Epiblast stem cell subpopulations represent mouse embryos of distinct pregastrulation stages

Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in?vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.     

A Novel Nodal Enhancer Dependent on Pluripotency Factors and Smad2/3 Signaling Conditions a Regulatory Switch During Epiblast Maturation

During early development, modulations in the expression of Nodal, a TGFβ family member, determine the specification of embryonic and extra-embryonic cell identities. Nodal has been extensively studied in the mouse, but aspects of its early expression remain unaccounted for. We identified a conserved hotspot for the binding of pluripotency factors at the Nodal locus and called this sequence “highly bound element” (HBE). Luciferase-based assays, the analysis of fluorescent HBE reporter transgenes, and a conditional mutation of HBE allowed us to establish that HBE behaves as an enhancer, is activated ahead of other Nodal enhancers in the epiblast, and is essential to Nodal expression in embryonic stem cells (ESCs) and in the mouse embryo. We also showed that HBE enhancer activity is critically dependent on its interaction with the pluripotency factor Oct4 and on Activin/Nodal signaling. Use of an in vitro model of epiblast maturation, relying on the differentiation of ESCs into epiblast stem cells (EpiSCs), revealed that this process entails a shift in the regulation of Nodal expression from an HBE-driven phase to an ASE-driven phase, ASE being another autoregulatory Nodal enhancer. Deletion of HBE in ESCs or in EpiSCs allowed us to show that HBE, although not necessary for Nodal expression in EpiSCs, is required in differentiating ESCs to activate the differentiation-promoting ASE and therefore controls this regulatory shift. Our findings clarify how early Nodal expression is regulated and suggest how this regulation can promote the specification of extra-embryonic precusors without inducing premature differentiation of epiblast cells. More generally, they open new perspectives on how pluripotency factors achieve their function.     

Embryonic stem cells require Wnt proteins to prevent differentiation to epiblast stem cells

Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref. 1). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor.     

Dynamic equilibrium and heterogeneity of mouse pluripotent stem cells with distinct functional and epigenetic states

Embryonic stem cells (ESCs) are apparently homogeneous self-renewing cells, but we observed heterogeneous expression of Stella in ESCs, which is a marker of pluripotency and germ cells. Here we show that, whereas Stella-positive ESCs were like the inner cell mass (ICM), Stella-negative cells were like the epiblast cells. These states were interchangeable, which reflects the metastability and plasticity of ESCs. The established equilibrium was skewed reversibly in the absence of signals from feeder cells, which caused a marked shift toward an epiblast-like state, while trichostatin A, an inhibitor of histone deactelylase, restored Stella-positive population. The two populations also showed different histone modifications and striking functional differences, as judged by their potential for differentiation. The Stella-negative ESCs were more like the postimplantation epiblast-derived stem cells (EpiSCs), albeit the stella locus was repressed by DNA methylation in the latter, which signifies a robust epigenetic boundary between ESCs and EpiSCs.     

Accessing naïve human pluripotency

Pluripotency manifests during mammalian development through formation of the epiblast, founder tissue of the embryo proper. Rodent pluripotent stem cells can be considered as two distinct states: na?ve and primed. Na?ve pluripotent stem cell lines are distinguished from primed cells by self-renewal in response to LIF signaling and MEK/GSK3 inhibition (LIF/2i conditions) and two active X chromosomes in female cells. In rodent cells, the na?ve pluripotent state may be accessed through at least three routes: explantation of the inner cell mass, somatic cell reprogramming by ectopic Oct4, Sox2, Klf4, and C-myc, and direct reversion of primed post-implantation-associated epiblast stem cells (EpiSCs). In contrast to their rodent counterparts, human embryonic stem cells and induced pluripotent stem cells more closely resemble rodent primed EpiSCs. A critical question is whether na?ve human pluripotent stem cells with bona fide features of both a pluripotent state and na?ve-specific features can be obtained. In this review, we outline current understanding of the differences between these pluripotent states in mice, new perspectives on the origins of na?ve pluripotency in rodents, and recent attempts to apply the rodent paradigm to capture na?ve pluripotency in human cells. Unraveling how to stably induce na?ve pluripotency in human cells will influence the full realization of human pluripotent stem cell biology and medicine.     

