MicroRNAs control the apoptotic threshold in primed pluripotent stem cells through regulation of BIM
ABSTRACT: Mammalian primed pluripotent stem cells have been shown to be highly susceptible to cell death stimuli due to their low apoptotic threshold, but how this threshold is regulated remains largely unknown. Here we identify miRNA-mediated regulation as a key mechanism controlling apoptosis in the post-implantation epiblast. Moreover, we find that three miRNA families, miR-20, miR-92 and miR-302, control the mitochondrial apoptotic machinery by fine-tuning the levels of expression of the pro-apoptotic protein BIM. These families therefore represent an essential buffer needed to maintain cell survival in stem cells that are not only primed for differentiation but also for cell death. We used microarrays to compare the gene expression profiles of Dicer conditional Epiblast stem cells (Dicer fx/fx EpiSCs, used as control cells) and Dicer deleted epiblast stem cells (Dicer-/- EpiSCs) five days after the induction of Dicer deletion Dicer fx/fx EpiSCs were left untreated (control cells) or treated with 0.3uM of 4-OH-Tamoxifen for three days and without Tamoxifen for two further days, until day 5 when RNA was extracted and used for microarray analysis. Three independent deletion experiments including a Dicer fx/fx sample and a Dicer -/- sample were analyzed as biological replicates.
Project description:Mammalian primed pluripotent stem cells have been shown to be highly susceptible to cell death stimuli due to their low apoptotic threshold, but how this threshold is regulated remains largely unknown. Here we identify miRNA-mediated regulation as a key mechanism controlling apoptosis in the post-implantation epiblast. Moreover, we find that three miRNA families, miR-20, miR-92 and miR-302, control the mitochondrial apoptotic machinery by fine-tuning the levels of expression of the pro-apoptotic protein BIM. These families therefore represent an essential buffer needed to maintain cell survival in stem cells that are not only primed for differentiation but also for cell death. We used microarrays to compare the gene expression profiles of Dicer conditional Epiblast stem cells (Dicer fx/fx EpiSCs, used as control cells) and Dicer deleted epiblast stem cells (Dicer-/- EpiSCs) five days after the induction of Dicer deletion Dicer fx/fx EpiSCs were left untreated (control cells) or treated with 0.3uM of 4-OH-Tamoxifen for three days and without Tamoxifen for two further days, until day 5 when RNA was extracted and used for microarray analysis. Three independent deletion experiments including a Dicer fx/fx sample and a Dicer -/- sample were analyzed as biological replicates.
Project description:To characterize the reprogramming of epiblast stem cells (EpiSCs) into embryonic stem cells (ESCs) induced by Esrrb, we performed microarray analysis of Tet-on Esrrb EpiSCs after treatment with doxycycline (Dox).
Project description:To investigate the molecular mechanisms underlying the reprogramming of epiblast stem cells (EpiSCs) into embryonic stem cells (ESCs) induced by Esrrb, we performed ChIP-seq analysis of Esrrb, Nanog, Oct4, and Sox2 in Tet-on Esrrb EpiSCs after treatment with doxycycline (Dox).
Project description:Transcriptional profiling of XiGFP Epiblast Stem Cells (EpiSCs) overexpressing Klf2, Prdm14, Prdm14+Klf2, or vector control. Cells cultured in activin and bFGF (day 0) or on day 2 and day 4 after transfer to serum and LIF on feeder cells and sorting for dsRed expression to remove feeder cells.
