offspring is concealed by embryonic temperature

    USGS Publications Warehouse

    Martin, Thomas E.; Arriero, Elena; Majewska, Ania

    2011-01-01

    Long embryonic periods are assumed to reflect slower intrinsic development that are thought to trade off to allow enhanced physiological systems, such as immune function. Yet, the relatively rare studies of this trade-off in avian offspring have not found the expected trade-off. Theory and tests have not taken into account the strong extrinsic effects of temperature on embryonic periods of birds. Here, we show that length of the embryonic period did not explain variation in two measures of immune function when temperature was ignored, based on studies of 34 Passerine species in tropical Venezuela (23 species) and north temperate Arizona (11 species). Variation in immune function was explained when embryonic periods were corrected for average embryonic temperature, in order to better estimate intrinsic rates of development. Immune function of offspring trades off with intrinsic rates of embryonic development once the extrinsic effects of embryonic temperatures are taken into account.

  12. Metabolic circadian rhythms in embryonic turtles.

    PubMed

    Loudon, Fiona Kay; Spencer, Ricky-John; Strassmeyer, Alana; Harland, Karen

    2013-07-01

    Oviparous species are model organisms for investigating embryonic development of endogenous physiological circadian rhythms without the influence of maternal biorhythms. Recent studies have demonstrated that heart rates and metabolic rates of embryonic turtles are not constant or always maximal and can be altered in response to the presence of embryos at a more advanced stage of development within the nest. A first step in understanding the physiological mechanisms underpinning these responses in embryonic ectothermic organisms is to develop metabolic profiles (e.g., heart rate) at different temperatures throughout incubation. Heart beat and rhythmic patterns or changes in development may represent important signals or cues within a nest and may be vital to coordinate synchronous hatching well in advance of the final stages of incubation. We developed baseline embryonic heart-rate profiles of embryos of the short-necked Murray River turtle (Emydura macquarii) to determine the stage of embryogenesis that metabolic circadian rhythms become established, if at all. Eggs were incubated at constant temperatures (26°C and 30°C) and heart rates were monitored at 6-h intervals over 24 h every 7-11 days until hatching. Circadian heart rate rhythms were detected at the mid-gestation period and were maintained until hatching. Heart rates throughout the day varied by up to 20% over 24 h and were not related to time of day. This study demonstrated that endogenous metabolic circadian rhythms in developing embryos in turtle eggs establish earlier in embryogenesis than those documented in other vertebrate taxa during embryogenesis. Early establishment of circadian rhythms in heart rates may be critical for communication among embryos and synchrony in hatching and emergence from the nest.

  13. Fibroblast growth factor (FGF) gene expression in the developing cerebellum suggests multiple roles for FGF signaling during cerebellar morphogenesis and development.

    PubMed

    Yaguchi, Yuichiro; Yu, Tian; Ahmed, Mohi U; Berry, Mary; Mason, Ivor; Basson, M Albert

    2009-08-01

    The cerebellum is derived from the anterior-most segment of the embryonic hindbrain, rhombomere 1 (r1). Previous studies have shown that the early development and patterning of r1 requires fibroblast growth factor (FGF) signaling. However, many of the developmental processes that shape cerebellar morphogenesis take place later in embryonic development and during the first 2 weeks of postnatal life in the mouse. Here, we present a more comprehensive analysis of the expression patterns of genes encoding FGF receptors and secreted FGF ligands during these later stages of cerebellar development. We show that these genes are expressed in multiple cell types in the developing cerebellum, in an astonishing array of distinct patterns. These data suggest that FGF signaling functions throughout cerebellar development to regulate many processes that shape the formation of a functional cerebellum.

  14. Epicardially-derived Fibroblasts Preferentially Contribute to the Parietal Leaflets of the Atrioventricular Valves in the Murine Heart

    PubMed Central

    Wessels, Andy; van den Hoff, Maurice J. B.; Adamo, Richard F.; Phelps, Aimee L.; Lockhart, Marie M.; Sauls, Kimberly; Briggs, Laura E.; Norris, Russell A.; van Wijk, Bram; Perez-Pomares, Jose M.; Dettman, Robert W.; Burch, John B. E.

    2012-01-01

    The importance of the epicardium for myocardial and valvuloseptal development has been well established; perturbation of epicardial development results in cardiac abnormalities, including thinning of the ventricular myocardial wall and malformations of the atrioventricular valvuloseptal complex. To determine the spatiotemporal contribution of epicardially derived cells to the developing fibroblast population in the heart we have used a mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED)10 until birth. EPDCs begin to populate the compact ventricular myocardium around ED12. The migration of epicardially-derived fibroblasts toward the interface between compact and trabecular myocardium is completed around ED14. Remarkably, epicardially-derived fibroblasts do not migrate into the trabecular myocardium until after ED17. Migration of EPDCs into the atrioventricular cushion mesenchyme commences around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets which derive from the lateral atrioventricular cushions. In these developing leaflets the epicardially-derived fibroblasts eventually largely replace the endocardially-derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the various leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in studies of heart development. PMID:22546693

  15. Do embryonic polar bodies commit suicide?

    PubMed

    Fabian, Dušan; Čikoš, Štefan; Rehák, Pavol; Koppel, Juraj

    2014-02-01

    The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

  16. Informing tendon tissue engineering with embryonic development.

    PubMed

    Glass, Zachary A; Schiele, Nathan R; Kuo, Catherine K

    2014-06-27

    Tendon is a strong connective tissue that transduces muscle-generated forces into skeletal motion. In fulfilling this role, tendons are subjected to repeated mechanical loading and high stress, which may result in injury. Tissue engineering with stem cells offers the potential to replace injured/damaged tissue with healthy, new living tissue. Critical to tendon tissue engineering is the induction and guidance of stem cells towards the tendon phenotype. Typical strategies have relied on adult tissue homeostatic and healing factors to influence stem cell differentiation, but have yet to achieve tissue regeneration. A novel paradigm is to use embryonic developmental factors as cues to promote tendon regeneration. Embryonic tendon progenitor cell differentiation in vivo is regulated by a combination of mechanical and chemical factors. We propose that these cues will guide stem cells to recapitulate critical aspects of tenogenesis and effectively direct the cells to differentiate and regenerate new tendon. Here, we review recent efforts to identify mechanical and chemical factors of embryonic tendon development to guide stem/progenitor cell differentiation toward new tendon formation, and discuss the role this work may have in the future of tendon tissue engineering.

  17. Embryonic development of Pelteobagrus fulvidraco (Richardson, 1846)

    NASA Astrophysics Data System (ADS)

    Wang, Weimin; Abbas, Khalid; Yan, Ansheng

    2006-12-01

    For production enhancement and procedure upgrade, the developmental phases of laboratory-reared eggs of catfish Pelteobagrus fulvidraco were investigated. Twenty mature females and 10 males were collected from Dadongmen wholesale fisheries market in Wuhan City on May 8, 2003. Zygotes were stripped from mature fish after hormone-induced ovulation, fertilized, and incubated through whole embryonic development. The fertilized eggs were stocked in density of 100 eggs/L in white square tanks of 10 L. Incubation water was dechlorinated tap water with continuous aeration. The tanks were lit directly with 60 W fluorescent bulbs with a 12 light: 12 dark photoperiod. Water temperature, dissolved oxygen and pH were 29.0±0.5°C, 6.7±0.4 mg/L and 7.4±2, respectively. The results showed that the eggs of P. fulvidraco were yellow, sticky and contained much yolk. The mean diameter of fertilized eggs was 2.03 mm. At the water temperature of 29.0±0.5°C, the ontogenesis spent about 33 h after fertilization. From fertilization to hatching, the embryonic development can be divided into 30 40 phases, which varies in the emphasis and direction of development. The detailed embryonic movement was also described.

  18. [Heart tissue from embryonic stem cells].

    PubMed

    Zimmermann, W-H

    2008-09-01

    Embryonic stem cells can give rise to all somatic cells, making them an attractive cell source for tissue engineering applications. The propensity of cells to form tissue-like structures in a culture dish has been well documented. We and others made use of this intrinsic property to generate bioartificial heart muscle. First proof-of-concept studies involved immature heart cells mainly from fetal chicken, neonatal rats and mice. They eventually provided evidence that force-generating heart muscle can be engineered in vitro. Recently, the focus shifted to the application of stem cells to eventually enable the generation of human heart muscle and reach following long-term goals: (1) development of a simplified in vitro model of heart muscle development; (2) generation of a human test-bed for drug screening and development; (3) allocation of surrogate heart tissue to myocardial repair applications. This overview will provide the background for cell-based myocardial repair, introduce the main myocardial tissue engineering concepts, discuss the use of embryonic and non-embryonic stem cells, and lays out the potential direct and indirect therapeutic use of human tissue engineered myocardium.

  19. Human embryonic stem cells and lung regeneration.

    PubMed

    Varanou, A; Page, C P; Minger, S L

    2008-10-01

    Human embryonic stem cells are pluripotent cells derived from the inner cell mass of preimplantation stage embryos. Their unique potential to give rise to all differentiated cell types has generated great interest in stem cell research and the potential that it may have in developmental biology, medicine and pharmacology. The main focus of stem cell research has been on cell therapy for pathological conditions with no current methods of treatment, such as neurodegenerative diseases, cardiac pathology, retinal dysfunction and lung and liver disease. The overall aim is to develop methods of application either of pure cell populations or of whole tissue parts to the diseased organ under investigation. In the field of pulmonary research, studies using human embryonic stem cells have succeeded in generating enriched cultures of type II pneumocytes in vitro. On account of their potential of indefinite proliferation in vitro, embryonic stem cells could be a source of an unlimited supply of cells available for transplantation and for use in gene therapy. Uncovering the ability to generate such cell types will expand our understanding of biological processes to such a degree that disease understanding and management could change dramatically.

  20. Mechanical signaling coordinates the embryonic heartbeat

    PubMed Central

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

    2016-01-01

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

  1. Informing tendon tissue engineering with embryonic development

    PubMed Central

    Glass, Zachary A.; Schiele, Nathan R.; Kuo, Catherine K.

    2014-01-01

    Tendon is a strong connective tissue that transduces muscle-generated forces into skeletal motion. In fulfilling this role, tendons are subjected to repeated mechanical loading and high stress, which may result in injury. Tissue engineering with stem cells offers the potential to replace injured/damaged tissue with healthy, new living tissue. Critical to tendon tissue engineering is the induction and guidance of stem cells towards the tendon phenotype. Typical strategies have relied on adult tissue homeostatic and healing factors to influence stem cell differentiation, but have yet to achieve tissue regeneration. A novel paradigm is to use embryonic developmental factors as cues to promote tendon regeneration. Embryonic tendon progenitor cell differentiation in vivo is regulated by a combination of mechanical and chemical factors. We propose that these cues will guide stem cells to recapitulate critical aspects of tenogenesis and effectively direct the cells to differentiate and regenerate new tendon. Here, we review recent efforts to identify mechanical and chemical factors of embryonic tendon development to guide stem/progenitor cell differentiation toward new tendon formation, and discuss the role this work may have in the future of tendon tissue engineering. PMID:24484642

  2. Embryonic and embryonic-like stem cells in heart muscle engineering.

    PubMed

    Zimmermann, Wolfram-Hubertus

    2011-02-01

    Cardiac muscle engineering is evolving rapidly and may ultimately be exploited to (1) model cardiac development, physiology, and pathology; (2) identify and validate drug targets; (3) assess drug safety and efficacy; and (4) provide therapeutic substitute myocardium. The ultimate success in any of these envisioned applications depends on the utility of human cells and their assembly into myocardial equivalents with structural and functional properties of mature heart muscle. Embryonic stem cells appear as a promising cell source in this respect, because they can be cultured reliably and differentiated robustly into cardiomyocytes. Despite their unambiguous cardiogenicity, data on advanced maturation and seamless myocardial integration of embryonic stem cell-derived cardiomyocytes in vivo are sparse. Additional concerns relate to the limited control over cardiomyogenic specification and cardiomyocyte maturation in vitro as well as the risk of teratocarcinoma formation and immune rejection of stem cell implants in vivo. Through the invent of embryonic-like stem cells - such as parthenogenetic stem cells, male germline stem cells, and induced pluripotent stem cells - some but certainly not all of these issues may be addressed, albeit at the expense of additional concerns. This review will discuss the applicability of embryonic and embryonic-like stem cells in myocardial tissue engineering and address issues that require particular attention before the potential of stem cell-based heart muscle engineering may be fully exploited. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited".

