RESUMEN
Recent years have seen exciting progress across human embryo research, including new methods for culturing embryos, transcriptional profiling of embryogenesis and gastrulation, mapping lineage trajectories, and experimenting on stem cell-based embryo models. These advances are beginning to define the dynamical principles of development across stages, tissues and organs, enabling a better understanding of human development before birth in health and disease, and potentially leading to improved treatments for infertility and developmental disorders. However, there are still significant roadblocks en route to this goal. Here, we highlight technical challenges to studying early human development and propose ways and means to overcome some of these constraints.
Asunto(s)
Desarrollo Embrionario , Gastrulación , Humanos , Desarrollo Embrionario/genética , Embrión de Mamíferos , Células MadreRESUMEN
Gene duplication events can drive evolution by providing genetic material for new gene functions, and they create opportunities for diverse developmental strategies to emerge between species. To study the contribution of duplicated genes to human early development, we examined the evolution and function of NANOGP1, a tandem duplicate of the transcription factor NANOG. We found that NANOGP1 and NANOG have overlapping but distinct expression profiles, with high NANOGP1 expression restricted to early epiblast cells and naïve-state pluripotent stem cells. Sequence analysis and epitope-tagging revealed that NANOGP1 is protein coding with an intact homeobox domain. The duplication that created NANOGP1 occurred earlier in primate evolution than previously thought and has been retained only in great apes, whereas Old World monkeys have disabled the gene in different ways, including homeodomain point mutations. NANOGP1 is a strong inducer of naïve pluripotency; however, unlike NANOG, it is not required to maintain the undifferentiated status of human naïve pluripotent cells. By retaining expression, sequence and partial functional conservation with its ancestral copy, NANOGP1 exemplifies how gene duplication and subfunctionalisation can contribute to transcription factor activity in human pluripotency and development.
Asunto(s)
Genes Homeobox , Células Madre Pluripotentes , Animales , Humanos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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 changes1-5. Global epigenetic reprogramming accompanies these changes6-8, 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.
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Metilación de ADN , Epigénesis Genética , Gástrula/citología , Gástrula/metabolismo , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , ARN/genética , Análisis de la Célula Individual , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Cromatina/genética , Cromatina/metabolismo , Desmetilación , Cuerpos Embrioides/citología , Endodermo/citología , Endodermo/embriología , Endodermo/metabolismo , Elementos de Facilitación Genéticos/genética , Epigenoma/genética , Eritropoyesis , Análisis Factorial , Gástrula/embriología , Gastrulación/fisiología , Mesodermo/citología , Mesodermo/embriología , Mesodermo/metabolismo , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN/análisis , Factores de Tiempo , Dedos de ZincRESUMEN
X chromosome inactivation (XCI) is an essential process, yet it initiates with remarkable diversity in various mammalian species. XIST, the main trigger of XCI, is controlled in the mouse by an interplay of lncRNA genes (LRGs), some of which evolved concomitantly to XIST and have orthologues across all placental mammals. Here, we addressed the functional conservation of human orthologues of two such LRGs, FTX and JPX. By combining analysis of single-cell RNA-seq data from early human embryogenesis with various functional assays in matched human and mouse pluripotent stem- or differentiated post-XCI cells, we demonstrate major functional differences for these orthologues between species, independently of primary sequence conservation. While the function of FTX is not conserved in humans, JPX stands as a major regulator of XIST expression in both species. However, we show that different entities of JPX control the production of XIST at various steps depending on the species. Altogether, our study highlights the functional versatility of LRGs across evolution, and reveals that functional conservation of orthologous LRGs may involve diversified mechanisms of action. These findings represent a striking example of how the evolvability of LRGs can provide adaptative flexibility to constrained gene regulatory networks.
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Placenta , ARN Largo no Codificante , Embarazo , Humanos , Femenino , Ratones , Animales , Placenta/metabolismo , Inactivación del Cromosoma X/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Mamíferos/genética , Embrión de Mamíferos/metabolismoRESUMEN
An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells.
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Heterocromatina/genética , Heterocromatina/metabolismo , Células Madre Embrionarias de Ratones/fisiología , Proteína Homeótica Nanog/metabolismo , Animales , Línea Celular , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Regulación hacia Abajo , Eliminación de Gen , Ratones , Proteína Homeótica Nanog/genética , Dominios Proteicos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Recent advances in human pluripotent stem cell (hPSC) research have uncovered different subpopulations within stem cell cultures and have captured a range of pluripotent states that hold distinct molecular and functional properties. At the two ends of the pluripotency spectrum are naïve and primed hPSC, whereby naïve hPSC grown in stringent conditions recapitulate features of the preimplantation human embryo, and the conventionally grown primed hPSC align closer to the early postimplantation embryo. Investigating these cell types will help to define the mechanisms that control early development and should provide new insights into stem cell properties such as cell identity, differentiation and reprogramming. Monitoring cell surface marker expression provides a valuable approach to resolve complex cell populations, to directly compare between cell types, and to isolate viable cells for functional experiments. This review discusses the discovery and applications of cell surface markers to study human pluripotent cell types with a particular focus on the transitions between naïve and primed states. Highlighted areas for future study include the potential functions for the identified cell surface proteins in pluripotency, the production of new high-quality monoclonal antibodies to naïve-specific protein epitopes and the use of cell surface markers to characterise subpopulations within pluripotent states.
