Recent reports that human pluripotent stem cells can be captured in a spectrum of states with variable properties has prompted a re‐evaluation of how pluripotency is acquired and stabilised. The ...latest additions to the stem cell hierarchy open up opportunities for understanding human development, reprogramming, and cell state transitions more generally. Many of the new cell lines have been collectively termed ‘naïve’ human pluripotent stem cells to distinguish them from the conventional ‘primed’ cells. Here, several transcriptional and epigenetic hallmarks of human pluripotent states in the recently described cell lines are reviewed and evaluated. Methods to derive and identify human naïve pluripotent stem cells are also discussed, with a focus on the uses and future developments of state‐specific reporter cell lines and cell‐surface proteins. Finally, opportunities and uncertainties in naïve stem cell biology are highlighted, and the current limitations of human naïve pluripotent stem cells considered, particularly in the context of differentiation.
Human pluripotent stem cells captured in a spectrum of states presents a challenge for unambiguously defining cell types. We review molecular hallmarks of human pluripotent states and evaluate methods to identify naïve pluripotent cells, with a focus on the uses and future developments of reporter cell lines and cell‐surface proteins.
Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through ...primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting.
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•Flow cytometry profiles cell surface proteins in naive and primed human PSCs•The human PSC state can be defined using robust state-specific protein markers•Identified cell surface proteins track the dynamics of naive-primed PSC conversions•Analyses of early-stage naive cells reveal transcription events during conversion
Collier et al. use profiling to identify cell surface proteins that are specific for naive versus primed human pluripotent cells and then use them to isolate and characterize live naive cells arising during primed-to-naive resetting.
In primates, the amnion emerges through cavitation of the epiblast during implantation, whereas in other species it does so later at gastrulation by the folding of the ectoderm. How the mechanisms of ...amniogenesis diversified during evolution remains unknown. Unexpectedly, single-cell analysis of primate embryos uncovered two transcriptionally and temporally distinct amniogenesis waves. To study this, we employed the naive-to-primed transition of human pluripotent stem cells (hPSCs) to model peri-implantation epiblast development. Partially primed hPSCs transiently gained the ability to differentiate into cavitating epithelium that transcriptionally and morphologically matched the early amnion, whereas fully primed hPSCs produced cells resembling the late amnion instead, thus recapitulating the two independent differentiation waves. The early wave follows a trophectoderm-like pathway and encompasses cavitation, whereas the late wave resembles an ectoderm-like route during gastrulation. The discovery of two independent waves explains how amniogenesis through cavitation could emerge during evolution via duplication of the pre-existing trophectoderm program.
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•The amnion develops in two distinct waves of epiblast differentiation in primates•The amniotic cavity is formed during the early wave via a trophectoderm-like route•Late amniogenesis follows a nonneural ectoderm-like transcriptional program•Differentiation of hPSC from different states models the two waves of amniogenesis
Lineage specification during human peri-implantation development remains poorly understood. Rostovskaya et al. discovered that in primate embryos the amnion is formed in two transcriptionally and temporally distinct waves of epiblast differentiation and established a human pluripotent stem cell-based model that closely recapitulates the timing and morphogenesis of both amniogenesis waves.
Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes
. Global ...epigenetic reprogramming accompanies these changes
, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.
Studies of mammalian development have advanced our understanding of the genetic, epigenetic, and cellular processes that orchestrate embryogenesis and have uncovered new insights into the unique ...aspects of human embryogenesis. Recent studies have now produced the first epigenetic maps of early human embryogenesis, stimulating new ideas about epigenetic reprogramming, cell fate control, and the potential mechanisms underpinning developmental plasticity in human embryos. In this review, we discuss these new insights into the epigenetic regulation of early human development and the importance of these processes for safeguarding development. We also highlight unanswered questions and key challenges that remain to be addressed.
Primordial germ cell (PGC) development is characterized by global epigenetic remodeling, which resets genomic potential and establishes an epigenetic ground state. Here we recapitulate PGC ...specification in vitro from naive embryonic stem cells and characterize the early events of epigenetic reprogramming during the formation of the human and mouse germline. Following rapid de novo DNA methylation during priming to epiblast-like cells, methylation is globally erased in PGC-like cells. Repressive chromatin marks (H3K9me2/3) and transposable elements are enriched at demethylation-resistant regions, while active chromatin marks (H3K4me3 or H3K27ac) are more prominent at regions that demethylate faster. The dynamics of specification and epigenetic reprogramming show species-specific differences, in particular markedly slower reprogramming kinetics in the human germline. Differences in developmental kinetics may be explained by differential regulation of epigenetic modifiers. Our work establishes a robust and faithful experimental system of the early events of epigenetic reprogramming and regulation in the germline.