  3. Morphology of the embryonic and hatchling American alligator ductus arteriosi and implications for embryonic cardiovascular shunting.

    PubMed

    Jacobs, Kimberley; Goy, Sarah K; Dzialowski, Edward M

    2012-02-01

    The ductus arteriosi (DA) are embryonic blood vessels found in amniotic vertebrates that shunt blood away from the pulmonary artery and lungs and toward the aorta. Here, we examine changes in morphology of the right and left DA (LDA), and right and left aorta (LAo) from embryonic and hatchling alligators. The developing alligator has two-patent DA that join the right and LAo. Both DA exhibit a muscular phenotype composed of an internal smooth muscle layer (2-4 cells thick). At hatching, the lumen diameter of both DA decreases as the vessels begin to close within the first 12 h of posthatch life. Between day 1 and day 12 posthatching, the vessel becomes fully occluded with endothelial and smooth muscle cells filling the lumen. A number of DA from hatchlings contained blood clots along their length. The lumen of the full term alligator DA is reduced in comparison with the full term chicken DA. The developing alligator embryo has an additional right-to-left shunt pathway in the LAo arising from the right ventricle. The embryonic LAo diameter is twice the diameter of either the right DA or LDA, providing a lower resistance pathway for blood leaving the right ventricle. On the basis of these findings, we propose that the paired DA of the embryonic alligator have a reduced role in the embryonic right-to-left shunt of blood from the right ventricle when compared with the avian DA.

  4. Hsp90 regulation of fibroblast activation in pulmonary fibrosis

    PubMed Central

    Sontake, Vishwaraj; Wang, Yunguan; Kasam, Rajesh K.; Sinner, Debora; Reddy, Geereddy B.; Naren, Anjaparavanda P.; McCormack, Francis X.; Jegga, Anil G.; Madala, Satish K.

    2017-01-01

    Idiopathic pulmonary fibrosis (IPF) is a severe fibrotic lung disease associated with fibroblast activation that includes excessive proliferation, tissue invasiveness, myofibroblast transformation, and extracellular matrix (ECM) production. To identify inhibitors that can attenuate fibroblast activation, we queried IPF gene signatures against a library of small-molecule-induced gene-expression profiles and identified Hsp90 inhibitors as potential therapeutic agents that can suppress fibroblast activation in IPF. Although Hsp90 is a molecular chaperone that regulates multiple processes involved in fibroblast activation, it has not been previously proposed as a molecular target in IPF. Here, we found elevated Hsp90 staining in lung biopsies of patients with IPF. Notably, fibroblasts isolated from fibrotic lesions showed heightened Hsp90 ATPase activity compared with normal fibroblasts. 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), a small-molecule inhibitor of Hsp90 ATPase activity, attenuated fibroblast activation and also TGF-β–driven effects on fibroblast to myofibroblast transformation. The loss of the Hsp90AB, but not the Hsp90AA isoform, resulted in reduced fibroblast proliferation, myofibroblast transformation, and ECM production. Finally, in vivo therapy with 17-AAG attenuated progression of established and ongoing fibrosis in a mouse model of pulmonary fibrosis, suggesting that targeting Hsp90 represents an effective strategy for the treatment of fibrotic lung disease. PMID:28239659

  5. Apathy in aging: are lack of interest and lack of initiative dissociable?

    PubMed

    Esposito, Fabienne; Rochat, Lucien; Juillerat Van der Linden, Anne-Claude; Lekeu, Françoise; Charnallet, Annik; Van der Linden, Martial

    2014-01-01

    Apathy is common in aging and generally defined on the basis of three dimensions: lack of initiative, lack of interest and emotional blunting. Curiously, no study until now has examined the associations and dissociations between these dimensions in elderly people (with or without dementia). These questions were addressed in two studies. In the first study, we explored the distribution of scores and the relationships between the three dimensions of apathy in 56 patients with dementia, focusing mainly on lack of initiative and lack of interest. Apathy was hetero-evaluated with the Apathy Inventory (AI), a scale widely used to assess the apathy dimensions in aging. In the second study, given the AI's limitations, we investigated in more detail the relationship between lack of initiative and interest in 115 elderly people using a new questionnaire specifically designed to assess these two dimensions. Results showed that lack of initiative was closely related to lack of interest (Study 1). Although we used a more specific questionnaire, these facets of apathy did not constitute two separable dimensions, but reflected a common main factor of apathy in aging (Study 2). Thus, the distinction between lack of initiative and lack of interest seems questionable. Only a multifactorial approach that includes the various psychological factors involved in apathy would enable one to gain a better understanding of the different manifestations of apathy and to highlight possible dissociations between them.

  6. Response of keloid fibroblasts to Vitamin D3 and quercetin treatment - in vitro study.

    PubMed

    Mathangi Ramakrishnan, K; Babu, M; Lakshmi Madhavi, M S

    2015-09-30

    Keloid scars continue to pose a challenge to clinicians as the treatment armamentarium lacks a formidable agent to tackle them. We have undertaken an in vitro study based on the mechanism of action of Vitamin D3 and quercetin on isolated keloid fibroblasts. Dose-dependent action on the reduction of cellular proliferation, collagen synthesis and induction of apoptosis by Vitamin D3 and quercetin are analyzed and probable mechanism of action is elaborated. This study thus opens up newer avenues in tackling keloid scars effectively.

  7. Suppression of fibroblast proliferation by oral spirochetes.

    PubMed Central

    Boehringer, H; Taichman, N S; Shenker, B J

    1984-01-01

    Soluble sonic extracts of several strains of Treponema denticola and Treponema vincentii were examined for their abilities to alter proliferation of both murine and human fibroblasts. We found that sonic extracts of all tested strains of T. denticola caused a dose-dependent inhibition of murine and human fibroblast proliferation when assessed by both DNA synthesis ([3H]thymidine incorporation) and direct cell counts. T. vincentii had only a minimal inhibitory effect at comparable doses. No inhibition was observed when sonic extracts were added simultaneously with [3H]thymidine, indicating that suppression was not due to the presence of excessive amounts of cold thymidine in the extract, nonspecific effects on thymidine utilization by the cells (transport and incorporation), or degradation of label. RNA ([3H]uridine incorporation) and protein ([3H]leucine incorporation) synthesis were similarly altered after exposure to the T. denticola sonic extracts. There was no effect on cell viability as measured by trypan blue exclusion. Inhibition could be reversed by extensive washing of the cells within the first few hours of exposure to sonic extracts. Preliminary characterization and purification indicated that the inhibitory factor(s) is not endotoxin since it is heat labile, and elutes in a single, well-defined peak on a Sephadex G-150 chromatography column corresponding to a molecular weight of approximately 50,000. Since oral spirochetes have been implicated in the pathogenesis of periodontal disorders, it is possible that they contribute to the disease process by inhibition of fibroblast growth and therefore may, at least in part, account for the loss of collagen seen in diseased tissue. PMID:6735466

  8. Characterization of human fibroblastic reticular cells as potential immunotherapeutic tools.

    PubMed

    Valencia, Jaris; Jiménez, Eva; Martínez, Víctor G; Del Amo, Beatriz G; Hidalgo, Laura; Entrena, Ana; Fernández-Sevilla, Lidia M; Del Río, Francisco; Varas, Alberto; Vicente, Ángeles; Sacedón, Rosa

    2017-05-01

    Fibroblastic reticular cells (FRCs) are essential players during adaptive immune responses not only as a structural support for the encounter of antigen-presenting cells and naive T lymphocytes but also as a source of modulatory signals. However, little is known about this cell population in humans. To address the phenotypical and functional analysis of human FRCs here we established splenic (SP) and mesenteric lymph node (LN) CD45(-)CD31(-)CD90(+)podoplanin(+) myofibroblastic cell cultures. They shared the phenotypical characteristics distinctive of FRCs, including the expression of immunomodulatory factors and peripheral tissue antigens. Nevertheless, human FRCs also showed particular features, some differing from mouse FRCs, like the lack of nitric oxide synthase (NOS2) expression after interferon (IFN)γstimulation. Interestingly, SP-FRCs expressed higher levels of interleukin (IL)-6, BMP4, CCL2, CXCL12 and Notch molecules, and strongly adapted their functional profile to lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (Poly I:C) and IFNγ stimulation. In contrast, we found higher expression of transforming growth factor (TGF)β and Activin A in LN-FRCs that barely responded via Toll-Like Receptor (TLR)3 and constitutively expressed retinaldehyde dehydrogenase 1 enzyme, absent in SP-FRCs. This study reveals human FRCs can be valuable models to increase our knowledge about the physiology of human secondary lymphoid organs in health and disease and to explore the therapeutic options of FRCs.

  9. Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer

    PubMed Central

    Handly-Santana, Abram; Biffi, Giulia; Elyada, Ela; Ponz-Sarvise, Mariano; Lee, Eun Jung; Chio, Iok In Christine; Tiriac, Hervé; Feig, Christine; Kultti, Anne; Crawford, James M.; Clevers, Hans; Park, Youngkyu

    2017-01-01

    Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development. PMID:28232471

  10. Immortalization of Werner syndrome and progeria fibroblasts

    SciTech Connect

    Saito, H.; Moses, R.E. )

    1991-02-01

    Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents.

  11. SV40-mediated immortalization of human fibroblasts.

    PubMed

    Ozer, H L; Banga, S S; Dasgupta, T; Houghton, J; Hubbard, K; Jha, K K; Kim, S H; Lenahan, M; Pang, Z; Pardinas, J R; Patsalis, P C

    1996-01-01

    We have identified a multistep mechanism by which the DNA virus SV40 overcomes cellular senescence. Expression of SV40 T antigen is required for both transient extension of life span and unlimited life span or immortalization. These effects are mediated through inactivation of function of growth suppressors pRB and p53 via complex formation with T antigen. However, immortalization additionally requires inactivation of a novel growth suppressor gene, which has recently been identified to be on the distal portion of the long arm of chromosome 6, designated SEN6. We propose that SEN6 is responsible for cellular senescence in fibroblasts and other cells.

  12. Mechanical properties of endothelialized fibroblast-derived vascular scaffolds stimulated in a bioreactor.

    PubMed

    Tondreau, Maxime Y; Laterreur, Véronique; Gauvin, Robert; Vallières, Karine; Bourget, Jean-Michel; Lacroix, Dan; Tremblay, Catherine; Germain, Lucie; Ruel, Jean; Auger, François A

    2015-05-01

    There is an ongoing clinical need for tissue-engineered small-diameter (<6mm) vascular grafts since clinical applications are restricted by the limited availability of autologous living grafts or the lack of suitability of synthetic grafts. The present study uses our self-assembly approach to produce a fibroblast-derived decellularized vascular scaffold that can then be available off-the-shelf. Briefly, scaffolds were produced using human dermal fibroblasts sheets rolled around a mandrel, maintained in culture to allow for the formation of cohesive and three-dimensional tubular constructs, and then decellularized by immersion in deionized water. Constructs were then endothelialized and perfused for 1week in an appropriate bioreactor. Mechanical testing results showed that the decellularization process did not influence the resistance of the tissue and an increase in ultimate tensile strength was observed following the perfusion of the construct in the bioreactor. These fibroblast-derived vascular scaffolds could be stored and later used to deliver readily implantable grafts within 4weeks including an autologous endothelial cell isolation and seeding process. This technology could greatly accelerate the clinical availability of tissue-engineered blood vessels.

  13. Sequence analysis of bovine C/EBPδ gene and its adipogenic effects on fibroblasts.

    PubMed

    Wang, Hong; Cheng, Gong; Fu, Changzhen; Wang, Hongbao; Yang, Wucai; Wang, Hongcheng; Zan, Linsen

    2014-01-01

    CCAAT/enhancer binding protein delta (C/EBPδ), an important transcriptional factor, regulates cell growth, differentiation and adipogenesis in humans and mice. However, we lack of directive information on the effects of C/EBPδ gene in bovine cells. In the present study, we cloned the CDS areas of bovine C/EBPδ gene and predicted its sequence characteristics. Moreover, we constructed the recombinant adenovirus plasmids of bovine C/EBPδ gene and harvested the subsequent adenoviruses to infect bovine primary fibroblasts. Oil Red O staining results showed lipid droplets accumulated gradually in the adenoviruses treated fibroblasts. Time course real-time PCR results indicated that over-expression of exogenous C/EBPδ regulated the mRNA expression levels of some key adipogenic genes, herein, activated the C/EBPα expression, increased lipoprotein lipase and fatty acid binding protein 4 mRNA expression levels, whereas inhibited leptin receptor gene. In conclusion, the present study demonstrates that the elevated C/EBPδ can induce the adipogenesis in the fibroblasts of cattle.

