Naive Human Pluripotent Cells Feature a Methylation Landscape Devoid of Blastocyst or Germline Memory.
ABSTRACT: 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 naive epiblast cells in human pre-implantation embryos. We have discovered that reverting primed hESCs to a hypomethylated naive state or deriving a new hESC line under naive 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 naive hESCs in vitro is distinct from that of 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 naive epiblast methylation landscape appears to be a consistent feature of self-renewing hypomethylated naive hESCs in vitro.
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. Overall design: This data includes 25 RNA-seq and 18 BS-seq datasets
Project description:Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.
Project description:Current challenges in capturing naive human pluripotent stem cells (hPSCs) suggest that the factors regulating human naive versus primed pluripotency remain incompletely defined. Here we demonstrate that the widely used Essential 8 minimal medium (E8) captures hPSCs at a naive-to-primed intermediate state of pluripotency expressing several naive-like developmental, bioenergetic, and epigenomic features despite providing primed-state-sustaining growth factor conditions. Transcriptionally, E8 hPSCs are marked by activated lipid biosynthesis and suppressed MAPK/TGF-? gene expression, resulting in endogenous ERK inhibition. These features are dependent on lipid-free culture conditions and are lost upon lipid exposure, whereas short-term pharmacological ERK inhibition restores naive-to-primed intermediate traits even in the presence of lipids. Finally, we identify de novo lipogenesis as a common transcriptional signature of E8 hPSCs and the pre-implantation human epiblast in vivo. These findings implicate exogenous lipid availability in regulating human pluripotency and define E8 hPSCs as a stable, naive-to-primed intermediate (NPI) pluripotent state.
Project description:Controlling responsiveness to prevailing signals is critical for robust transitions between cell states during development. For example, fibroblast growth factor (FGF) drives naive pluripotent cells into extraembryonic lineages before implantation but sustains pluripotency in primed cells of the post-implantation epiblast. Nanog supports pluripotency in naive cells, while Nodal supports pluripotency in primed cells, but the handover from Nanog to Nodal does not proceed seamlessly, opening up the risk of aberrant differentiation if FGF is activated before Nodal. Here, we report that Id1 acts as a sensor to detect delays in Nodal activation after the downregulation of Nanog. Id1 then suppresses FGF activity to delay differentiation. Accordingly, Id1 is not required for naive or primed pluripotency but rather stabilizes epiblast identity during the transition between these states. These findings help explain how development proceeds robustly in the face of imprecise signals and highlight the importance of mechanisms that stabilize cell identity during developmental transitions.
Project description:Human embryonic stem cells (hESCs) are more similar to "primed" mouse epiblast stem cells (mEpiSCs). mEpiSCs, which are derived in Activin A, show an increased propensity to form primordial germ cell (PGC)-like cells in response to bone morphogenic protein 4 (BMP4). Hence, we hypothesized that hESCs derived in the presence of Activin A may be more competent in differentiating towards PGC-like cells after supplementation with BMP4 compared to standard hESC lines. We were able to successfully derive two hESC lines in the presence of Activin A, which were pluripotent and showed higher base levels of STELLA and cKIT compared to standard hESC lines derived without Activin A addition. Furthermore, upon differentiation as embryoid bodies in the presence of BMP4, we observed upregulation of VASA at day 7, both at the transcript and protein level compared to standard hESC lines, which appeared to take longer time for PGC specification. Unlike other hESC lines, nuclear pSMAD2/3 presence confirmed that Activin signalling was switched on in Activin A-derived hESC lines. They were also responsive to BMP4 based on nuclear detection of pSMAD1/5/8 and showed endodermal differentiation as a result of GATA-6 expression. Hence, our results provide novel insights into the impact of hESC derivation in the presence of Activin A and its subsequent influence on germ cell differentiation potential in vitro.
Project description:Naive and primed pluripotent human embryonic stem cells bear transcriptional similarity to pre- and post-implantation epiblast and thus constitute a developmental model for understanding the pluripotent stages in human embryo development. To identify new transcription factors that differentially regulate the unique pluripotent stages, we mapped open chromatin using ATAC-seq and found enrichment of the activator protein-2 (AP2) transcription factor binding motif at naive-specific open chromatin. We determined that the AP2 family member TFAP2C is upregulated during primed to naive reversion and becomes widespread at naive-specific enhancers. TFAP2C functions to maintain pluripotency and repress neuroectodermal differentiation during the transition from primed to naive by facilitating the opening of enhancers proximal to pluripotency factors. Additionally, we identify a previously undiscovered naive-specific POU5F1 (OCT4) enhancer enriched for TFAP2C binding. Taken together, TFAP2C establishes and maintains naive human pluripotency and regulates OCT4 expression by mechanisms that are distinct from mouse.
Project description:The naïve pluripotent state has been shown in mice to lead to broad and more robust developmental potential relative to primed mouse epiblast cells. The human naïve ES cell state has eluded derivation without the use of transgenes, and forced expression of OCT4, KLF4, and KLF2 allows maintenance of human cells in a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107(20):9222-9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling of preexisting primed hESC lines by preculture in the histone deacetylase inhibitors butyrate and suberoylanilide hydroxamic acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the context of teratomas. hESCs can exist in a naïve state without the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells to naïve is efficient and reproducible.
Project description:For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs). Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development.
Project description:We report a novel culture condition inducing naive pluripotency in hESCs in a rapid, robust and reproducible way. These naive hESCs were similar to mESCs exhibiting domed colony morphology, increased single survival, reduced doubling time, upregulation of naive pluripotency-specific genes, unbiased lineage-specific differentiation, hypomethylation, separate clustering profile from parental primed hESCs and were dependent on signalling pathways similar to naive mESCs. Global DNA methylation analysis of naive hESCs and their parental primed hESC counterparts.
Project description:Following implantation, mouse epiblast cells transit from a naive to a primed state in which they are competent for both somatic and primordial germ cell (PGC) specification. Using mouse embryonic stem cells as an in vitro model to study the transcriptional regulatory principles orchestrating peri-implantation development, here we show that the transcription factor Foxd3 is necessary for exit from naive pluripotency and progression to a primed pluripotent state. During this transition, Foxd3 acts as a repressor that dismantles a significant fraction of the naive pluripotency expression program through decommissioning of active enhancers associated with key naive pluripotency and early germline genes. Subsequently, Foxd3 needs to be silenced in primed pluripotent cells to allow re-activation of relevant genes required for proper PGC specification. Our findings therefore uncover a cycle of activation and deactivation of Foxd3 required for exit from naive pluripotency and subsequent PGC specification.