Human embryonic stem cells contribute to embryonic and extraembryonic lineages in mouse embryos through inhibition of apoptosis
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE100862 | GEO | 2017/11/10
SECONDARY ACCESSION(S): PRJNA393309
REPOSITORIES: GEO
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Project description:BackgroundPrevious attempts to isolate pluripotent cell lines from rat preimplantation embryo in mouse embryonic stem (ES) cell culture conditions (serum and LIF) were unsuccessful, however the resulting cells exhibited the expression of such traditional pluripotency markers as SSEA-1 and alkaline phosphatase. We addressed the question, which kind of cell lineages are produced from rat preimplantation embryo under "classical" mouse ES conditions.ResultsWe characterized two cell lines (C5 and B10) which were obtained from rat blastocysts in medium with serum and LIF. In the B10 cell line we found the expression of genes known to be expressed in trophoblast, Cdx-2, cytokeratin-7, and Hand-1. Also, B10 cells invaded the trophectodermal layer upon injection into rat blastocysts. In contrast to mouse Trophoblast Stem (TS) cells proliferation of B10 cells occurred independently of FGF4. Cells of the C5 line expressed traditional markers of extraembryonic-endoderm (XEN) cells, in particular, GATA-4, but also the pluripotency markers SSEA-1 and Oct-4. C5 cell proliferation exhibited dependence on LIF, which is not known to be required by mouse XEN cells.ConclusionsOur results confirm and extend previous findings about differences between blastocyst-derived cell lines of rat and mice. Our data show, that the B10 cell line represents a population of FGF4-independent rat TS-like cells. C5 cells show features that have recently become known as characteristic of rat XEN cells. Early passages of C5 and B10 cells contained both, TS and XEN cells. We speculate, that mechanisms maintaining self-renewal of cell lineages in rat preimplantation embryo and their in vitro counterparts, including ES, TS and XEN cells are different than in respective mouse lineages.
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Project description:The recently emerged CRISPR/Cas9 technique has opened a new perspective on readily editing specific genes. When combined with transcription activators, it can precisely manipulate endogenous gene expression. Here, we enhanced the expression of endogenous Cdx2 and Gata6 genes by CRISPR-mediated activators, thus mouse embryonic stem cells (ESCs) were directly converted into two extraembryonic lineages, i.e., typical trophoblast stem cells (TSCs) and extraembryonic endoderm cells (XENCs), which exhibited characters of TSC or XENC derived from the blastocyst extraembryonic lineages such as cell morphology, specific gene expression, and differentiation ability in vitro and in vivo. This study demonstrates that the cell fate can be effectively manipulated by directly activating of specific endogenous gene expression with CRISPR-mediated activator.
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Project description:At the time of implantation in the maternal uterus, the mouse blastocyst possesses an inner cell mass comprising two lineages: epiblast (Epi) and primitive endoderm (PrE). Representative stem cells derived from these two cell lineages can be expanded and maintained indefinitely in vitro as either embryonic stem (ES) or XEN cells, respectively. Here we describe protocols that can be used to establish XEN cell lines. These include the establishment of XEN cells from blastocyst-stage embryos in either standard embryonic or trophoblast stem (TS) cell culture conditions. We also describe protocols for establishing XEN cells directly from ES cells by either retinoic acid and activin-based conversion or by overexpression of the GATA transcription factor Gata6. XEN cells are a useful model of PrE cells, with which they share gene expression, differentiation potential and lineage restriction. The robust protocols for deriving XEN cells described here can be completed within 2-3 weeks.
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Project description:Most of our current knowledge regarding early lineage specification and embryo-derived stem cells comes from studies in rodent models. However, key gaps remain in our understanding of these developmental processes from nonrodent species. Here, we report the detailed characterization of pig extraembryonic endoderm (pXEN) cells, which can be reliably and reproducibly generated from primitive endoderm (PrE) of blastocyst. Highly expandable pXEN cells express canonical PrE markers and transcriptionally resemble rodent XENs. The pXEN cells contribute both to extraembryonic tissues including visceral yolk sac as well as embryonic gut when injected into host blastocysts, and generate live offspring when used as a nuclear donor in somatic cell nuclear transfer (SCNT). The pXEN cell lines provide a novel model for studying lineage segregation, as well as a source for genome editing in livestock.
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Project description:Retinoblastoma (Rb)-deficient embryos show severe defects in neurogenesis, erythropoiesis, and lens development and die at embryonic day 14.5. Our recent results demonstrated a drastic disorganization of the labyrinth layer in the placenta of Rb-deficient embryos, accompanied by reduced placental transport function. When these Rb-/- embryos were supplied with a wild-type placenta by using either tetraploid aggregation or genetic approaches, animals survived until birth. Here we analyze the role of extraembryonic Rb in regulating proliferation, apoptosis, and differentiation in the rescued animals at different developmental stages. Many of the neurological and erythroid abnormalities thought to be responsible for the embryonic lethality of Rb-/- animals, including the ectopic apoptosis in the CNS, were virtually absent in rescued Rb-/- pups. However, rescued animals died at birth with severe skeletal muscle defects. Like in Rb knockout embryos, rescued animals showed a marked increase in DNA replication and cell division in the CNS. In sharp contrast, the typical widespread neuronal apoptosis was absent in Rb-deficient embryos reconstituted with a normal placenta. In lens fiber cells, however, the inappropriate proliferation and apoptosis that is normally observed in Rb-/- embryos continued unabated in rescued animals. These results demonstrate that Rb function in extraembryonic lineages plays an important role in the survival of neuronal cells and in the differentiation of the erythroid lineage, providing mechanistic insight into the cell autonomous and nonautonomous functions of Rb during development.