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

    NASA Astrophysics Data System (ADS)

    Rajanahalli Krishnamurthy, Pavan

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

  15. Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts

    SciTech Connect

    Cockayne, D.; Cutroneo, K.R.

    1988-04-19

    Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro..cap alpha..1(I) and pro..cap alpha..2(I) mRNAs but not ..beta..-actin mRNA. Fibroblasts receiving dexamethasone and (5,6-/sup 3/H)uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not ..beta..-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, the authors determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the ..cap alpha..2(I) procollagen gene. Nuclear protein blots were probed with the /sup 32/P-end-labeled pBR322 vector DNA and /sup 32/P-end-labeled ..cap alpha..2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the ..cap alpha..2(I) procollagen promoter containing DNA were calculated.

  16. LRRK2 Expression Is Deregulated in Fibroblasts and Neurons from Parkinson Patients with Mutations in PINK1.

    PubMed

    Azkona, Garikoitz; López de Maturana, Rakel; Del Rio, Patricia; Sousa, Amaya; Vazquez, Nerea; Zubiarrain, Amaia; Jimenez-Blasco, Daniel; Bolaños, Juan P; Morales, Blas; Auburger, Georg; Arbelo, José Matias; Sánchez-Pernaute, Rosario

    2016-12-14

    Mutations in PINK1 (PARK6), a serine/threonine kinase involved in mitochondrial homeostasis, are associated with early onset Parkinson's disease. Fibroblasts from Parkinson's disease patients with compound heterozygous mutations in exon 7 (c.1488 + 1G > A; c.1252_1488del) showed no apparent signs of mitochondrial impairment. To elucidate changes primarily caused by lack of functional PINK1, we over-expressed wild-type PINK1, which induced a significant downregulation of LRRK2 (PARK8). Indeed, we found that LRRK2 protein basal levels were significantly higher in the mutant PINK1 fibroblasts. To examine the interaction between the two PARK genes in a disease-relevant cell context, we generated induced pluripotent stem cell (iPSC) lines from mutant, carrier and control fibroblasts by lentiviral-mediated re-programming. Efficiency of neural induction and dopamine differentiation using a floor-plate induction protocol was similar in all genotypes. As observed in fibroblasts, PINK1 mutant neurons showed increased LRRK2 expression both at the RNA and protein level and transient over-expression of wild-type PINK1 efficiently downregulated LRRK2 levels. Additionally, we confirmed a dysregulation of LRRK2 expression in fibroblasts from patients with a different homozygous mutation in PINK1 exon 4, c.926G > A (G309D). Thus, our results identify a novel role of PINK1 modulating the levels of LRRK2 in Parkinson's disease fibroblasts and neurons, suggest a convergent pathway for these PARK genes, and broaden the role of LRRK2 in the pathogenesis of Parkinson's disease.

  17. Fibroblast Growth Factor Receptor-Dependent and -Independent Paracrine Signaling by Sunitinib-Resistant Renal Cell Carcinoma

    PubMed Central

    Tran, Tram Anh; Leong, Hon Sing; Pavia-Jimenez, Andrea; Fedyshyn, Slavic; Yang, Juan; Kucejova, Blanka; Sivanand, Sharanya; Spence, Patrick; Xie, Xian-Jin; Peña-Llopis, Samuel; Power, Nicholas

    2016-01-01

    Antiangiogenic therapies, such as sunitinib, have revolutionized renal cell carcinoma (RCC) treatment. However, a precarious understanding of how resistance emerges and a lack of tractable experimental systems hinder progress. We evaluated the potential of primary RCC cultures (derived from tumors and tumor grafts) to signal to endothelial cells (EC) and fibroblasts in vitro and to stimulate angiogenesis ex vivo in chorioallantoic membrane (CAM) assays. From 65 patients, 27 primary cultures, including several from patients with sunitinib-resistant RCC, were established. RCC cells supported EC survival in coculture assays and induced angiogenesis in CAM assays. RCC-induced EC survival was sensitive to sunitinib in half of the tumors and was refractory in tumors from resistant patients. Sunitinib sensitivity correlated with vascular endothelial growth factor (VEGF) production. RCC induced paracrine extracellular signal-regulated kinase (ERK) activation in EC which was inhibited by sunitinib in sensitive but not in resistant tumors. As determined by fibroblast growth factor receptor substrate 2 (FRS2) phosphorylation in fibroblasts, RCC broadly induced low-level fibroblast growth factor receptor (FGFR) signaling. Whereas ERK activation in EC was uniformly inhibited by combined VEGF/platelet-derived growth factor (PDGF)/FGF receptor inhibitors, paracrine ERK activation in fibroblasts was blocked in only a fraction of tumors. Our data show that RCC activates EC through VEGF-dependent and -independent pathways, that sunitinib sensitivity correlates with VEGF-mediated ERK activation, and that combined inhibition of VEGF/PDGF/FGF receptors is sufficient to inhibit mitogenic signaling in EC but not in fibroblasts. PMID:27141054

  18. New mouse xenograft model modulated by tumor-associated fibroblasts for human multi-drug resistance in cancer

    PubMed Central

    MA, YAN; LIN, ZHIQIANG; FALLON, JOHN K.; ZHAO, QIANG; LIU, DAN; WANG, YONGJUN; LIU, FENG

    2015-01-01

    We developed an MDR tumor model that is modulated by tumor-associated fibroblasts. Studies on proliferation of tumor cell lines including paclitaxel-sensitive and resistant cell lines were performed. The expressions of P-gp and α-smooth muscle actin (α-SMA) antigen were evaluated by immunohistochemistry and western blot analysis. Quantitative P-gp analyses of different cell lines were accomplished by nanoUPLC-MS/MS. Tumor cell colony formation assay and established xenograft model was used to investigate the relationship between P-gp expression, fibroblast levels and tumorigenesis. The mouse xenograft model was developed after co-inoculation with MDR tumor cells and NIH/3T3 fibroblast cells. There was no correlation between tumorigenesis in vivo and the growth rate of cells in vitro. The proliferation among different cell lines had no significant differences, but the P-gp expression and tumor growth in the xenograft model were fairly different. P-gp determination and α-SMA immunofluorescence staining clarified the relationship between P-gp expression, fibroblast levels and tumorigenesis. It was more difficult for tumor cells with higher P-gp levels to recruit fibroblasts in vivo, resulting in lower tumorigenesis due to the lack of structural and chemical support during tumor progression. In the established paclitaxel-resistant mouse xenograft model, no obvious antitumor effect was observed after Taxol treatment, but a significant decrease in tumor size for the group treated with gemcitabine sensitive to the model. The results show that the added fibroblasts do not disturb the applicability of the model in MDR. Therefore, this mouse xenograft MDR model could serve as an effective tool for MDR research. PMID:26352907

  19. Melanoma Cells Block PEDF Production in Fibroblasts to Induce the Tumor-Promoting Phenotype of Cancer-Associated Fibroblasts.

    PubMed

    Nwani, Nkechiyere G; Deguiz, Maria L; Jimenez, Benilde; Vinokour, Elena; Dubrovskyi, Oleksii; Ugolkov, Andrey; Mazar, Andrew P; Volpert, Olga V

    2016-04-15

    Loss of pigment epithelium-derived factor (PEDF, SERPINF1) in cancer cells is associated with poor prognosis and metastasis, but the contribution of stromal PEDF to cancer evolution is poorly understood. Therefore, we investigated the role of fibroblast-derived PEDF in melanoma progression. We demonstrate that normal dermal fibroblasts expressing high PEDF levels attenuated melanoma growth and angiogenesis in vivo, whereas PEDF-depleted fibroblasts exerted tumor-promoting effects. Accordingly, mice with global PEDF knockout were more susceptible to melanoma metastasis. We also demonstrate that normal fibroblasts in close contact with PEDF-null melanoma cells lost PEDF expression and tumor-suppressive properties. Further mechanistic investigations underlying the crosstalk between tumor and stromal cells revealed that melanoma cells produced PDGF-BB and TGFβ, which blocked PEDF production in fibroblasts. Notably, cancer-associated fibroblasts (CAF) isolated from patient-derived tumors expressed markedly low levels of PEDF. Treatment of patient CAF and TGFβ-treated normal fibroblasts with exogenous PEDF decreased the expression of CAF markers and restored PEDF expression. Finally, expression profiling of PEDF-depleted fibroblasts revealed induction of IL8, SERPINB2, hyaluronan synthase-2, and other genes associated with tumor promotion and metastasis. Collectively, our results demonstrate that PEDF maintains tumor-suppressive functions in fibroblasts to prevent CAF conversion and illustrate the mechanisms by which melanoma cells silence stromal PEDF to promote malignancy. Cancer Res; 76(8); 2265-76. ©2016 AACR.

  20. Direct Conversion of Fibroblasts to Megakaryocyte Progenitors.

    PubMed

    Pulecio, Julian; Alejo-Valle, Oriol; Capellera-Garcia, Sandra; Vitaloni, Marianna; Rio, Paula; Mejía-Ramírez, Eva; Caserta, Ilaria; Bueren, Juan A; Flygare, Johan; Raya, Angel

    2016-10-11

    Current sources of platelets for transfusion are insufficient and associated with risk of alloimmunization and blood-borne infection. These limitations could be addressed by the generation of autologous megakaryocytes (MKs) derived in vitro from somatic cells with the ability to engraft and differentiate in vivo. Here, we show that overexpression of a defined set of six transcription factors efficiently converts mouse and human fibroblasts into MK-like progenitors. The transdifferentiated cells are CD41(+), display polylobulated nuclei, have ploidies higher than 4N, form MK colonies, and give rise to platelets in vitro. Moreover, transplantation of MK-like murine progenitor cells into NSG mice results in successful engraftment and further maturation in vivo. Similar results are obtained using disease-corrected fibroblasts from Fanconi anemia patients. Our results combined demonstrate that functional MK progenitors with clinical potential can be obtained in vitro, circumventing the use of hematopoietic progenitors or pluripotent stem cells.

  1. Altered chromosome 6 in immortal human fibroblasts.

    PubMed

    Hubbard-Smith, K; Patsalis, P; Pardinas, J R; Jha, K K; Henderson, A S; Ozer, H L

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene.

  2. Altered chromosome 6 in immortal human fibroblasts.

    PubMed Central

    Hubbard-Smith, K; Patsalis, P; Pardinas, J R; Jha, K K; Henderson, A S; Ozer, H L

    1992-01-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene. Images PMID:1373811

  3. Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila post-embryonic brain

    PubMed Central

    Avet-Rochex, Amélie; Kaul, Aamna K.; Gatt, Ariana P.; McNeill, Helen; Bateman, Joseph M.