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Biomarcadores/metabolismo , Proteínas de la Membrana/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Diferenciación Celular/fisiología , Desarrollo Embrionario/fisiología , Células Madre Embrionarias/metabolismo , HumanosRESUMEN
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.
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Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Epigenómica , HumanosRESUMEN
We examined the allele-specific expression of six imprinted genes and the methylation profiles of three imprinting control regions to assess the epigenetic status of human embryonic stem cells. We identified generally monoallelic gene expression and normal methylation patterns. During prolonged passage, one cell line became biallelic with respect to H19, but without loss of the gametic methylation imprint. These data argue for a substantial degree of epigenetic stability in human embryonic stem cells.
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Epigénesis Genética , Impresión Genómica , Células Madre , Alelos , Línea Celular , Células Cultivadas , Metilación de ADN , Embrión de Mamíferos , Expresión Génica , Genes Reguladores , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , ARN Largo no Codificante , ARN no Traducido/metabolismoRESUMEN
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.
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Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Células Madre Embrionarias/citología , Endodermo/citología , Endodermo/embriología , Endodermo/metabolismo , Epigénesis Genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Histonas/química , Histonas/genética , Técnicas In Vitro , Metilación , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Embarazo , Trofoblastos/citología , Trofoblastos/metabolismoRESUMEN
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.
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Metilación de ADN , Epigénesis Genética , Humanos , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/genéticaRESUMEN
In the adult mouse brain, the subventricular zone (SVZ) underlying the lateral ventricles harbours a population of quiescent neural stem cells, which can be activated (aNSCs) to initiate proliferation and generate a neurogenic lineage consisting of transit amplifying progenitors (TAPs), neuroblasts (NBs) and newborn neurons. This process is markedly reduced during aging. Recent studies suggest that the aged SVZ niche decreases the pool of proliferating neural/stem progenitor cells (NSPCs), and hence adult neurogenesis, by causing transcriptomic changes that promote NSC quiescence. The transcription factors that mediate these changes, however, remain unclear. We previously found that the homeobox gene Dbx2 is upregulated in NSPCs of the aged mouse SVZ and can inhibit the growth of NSPC cultures. Here, we further investigate its role as a candidate transcriptional regulator of neurogenic decline. We show that Dbx2 expression is downregulated by Epidermal Growth Factor receptor signaling, which promotes NSPC proliferation and decreases in the aged SVZ. By means of transgenic NSPC lines overexpressing Dbx2, we also show that this gene inhibits NSPC proliferation by hindering the G2/M transition. Furthermore, we exploit RNA sequencing of transgenic NSPCs to elucidate the transcriptomic networks modulated by Dbx2. Among the top hits, we report the downregulation of the molecular pathways implicated in cell cycle progression. Accordingly, we find that Dbx2 function is negatively correlated with the transcriptional signatures of proliferative NSPCs (aNSCs, TAPs and early NBs). These results point to Dbx2 as a transcription factor relaying the anti-neurogenic input of the aged niche to the NSPC transcriptome.
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Genes Homeobox , Neurogénesis , Animales , Ratones , Envejecimiento/genética , División Celular , Neurogénesis/genética , Neuronas , Factores de TranscripciónRESUMEN
Trophoblast cells are required for the growth and survival of the fetus during pregnancy, and failure to maintain appropriate trophoblast regulation is associated with placental insufficiencies and intrauterine growth restriction. Development of the trophoblast lineage is mediated by interactions between genetic and epigenetic factors. This review will focus on new insights that have been gained from analysis of mouse models into the epigenetic mechanisms that are required for the early establishment of the trophoblast lineage and for the development of specialized cell types of the fetal placenta. In particular, the importance of DNA methylation, 5-hydroxymethylcytosine and histone modifications in orchestrating trophoblast gene expression and functional outcome will be discussed. These insights are beginning to be extended towards human studies and initial results suggest that the causes and consequences of a variety of placental pathologies are related to epigenetic processes. Furthermore, the epigenetic landscape that regulates trophoblast cells seems to be particularly vulnerable to perturbation during development. This has major implications for diet and other environmental factors during pregnancy.