Search for nave human pluripotent stem cells

Normal mouse pluripotent stem cells were originally derived from the inner cell mass(ICM) of blastocysts and shown to be the in vitro equivalent of those pre-implantation embryonic cells, and thus were called embryonic stem cells(ESCs). More than a decade later, pluripotent cells were isolated from the ICM of human blastocysts. Despite being called human ESCs, these cells differ significantly from mouse ESCs, including different morphology and mechanisms of control of pluripotency, suggesting distinct embryonic origins of ESCs from the two species. Subsequently, mouse pluripotent stem cells were established from the ICMderived epiblast of post-implantation embryos. These mouse epiblast stem cells(Epi SCs) are morphological and epigenetically more similar to human ESCs. This raised the question of whether cells from the human ICM are in a more advanced differentiation stage than their murine counterpart, or whether the available culture conditions were not adequate to maintain those human cells in their in vivo state, leading to a transition into Epi SC-like cells in vitro. More recently, novel culture conditions allowed the conversion of human ESCs into mouse ESC-like cells called nave(or ground state) human ESCs, and the derivation of nave human ESCs from blastocysts. Here we will review the characteristics of each type of pluripotent stem cells, how(and whether) these relate to different stages of embryonic development, and discuss the potential implications of nave human ESCs in research and therapy.     

A Modified EpiSC Culture Condition Containing a GSK3 Inhibitor Can Support Germline-Competent Pluripotency in Mice

Embryonic stem cells (ESCs) can contribute to the tissues of chimeric animals, including the germline. By contrast, epiblast stem cells (EpiSCs) barely contribute to chimeras. These two types of cells are established and maintained under different culture conditions. Here, we show that a modified EpiSC culture condition containing the GSK3 inhibitor CHIR99021 can support a germline-competent pluripotent state that is intermediate between ESCs and EpiSCs. When ESCs were cultured under a modified condition containing bFGF, Activin A, and CHIR99021, they converted to intermediate pluripotent stem cells (INTPSCs). These INTPSCs were able to form teratomas in vivo and contribute to chimeras by blastocyst injection. We also induced formation of INTPSCs (iINTPSCs) from mouse embryonic fibroblasts by exogenous expression of four reprogramming factors, Oct3/4, Sox2, Klf4, and c-Myc, under the INTPSC culture condition. These iINTPSCs contributed efficiently to chimeras, including the germline, by blastocyst injection. The INTPSCs exhibited several characteristic properties of both ESCs and EpiSCs. Our results suggest that the modified EpiSC culture condition can support growth of cells that meet the most stringent criteria for pluripotency, and that germline-competent pluripotency may depend on the activation state of Wnt signaling.     

Different flavors of pluripotency, molecular mechanisms, and practical implications

Pluripotent stem cells (PSCs) have been classified into two distinct states: a primitive, naive LIF-dependent state represented by murine ESCs, and a primed bFGF-dependent state observed in murine and rat epiblast stem cells (EpiSCs). The vast similarities between EpiSCs and human ESCs suggest that, despite their blastocyst origin, human ESCs exist in a primed pluripotent state. Recent findings demonstrate that the naive and primed pluripotent states are interconvertible, even in human cells, and hint that growth factor-mediated Nanog expression may be an important factor regulating the balance between them.     

Epigenetic reprogramming: Prdm14 hits the accelerator

Cell Stem Cell 10 4, 425–439 (2012); published online April062012The release of epigenetic boundaries during epigenetic reprogramming is poorly understood. In the recent issue of Cell Stem Cell Journal, Gillich and colleagues identify a unique role for Prdm14 in the acceleration of this process (Gillich et al, 2012).Pluripotent stem cells can be established from pre-implantation blastocyst embryos (embryonic stem cells, ESCs) as well as from the post-implantation epiblast stem cells (EpiSCs; Chenoweth et al, 2010). Murine ESCs and EpiSCs both express central pluripotency factors such as Oct4, Nanog and Sox2, yet the different developmental origins of these two cell types is clearly reflected in their molecular, epigenetic and functional properties. Murine ESCs appear to exist in a unique ‘naive'' state reminiscent of the pre-implantation epiblast. They are characterized by the expression of germ cell–related genes, a remarkably open chromatin structure with two active X chromosomes, and the functional ability to contribute to chimera formation upon blastocyst complementation (Nichols and Smith, 2011). In contrast, EpiSCs reflect the properties of the post-implantation epiblast, characterized by low-level expression of early determinants of somatic differentiation, a near-absence of germ cell gene expression, inactivation of one of the X chromosomes and negligible ability to support the development of chimeric mice. The conversion of primed to naive pluripotent state requires the release of epigenetic restrictions that are established in the post-implantation epiblast. It is thus a reprogramming process akin to the derivation of induced pluripotent stem cells (iPSCs) from somatic cells. The results on Prdm14 from Gillich and colleagues offer new insights into the underlying molecular mechanisms governing epigenetic reprogramming.     