    2012-01-01

    Glial cells are essential for the development and function of the nervous system. In the mammalian brain, vast numbers of glia of several different functional types are generated during late embryonic and early foetal development. However, the molecular cues that instruct gliogenesis and determine glial cell type are poorly understood. During post-embryonic development, the number of glia in the Drosophila larval brain increases dramatically, potentially providing a powerful model for understanding gliogenesis. Using glial-specific clonal analysis we find that perineural glia and cortex glia proliferate extensively through symmetric cell division in the post-embryonic brain. Using pan-glial inhibition and loss-of-function clonal analysis we find that Insulin-like receptor (InR)/Target of rapamycin (TOR) signalling is required for the proliferation of perineural glia. Fibroblast growth factor (FGF) signalling is also required for perineural glia proliferation and acts synergistically with the InR/TOR pathway. Cortex glia require InR in part, but not downstream components of the TOR pathway, for proliferation. Moreover, cortex glia absolutely require FGF signalling, such that inhibition of the FGF pathway almost completely blocks the generation of cortex glia. Neuronal expression of the FGF receptor ligand Pyramus is also required for the generation of cortex glia, suggesting a mechanism whereby neuronal FGF expression coordinates neurogenesis and cortex gliogenesis. In summary, we have identified two major pathways that control perineural and cortex gliogenesis in the post-embryonic brain and have shown that the molecular circuitry required is lineage specific. PMID:22745312

  4. Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

    PubMed

    Chang, Tien-Cheng; Liu, Ya-Guang; Eddy, Carlton A; Jacoby, Ethan S; Binkley, Peter A; Brzyski, Robert G; Schenken, Robert S

    2011-06-01

    The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

  5. Transcriptional Silencing of Moloney Murine Leukemia Virus in Human Embryonic Carcinoma Cells.

    PubMed

    Wang, Gary Z; Goff, Stephen P

    2017-01-01

    Embryonic carcinoma (EC) cells are malignant counterparts of embryonic stem (ES) cells and serve as useful models for investigating cellular differentiation and human embryogenesis. Though the susceptibility of murine EC cells to retroviral infection has been extensively analyzed, few studies of retrovirus infection of human EC cells have been performed. We tested the susceptibility of human EC cells to transduction by retroviral vectors derived from three different retroviral genera. We show that human EC cells efficiently express reporter genes delivered by vectors based on human immunodeficiency virus type 1 (HIV-1) and Mason-Pfizer monkey virus (M-PMV) but not Moloney murine leukemia virus (MLV). In human EC cells, MLV integration occurs normally, but no viral gene expression is observed. The block to MLV expression of MLV genomes is relieved upon cellular differentiation. The lack of gene expression is correlated with transcriptional silencing of the MLV promoter through the deposition of repressive histone marks as well as DNA methylation. Moreover, depletion of SETDB1, a histone methyltransferase, resulted in a loss of transcriptional silencing and upregulation of MLV gene expression. Finally, we provide evidence showing that the lack of MLV gene expression may be attributed in part to the lack of MLV enhancer function in human EC cells.

  6. [Microglial cells and development of the embryonic central nervous system].

    PubMed

    Legendre, Pascal; Le Corronc, Hervé

    2014-02-01

    Microglia cells are the macrophages of the central nervous system with a crucial function in the homeostasis of the adult brain. However, recent studies showed that microglial cells may also have important functions during early embryonic central nervous system development. In this review we summarize recent works on the extra embryonic origin of microglia, their progenitor niche, the pattern of their invasion of the embryonic central nervous system and on interactions between embryonic microglia and their local environment during invasion. We describe microglial functions during development of embryonic neuronal networks, including their roles in neurogenesis, in angiogenesis and developmental cell death. These recent discoveries open a new field of research on the functions of neural-microglial interactions during the development of the embryonic central nervous system.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. ...

  8. Ultraviolet germicidal irradiation and its effects on elemental distributions in mouse embryonic fibroblast cells in x-ray fluorescence microanalysis.

    PubMed

    Jin, Qiaoling; Vogt, Stefan; Lai, Barry; Chen, Si; Finney, Lydia; Gleber, Sophie-Charlotte; Ward, Jesse; Deng, Junjing; Mak, Rachel; Moonier, Nena; Jacobsen, Chris

    2015-01-01

    Rapidly-frozen hydrated (cryopreserved) specimens combined with cryo-scanning x-ray fluorescence microscopy provide an ideal approach for investigating elemental distributions in biological cells and tissues. However, because cryopreservation does not deactivate potentially infectious agents associated with Risk Group 2 biological materials, one must be concerned with contamination of expensive and complicated cryogenic x-ray microscopes when working with such materials. We employed ultraviolet germicidal irradiation to decontaminate previously cryopreserved cells under liquid nitrogen, and then investigated its effects on elemental distributions under both frozen hydrated and freeze dried states with x-ray fluorescence microscopy. We show that the contents and distributions of most biologically important elements remain nearly unchanged when compared with non-ultraviolet-irradiated counterparts, even after multiple cycles of ultraviolet germicidal irradiation and cryogenic x-ray imaging. This provides a potential pathway for rendering Risk Group 2 biological materials safe for handling in multiuser cryogenic x-ray microscopes without affecting the fidelity of the results.

  9. Ultraviolet Germicidal Irradiation and Its Effects on Elemental Distributions in Mouse Embryonic Fibroblast Cells in X-Ray Fluorescence Microanalysis

    PubMed Central

    Jin, Qiaoling; Vogt, Stefan; Lai, Barry; Chen, Si; Finney, Lydia; Gleber, Sophie-Charlotte; Ward, Jesse; Deng, Junjing; Mak, Rachel; Moonier, Nena; Jacobsen, Chris

    2015-01-01

    Rapidly-frozen hydrated (cryopreserved) specimens combined with cryo-scanning x-ray fluorescence microscopy provide an ideal approach for investigating elemental distributions in biological cells and tissues. However, because cryopreservation does not deactivate potentially infectious agents associated with Risk Group 2 biological materials, one must be concerned with contamination of expensive and complicated cryogenic x-ray microscopes when working with such materials. We employed ultraviolet germicidal irradiation to decontaminate previously cryopreserved cells under liquid nitrogen, and then investigated its effects on elemental distributions under both frozen hydrated and freeze dried states with x-ray fluorescence microscopy. We show that the contents and distributions of most biologically important elements remain nearly unchanged when compared with non-ultraviolet-irradiated counterparts, even after multiple cycles of ultraviolet germicidal irradiation and cryogenic x-ray imaging. This provides a potential pathway for rendering Risk Group 2 biological materials safe for handling in multiuser cryogenic x-ray microscopes without affecting the fidelity of the results. PMID:25706293

  10. Ultraviolet germicidal irradiation and its effects on elemental distributions in mouse embryonic fibroblast cells in x-ray fluorescence microanalysis

    DOE PAGES

    Jin, Qiaoling; Vogt, Stefan; Lai, Barry; ...

    2015-02-23

    Rapidly-frozen hydrated (cryopreserved) specimens combined with cryo-scanning x-ray fluorescence microscopy provide an ideal approach for investigating elemental distributions in biological cells and tissues. However, because cryopreservation does not deactivate potentially infectious agents associated with Risk Group 2 biological materials, one must be concerned with contamination of expensive and complicated cryogenic x-ray microscopes when working with such materials. We employed ultraviolet germicidal irradiation to decontaminate previously cryopreserved cells under liquid nitrogen, and then investigated its effects on elemental distributions under both frozen hydrated and freeze dried states with x-ray fluorescence microscopy. We show that the contents and distributions of most biologicallymore » important elements remain nearly unchanged when compared with non-ultraviolet-irradiated counterparts, even after multiple cycles of ultraviolet germicidal irradiation and cryogenic x-ray imaging. This provides a potential pathway for rendering Risk Group 2 biological materials safe for handling in multiuser cryogenic x-ray microscopes without affecting the fidelity of the results.« less

  11. Ultraviolet germicidal irradiation and its effects on elemental distributions in mouse embryonic fibroblast cells in x-ray fluorescence microanalysis

    SciTech Connect

    Jin, Qiaoling; Vogt, Stefan; Lai, Barry; Chen, Si; Finney, Lydia; Gleber, Sophie-Charlotte; Ward, Jesse; Deng, Junjing; Mak, Rachel; Moonier, Nena; Jacobsen, Chris; Brody, James P.

    2015-02-23

    Rapidly-frozen hydrated (cryopreserved) specimens combined with cryo-scanning x-ray fluorescence microscopy provide an ideal approach for investigating elemental distributions in biological cells and tissues. However, because cryopreservation does not deactivate potentially infectious agents associated with Risk Group 2 biological materials, one must be concerned with contamination of expensive and complicated cryogenic x-ray microscopes when working with such materials. We employed ultraviolet germicidal irradiation to decontaminate previously cryopreserved cells under liquid nitrogen, and then investigated its effects on elemental distributions under both frozen hydrated and freeze dried states with x-ray fluorescence microscopy. We show that the contents and distributions of most biologically important elements remain nearly unchanged when compared with non-ultraviolet-irradiated counterparts, even after multiple cycles of ultraviolet germicidal irradiation and cryogenic x-ray imaging. This provides a potential pathway for rendering Risk Group 2 biological materials safe for handling in multiuser cryogenic x-ray microscopes without affecting the fidelity of the results.

  12. Comparison of diploid fibroblast and rabbit kidney tissue cultures and a diploid fibroblast microtiter plate system for the isolation of herpes simplex virus.

    PubMed Central

    Langenberg, A; Zbanyszek, R; Dragavon, J; Ashley, R; Corey, L

    1988-01-01

    We evaluated the relative sensitivities of two cell systems (rabbit kidney [RK] and human diploid fibroblast [DF; human embryonic tonsil]) in standard tube cultures versus DF cells in a 48-well microtiter plate system for the detection of both symptomatic and asymptomatic herpes simplex virus (HSV) infection. At least one system isolated HSV in 111 of 809 specimens (13.7%). HSV was isolated in RK tube cultures from 110 specimens (99%), in DF tube cultures from 91 specimens (82%), and in DF microtiter plates from 95 specimens (86%). The frequency of HSV isolation varied with the anatomic site and the presence or absence of a herpetic lesion. The sensitivities of the three culture systems remained similar whether the specimens were obtained from lesions or whether the specimens were taken to determine if asymptomatic excretion of HSV was present. While RK tube cultures were more sensitive than DF tube cultures, the DF microtiter plate system was as sensitive as DF tube cultures and its use is supported as a cheaper and less labor-intensive method for the detection of HSV. PMID:2846647

  13. Fibroblast growth factor 8 signaling through fibroblast growth factor receptor 1 is required for the emergence of gonadotropin-releasing hormone neurons.

    PubMed

    Chung, Wilson C J; Moyle, Sarah S; Tsai, Pei-San

    2008-10-01

    GnRH neurons are essential for the onset and maintenance of reproduction. Mutations in both fibroblast growth factor receptor (Fgfr1) and Fgf8 have been shown to cause Kallmann syndrome, a disease characterized by hypogonadotropic hypogonadism and anosmia, indicating that FGF signaling is indispensable for the formation of a functional GnRH system. Presently it is unclear which stage of GnRH neuronal development is most impacted by FGF signaling deficiency. GnRH neurons express both FGFR1 and -3; thus, it is also unclear whether FGFR1 or FGFR3 contributes directly to GnRH system development. In this study, we examined the developing GnRH system in mice deficient in FGF8, FGFR1, or FGFR3 to elucidate the individual contribution of these FGF signaling components. Our results show that the early emergence of GnRH neurons from the embryonic olfactory placode requires FGF8 signaling, which is mediated through FGFR1, not FGFR3. These data provide compelling evidence that the developing GnRH system is exquisitely sensitive to reduced levels of FGF signaling. Furthermore, Kallmann syndrome stemming from FGF signaling deficiency may be due primarily to defects in early GnRH neuronal development prior to their migration into the forebrain.

  14. Mesenchymal stem cells induce dermal fibroblast responses to injury

    SciTech Connect

    Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

    2010-01-01

    Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

  15. Differential effects of planktonic and biofilm MRSA on human fibroblasts.

    PubMed

    Kirker, Kelly R; James, Garth A; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2012-01-01

    Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin-resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell-culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin-resistant S. aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin-resistant S. aureus, namely limited migration followed by death. Enzyme-linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm-conditioned medium. Planktonic-conditioned medium induced more interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-β1, heparin-bound epidermal growth factor, matrix metalloproteinase-1, and metalloproteinase-3 production in fibroblasts than the biofilm-conditioned medium. Biofilm-conditioned medium induced more tumor necrosis factor-α production in fibroblasts compared with planktonic-conditioned medium, and suppressed metalloproteinase-3 production compared with controls.