Project description:Transcription factor/enhancer interactions determine cell specific gene expression. Here, we followed enhancers during differentiations of embryonic stem (ESCs) to epiblast like cells (EpiLCs). There were highly dynamic changes in histone lysine 27 acetylation at enhancer sites throughout the genome. These sites were enriched for a Foxd3 binding motif, a forkhead transcription factor essential in early embryonic development. Surprisingly, Foxd3 occupied largely mutually exclusive sites in the ESCs versus EpiLCs. Foxd3 bound to nucleosome occupied regions, simultaneously evicting the histones while inhibiting full gene expression through the recruitment of histone deacetylases. Knockout of Foxd3 resulted in hyperacetylation and transcriptional upregulation of neighboring genes, many of which were further upregulated at later stages of differentiation. These data show that Foxd3 primes enhancer sites during pregastrulation by removing nucleosomes, yet suppresses neighboring histone hyperacetylation. Such a mechanism may be common to many transcription factors that prepare enhancers for later gene activation during development. ChIP-seq of H3K4me1, H3K27ac, H3K27me3, p300, H3K4me3, RNA Pol2 and Oct4 in four pluripotent states: embryonic stem cells (ESCs) day 1 ESC differentiation, Epi-like stem cells (EpiLCs), and epiblast stem cells (EpiSCs); ChIP-seq of 3XFlag tagged Foxd3 in ESCs and EpiLCs; ChIP-seq of H3K4me1, H3K27ac, H3K27me3, p300 and H3K4me3 in Foxd3 conditional knockout cells (tamoxifen-inducible) -/+ 36h Tamoxifen treatemnt. ChIP seq of Flag-Foxd3 (third replicate), ChIP-seq of HDAC1 and Brg1 in WT and Foxd3 KO cells and MNase-ChIP-seq of H3K4me1
Project description:Epiblast stem cells (EpiSCs) were derived from the epiblast or the ectoderm (epi/ect) of pre-gastrula stage to late-bud stage mouse embryos. To identify if the EpiSCs retain any original stage specific characteristics or which developmental stage of epi/ect they most closely related to, we performed microarray analysis to compare the gene expression profile of multiple EpiSC lines with that of epi/ect of 7 different stages. Eighteen EpiSC lines established from R1-129 embryos of different stages and 1 line (EpiSC9) imported were harvested in triplicate cultured separately for 3 days. The * marks the subline thawed at a different time and harvested but originated from the same embryo. EpiSC9_T is an RNA sample provided by Dr Tesar. ESCs, iPSCs and MEFs were prepared accordingly. For the epiblast samples, in order to avoid the averaging effect by pooling samples, we linearly amplified the RNA starting from a single epiblast or ectoderm.
Project description:Epiblast stem cells (EpiSCs) are pluripotent cells that can be isolated and cultured from post implantation embryos. In contrast to embryonic stem cells (ESCs), systematic studies to investigate the genes that maintain pluripotency in EpiSCs have not been reported. Here we combine a genome-wide RNAi screen with genetic interaction, protein localization and protein-level dependency studies to delineate connectivity between factors that control Oct4 expression in EpiSCs and compare the role of these factors to their function in ES cells. We demonstrate the power of this integrative approach by the identification of Tox4 as an interactor of PP1 (Protein Phosphatase 1) and Paf1C, a complex that acts in multiple aspects of RNAPII regulation. Our results indicate that Tox4 cooperates with Paf1C and PP1 to influence the phosphorylation status of the RNAPII CTD tail during transcription and that this function is vital for maintenance of pluripotent cell identity. RNA-seq of Tox4 knockdown in mouse EpiSCs
Project description:Stem cell biology has garnered much attention due to its potential to impact human health through disease modeling and cell replacement therapy. This is especially pertinent to myelin-related disorders such as multiple sclerosis and leukodystrophies where restoration of normal oligodendrocyte function could provide an effective treatment. Progress in myelin repair has been constrained by the difficulty in generating pure populations of oligodendrocyte progenitor cells (OPCs) in sufficient quantities. Pluripotent stem cells theoretically provide an unlimited source of OPCs but significant advances are currently hindered by heterogeneous differentiation strategies that lack reproducibility. Here we provide a platform for the directed differentiation of pluripotent mouse epiblast stem cells (EpiSCs) through a defined series of developmental transitions into a pure population of highly expandable OPCs in ten days. These OPCs robustly differentiate into myelinating oligodendrocytes both in vitro and in vivo. Our results demonstrate that pluripotent stem cells can provide a pure population of clinically-relevant, myelinogenic oligodendrocytes and offer a tractable platform for defining the molecular regulation of oligodendrocyte development, drug screening, and potential cell-based remyelinating therapies. 6 total samples were analyzed. Pluripotent epiblast stem cells (EpiSCs) were differentiated to patterned neural rosettes, oligodendrocyte progenitor cells (OPCs), and oligodendrocytes. OPCs and oligodedrocytes were analyzed at two separate passages (3 and 11).