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Project description:The cell movements underlying the morphogenesis of the embryonic endoderm, the tissue that will give rise to the respiratory and digestive tracts, are complex and not well understood. Using live imaging combined with genetic labeling, we investigated the cell behaviors and fate of the visceral endoderm during gut endoderm formation in the mouse gastrula. Contrary to the prevailing view, our data reveal no mass displacement of visceral endoderm to extraembryonic regions concomitant with the emergence of epiblast-derived definitive endoderm. Instead, we observed dispersal of the visceral endoderm epithelium and extensive mixing between cells of visceral endoderm and epiblast origin. Visceral endoderm cells remained associated with the epiblast and were incorporated into the early gut tube. Our findings suggest that the segregation of extraembryonic and embryonic tissues within the mammalian embryo is not as strict as believed and that a lineage previously defined as exclusively extraembryonic contributes cells to the embryo.
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Project description:Small molecule inhibitors of the bromodomain and extraterminal (BET) family of proteins are in clinical trials for a variety of cancers, but patient selection strategies are limited. This is due in part to the heterogeneity of response following BET inhibition (BETi), which includes differentiation, senescence, and cell death in subsets of cancer cell lines. To elucidate the dominant features defining response to BETi, we carried out phenotypic and gene expression analysis of both treatment naïve cell lines and engineered tolerant lines. We found that both de novo and acquired tolerance to BET inhibition are driven by the robustness of the apoptotic response and that genetic or pharmacological manipulation of the apoptotic signaling network can modify the phenotypic response to BETi. We further identify that ordered expression of the apoptotic genes BCL2, BCL2L1, and BAD significantly predicts response to BETi. Our findings highlight the role of the apoptotic network in response to BETi, providing a molecular basis for patient stratification and combination therapies. Gene expression profiling of A375 melanoma cells or NOMO-1 AML cells treated with DMSO or the BET inhibitor, CPI203. Also, gene expression profiling of the respective derived BETi-tolerant cells treated with DMSO or CPI203.
2016-01-07 | E-GEOD-69383 | biostudies-arrayexpress
Project description:Our study examined the in vivo chimeric and survival capacities of chimeras created by injecting tetraploid embryonic stem cells (ESCs) expressing green fluorescent protein (GFP) into diploid embryos. At 3.5 days post-coitum (dpc) and 4.5?dpc, the tetraploid ESCs were able to contribute to the inner cell mass (ICM) just as diploid ESCs tagged with GFP. At 6.5?dpc, 8.0?dpc and 10.5?dpc, the tetraploid ESCs manifested in the same location as the diploid ESCs. The GFP cells in the extraembryonic tissues and fetuses of tetraploid ESC chimeras were tetraploid as determined by fluorescence activated cell sorting (FACS). Furthermore, tetraploid ESCs contributed to the development of the placenta, embryolemma and umbilical cord at 13.5?dpc and 16.5?dpc; however, very less GFP cells were found in the fetuses of tetraploid ESC chimeras. We further found that the proliferation of tetraploid ESCs was slower than that of diploid ESCs. In addition, the relative mRNA expression in the three germ layers and the trophoblast was abnormal in the EBs of tetraploid ESCs compared with diploid ESCs. In short, slower proliferation and abnormal differentiation potential of tetraploid ESCs might be two of the reasons for their poor survival and chimeric capacities.
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Project description:Cell death is an important host defense in response to viral infections. In this study,we performed an unbiased whole-genome CRISPR/Cas9 screen in A549 lung adenocarcinoma cells to identify potential regulators involved in cell death triggered dsRNA, a common byproduct of viral replication. Of several top candidate genes, we identified the RNA binding protein ELAV like protein 1 (ELAVL1) (ELAVL1, also called HuR) that encodes Hu antigen R (HuR). Depletion of HuR by gene editing led to less cell death induced by dsRNA. We further demonstrated that HuR regulated apoptosis, and the RNA recognition motif (RRM) 3 of HuR was essential for its proapoptotic function. HuR bound mRNA of anti-apoptotic gene BCL2. HuR depletion had no influence on BCL2 mRNA levels, but instead downregulated the BCL2 translation. Polysome fractionation studies showed that HuR retarded the BCL2 mRNA in the non-translating pool of polysomes. Moreover, protection from dsRNA-induced apoptosis by HuR depletion required the presence of BCL2, indicating that the proapoptotic function of HuR is executed by suppressing BCL2. Consistently, HuR regulated apoptosis induced by infection of encephalomyocarditis or Semliki Forest virus, two unrelated positive strand RNA viruses. Collectively, our work identified a suite of proteins that regulate dsRNA-induced cell death, and elucidated the mechanism by which HuR acts as a pro-apoptotic factor.
2021-01-28 | GSE165625 | GEO
Project description:BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.
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