  16. Catalytically inactive human cathepsin D triggers fibroblast invasive growth

    PubMed Central

    Laurent-Matha, Valérie; Maruani-Herrmann, Sharon; Prébois, Christine; Beaujouin, Mélanie; Glondu, Murielle; Noël, Agnès; Alvarez-Gonzalez, Marie Luz; Blacher, Sylvia; Coopman, Peter; Baghdiguian, Stephen; Gilles, Christine; Loncarek, Jadranka; Freiss, Gilles; Vignon, Françoise; Liaudet-Coopman, Emmanuelle

    2005-01-01

    The aspartyl-protease cathepsin D (cath-D) is overexpressed and hypersecreted by epithelial breast cancer cells and stimulates their proliferation. As tumor epithelial–fibroblast cell interactions are important events in cancer progression, we investigated whether cath-D overexpression affects also fibroblast behavior. We demonstrate a requirement of cath-D for fibroblast invasive growth using a three-dimensional (3D) coculture assay with cancer cells secreting or not pro-cath-D. Ectopic expression of cath-D in cath-D–deficient fibroblasts stimulates 3D outgrowth that is associated with a significant increase in fibroblast proliferation, survival, motility, and invasive capacity, accompanied by activation of the ras–MAPK pathway. Interestingly, all these stimulatory effects on fibroblasts are independent of cath-D proteolytic activity. Finally, we show that pro-cath-D secreted by cancer cells is captured by fibroblasts and partially mimics effects of transfected cath-D. We conclude that cath-D is crucial for fibroblast invasive outgrowth and could act as a key paracrine communicator between cancer and stromal cells, independently of its catalytic activity. PMID:15668295

  17. Upregulation of Mitochondrial Content in Cytochrome c Oxidase Deficient Fibroblasts.

    PubMed

    Kogot-Levin, Aviram; Saada, Ann; Leibowitz, Gil; Soiferman, Devorah; Douiev, Liza; Raz, Itamar; Weksler-Zangen, Sarah

    2016-01-01

    Cytochrome-c-oxidase (COX) deficiency is a frequent cause of mitochondrial disease and is associated with a wide spectrum of clinical phenotypes. We studied mitochondrial function and biogenesis in fibroblasts derived from the Cohen (CDs) rat, an animal model of COX deficiency. COX activity in CDs-fibroblasts was 50% reduced compared to control rat fibroblasts (P<0.01). ROS-production in CDs fibroblasts increased, along with marked mitochondrial fragmentation and decreased mitochondrial membrane-potential, indicating mitochondrial dysfunction. Surprisingly, cellular ATP content, oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were unchanged. To clarify the discrepancy between mitochondrial dysfunction and ATP production, we studied mitochondrial biogenesis and turnover. The content of mitochondria was higher in CDs-fibroblasts. Consistently, AMPK activity and the expression of NRF1-target genes, NRF2 and PGC1-α that mediate mitochondrial biogenesis were increased (P<0.01 vs control fibroblast). In CDs-fibrobalsts, the number of autophagosomes (LC3+ puncta) containing mitochondria in CDs fibroblasts was similar to that in control fibroblasts, suggesting that mitophagy was intact. Altogether, our findings demonstrate that mitochondrial dysfunction and oxidative stress are associated with an increase in mitochondrial biogenesis, resulting in preservation of ATP generation.

  18. Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

    SciTech Connect

    Nobe, Koji; Nobe, Hiromi; Yoshida, Hiroko; Kolodney, Michael S.; Paul, Richard J.; Honda, Kazuo

    2010-08-20

    Research highlights: {yields} Mechanisms of fibroblast cell contraction in collagen matrix. {yields} Assessed an isometric force development using 3D-reconstituted-fibroblast fiber. {yields} Constitutively active Rho A induced the over-contraction of fibroblast cells. {yields} Rho A and Rho kinase pathway has a central role in fibroblast cell contraction. -- Abstract: Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibers and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor ( (Y27632)) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.

  19. Transplantation of Embryonic and Induced Pluripotent Stem Cell-Derived 3D Retinal Sheets into Retinal Degenerative Mice

    PubMed Central

    Assawachananont, Juthaporn; Mandai, Michiko; Okamoto, Satoshi; Yamada, Chikako; Eiraku, Mototsugu; Yonemura, Shigenobu; Sasai, Yoshiki; Takahashi, Masayo

    2014-01-01

    Summary In this article, we show that mouse embryonic stem cell- or induced pluripotent stem cell-derived 3D retinal tissue developed a structured outer nuclear layer (ONL) with complete inner and outer segments even in an advanced retinal degeneration model (rd1) that lacked ONL. We also observed host-graft synaptic connections by immunohistochemistry. This study provides a “proof of concept” for retinal sheet transplantation therapy for advanced retinal degenerative diseases. PMID:24936453

  20. Bioenergetic Changes during Differentiation of Human Embryonic Stem Cells along the Hepatic Lineage

    PubMed Central

    Hopkinson, Branden M.; Kalisz, Mark; Vestentoft, Peter Siig; Juel Rasmussen, Lene; Bisgaard, Hanne Cathrine

    2017-01-01

    Mitochondrial dysfunction has been demonstrated to result in premature aging due to its effects on stem cells. Nevertheless, a full understanding of the role of mitochondrial bioenergetics through differentiation is still lacking. Here we show the bioenergetics profile of human stem cells of embryonic origin differentiating along the hepatic lineage. Our study reveals especially the transition between hepatic specification and hepatic maturation as dependent on mitochondrial respiration and demonstrates that even though differentiating cells are primarily dependent on glycolysis until induction of hepatocyte maturation, oxidative phosphorylation is essential at all stages of differentiation. PMID:28265337

  1. Salmonella typhimurium mutants lacking NAD pyrophosphatase.

    PubMed Central

    Park, U E; Roth, J R; Olivera, B M

    1988-01-01

    NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome. PMID:2841298

  2. Influence of three laser wavelengths on human fibroblasts cell culture.

    PubMed

    Crisan, Bogdan; Soritau, Olga; Baciut, Mihaela; Campian, Radu; Crisan, Liana; Baciut, Grigore

    2013-02-01

    Although experimental studies in vitro and vivo have been numerous, the effect of laser wavelength irradiation on human fibroblast cell culture is poorly understood. This emphasizes the need of additional cellular and molecular research into laser influence with low energy and power. The aim of this study was to assess the influence of three different laser wavelengths on the human skin fibroblasts cell culture. We wanted to evaluate if near infrared lasers had any influence in healing of wounds by stimulating mitochondrial activity of fibroblasts. The cells were irradiated using 830-, 980- and 2,940-nm laser wavelengths. The irradiated cells were incubated and their mitochondrial activity was assessed by the MTT assay at 24, 48 and 72 h. Simultaneously, an apoptosis assay was assessed on the irradiated fibroblasts. It can be concluded that laser light of the near-infrared region (830 and 980 nm) influences fibroblasts mitochondrial activity compared to the 2,940-nm wavelength which produces apoptosis.

  3. Novel therapeutic strategies targeting fibroblasts and fibrosis in heart disease.

    PubMed

    Gourdie, Robert G; Dimmeler, Stefanie; Kohl, Peter

    2016-09-01

    Our understanding of the functions of cardiac fibroblasts has moved beyond their roles in heart structure and extracellular matrix generation and now includes their contributions to paracrine, mechanical and electrical signalling during ontogenesis and normal cardiac activity. Fibroblasts also have central roles in pathogenic remodelling during myocardial ischaemia, hypertension and heart failure. As key contributors to scar formation, they are crucial for tissue repair after interventions including surgery and ablation. Novel experimental approaches targeting cardiac fibroblasts are promising potential therapies for heart disease. Indeed, several existing drugs act, at least partially, through effects on cardiac connective tissue. This Review outlines the origins and roles of fibroblasts in cardiac development, homeostasis and disease; illustrates the involvement of fibroblasts in current and emerging clinical interventions; and identifies future targets for research and development.

  4. Collagen Expression in Fibroblasts with a Novel LMNA Mutation

    PubMed Central

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

    2007-01-01

    Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy. PMID:17150192

  5. Collagen expression in fibroblasts with a novel LMNA mutation

    SciTech Connect

    Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko . E-mail: picard@u.washington.edu

    2007-01-19

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

  6. Maternal diet programs embryonic kidney gene expression.

    PubMed

    Welham, Simon J M; Riley, Paul R; Wade, Angie; Hubank, Mike; Woolf, Adrian S

    2005-06-16

    Human epidemiological data associating birth weight with adult disease suggest that organogenesis is "programmed" by maternal diet. In rats, protein restriction in pregnancy produces offspring with fewer renal glomeruli and higher systemic blood pressures than controls. We tested the hypothesis that maternal diet alters gene expression in the metanephros, the precursor of the definitive mammalian kidney. We demonstrated that maternal low-protein diet initiated when pregnancy starts and maintained to embryonic day 13, when the metanephros consists of mesenchyme surrounding a once-branched ureteric bud, is sufficient to significantly reduce glomerular numbers in offspring by about 20%. As assessed by representational difference analyses and real-time quantitative polymerase chain reactions, low-protein diet modulated gene expression in embryonic day 13 metanephroi. In particular, levels of prox-1, the ortholog of Drosophila transcription factor prospero, and cofilin-1, a regulator of the actin cytoskeleton, were reduced. During normal metanephrogenesis, prox-1 protein was first detected in mesenchymal cells around the ureteric tree and thereafter in nascent nephron epithelia, whereas cofilin-1 immunolocalized to bud derivatives and condensing mesenchyme. Previously, we reported that low-protein diets increased mesenchymal apoptosis cells when metanephrogenesis began and thereafter reduced numbers of precursor cells. Collectively, these studies prove that the maternal diet programs the embryonic kidney, altering cell turnover and gene expression at a time when nephrons and glomeruli have yet to form. The human implication is that the maternal diet ingested between conception and 5- 6-wk gestation contributes to the variation in glomerular numbers that are known to occur between healthy and hypertensive populations.

  7. Analysis of circadian rhythms in embryonic stem cells.

    PubMed

    Paulose, Jiffin K; Rucker, Edmund B; Cassone, Vincent M

    2015-01-01

    Recent attention on the early development of circadian rhythms has yielded several avenues of potential study regarding molecular and physiological rhythms in embryonic stem cells (ESCs) and their derivatives. While general guidelines of experimental design are-as always-applicable, there are certain idiosyncrasies with respect to experiments involving circadian rhythms that will be addressed. ESCs provide a number of challenges to the circadian biologist: growth rates are normally much higher than in established cell culture systems, the cells' innate drive towards differentiation and the lack of known synchronizing input pathways are a few examples. Some of these challenges can be addressed post hoc, such as normalization to total RNA or protein for transcript abundance studies. Most others, as outlined here, require special handling of the samples before and during experimentation in order to preserve any potential circadian oscillation that is present. Failure to do so may result in a disruption of endogenous oscillation(s) or, potentially worse, generation of an artificial oscillation that has no biological basis. This chapter begins with cultured ESCs, derived from primary blastocysts or in the form of cell lines, and outlines two methods of measuring circadian rhythms: the 2DG method of measuring glucose uptake (Sokoloff et al. J Neurochem 28:897-916, 1977) and real-time measurement of molecular rhythms using transgenic bioluminescence (Yoo et al. Proc Natl Acad Sci U S A 101:5339-5346, 2004).

  8. Gamete Donor Consent and Human Embryonic Stem Cell Research.

    PubMed

    Siegel, Andrew W

    2015-06-01

    There is a lack of consensus on whether the derivation and use of human embryonic stem cells (hESCs) from embryos remaining after infertility treatment morally require the informed consent of third-party gamete donors who contributed to the creation of the embryos. The principal guidelines for oversight and funding of hESC research in the United States make minimal or no demands for consent from gamete donors. In this article, I consider the arguments supporting and opposing gamete donor consent for hESC research and embryo research more broadly. I argue that it is not morally permissible to use leftover embryos in research without the informed consent of gamete donors, and that we should place restrictions on the use of existing hESC lines that may have been derived without informed consent. While the standard argument for this position relies on an appeal to gamete donors' interest in controlling what happens with their genetic material, I identify shortcomings with the standard approach and seek instead to locate the deeper moral foundations for gamete donor consent in rights to bodily integrity.

  9. The Ultrastructural Signature of Human Embryonic Stem Cells.

    PubMed

    Underwood, Jean M; Becker, Klaus A; Stein, Gary S; Nickerson, Jeffrey A

    2017-04-01

    The epigenetics and molecular biology of human embryonic stem cells (hES cells) have received much more attention than their architecture. We present a more complete look at hES cells by electron microscopy, with a special emphasis on the architecture of the nucleus. We propose that there is an ultrastructural signature of pluripotent human cells. hES cell nuclei lack heterochromatin, including the peripheral heterochromatin, that is common in most somatic cell types. The absence of peripheral heterochromatin may be related to the absence of lamins A and C, proteins important for linking chromatin to the nuclear lamina and envelope. Lamins A and C expression and the development of peripheral heterochromatin were early steps in the development of embryoid bodies. While hES cell nuclei had abundant nuclear pores, they also had an abundance of nuclear pores in the cytoplasm in the form of annulate lamellae. These were not a residue of annulate lamellae from germ cells or the early embryos from which hES cells were derived. Subnuclear structures including nucleoli, interchromatin granule clusters, and Cajal bodies were observed in the nuclear interior. The architectural organization of human ES cell nuclei has important implications for cell structure-gene expression relationships and for the maintenance of pluripotency. J. Cell. Biochem. 118: 764-774, 2017. © 2016 Wiley Periodicals, Inc.

