RESUMEN
Mammalian blastocyst formation involves the specification of the trophectoderm followed by the differentiation of the inner cell mass into embryonic epiblast and extra-embryonic primitive endoderm (PrE). During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the PrE alone was sufficient to regenerate a complete blastocyst and continue post-implantation development. We identify an in vitro population similar to the early PrE in vivo that exhibits the same embryonic and extra-embryonic potency and can form complete stem cell-based embryo models, termed blastoids. Commitment in the PrE is suppressed by JAK/STAT signaling, collaborating with OCT4 and the sustained expression of a subset of pluripotency-related transcription factors that safeguard an enhancer landscape permissive for multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlight the importance of the PrE in perturbed development.
Asunto(s)
Blastocisto , Diferenciación Celular , Endodermo , Animales , Endodermo/metabolismo , Endodermo/citología , Ratones , Blastocisto/metabolismo , Blastocisto/citología , Linaje de la Célula , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Transducción de Señal , Desarrollo Embrionario , Quinasas Janus/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción STAT/metabolismo , Factores de Transcripción/metabolismo , Femenino , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/citologíaRESUMEN
In early mammalian development, cleavage stage blastomeres and inner cell mass (ICM) cells co-express embryonic and extra-embryonic transcriptional determinants. Using a protein-based double reporter we identify an embryonic stem cell (ESC) population that co-expresses the extra-embryonic factor GATA6 alongside the embryonic factor SOX2. Based on single cell transcriptomics, we find this population resembles the unsegregated ICM, exhibiting enhanced differentiation potential for endoderm while maintaining epiblast competence. To relate transcription factor binding in these cells to future fate, we describe a complete enhancer set in both ESCs and naive extra-embryonic endoderm stem cells and assess SOX2 and GATA6 binding at these elements in the ICM-like ESC sub-population. Both factors support cooperative recognition in these lineages, with GATA6 bound alongside SOX2 on a fraction of pluripotency enhancers and SOX2 alongside GATA6 more extensively on endoderm enhancers, suggesting that cooperative binding between these antagonistic factors both supports self-renewal and prepares progenitor cells for later differentiation.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción , Animales , Linaje de la Célula/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Diferenciación Celular/genética , Estratos Germinativos , Endodermo , Blastocisto , Mamíferos/metabolismoRESUMEN
The primitive endoderm (PrE, also named hypoblast), a predominantly extraembryonic epithelium that arises from the inner cell mass (ICM) of the mammalian pre-implantation blastocyst, plays a fundamental role in embryonic development, giving rise to the yolk sac, establishing the anterior-posterior axis and contributing to the gut. PrE is specified from the ICM at the same time as the epiblast (Epi) that will form the embryo proper. While in vitro cell lines resembling the pluripotent Epi have been derived from a variety of conditions, only one model system currently exists for the PrE, naïve extraembryonic endoderm (nEnd). As a result, considerably more is known about the gene regulatory networks and signalling requirements of pluripotent stem cells than nEnd. In this review, we describe the ontogeny and differentiation of the PrE or hypoblast in mouse and primate and then discuss in vitro cell culture models for different extraembryonic endodermal cell types.
Asunto(s)
Endodermo , Estratos Germinativos , Embarazo , Femenino , Humanos , Ratones , Animales , Endodermo/metabolismo , Diferenciación Celular/genética , Embrión de Mamíferos , Blastocisto , MamíferosRESUMEN
Modified parental histones are segregated symmetrically to daughter DNA strands during replication and can be inherited through mitosis. How this may sustain the epigenome and cell identity remains unknown. Here we show that transmission of histone-based information during DNA replication maintains epigenome fidelity and embryonic stem cell plasticity. Asymmetric segregation of parental histones H3-H4 in MCM2-2A mutants compromised mitotic inheritance of histone modifications and globally altered the epigenome. This included widespread spurious deposition of repressive modifications, suggesting elevated epigenetic noise. Moreover, H3K9me3 loss at repeats caused derepression and H3K27me3 redistribution across bivalent promoters correlated with misexpression of developmental genes. MCM2-2A mutation challenged dynamic transitions in cellular states across the cell cycle, enhancing naïve pluripotency and reducing lineage priming in G1. Furthermore, developmental competence was diminished, correlating with impaired exit from pluripotency. Collectively, this argues that epigenetic inheritance of histone modifications maintains a correctly balanced and dynamic chromatin landscape able to support mammalian cell differentiation.
Asunto(s)
Epigenoma , Histonas , Animales , Histonas/genética , Cromatina/genética , Células Madre Embrionarias , Mitosis , MamíferosRESUMEN
Cooperative DNA binding of transcription factors (TFs) integrates the cellular context to support cell specification during development. Naive mouse embryonic stem cells are derived from early development and can sustain their pluripotent identity indefinitely. Here, we ask whether TFs associated with pluripotency evolved to directly support this state or if the state emerges from their combinatorial action. NANOG and ESRRB are key pluripotency factors that co-bind DNA. We find that when both factors are expressed, ESRRB supports pluripotency. However, when NANOG is absent, ESRRB supports a bistable culture of cells with an embryo-like primitive endoderm identity ancillary to pluripotency. The stoichiometry between NANOG and ESRRB allows quantitative titration of this differentiation, and in silico modeling of bipartite ESRRB activity suggests it safeguards plasticity in differentiation. Thus, the concerted activity of cooperative TFs can transform their effect to sustain intermediate cell identities and allow ex vivo expansion of immortal stem cells. A record of this paper's transparent peer review process is included in the supplemental information.
