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  • Bovine induced pluripotent stem cells are more resistant to apoptosis than testicular cells in response to mono-(2-ethylhexyl) phthalate. 24658443

    Although the androgen receptor (AR) has been implicated in the promotion of apoptosis in testicular cells (TSCs), the molecular pathway underlying AR-mediated apoptosis and its sensitivity to environmental hormones in TSCs and induced pluripotent stem cells (iPSCs) remain unclear. We generated the iPSCs from bovine TSCs via the electroporation of OCT4. The established iPSCs were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4 to maintain and stabilize the expression of stemness genes and their pluripotency. Apoptosis signaling was assessed after exposure to mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of di-(2-ethylhexyl) phthalate. Here, we report that iPSCs were more resistant to MEHP-induced apoptosis than were original TSCs. MEHP also repressed the expression of AR and inactivated WNT signaling, and then led to the commitment of cells to apoptosis via the cyclin dependent kinase inhibitor p21CIP1. The loss of the frizzed receptor 7 and the gain of p21CIP were responsible for the stimulatory effect of MEHP on AR-mediated apoptosis. Our results suggest that testicular iPSCs can be used to study the signaling pathways involved in the response to environmental disruptors, and to assess the toxicity of environmental endocrine disruptors in terms of the maintenance of stemness and pluripotency.
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  • Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated status of hESCs in xeno-free culture conditions. 19202140

    BACKGROUND: Clinical-grade human embryonic stem cells (hESCs) ideally should be derived and maintained in xeno-free culture conditions using defined chemicals or materials of human origin. This will reduce the possibility of xeno-derived pathogenic infection and/or unfavorable immune reaction in clinical application. The present study therefore aimed to derive autogenic feeders from hESCs and evaluate their capability to support the pluripotency of hESCs in xeno-free culture conditions. METHODS AND RESULTS: H9 hESCs were cultured in media containing human serum (HS), serum replacement (SR) or KFM combination, to generate autogenic feeders (named HSdF, SRdF and KFMdF, respectively). Reverse transcription polymerase chain reaction, flow cytometry and immunofluorescence analysis using pluripotent stem cell markers, markers of early cell lineages and surface markers revealed that HSdF, SRdF and KFMdF likely belonged to different cellular subpopulations. The efficiency of the autogenic feeders in maintaining pluripotency of H9 hESCs using media containing SR, fetal bovine serum, HS or 1% HS plus various combinations of growth factors was evaluated by flow cytometric analysis of Oct4 expression. All three autogenic feeders were shown to be capable of maintaining the undifferentiated status of H9 hESCs in SR-containing media in long-term culture. When supplemented with bFGF, activin A and noggin, hESCs could also be maintained favorably on KFMdF in a medium containing 1% HS without losing their pluripotent potentials both in vitro and in vivo. CONCLUSIONS: Novel autogenic feeders can be derived from hESCs under xeno-free conditions and they can robustly maintain the pluripotent identity of hESCs in xeno-free media containing a low concentration of HS.
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  • Efficient derivation of embryonic stem cells from nuclear transfer and parthenogenetic embryos derived from cryopreserved oocytes. 20677934

    Deriving histocompatible embryonic stem (ES) cells by somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA) requires fresh oocytes, which prevents their applications in humans. Here, we evaluated the efficiency of deriving ES cells from mature metaphase II (MII) and immature metaphase I (MI) vitrified oocytes, by PA or SCNT, in a mouse model. We successfully generated ES cell lines from PA (MII and MI) and SCNT (MII and MI) blastocysts. These cell lines expressed genes and antigens characteristic of pluripotent ES cells and produced full-term pups upon tetraploid embryo complementation. This study established an animal model for efficient generation of patient-specific ES cell lines using cryopreserved oocytes. This is a major step forward in the application of therapeutic cloning and parthenogenetic technology in human regenerative medicine and will serve as an important alternative to the iPS cell technology in countries/regions where these technologies are permitted.
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  • Myc regulates the transcription of the PRC2 gene to control the expression of developmental genes in embryonic stem cells. 22184065

    Myc family members are critical to maintain embryonic stem cells (ESC) in the undifferentiated state. However, the mechanism by which they perform this task has not yet been elucidated. Here we show that Myc directly upregulates the transcription of all core components of the Polycomb repressive complex 2 (PRC2) as well as the ESC-specific PRC2-associated factors. By expressing Myc protein fused with the estrogen receptor (Myc-ER) in fibroblasts, we observed that Myc, binding to the regulatory elements of Suz12, Ezh2, and Eed, induces the acetylation of histones H3 and H4 and the recruitment of elongating RNA polymerase II at their promoters. The silencing of both c-Myc and N-Myc in ESC results in reduced expression of PRC2 and H3K27me3 at Polycomb target developmental regulators and upregulation of genes involved in primitive endoderm differentiation. The ectopic expression of PRC2 in ESC, either silenced for c-Myc and N-Myc or induced to differentiate by leukemia inhibitory factor (LIF) withdrawal, is sufficient to maintain the H3K27me3 mark at genes with bivalent histone modifications and keep repressed the genes involved in ESC differentiation. Thus, Myc proteins control the expression of developmental regulators via the upregulation of the Polycomb PRC2 complex.
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  • Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb. 19136965

    The dominant effect of transcription factors in imparting expanded potency is best exemplified by the reprogramming of fibroblasts to pluripotent cells using retrovirus-mediated transduction of defined transcription factors. In the murine system, Oct4, Sox2, c-Myc and Klf4 are sufficient to convert fibroblasts to induced pluripotent stem (iPS) cells that have many characteristics of embryonic stem (ES) cells. Here we show that the orphan nuclear receptor Esrrb functions in conjunction with Oct4 and Sox2 to mediate reprogramming of mouse embryonic fibroblasts (MEFs) to iPS cells. Esrrb-reprogrammed cells share similar expression and epigenetic signatures as ES cells. These cells are also pluripotent and can differentiate in vitro and in vivo into the three major embryonic cell lineages. Furthermore, these cells contribute to mouse chimaeras and are germline transmissible. In ES cells, Esrrb targets many genes involved in self-renewal and pluripotency. This suggests that Esrrb may mediate reprogramming through the upregulation of ES-cell-specific genes. Our findings also indicate that it is possible to reprogram MEFs without exogenous Klf transcription factors and link a nuclear receptor to somatic cell reprogramming.
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