  10. Urine excretion strategy for stem cell-generated embryonic kidneys.

    PubMed

    Yokote, Shinya; Matsunari, Hitomi; Iwai, Satomi; Yamanaka, Shuichiro; Uchikura, Ayuko; Fujimoto, Eisuke; Matsumoto, Kei; Nagashima, Hiroshi; Kobayashi, Eiji; Yokoo, Takashi

    2015-10-20

    There have been several recent attempts to generate, de novo, a functional whole kidney from stem cells using the organogenic niche or blastocyst complementation methods. However, none of these attempts succeeded in constructing a urinary excretion pathway for the stem cell-generated embryonic kidney. First, we transplanted metanephroi from cloned pig fetuses into gilts; the metanephroi grew to about 3 cm and produced urine, although hydronephrosis eventually was observed because of the lack of an excretion pathway. Second, we demonstrated the construction of urine excretion pathways in rats. Rat metanephroi or metanephroi with bladders (developed from cloacas) were transplanted into host rats. Histopathologic analysis showed that tubular lumina dilation and interstitial fibrosis were reduced in kidneys developed from cloacal transplants compared with metanephroi transplantation. Then we connected the host animal's ureter to the cloacal-developed bladder, a technique we called the "stepwise peristaltic ureter" (SWPU) system. The application of the SWPU system avoided hydronephrosis and permitted the cloacas to differentiate well, with cloacal urine being excreted persistently through the recipient ureter. Finally, we demonstrated a viable preclinical application of the SWPU system in cloned pigs. The SWPU system also inhibited hydronephrosis in the pig study. To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney.

  11. Description of embryonic development of spotted green pufferfish (Tetraodon nigroviridis).

    PubMed

    Zaucker, Andreas; Bodur, Türker; Roest Crollius, Hugues; Hadzhiev, Yavor; Gehrig, Jochen; Loosli, Felix; Watson, Craig; Müller, Ferenc

    2014-12-01

    Pufferfish species of the Tetraodontidae family carry the smallest genomes among vertebrates. Their compressed genomes are thought to be enriched for functional DNA compared to larger vertebrate genomes, and they are important models for comparative genomics. The significance of pufferfish as model organisms in comparative genomics is due to the availability of two sequenced genomes, that of spotted green pufferfish (Tetraodon nigroviridis) and fugu (Takifugu rubripes). However, there is only a very limited utilization of pufferfish as an experimental model organism, due to the lack of established husbandry and developmental genetics protocols. In this study, we provide the first description of the normal embryonic development of Tetraodon nigroviridis. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored by brightfield microscopy at constant temperature. Tetraodon development was divided into distinct stages based on diagnostic morphological features, which were adopted from published literature on normal development of other fish species like medaka (Oryzias latipes), zebrafish (Danio rerio), and fugu. Tetraodon embryos show more similar morphologies to medaka than to zebrafish, reflecting its phylogenetic position. The early developmental stage series described in this study forms the foundation for the utilization of tetraodon as an experimental model organism for comparative developmental studies.

  12. Description of Embryonic Development of Spotted Green Pufferfish (Tetraodon nigroviridis)

    PubMed Central

    Zaucker, Andreas; Bodur, Türker; Roest Crollius, Hugues; Hadzhiev, Yavor; Gehrig, Jochen; Loosli, Felix; Watson, Craig

    2014-01-01

    Abstract Pufferfish species of the Tetraodontidae family carry the smallest genomes among vertebrates. Their compressed genomes are thought to be enriched for functional DNA compared to larger vertebrate genomes, and they are important models for comparative genomics. The significance of pufferfish as model organisms in comparative genomics is due to the availability of two sequenced genomes, that of spotted green pufferfish (Tetraodon nigroviridis) and fugu (Takifugu rubripes). However, there is only a very limited utilization of pufferfish as an experimental model organism, due to the lack of established husbandry and developmental genetics protocols. In this study, we provide the first description of the normal embryonic development of Tetraodon nigroviridis. Embryos were obtained by in vitro fertilization of eggs, and subsequent development was monitored by brightfield microscopy at constant temperature. Tetraodon development was divided into distinct stages based on diagnostic morphological features, which were adopted from published literature on normal development of other fish species like medaka (Oryzias latipes), zebrafish (Danio rerio), and fugu. Tetraodon embryos show more similar morphologies to medaka than to zebrafish, reflecting its phylogenetic position. The early developmental stage series described in this study forms the foundation for the utilization of tetraodon as an experimental model organism for comparative developmental studies. PMID:25243591

  13. Urine excretion strategy for stem cell-generated embryonic kidneys

    PubMed Central

    Yokote, Shinya; Matsunari, Hitomi; Iwai, Satomi; Yamanaka, Shuichiro; Uchikura, Ayuko; Fujimoto, Eisuke; Matsumoto, Kei; Nagashima, Hiroshi; Kobayashi, Eiji; Yokoo, Takashi

    2015-01-01

    There have been several recent attempts to generate, de novo, a functional whole kidney from stem cells using the organogenic niche or blastocyst complementation methods. However, none of these attempts succeeded in constructing a urinary excretion pathway for the stem cell-generated embryonic kidney. First, we transplanted metanephroi from cloned pig fetuses into gilts; the metanephroi grew to about 3 cm and produced urine, although hydronephrosis eventually was observed because of the lack of an excretion pathway. Second, we demonstrated the construction of urine excretion pathways in rats. Rat metanephroi or metanephroi with bladders (developed from cloacas) were transplanted into host rats. Histopathologic analysis showed that tubular lumina dilation and interstitial fibrosis were reduced in kidneys developed from cloacal transplants compared with metanephroi transplantation. Then we connected the host animal’s ureter to the cloacal-developed bladder, a technique we called the “stepwise peristaltic ureter” (SWPU) system. The application of the SWPU system avoided hydronephrosis and permitted the cloacas to differentiate well, with cloacal urine being excreted persistently through the recipient ureter. Finally, we demonstrated a viable preclinical application of the SWPU system in cloned pigs. The SWPU system also inhibited hydronephrosis in the pig study. To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney. PMID:26392557

  14. Cell Cycle Regulation and Apoptotic Responses of the Embryonic Chick Retina by Ionizing Radiation

    PubMed Central

    Layer, Paul G.; Frohns, Florian

    2016-01-01

    Ionizing radiation (IR) exerts deleterious effects on the developing brain, since proliferative neuronal progenitor cells are highly sensitive to IR-induced DNA damage. Assuming a radiation response that is comparable to mammals, the chick embryo would represent a lower vertebrate model system that allows analysis of the mechanisms underlying this sensitivity, thereby contributing to the reduction, refinement and replacement of animal experiments. Thus, this study aimed to elucidate the radiation response of the embryonic chick retina in three selected embryonic stages. Our studies reveal a lack in the radiation-induced activation of a G1/S checkpoint, but rapid abrogation of G2/M progression after IR in retinal progenitors throughout development. Unlike cell cycle control, radiation-induced apoptosis (RIA) showed strong variations between its extent, dose dependency and temporal occurrence. Whereas the general sensitivity towards RIA declined with ongoing differentiation, its dose dependency constantly increased with age. For all embryonic stages RIA occurred during comparable periods after irradiation, but in older animals its maximum shifted towards earlier post-irradiation time points. In summary, our results are in good agreement with data from the developing rodent retina, strengthening the suitability of the chick embryo for the analysis of the radiation response in the developing central nervous system. PMID:27163610

  15. Tbx18 targets dermal condensates for labeling, isolation, and gene ablation during embryonic hair follicle formation.

    PubMed

    Grisanti, Laura; Clavel, Carlos; Cai, Xiaoqiang; Rezza, Amelie; Tsai, Su-Yi; Sennett, Rachel; Mumau, Melanie; Cai, Chen-Leng; Rendl, Michael

    2013-02-01

    How cell fate decisions of stem and progenitor cells are regulated by their microenvironment or niche is a central question in stem cell and regenerative biology. Although functional analysis of hair follicle epithelial stem cells by gene targeting is well established, the molecular and genetic characterization of the dermal counterpart during embryonic morphogenesis has been lacking because of the absence of cell type-specific drivers. Here, we report that T-box transcription factor Tbx18 specifically marks dermal papilla (DP) precursor cells during embryonic hair follicle morphogenesis. With Tbx18(LacZ), Tbx18(H2BGFP), and Tbx18(Cre) knock-in mouse models, we demonstrate LacZ and H2BGFP (nuclear green fluorescent protein) expression and Cre activity in dermal condensates of nascent first-wave hair follicles at E14.5. As Tbx18 expression becomes more widespread throughout the dermis at later developmental stages, we use tamoxifen-inducible Cre-expressing mice, Tbx18(MerCreMer), to exclusively target DP precursor cells and their progeny. Finally, we ablate Tbx18 in full knockout mice, but find no perturbations in hair follicle formation, suggesting that Tbx18 is dispensable for normal DP function. In summary, our study establishes Tbx18 as a genetic driver to target for the first time embryonic DP precursors for labeling, isolation, and gene ablation that will greatly enhance investigations into their molecular functions during hair follicle morphogenesis.

  16. Teratogenic effects of pyridoxine on the spinal cord and dorsal root ganglia of embryonic chickens.

    PubMed

    Sharp, A A; Fedorovich, Y

    2015-03-19

    Our understanding of the role of somatosensory feedback in regulating motility during chicken embryogenesis and fetal development in general has been hampered by the lack of an approach to selectively alter specific sensory modalities. In adult mammals, pyridoxine overdose has been shown to cause a peripheral sensory neuropathy characterized by a loss of both muscle and cutaneous afferents, but predominated by a loss of proprioception. We have begun to explore the sensitivity of the nervous system in chicken embryos to the application of pyridoxine on embryonic days 7 and 8, after sensory neurons in the lumbosacral region become post-mitotic. Upon examination of the spinal cord, dorsal root ganglion and peripheral nerves, we find that pyridoxine causes a loss of neurotrophic tyrosine kinase receptor type 3-positive neurons, a decrease in the diameter of the muscle innervating nerve tibialis, and a reduction in the number of large diameter axons in this nerve. However, we found no change in the number of Substance P or calcitonin gene-related peptide-positive neurons, the number of motor neurons or the diameter or axonal composition of the femoral cutaneous nerve. Therefore, pyridoxine causes a peripheral sensory neuropathy in embryonic chickens largely consistent with its effects in adult mammals. However, the lesion may be more restricted to proprioception in the chicken embryo. Therefore, pyridoxine lesion induced during embryogenesis in the chicken embryo can be used to assess how the loss of sensation, largely proprioception, alters spontaneous embryonic motility and subsequent motor development.

  17. Embryonic in vivo electroporation in the mouse.

    PubMed

    Saito, Tetsuichiro

    2010-01-01

    Electroporation combined with surgery is a quick and highly efficient method to transfect nucleic acids into various embryonic tissues in a spatiotemporally restricted manner. Forceps-type electrodes facilitate transfection by delivering electric pulses from outside of the embryo. Many electroporated embryos survive in the pregnant mouse, are born, and are reared. The developing central nervous system (CNS) is a good target for transfection, because there are many neural progenitors adjacent to the ventricle, into which nucleic acids are relatively easily injected. The expression of transfected genes persists in neurons for months.

  18. [Ethical aspects of embryonic stem cell research].

    PubMed

    Kostka, U

    2002-11-01

    Research using embryonic stem cells raises a variety of ethical questions, which will be explored in this article. At the core of the ethical controversy is the question of the status of the embryo and its availability for research. A range of countries have approved the use of "supernumerous" embryos from in-vitro fertilization. But ethical problems also arise in reproduction medicine, the informed consent of affected couples, and the targeted production of embryos and egg cell donation for research. The author discuss some of these neglected issues and develops suggestions for comprehensive ethical reflection.

