Project description:Human embryonic stem cells (hESCs) typically exhibit "primed" pluripotency, analogous to stem cells derived from the mouse post-implantation epiblast. This has led to a search for growth conditions that support self-renewal of hESCs akin to hypomethylated naïve epiblast cells in human pre-implantation embryos. We have discovered that reverting primed hESCs to a hypomethylated naïve state or deriving a new hESC line under naïve conditions results in the establishment of Stage Specific Embryonic Antigen 4 (SSEA4) negative hESC lines with a transcriptional program resembling the human pre-implantation epiblast. In contrast, we discovered that the methylome of naïve hESCs in vitro is distinct from the human epiblast in vivo with loss of DNA methylation at primary imprints and a lost "memory" of the methylation state of the human oocyte. This failure to recover the naïve epiblast methylation landscape appears to be a consistent feature of self-renewing hypomethylated naïve hESCs in vitro.
Project description:The TET dioxygenases erase mediate DNA dedemethylation in pre-implantation embryos and in primordial germ cells, yet limited studies address their contribution to the global gain of DNA methylation following implantation. Here, we show that Tet1 is expressed and non-redundantly contributes to 5-hydroxymethylctyosine (5hmC) non-redundantly in the pre-gastrulation mouse epiblast. Ablation of Tet1 in primed epiblast cells results in widespread loss of 5hmC associated with gain of 5-methylcytosine at CpG islands and promoters. Moreover, Tet1 is expressed, albeit at lower levels, in the extra-embryonic ectoderm. Tet1-deficiency in the pre-streak mouse embryos causes dysregulation of early lineage regulators in the epiblast and increased expression of metabolic genes in the extra-embryonic ectoderm. Our studies reveal a distinct role of Tet1 in regulating the methylome landscape of the post-implantation mammalian epiblast and a hitherto unknown gene repressive effect in the extra-embryonic lineage, providing insights into the early developmental origins of epigenetic-based basis of imprinting and developmental disorders.
Project description:During mammalian pre-implantation development, the cells of the blastocyst’s inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived extra-embryonic endoderm (XEN) cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and extra-embryonic endoderm differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the re-organization of membrane trafficking machinery and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes.
Project description:Primitive ectoderm cells (PE) in blastocysts represent the foundation of the pluripotent state, which is lost progressively during development. For example, development of epiblast cells from PE in postimplantation embryos is accompanied by transcriptional and epigenetic changes, including DNA methylation and X inactivation (Refs); these changes alter the nature of epiblast cells fundamentally, affecting their responsiveness to signaling molecules, and constitute a robust boundary that prevents their reversion to a PE-like state. Notably, epiblast cells unlike PE, are refractory to leukaemia inhibitory factor (LIF)/STAT3 signalling in vitro; instead, they respond to FGF/Activin to form self-renewing epiblast stem cells (EpiSCs) that are like human ES cells, which differ significantly from mouse embryonic stem cells (ES) derived from PE. However, here we show that under appropriate conditions, epiblast cells from postimplantation embryos can respond to LIF/STAT3/fetal calf serum (FCS), and undergo reprogramming to form embryonic stem cell -like cells (repiES: reprogrammed epiblast ES-like cells). Reprogramming of epiblast cells occurs progressively. First, they form colonies that retain key properties of epiblast cells (cEpi: cultured epiblast), which subsequently show erasure of epigenetic modifications, including DNA demethylation and X-reactivation to generate repiES. Analysis also revealed that repiES progressively acquire a transcriptome profile of ES cells that is distinct from cEpi and EpiSCs. In chimeras, repiES contributed to all the tissues, including germ cells. Thus, we show for the first time that reversion of epiblast cells to repiES phenotype entails progressive loss of phenotypic and epigenetic memory of epiblast cells. Our study provides insights into underlying mechanisms, and a tractable model for how signaling molecules induce epigenetic reprogramming of cells leading to an elemental pluripotent state.
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:Pluripotency is highly dynamic and progresses through a continuum of pluripotent stem-cell states. The two states that bookend the pluripotency continuum, naïve and primed, are well characterized, but our understanding of the intermediate states and transitions between them remain incomplete. Here, we dissect the dynamics of pluripotent state transitions underlying pre- to post-implantation epiblast differentiation. Through comprehensive mapping of the proteome, phosphoproteome, transcriptome, and epigenome of embryonic stem cells transitioning from naïve to primed pluripotency, we find that rapid, acute, and widespread changes to the phosphoproteome precede ordered changes to the epigenome, transcriptome, and proteome. Reconstruction of kinase-substrate networks reveals signaling cascades, dynamics, and crosstalk. Distinct waves of global proteomic changes mark discrete phases of pluripotency, with cell state-specific surface markers tracking pluripotent state transitions. Our data provide new insights into the multi-layered control of the phased progression of pluripotency and a foundation for modeling mechanisms regulating pluripotent state transitions (www.stemcellatlasorg).
Project description:Pluripotent stem cells can give rise to the three embryonic germ layers and the characterization of their properties is crucial to exploit their therapeutic potential. Mouse embryonic stem cells (mESCs) are isolated and usually maintained in vitro in a primed state that resembles the post-implantation epiblast features. Furthermore, primed mESCs can be de-differentiated to a naive state that resembles the pre-implantation inner cell mass (ICM). Cell differentiation or genotoxic stress, among others, can alter DNA replication, which is a flexible process able to adapt to different cellular contexts. Here, we demonstrate that primed-to-naive mESC reprogramming triggers replication fork slowdown, increased fork asymmetry and a compensatory activation of dormant origins. Using iPOND (“isolation of proteins on nascent DNA”) coupled to mass spectrometry we have characterized the changes in replisome composition between naive and primed mESCs. Several DNA repair factors, including MRE11 nuclease, are enriched in naive mESCs forks, while factors involved in ubiquitin-dependent protein metabolism are enriched in primed mESC forks. We report that primed-to-naive mESC de-differentiation promotes recruitment of MRE11 to the forks in response to transcription-replication conflicts, underlying the DNA replication rewiring required for efficient mESC reprogramming.