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•Defined specification of human in vitro PGCLCs from naive ESCs•Human and mouse epigenetic germline reprogramming tempo differs significantly•Demethylation-resistant regions are enriched in TEs and repressive chromatin marks•Mouse in vitro PGCLCs show expression of transposon-derived piRNAs
von Meyenn et al. establish a system for human primordial germ cell specification in vitro to characterize early events of epigenetic reprogramming during formation of the human germline relative to the mouse. Reprogramming in human versus mouse shows significantly different developmental timing that may be explained by differential DNMT3 and UHRF1 regulation.
A unique property of the mammalian embryo is that stem cells can be derived from its early tissue lineages. These lineages will give rise to the fetus as well as essential extraembryonic tissues. ...Understanding how chromatin regulation participates in establishment of these lineages in the embryo and their derived stem cells provides insight that will critically inform our understanding of embryogenesis and stem cell biology. Here, we compare the genomewide location of active and repressive histone modifications in embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm stem cells from the mouse. Our results show that the active modification H3K4me3 has a similar role in the three stem cell types, but the repressive modification H3K27me3 varies in abundance and genomewide distribution. Thus, alternative mechanisms mediate transcriptional repression in stem cells from the embryo. In addition, using carrier chromatin immunoprecipitation we show that bivalent histone domains seen in embryonic stem cells exist in pluripotent cells of the early embryo. However, the epigenetic status of extraembryonic progenitor cells in the embryo did not entirely reflect the extraembryonic stem cell lines. These studies indicate that histone modification mechanisms may differ between early embryo lineages and emphasize the importance of examining in vivo and in vitro progenitor cells.
Through the histone methyltransferase EZH2, the Polycomb complex PRC2 mediates H3K27me3 and is associated with transcriptional repression. PRC2 regulates cell-fate decisions in model organisms; ...however, its role in regulating cell differentiation during human embryogenesis is unknown. Here, we report the characterization of EZH2-deficient human embryonic stem cells (hESCs). H3K27me3 was lost upon EZH2 deletion, identifying an essential requirement for EZH2 in methylating H3K27 in hESCs, in contrast to its non-essential role in mouse ESCs. Developmental regulators were derepressed in EZH2-deficient hESCs, and single-cell analysis revealed an unexpected acquisition of lineage-restricted transcriptional programs. EZH2-deficient hESCs show strongly reduced self-renewal and proliferation, thereby identifying a more severe phenotype compared to mouse ESCs. EZH2-deficient hESCs can initiate differentiation toward developmental lineages; however, they cannot fully differentiate into mature specialized tissues. Thus, EZH2 is required for stable ESC self-renewal, regulation of transcriptional programs, and for late-stage differentiation in this model of early human development.
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•Comprehensive examination of EZH2 function in human ESC regulation•EZH2 deficiency causes lineage-restricted derepression of developmental regulators•More severe self-renewal and growth defects in EZH2-deficient hESCs than in mESCs•EZH2-deficient hESCs can differentiate to early lineages but cannot form mature tissues
Collinson et al. use EZH2-deficient human ESCs to demonstrate the broad conservation of Polycomb-group protein function in controlling cell-fate decisions and transcriptional programs during early human development. The authors also uncover unexpected human-specific differences that result in a more severe self-renewal and proliferation phenotype than that of PRC2-deficient mouse ESCs.
The transition from naive to primed pluripotency is accompanied by an extensive reorganisation of transcriptional and epigenetic programmes. However, the role of transcriptional enhancers and ...three-dimensional chromatin organisation in coordinating these developmental programmes remains incompletely understood. Here, we generate a high-resolution atlas of gene regulatory interactions, chromatin profiles and transcription factor occupancy in naive and primed human pluripotent stem cells, and develop a network-graph approach to examine the atlas at multiple spatial scales. We uncover highly connected promoter hubs that change substantially in interaction frequency and in transcriptional co-regulation between pluripotent states. Small hubs frequently merge to form larger networks in primed cells, often linked by newly-formed Polycomb-associated interactions. We identify widespread state-specific differences in enhancer activity and interactivity that correspond with an extensive reconfiguration of OCT4, SOX2 and NANOG binding and target gene expression. These findings provide multilayered insights into the chromatin-based gene regulatory control of human pluripotent states.
Human pluripotent stem cells exist in naïve and primed states that recapitulate the distinct molecular and cellular properties of pre- and post-implantation epiblast cells, respectively. Naïve ...pluripotent stem cells can be captured directly from blastocysts but, more commonly, the cells are reprogrammed from primed cells in a process called "resetting". Several methods to achieve resetting have been described. Chemical resetting of primed cells to a naïve pluripotent state is one such method and has come to the forefront as a simple, efficient, and transgene-free method to induce naïve pluripotency. The process involves the transient application of a histone deacetylase inhibitor to initiate resetting, followed by the emergence of nascent naïve pluripotent stem cells in supportive conditions, and finally the stabilization and expansion of naïve pluripotent stem cell cultures. Here, a detailed protocol is provided for chemical resetting starting from plating primed cells until a stable culture of naïve pluripotent stem cells is established.
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