  19. Imaging embryonic morphogenesis in C. elegans.

    PubMed

    Hardin, Jeff

    2011-01-01

    The Caenorhabditis elegans embryo is well suited to morphogenetic analysis via modern microscopy, due to its short generation time, transparency, invariant lineage, and the ability to generate transgenic embryos expressing various fluorescent proteins. This chapter provides an overview of microscopy techniques for imaging embryonic morphogenesis, including making agar mounts, capturing four-dimensional (4D) data using Nomarski microscopy, imaging of actin in embryos, factors important for optimizing 4D fluorescence microscopy, and recent techniques that leverage fluorescence microscopy for intracellular imaging of cellular components during morphogenesis.

  20. Phenotypic markers in human skin fibroblasts as possible diagnostic indices of hereditary adenomatosis of the colon and rectum.

    PubMed

    Kopelovich, L

    1977-11-01

    Hereditary adenomatosis of the colon and rectum (ACR) and its Gardner's syndrome variant, an autosomal dominant trait, indicate a propensity for neoplasia. The present study describes the growth abnormalities of cultured human skin fibroblasts derived from normal-appearing cutaneous biopsies of ACR genotypes and a portion of the clinically asymptomatic ACR progeny, first filial generation, and their differential susceptibility to transformation by Kirsten murine sarcoma virus. These skin fibroblasts, but not cells derived from unaffected individuals, showed lack of contact inhibition, decreased serum requirement for growth, elevated levels of plasminogen activator, and alterations in the intracellular distribution of actin cables; they did not, however, grow in the absence of anchorage, nor did they form palpable tumors in congenitally athymic BALB/c nu/nu mice, and they were normal with regard to cholesterol feedback regulation. Skin fibroblasts from ACR subjects were 100- to 1000-fold more susceptible to transformation by the Kirsten murine sarcoma virus than were normal cells. The virally transformed skin fibroblasts were anchorage-independent and formed tumors in athymic mice. These growth abnormalities represent steps in the changing phenotypic expression of cells undergoing neoplastic transformation. Identification of abnormal expressions associated with oncogenesis may facilitate their use as diagnostic indices for the detection of latent forms of colon cancer in man.

  1. Replacement of α-galactosidase A in Fabry disease: effect on fibroblast cultures compared with biopsied tissues of treated patients

    PubMed Central

    Keslová-Veselíková, Jana; Hůlková, Helena; Dobrovolný, Robert; Asfaw, Befekadu; Poupětová, Helena; Berná, Linda; Sikora, Jakub; Goláň, Lubor

    2008-01-01

    The function and intracellular delivery of enzyme therapeutics for Fabry disease were studied in cultured fibroblasts and in the biopsied tissues of two male patients to show diversity of affected cells in response to treatment. In the mutant fibroblasts cultures, the final cellular level of endocytosed recombinant α-galactosidases A (agalsidases, FabrazymeTM, and ReplagalTM) exceeded, by several fold, the amount in control fibroblasts and led to efficient direct intra-lysosomal hydrolysis of (3H)Gb3Cer. In contrast, in the samples from the heart and some other tissues biopsied after several months of enzyme replacement therapy (ERT) with FabrazymeTM, only the endothelial cells were free of storage. Persistent Gb3Cer storage was found in cardiocytes (accompanied by increase of lipopigment), smooth muscle cells, fibroblasts, sweat glands, and skeletal muscle. Immunohistochemistry of cardiocytes demonstrated, for the first time, the presence of a considerable amount of the active enzyme in intimate contact with the storage compartment. Factors responsible for the limited ERT effectiveness are discussed, namely post-mitotic status of storage cells preventing their replacement by enzyme supplied precursors, modification of the lysosomal system by longstanding storage, and possible relative lack of Sap B. These observations support the strategy of early treatment for prevention of lysosomal storage. PMID:18351385

  2. Tumor-secreted LOXL2 Activates Fibroblasts Through FAK Signaling

    PubMed Central

    Barker, Holly E.; Bird, Demelza; Lang, Georgina; Erler, Janine T.

    2013-01-01

    Cancer-associated fibroblasts enhance cancer progression when activated by tumor cells through mechanisms not yet fully understood. Blocking mammary tumor cell-derived lysyl oxidase-like 2 (LOXL2) significantly inhibited mammary tumor cell invasion and metastasis in transgenic and orthotopic mouse models. Here we discovered that tumor-derived LOXL2 directly activated stromal fibroblasts in the tumor microenvironment. Genetic manipulation or antibody inhibition of LOXL2 in orthotopically grown mammary tumors reduced the expression of α-smooth muscle actin (α-SMA). Using a marker for reticular fibroblasts, it was determined that expression of α-SMA was localized to fibroblasts recruited from the host tissue. This marker also revealed that the matrix present in tumors with reduced levels of LOXL2 was more scattered compared to control tumors which exhibited matrices with dense, parallel alignments. Importantly, in vitro assays revealed that tumor-derived LOXL2 and a recombinant LOXL2 protein induced fibroblast branching on collagen matrices, as well as increased fibroblast-mediated collagen contraction and invasion of fibroblasts through extracellular matrix (ECM). Moreover, LOXL2 induced the expression of α-SMA in fibroblasts grown on collagen matrices. Mechanistically, it was determined that LOXL2 activated fibroblasts through integrin-mediated FAK activation. These results indicate that inhibition of LOXL2 in tumors not only reduces tumor cell invasion but also attenuates the activation of host cells in the tumor microenvironment. Implications: These findings reveal new insight into the mechanisms of fibroblast activation, a novel function of LOXL2, and further highlight the importance of generating LOXL2-targeted therapies for the prevention of tumor progression and metastasis. PMID:24008674

  3. Does the Autistic Brain Lack Core Modules?

    PubMed Central

    Gernsbacher, Morton Ann; Frymiare, Jennifer L.

    2014-01-01

    Researchers have hypothesized that autistics are missing core modules of the brain, critical neural tissue necessary for accomplishing various processes. In this article, we critically review the evidence supporting two such hypothesized deficits. We ask whether autistic brains lack a module for understanding the behavior of others (i.e., theory of mind) and whether they lack a module for processing faces. We illustrate that successful performance on theory of mind tasks depends on linguistic ability; therefore, it is not surprising that autistics are more likely to fail theory of mind tasks because a qualitative impairment in communication is one of the primary diagnostic criteria for autism. Similarly, we illustrate that autistics are less likely to fixate the eye region of facial photographs and that the amount of time spent fixating the eye region correlates with activation in the face processing “module”; therefore, it is not surprising that autistics are less likely to activate the putative face processing area. These illustrations cast doubt on the arguments that the autistic brain is missing the core modules responsible for understanding theory of mind and for processing faces. PMID:25520587

  4. Analysing the lack of Demand Organisation

    NASA Astrophysics Data System (ADS)

    Boxer, Philip; Cohen, Bernard

    1998-07-01

    We seek to develop means of intervention in Enterprises that will enable them to react in an effective, sustainable and timely fashion to changes in the ways that markets and demand are organized; that is, to act strategically. We take an enterprise to be some entity that seeks to provide its clients with services that they value while maintaining its ability to do so in the face of changes in the demands of its clients and in the resources at its disposal. The services that clients value form around what the organization of their demands lack. The concept of strategy therefore rests on critically evaluating the ontology and semantics of the Enterprise in relation to these holes in demand organization. We access ontology and semantics by constructing and manipulating hypothetical, first-order, mathematical models of the Enterprise's services and of its value-adding processes. Because an enterprise is an anticipatory system, its semantic domain must include representations of the enterprise's model of itself and of the market and demand organizations within which it competes. First-order (set) theory provides adequate expressive power here, but alternative, higher order, mathematical frameworks, such as Dubois' hyperincursion, provide inadequate power, particularly in relation to the analysis of the properties of emergence. Knowing exactly why and where this mathematical lack manifests in the analysis process enables effective collaboration between systems analysts and psychoanalysts, and suggest directions for mathematical research.

  5. Quantum preparation uncertainty and lack of information

    NASA Astrophysics Data System (ADS)

    Rozpędek, Filip; Kaniewski, Jędrzej; Coles, Patrick J.; Wehner, Stephanie

    2017-02-01

    The quantum uncertainty principle famously predicts that there exist measurements that are inherently incompatible, in the sense that their outcomes cannot be predicted simultaneously. In contrast, no such uncertainty exists in the classical domain, where all uncertainty results from ignorance about the exact state of the physical system. Here, we critically examine the concept of preparation uncertainty and ask whether similarly in the quantum regime, some of the uncertainty that we observe can actually also be understood as a lack of information (LOI), albeit a lack of quantum information. We answer this question affirmatively by showing that for the well known measurements employed in BB84 quantum key distribution (Bennett and Brassard 1984 Int. Conf. on Computer System and Signal Processing), the amount of uncertainty can indeed be related to the amount of available information about additional registers determining the choice of the measurement. We proceed to show that also for other measurements the amount of uncertainty is in part connected to a LOI. Finally, we discuss the conceptual implications of our observation to the security of cryptographic protocols that make use of BB84 states.

  6. Ectopic expression of Cripto-1 in transgenic mouse embryos causes hemorrhages, fatal cardiac defects and embryonic lethality

    PubMed Central

    Lin, Xiaolin; Zhao, Wentao; Jia, Junshuang; Lin, Taoyan; Xiao, Gaofang; Wang, Shengchun; Lin, Xia; Liu, Yu; Chen, Li; Qin, Yujuan; Li, Jing; Zhang, Tingting; Hao, Weichao; Chen, Bangzhu; Xie, Raoying; Cheng, Yushuang; Xu, Kang; Yao, Kaitai; Huang, Wenhua; Xiao, Dong; Sun, Yan

    2016-01-01

    Targeted disruption of Cripto-1 in mice caused embryonic lethality at E7.5, whereas we unexpectedly found that ectopic Cripto-1 expression in mouse embryos also led to embryonic lethality, which prompted us to characterize the causes and mechanisms underlying embryonic death due to ectopic Cripto-1 expression. RCLG/EIIa-Cre embryos displayed complex phenotypes between embryonic day 14.5 (E14.5) and E17.5, including fatal hemorrhages (E14.5-E15.5), embryo resorption (E14.5-E17.5), pale body surface (E14.5-E16.5) and no abnormal appearance (E14.5-E16.5). Macroscopic and histological examination revealed that ectopic expression of Cripto-1 transgene in RCLG/EIIa-Cre embryos resulted in lethal cardiac defects, as evidenced by cardiac malformations, myocardial thinning, failed assembly of striated myofibrils and lack of heartbeat. In addition, Cripto-1 transgene activation beginning after E8.5 also caused the aforementioned lethal cardiac defects in mouse embryos. Furthermore, ectopic Cripto-1 expression in embryonic hearts reduced the expression of cardiac transcription factors, which is at least partially responsible for the aforementioned lethal cardiac defects. Our results suggest that hemorrhages and cardiac abnormalities are two important lethal factors in Cripto-1 transgenic mice. Taken together, these findings are the first to demonstrate that sustained Cripto-1 transgene expression after E11.5 causes fatal hemorrhages and lethal cardiac defects, leading to embryonic death at E14.5-17.5. PMID:27687577

  7. Generation of HEXA-deficient hiPSCs from fibroblasts of a Tay-Sachs disease patient.

    PubMed

    Liu, Zhong; Zhao, Rui

    2016-09-01

    Human iPSC line TSD-01-hiPSC was generated from fibroblasts of a patient with infantile Tay-Sachs disease (TSD). The patient is compound heterozygous at the HEXA gene by carrying a 1278insTATC allele and an IVS12+1G>C allele. STEMCCA lentivirus, which expresses OCT4, SOX2, KLF4, and c-MYC from a polycistronic transcript, were used for reprogramming. TSD-01-hiPSC express pluripotency markers such as OCT4, SOX2, NANOG, Tra-1-60, and alkaline phosphatase, and can differentiate into tissues from all the three embryonic germ layers. This TSD patient-derived hiPSC line may serve as a valuable in vitro tool for disease modeling and drug test.

