Stat3 activation is limiting for reprogramming to ground state pluripotency.
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ABSTRACT:
SUBMITTER: Yang J
PROVIDER: S-EPMC3459098 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Induced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming. We report that brain-derived neural stem (NS) cells acquire undifferentiated morphology rapidly and at high frequency after a single round of transduction with reprogramming factors. However, critical attributes of true pluripotency--including stable expression of endogenous Oct4 and Nanog, epigenetic erasure of X chromosome silencing in female cells, and ability to colonise chimaeras--were not attained. We therefore applied molecularly defined conditions for the derivation and propagation of authentic pluripotent stem cells from embryos. We combined dual inhibition (2i) of mitogen-activated protein kinase signalling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukaemia inhibitory factor (LIF). The 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimaerism. Using 2i /LIF, NS cell reprogramming required only 1-2 integrations of each transgene. Furthermore, transduction with Sox2 and c-Myc is dispensable, and Oct4 and Klf4 are sufficient to convert NS cells into chimaera-forming iPS cells. These findings demonstrate that somatic cell state influences requirements for reprogramming and delineate two phases in the process. The ability to capture pre-pluripotent cells that can advance to ground state pluripotency simply and with high efficiency opens a door to molecular dissection of this remarkable phenomenon.
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Project description:Translational control plays a central role in regulation of gene expression and can lead to significant divergence between mRNA- and protein-abundance. Here, we used genome-wide approaches combined with time-course analysis to measure the mRNA-abundance, mRNA-translation rate and protein expression during the transition of naïve-to-primed mouse embryonic stem cells (ESCs). We find that the ground state ESCs cultured with GSK3-, MEK-inhibitors and LIF (2iL) display higher ribosome density on a selective set of mRNAs. This set of mRNAs undergo strong translational buffering to maintain stable protein expression levels in 2iL-ESCs. Importantly, we show that the global alteration of cellular proteome during the transition of naïve-to-primed pluripotency is largely accompanied by transcriptional rewiring. Thus, we provide a comprehensive and detailed overview of the global changes in gene expression in different states of ESCs and dissect the relative contributions of mRNA-transcription, translation and regulation of protein stability in controlling protein abundance.
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Project description:Mouse embryonic stem (ES) cells grown in serum exhibit greater heterogeneity in morphology and expression of pluripotency factors than ES cells cultured in defined medium with inhibitors of two kinases (Mek and GSK3), a condition known as "2i" postulated to establish a naive ground state. We show that the transcriptome and epigenome profiles of serum- and 2i-grown ES cells are distinct. 2i-treated cells exhibit lower expression of lineage-affiliated genes, reduced prevalence at promoters of the repressive histone modification H3K27me3, and fewer bivalent domains, which are thought to mark genes poised for either up- or downregulation. Nonetheless, serum- and 2i-grown ES cells have similar differentiation potential. Precocious transcription of developmental genes in 2i is restrained by RNA polymerase II promoter-proximal pausing. These findings suggest that transcriptional potentiation and a permissive chromatin context characterize the ground state and that exit from it may not require a metastable intermediate or multilineage priming.
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Project description:Mouse embryonic stem (ES) cells derived from pluripotent early epiblast contribute functionally differentiated progeny to all foetal lineages of chimaeras. By contrast, epistem cell (EpiSC) lines from post-implantation epithelialised epiblast are unable to colonise the embryo even though they express the core pluripotency genes Oct4, Sox2 and Nanog. We examined interconversion between these two cell types. ES cells can readily become EpiSCs in response to growth factor cues. By contrast, EpiSCs do not change into ES cells. We exploited PiggyBac transposition to introduce a single reprogramming factor, Klf4, into EpiSCs. No effect was apparent in EpiSC culture conditions, but in ground state ES cell conditions a fraction of cells formed undifferentiated colonies. These EpiSC-derived induced pluripotent stem (Epi-iPS) cells activated expression of ES cell-specific transcripts including endogenous Klf4, and downregulated markers of lineage specification. X chromosome silencing in female cells, a feature of the EpiSC state, was erased in Epi-iPS cells. They produced high-contribution chimaeras that yielded germline transmission. These properties were maintained after Cre-mediated deletion of the Klf4 transgene, formally demonstrating complete and stable reprogramming of developmental phenotype. Thus, re-expression of Klf4 in an appropriate environment can regenerate the naïve ground state from EpiSCs. Reprogramming is dependent on suppression of extrinsic growth factor stimuli and proceeds to completion in less than 1% of cells. This substantiates the argument that EpiSCs are developmentally, epigenetically and functionally differentiated from ES cells. However, because a single transgene is the minimum requirement to attain the ground state, EpiSCs offer an attractive opportunity for screening for unknown components of the reprogramming process.
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Project description:The transition of mouse embryonic stem cells (ESCs) between serum/LIF and 2i(MEK and GSK3 kinase inhibitor)/LIF culture conditions serves as a valuable model for exploring the mechanisms underlying ground and confused pluripotent states. Regulatory networks comprising core and ancillary pluripotency factors drive the gene expression programs defining stable naïve pluripotency. In our study, we systematically screened factors essential for ESC pluripotency, identifying TEAD2 as an ancillary factor maintaining ground-state pluripotency in 2i/LIF ESCs and facilitating the transition from serum/LIF to 2i/LIF ESCs. TEAD2 exhibits increased binding to chromatin in 2i/LIF ESCs, targeting active chromatin regions to regulate the expression of 2i-specific genes. Additionally, TEAD2 facilitates the expression of 2i-specific genes by mediating enhancer-promoter interactions during the serum/LIF to 2i/LIF transition. Notably, deletion of Tead2 results in reduction of a specific set of enhancer-promoter interactions without significantly affecting binding of chromatin architecture proteins, CCCTC-binding factor (CTCF), and Yin Yang 1 (YY1). In summary, our findings highlight a novel prominent role of TEAD2 in orchestrating higher-order chromatin structures of 2i-specific genes to sustain ground-state pluripotency.
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Project description:Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.
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Project description:Pluripotent stem cells (PSCs) occupy a spectrum of reversible molecular states ranging from a naive ground-state in 2i, to metastable embryonic stem cells (ESCs) in serum, to lineage-primed epiblast stem cells (EpiSCs). To investigate the role of DNA methylation (5mC) across distinct pluripotent states, we mapped genome-wide 5mC and 5-hydroxymethycytosine (5hmC) in multiple PSCs. Ground-state ESCs exhibit an altered distribution of 5mC and 5hmC at regulatory elements and dramatically lower absolute levels relative to ESCs in serum. By contrast, EpiSCs exhibit increased promoter 5mC coupled with reduced 5hmC, which contributes to their developmental restriction. Switch to 2i triggers rapid onset of both the ground-state gene expression program and global DNA demethylation. Mechanistically, repression of de novo methylases by PRDM14 drives DNA demethylation at slow kinetics, whereas TET1/TET2-mediated 5hmC conversion enhances both the rate and extent of hypomethylation. These processes thus act synergistically during transition to ground-state pluripotency to promote a robust hypomethylated state.
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