Naive and primed murine pluripotent stem cells have distinct miRNA expression profiles

Over the last years, the microRNA (miRNA) pathway has emerged as a key component of the regulatory network of pluripotency. Although clearly distinct states of pluripotency have been described in vivo and ex vivo, differences in miRNA expression profiles associated with the developmental modulation of pluripotency have not been extensively studied so far. Here, we performed deep sequencing to profile miRNA expression in naive (embryonic stem cell [ESC]) and primed (epiblast stem cell [EpiSC]) pluripotent stem cells derived from mouse embryos of identical genetic background. We developed a graphical representation method allowing the rapid identification of miRNAs with an atypical profile including mirtrons, a small nucleolar RNA (snoRNA)-derived miRNA, and miRNAs whose biogenesis may differ between ESC and EpiSC. Comparison of mature miRNA profiles revealed that ESCs and EpiSCs exhibit very different miRNA signatures with one third of miRNAs being differentially expressed between the two cell types. Notably, differential expression of several clusters, including miR290-295, miR17-92, miR302/367, and a large repetitive cluster on chromosome 2, was observed. Our analysis also showed that differentiation priming of EpiSC compared to ESC is evidenced by changes in miRNA expression. These dynamic changes in miRNAs signature are likely to reflect both redundant and specific roles of miRNAs in the fine-tuning of pluripotency during development.     

Isolation of epiblast stem cells from preimplantation mouse embryos

Pluripotent stem cells provide a platform to interrogate control elements that function to generate all cell types of the body. Despite their utility for modeling development and disease, the relationship of mouse and human pluripotent stem cell states to one another remains largely undefined. We have shown that mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) are distinct, pluripotent states isolated from pre- and post-implantation embryos respectively. Human ES cells are different than mouse ES cells and share defining features with EpiSCs, yet are derived from pre-implantation human embryos. Here we show that EpiSCs can be routinely derived from pre-implantation mouse embryos. The preimplantation-derived EpiSCs exhibit molecular features and functional properties consistent with bona fide EpiSCs. These results provide a simple method for isolating EpiSCs and offer direct insight into the intrinsic and extrinsic mechanisms that regulate the acquisition of distinct pluripotent states.     

DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation

Epiblast stem cells (EpiSCs), which are pluripotent cells isolated from early post-implantation mouse embryos (E5.5), show both similarities and differences compared to mouse embryonic stem cells (mESCs), isolated earlier from the inner cell mass (ICM) of the E3.5 embryo. Previously, we have observed that while chromatin is very dispersed in E3.5 ICM, compact chromatin domains and chromocentres appear in E5.5 epiblasts after embryo implantation. Given that the observed chromatin re-organization in E5.5 epiblasts coincides with an increase in DNA methylation, in this study, we aimed to examine the role of DNA methylation in chromatin re-organization during the in vitro conversion of ESCs to EpiSCs. The requirement for DNA methylation was determined by converting both wild-type and DNA methylation-deficient ESCs to EpiSCs, followed by structural analysis with electron spectroscopic imaging (ESI). We show that the chromatin re-organization which occurs in vivo can be re-capitulated in vitro during the ESC to EpiSC conversion. Indeed, after 7 days in EpiSC media, compact chromatin domains begin to appear throughout the nuclear volume, creating a chromatin organization similar to E5 epiblasts and embryo-derived EpiSCs. Our data demonstrate that DNA methylation is dispensable for this global chromatin re-organization but required for the compaction of pericentromeric chromatin into chromocentres.