  8. Actin dynamics in mouse fibroblasts in microgravity

    NASA Astrophysics Data System (ADS)

    Moes, Maarten J. A.; Bijvelt, Jose J.; Boonstra, Johannes

    2007-09-01

    After stimulating with the growth factor PDGF, cells exhibit abundant membrane ruffling and other morphological changes under normal gravity conditions. These morphological changes are largely determined by the actin microfilament system. Now these actin dynamics were studied under microgravity conditions in mouse fibroblasts during the DELTA mission. The aim of the present study was to describe the actin morphology in detail, to establish the effect of PDGF on actin morphology and to study the role of several actin-interacting proteins involved in introduced actin dynamics in microgravity. Identical experiments were conducted at 1G on earth as a reference. No results in microgravity were obtained due to a combination of malfunctioning hardware and unfulfilled temperature requirements.

  9. Two distinct mechanisms of fibroblast adhesion

    NASA Astrophysics Data System (ADS)

    Harper, P. A.; Juliano, R. L.

    1981-03-01

    The adhesion of cells to the connective tissue matrix is commonly thought to be governed by fibronectin, a pericellular glycoprotein with binding sites for cell surfaces, collagen and glycosaminoglycans. Here we report evidence that Chinese hamster ovary (CHO) cells possess an alternative mechanism for adhesion which is independent of fibronectin. Cells of a variant CHO clone called ADVF11 are defective in their ability to adhere to fibronectin-coated substrata, but can adhere to a substratum coated with SAM (substrate-attached material), a pericellular material produced by fibroblasts. The adhesion of wild-type CHO cells to fibronectin-coated substrata and adhesion of ADVF11 cells to SAM-coated substrata are differentially sensitive to proteolytic treatment. This suggests that there are two distinct adhesion mechanisms for CHO cells, only one of which is dependent on fibronectin.

  10. Regulation of sulfated glycosaminoglycan production by prostaglandin E2 in cultured lung fibroblasts

    SciTech Connect

    Karlinsky, J.B.; Goldstein, R.H. )

    1989-08-01

    Prostaglandin E2 (PGE2) has been shown to increase the synthesis of hyaluronic acid in cultured fibroblasts by increasing the activity of hyaluronate synthetase, a group of plasma membrane-bound synthetic enzymes. We examined whether PGE2 also increased the activity of those enzyme systems involved in the synthesis of sulfated glycosaminoglycan in the human embryonic lung fibroblast. Exposure of cells to PGE2 resulted in dose-dependent increases in glucosamine incorporation into all sulfated glycosaminoglycan subtypes. PGE2 at 10(-7) mol/L increased total glycosaminoglycan per dish to 21.6 +/- 3.1 micrograms versus 12.0 +/- 2.5 micrograms in control untreated cultures. Stimulation of endogenous PGE2 production by bradykinin had a similar effect on glycosaminoglycan synthesis. To examine whether PGE2 affected sulfated glycosaminoglycan protein core production, cells were labeled with tritiated glucosamine in the presence of cycloheximide. Under these conditions, incorporation of radiolabel into all glycosaminoglycan subtypes was reduced. However, when exogenous sulfated glycosaminoglycan chain initiator (p-nitrophenyl beta-D-xyloside) was added, incorporation of tritiated glucosamine into sulfated glycosaminoglycan increased but not to levels found in control cultures. Application of PGE2 to cultures treated with cycloheximide alone, or to cultures treated with cycloheximide plus xyloside, increased tritiated glucosamine incorporation into chondroitin, dermatan sulfate, and to a lesser extent into heparan sulfate. We conclude that PGE2 stimulates synthesis of all sulfated glycosaminoglycan even in the absence of new protein core production, probably by increasing activities of sulfated glycosaminoglycan synthetase enzymes. PGE2 stimulation of heparan sulfate synthesis is partially dependent on the availability of heparan sulfate-specific protein core.

  11. ALK1 heterozygosity increases extracellular matrix protein expression, proliferation and migration in fibroblasts.

    PubMed

    Muñoz-Félix, José M; Perretta-Tejedor, Nuria; Eleno, Nélida; López-Novoa, José M; Martínez-Salgado, Carlos

    2014-06-01

    Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1(+/+) and ALK1(+/-) mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.

  12. Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity: EFFECTS ON EMBRYO SURVIVAL.

    PubMed

    Fresco, Victor M; Kern, Christine B; Mohammadi, Moosa; Twal, Waleed O

    2016-09-02

    Fibulin-1 (FBLN1) is a member of a growing family of extracellular matrix glycoproteins that includes eight members and is involved in cellular functions such as adhesion, migration, and differentiation. FBLN1 has also been implicated in embryonic heart and valve development and in the formation of neural crest-derived structures, including aortic arch, thymus, and cranial nerves. Fibroblast growth factor 8 (FGF8) is a member of a large family of growth factors, and its functions include neural crest cell (NCC) maintenance, specifically NCC migration as well as patterning of structures formed from NCC such as outflow tract and cranial nerves. In this report, we sought to investigate whether FBLN1 and FGF8 have cooperative roles in vivo given their influence on the development of the same NCC-derived structures. Surface plasmon resonance binding data showed that FBLN1 binds tightly to FGF8 and prevents its enzymatic degradation by ADAM17. Moreover, overexpression of FBLN1 up-regulates FGF8 gene expression, and down-regulation of FBLN1 by siRNA inhibits FGF8 expression. The generation of a double mutant Fbln1 and Fgf8 mice (Fbln1(-/-) and Fgf8(-/-)) showed that haplo-insufficiency (Fbln1(+/-) and Fgf8(+/-)) resulted in increased embryonic mortality compared with single heterozygote crosses. The mortality of the FGF8/Fbln1 double heterozygote embryos occurred between 14.5 and 16.5 days post-coitus. In conclusion, FBLN1/FGF8 interaction plays a role in survival of vertebrate embryos, and reduced levels of both proteins resulted in added mortality in utero The FBLN1/FGF8 interaction may also be involved in the survival of neural crest cell population during development.

  13. Glypican-1 controls brain size through regulation of fibroblast growth factor signaling in early neurogenesis

    PubMed Central

    Jen, Yi-Huei Linda; Musacchio, Michele; Lander, Arthur D

    2009-01-01

    Background Cell surface heparan sulfate proteoglycans (HSPGs) act as co-receptors for multiple families of growth factors that regulate animal cell proliferation, differentiation and patterning. Elimination of heparan sulfate during brain development is known to produce severe structural abnormalities. Here we investigate the developmental role played by one particular HSPG, glypican-1 (Gpc1), which is especially abundant on neuronal cell membranes, and is the major HSPG of the adult rodent brain. Results Mice with a null mutation in Gpc1 were generated and found to be viable and fertile. The major phenotype associated with Gpc1 loss is a highly significant reduction in brain size, with only subtle effects on brain patterning (confined to the anterior cerebellum). The brain size difference emerges very early during neurogenesis (between embryonic days 8.5 and 9.5), and remains roughly constant throughout development and adulthood. By examining markers of different signaling pathways, and the differentiation behaviors of cells in the early embryonic brain, we infer that Gpc1-/- phenotypes most likely result from a transient reduction in fibroblast growth factor (FGF) signaling. Through the analysis of compound mutants, we provide strong evidence that Fgf17 is the FGF family member through which Gpc1 controls brain size. Conclusion These data add to a growing literature that implicates the glypican family of HSPGs in organ size control. They also argue that, among heparan sulfate-dependent signaling molecules, FGFs are disproportionately sensitive to loss of HSPGs. Finally, because heterozygous Gpc1 mutant mice were found to have brain sizes half-way between homozygous and wild type, the data imply that endogenous HSPG levels quantitatively control growth factor signaling, a finding that is both novel and relevant to the general question of how the activities of co-receptors are exploited during development. PMID:19732411

  14. Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts

    PubMed Central

    Albrengues, Jean; Bertero, Thomas; Grasset, Eloise; Bonan, Stephanie; Maiel, Majdi; Bourget, Isabelle; Philippe, Claude; Herraiz Serrano, Cecilia; Benamar, Samia; Croce, Olivier; Sanz-Moreno, Victoria; Meneguzzi, Guerrino; Feral, Chloe C.; Cristofari, Gael; Gaggioli, Cedric

    2015-01-01

    Carcinoma-associated fibroblasts (CAF) mediate the onset of a proinvasive tumour microenvironment. The proinflammatory cytokine LIF reprograms fibroblasts into a proinvasive phenotype, which promotes extracellular matrix remodelling and collective invasion of cancer cells. Here we unveil that exposure to LIF initiates an epigenetic switch leading to the constitutive activation of JAK1/STAT3 signalling, which results in sustained proinvasive activity of CAF. Mechanistically, p300-histone acetyltransferase acetylates STAT3, which, in turn, upregulates and activates the DNMT3b DNA methyltransferase. DNMT3b methylates CpG sites of the SHP-1 phosphatase promoter, which abrogates SHP-1 expression, and results in constitutive phosphorylation of JAK1. Sustained JAK1/STAT3 signalling is maintained by DNA methyltransferase DNMT1. Consistently, in human lung and head and neck carcinomas, STAT3 acetylation and phosphorylation are inversely correlated with SHP-1 expression. Combined inhibition of DNMT activities and JAK signalling, in vitro and in vivo, results in long-term reversion of CAF-associated proinvasive activity and restoration of the wild-type fibroblast phenotype. PMID:26667266

  15. Estimation of cellular fabric in embryonic epithelia.

    PubMed

    Iles, Peter J W; Brodland, G Wayne; Clausi, David A; Puddister, Shannon M

    2007-02-01

    Recent computational and analytical studies have shown that cellular fabric-as embodied by average cell size, aspect ratio and orientation-is a key indicator of the stresses acting in an embryonic epithelium. Cellular fabric in real embryonic tissues could not previously be measured automatically because the cell boundaries tend to be poorly defined, significant lighting and cell pigmentation differences occur and tissues contain a variety of cell geometries. To overcome these difficulties, four algorithms were developed: least squares ellipse fitting (LSEF), area moments (AM), correlation and axes search (CAS) and Gabor filters (GF). The AM method was found to be the most reliable of these methods, giving typical cell size, aspect ratio and orientation errors of 18%, 0.10 and 7.4 degrees, respectively, when evaluated against manually segmented images. The power of the AM algorithm to provide new insights into the mechanics of morphogenesis is demonstrated through a brief investigation of gastrulation, where fabric data suggest that key gastrulation movements are driven by epidermal tensions circumferential to the blastopore.

  16. Organotypic slice culture of embryonic brain tissue.

    PubMed

    Daza, Ray A M; Englund, Chris; Hevner, Robert F

    2007-12-01

    INTRODUCTIONThis protocol describes how to dissect, assemble, and cultivate mouse embryonic (E) brain tissue from age E11.5 to E18.5 (days) for organotypic slice culture. These preparations can be used for a variety of assays and studies including coculture of different brain regions, cell migration assays, axon guidance assays, and DNA electroporation experiments. During electroporation, an electric current is applied to the surface of a specific target area of the brain slice in order to open holes in the plasma membrane and introduce a plasmid of coding DNA. The floating slice-on-membrane construct helps to preserve the structural integrity of the brain slices, while maintaining easy experimental access and optimal viability. Experiments can be monitored in living slices (e.g., with confocal imaging), and further studies can be completed using slices that have been fixed and cryosectioned at the end of the experiment. Any region of embryonic brain or spinal tissue can be used in this protocol.

  17. Embryonic paratesticular rhabdomyosarcoma: a case report

    PubMed Central

    2013-01-01

    Introduction An embryonic paratesticular rhabdomyosarcoma is a very rare mesenchymal tumor. It is an intrascrotal tumor that is localized in paratesticular structures such as the epididymis or spermatic cord. Rhabdomyosarcoma is most often observed in children and adolescents, presenting as a painless scrotal mass. Case presentation Our patient was an 18-year-old Moroccan man who presented with a painless left scrotal mass that had evolved over four months. An inguinal orchiectomy was performed. A histological examination of the excised tissue revealed an embryonic rhabdomyosarcoma. Our patient had three sessions of chemotherapy with vincristine, actinomycin C and cyclophosphamide. Each chemotherapy session was conducted over five days, with a cycle of 21 days. Our patient was assessed two months after the last chemotherapy session and demonstrated good clinical improvement. Conclusion Paratesticular rhabdomyosarcoma is a rare aggressive tumor manifesting in children and very young adults. Localized forms have a good prognosis whereas metastatic tumors show very poor results. A well-defined treatment based on surgery and chemotherapy yields good results. PMID:23561643

  18. Embryonic stem cells: from markers to market.<