Project description:The generation of properly functioning gametes in vitro, a key goal in developmental/reproductive biology, requires multi-step reconstitutions of complex germ cell development. Based on the logic of primordial germ cell (PGC)-specification, we demonstrate here the generation of PGC-like cells (PGCLCs) in mice with robust capacity for spermatogenesis from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) through epiblast-like cells (EpiLCs), a cellular state highly similar to pre-gastrulating epiblasts, but distinct from epiblast stem cells (EpiSCs). The global transcription profiles, epigenetic reprogramming, and cellular dynamics during PGCLC induction from EpiLCs are a meticulous capture of those associated with PGC specification from the epiblasts. Furthermore, we identify Integrin-beta 3 and SSEA1 as markers that purify PGCLCs with spermatogenic capacity free from tumorigenic undifferentiated cells. With the reconstitution of PGC specification pathway from the naive inner cell mass state, our study defines a paradigm for the essential step of in vitro gametogenesis. We performed this analysis to reveal the characters of the cells that we created in this study, epiblast-like cells (EpiLCs) and primordial germ cells-like cells (PGCLCs). Because EpiLCs were induced from embryonic stem cells (ESCs), and equivalent to pre-gastrulating epiblast (embryonic day [E] 5.5-6.0) in vivo (embryo), ESCs and epiblast were included in this analysis. Epiblast stem cells (EpiSCs) are a culture cell type derived from epiblast, and were also included. PGCLCs were supposed to be equivalent to E9.5 PGCs based on reporter fluorescent transgene expressions and epigenetic properties, and therefore E9.5 PGCs were also inckuded in this analysis. Because epiblast and E9.5 PGCs are of a small number of cells in embryos (a few hundred to thousand cells), cDNAs were amplified with a quantitative global PCR method (Kurimoto et al., 2006, Nucleic Acids Research) for microarray analyses. We took two biological replicate for each cell type.
Project description:Recently, (in vitro) pluripotent EpiSCs were derived from the post-implantation egg cylinder stage epiblasts of mouse and rat. These EpiSCs resemble and correspond very closely to the conventional human embryonic stem cells (hESCs) in the colony morphology and culture/signaling requirements for maintaining pluripotency, but exhibit a range of significant phenotypic and signaling response differences from the conventional mouse ES cells (mESCs). These observations strongly support the notion that EpiSCs and hESCs are intrinsically similar, and raise an attractive hypothesis: as mESCs and EpiSCs/hESCs represent two distinct pluripotency states: the mESC-like state representing the ICM of pre-implantation blastcyst and the EpiSC-like state representing the post-implantation epiblasts, whether the epiblast state (including conventional hESCs) can be converted back to the ICM state. Despite studies providing evidence that epiblast-like cells exist and transition back and forth within colony of conventional mESCs; mESCs and EpiSCs share substantial set of pluripotency transcriptional factors, including Oct4, Sox2 and Nanog; and mESCs are more stable in culture, in the present study we found that EpiSCs differentiated rapidly under mESC culture conditions and no “spontaneously” converted mESC could be readily identified and isolated over serial passages at the population or clonal level. Remarkably, we found that blockage of the TGFβ pathway or inhibition of the H3K4 demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes of EpiSCs towards mESC phenotypes with activation of some ICM-specific gene expression. However, full conversion of EpiSCs to a mESC-like state with competence to chimeric contribution can only be readily generated with a combination of inhibitors of LSD1 and ALK. These observations underscore a powerful and direct induction of reprogramming from the developmentally later-stage EpiSCs to a mESC-like stage by a synergy of signaling and direct epigenetic modulations. It also highlights a significant role for TGFβ pathway inhibition in promoting reprogramming to and sustaining true pluripotency, which further supports our recent studies in generating chimerism-competent rat pluripotent cells. Collectively, our studies provide a proof-of-concept demonstration that pluripotency-restricted EpiSCs can be readily converted to a mESC-like state in the absence of any genetic manipulation by precise pharmacological control of signaling pathways that distinguish the two pluripotency states and an epigenetic target simultaneously, and offer a convenient experimental system to further study the mechanism. Such method and concept may also provide an avenue for generating a new type of mESC-like human pluripotent cell. Global gene-expression analyses of the parnate/mAMFGi cells