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Epigénesis Genética/fisiología , Retardo del Crecimiento Fetal/fisiopatología , Trofoblastos/fisiología , 5-Metilcitosina/análogos & derivados , Animales , Citosina/análogos & derivados , Citosina/metabolismo , Metilación de ADN , Femenino , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Ratones , Modelos Animales , Embarazo , Complicaciones del Embarazo/fisiopatologíaRESUMEN
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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Células Madre Pluripotentes , Blastocisto , Diferenciación Celular , Estratos Germinativos , HumanosRESUMEN
Cell-surface proteins provide excellent biomarkers to identify specific cell types and resolve heterogeneous cell populations. The analysis of cell-surface proteins by flow cytometry produces robust and quantitative information with single-cell resolution, and allows live target cells to be purified and characterized or re-cultured. Studies using antibody screens, proteomics, and candidate analysis have identified a comprehensive set of proteins that are expressed on the surface of naïve and primed human pluripotent stem cells. These findings have led to the development of suitable protein markers and antibodies to accurately distinguish between these two cell types. Here, a detailed protocol is provided that uses multi-color flow cytometry to analyze cell-surface protein expression in naïve and primed human pluripotent stem cells. This method enables the unambiguous identification of pluripotent cell types and the opportunity to sort target cells including during cell state transitions. The protocol can be combined to additionally investigate the expression of reporter genes and other informative features, such as DNA content.
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Células Madre Pluripotentes , Biomarcadores , Diferenciación Celular , Citometría de Flujo , Humanos , Proteínas de la MembranaRESUMEN
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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Células Madre Pluripotentes , Animales , Diferenciación Celular , Ectodermo , Estratos Germinativos , PrimatesRESUMEN
Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1É. Mouse heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs). Here, we report that MSR transcripts can drive the formation of HP1É droplets in vitro, and modulate heterochromatin into dynamic condensates in ESCs, contributing to the formation of large nuclear domains that are characteristic of pluripotent cells. Depleting MSR transcripts causes heterochromatin to transition into a more compact and static state. Unexpectedly, changing heterochromatin's biophysical properties has severe consequences for ESCs, including chromosome instability and mitotic defects. These findings uncover an essential role for MSR transcripts in modulating the organisation and properties of heterochromatin to preserve genome stability. They also provide insights into the processes that could regulate phase separation and the functional consequences of disrupting the properties of heterochromatin condensates.
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Heterocromatina , Células Madre Embrionarias de Ratones , Animales , Inestabilidad Cromosómica/genética , Células Madre Embrionarias , Heterocromatina/genética , Histonas/genética , RatonesRESUMEN
Uncovering the mechanisms that establish naïve pluripotency in humans is crucial for the future applications of pluripotent stem cells including the production of human blastoids. However, the regulatory pathways that control the establishment of naïve pluripotency by reprogramming are largely unknown. Here, we use genome-wide screening to identify essential regulators as well as major impediments of human primed to naïve pluripotent stem cell reprogramming. We discover that factors essential for cell state change do not typically undergo changes at the level of gene expression but rather are repurposed with new functions. Mechanistically, we establish that the variant Polycomb complex PRC1.3 and PRDM14 jointly repress developmental and gene regulatory factors to ensure naïve cell reprogramming. In addition, small-molecule inhibitors of reprogramming impediments improve naïve cell reprogramming beyond current methods. Collectively, this work defines the principles controlling the establishment of human naïve pluripotency and also provides new insights into mechanisms that destabilize and reconfigure cell identity during cell state transitions.
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Reprogramación Celular , Células Madre Pluripotentes , Complejo Represivo Polycomb 1 , Diferenciación Celular , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes/citología , Complejo Represivo Polycomb 1/metabolismoRESUMEN
Senescence is a fate-determined state, accompanied by reorganization of heterochromatin. Although lineage-appropriate genes can be temporarily repressed through facultative heterochromatin, stable silencing of lineage-inappropriate genes often involves the constitutive heterochromatic mark, histone H3 lysine 9 trimethylation (H3K9me3). The fate of these heterochromatic genes during senescence is unclear. In the present study, we show that a small number of lineage-inappropriate genes, exemplified by the LCE2 skin genes, are derepressed during senescence from H3K9me3 regions in fibroblasts. DNA FISH experiments reveal that these gene loci, which are condensed at the nuclear periphery in proliferative cells, are decompacted during senescence. Decompaction of the locus is not sufficient for LCE2 expression, which requires p53 and C/EBPß signaling. NLRP3, which is predominantly expressed in macrophages from an open topologically associated domain (TAD), is also derepressed in senescent fibroblasts due to the local disruption of the H3K9me3-rich TAD that contains it. NLRP3 has been implicated in the amplification of inflammatory cytokine signaling in senescence and aging, highlighting the functional relevance of gene induction from 'permissive' H3K9me3 regions in senescent cells.
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Heterocromatina , Histonas , Heterocromatina/genética , Histonas/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Senescencia Celular/genética , Expresión GénicaRESUMEN
Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.