Asunto(s)
Células Madre Embrionarias de Ratones , Factores de Transcripción , Animales , Ratones , Diferenciación Celular , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Cell proliferation is fundamental for almost all stages of development and differentiation that require an increase in cell number. Although cell cycle phase has been associated with differentiation, the actual process of proliferation has not been considered as having a specific role. Here we exploit human embryonic stem cell-derived endodermal progenitors that we find are an in vitro model for the ventral foregut. These cells exhibit expansion-dependent increases in differentiation efficiency to pancreatic progenitors that are linked to organ-specific enhancer priming at the level of chromatin accessibility and the decommissioning of lineage-inappropriate enhancers. Our findings suggest that cell proliferation in embryonic development is about more than tissue expansion; it is required to ensure equilibration of gene regulatory networks allowing cells to become primed for future differentiation. Expansion of lineage-specific intermediates may therefore be an important step in achieving high-fidelity in vitro differentiation.
Asunto(s)
Cromatina , Páncreas , Humanos , Linaje de la Célula/genética , Diferenciación Celular/genética , Cromatina/genética , Cromatina/metabolismo , Páncreas/metabolismo , Elementos de Facilitación Genéticos/genéticaRESUMEN
Development of multicellular organisms is orchestrated by persistent cell-cell communication between neighboring partners. Direct interaction between different cell types can induce molecular signals that dictate lineage specification and cell fate decisions. Current single-cell RNA-seq technology cannot adequately analyze cell-cell contact-dependent gene expression, mainly due to the loss of spatial information. To overcome this obstacle and resolve cell-cell contact-specific gene expression during embryogenesis, we performed RNA sequencing of physically interacting cells (PIC-seq) and assessed them alongside similar single-cell transcriptomes derived from developing mouse embryos between embryonic day (E) 7.5 and E9.5. Analysis of the PIC-seq data identified gene expression signatures that were dependent on the presence of specific neighboring cell types. Our computational predictions, validated experimentally, demonstrated that neural progenitor (NP) cells upregulate Lhx5 and Nkx2-1 genes, when exclusively interacting with definitive endoderm (DE) cells. Moreover, there was a reciprocal impact on the transcriptome of DE cells, as they tend to upregulate Rax and Gsc when in contact with NP cells. Using individual cell transcriptome data, we formulated a means of computationally predicting the impact of one cell type on the transcriptome of its neighboring cell types. We have further developed a distinctive spatial-t-distributed stochastic neighboring embedding to display the pseudospatial distribution of cells in a 2-dimensional space. In summary, we describe an innovative approach to study contact-specific gene regulation during embryogenesis.
Asunto(s)
Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Animales , Ratones , Desarrollo Embrionario/genética , Diferenciación Celular/genética , Transcriptoma , Análisis de Secuencia de ARN , Análisis de la Célula Individual/métodos , Perfilación de la Expresión GénicaRESUMEN
The support of pluripotent cells over time is an essential feature of development. In eutherian embryos, pluripotency is maintained from naïve states in peri-implantation to primed pluripotency at gastrulation. To understand how these states emerged, we reconstruct the evolutionary trajectory of the Pou5 gene family, which contains the central pluripotency factor OCT4. By coupling evolutionary sequence analysis with functional studies in mouse embryonic stem cells, we find that the ability of POU5 proteins to support pluripotency originated in the gnathostome lineage, prior to the generation of two paralogues, Pou5f1 and Pou5f3 via gene duplication. In osteichthyans, retaining both genes, the paralogues differ in their support of naïve and primed pluripotency. The specialization of these duplicates enables the diversification of function in self-renewal and differentiation. By integrating sequence evolution, cell phenotypes, developmental contexts and structural modelling, we pinpoint OCT4 regions sufficient for naïve pluripotency and describe their adaptation over evolutionary time.
Asunto(s)
Células Madre Pluripotentes , Animales , Diferenciación Celular/genética , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Ratones , Células Madre Embrionarias de Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismoRESUMEN
During embryonic development cells acquire identity as they proliferate, implying that an intrinsic facet of cell fate choice requires coupling lineage decisions to cell division. How is the cell cycle regulated to promote or suppress heterogeneity and differentiation? We explore this question combining time lapse imaging with single-cell RNA-seq in the contexts of self-renewal, priming, and differentiation of mouse embryonic stem cells (ESCs) towards the Primitive Endoderm (PrE) lineage. Since ESCs are derived from the inner cell mass (ICM) of the mammalian blastocyst, ESCs in standard culture conditions are transcriptionally heterogeneous containing dynamically interconverting subfractions primed for either of the two ICM lineages, Epiblast and PrE. Here, we find that differential regulation of cell cycle can tip the balance between these primed populations, such that naïve ESC culture promotes Epiblast-like expansion and PrE differentiation stimulates the selective survival and proliferation of PrE-primed cells. In endoderm differentiation, this change is accompanied by a counter-intuitive increase in G1 length, also observed in vivo. While fibroblast growth factor/extracellular signal-regulated kinase (FGF/ERK) signalling is a key regulator of ESC differentiation and PrE specification, we find it is not just responsible for ESCs heterogeneity, but also the inheritance of similar cell cycles between sisters and cousins. Taken together, our results indicate a tight relationship between transcriptional heterogeneity and cell cycle regulation in lineage specification, with primed cell populations providing a pool of flexible cell types that can be expanded in a lineage-specific fashion while allowing plasticity during early determination.
Asunto(s)
Endodermo , Regulación del Desarrollo de la Expresión Génica , Animales , Blastocisto , Puntos de Control del Ciclo Celular , Diferenciación Celular/fisiología , Linaje de la Célula/genética , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Estratos Germinativos , Mamíferos/metabolismo , Ratones , EmbarazoRESUMEN
High-resolution maps of embryonic development suggest that acquisition of cell identity is not limited to canonical germ layers but proceeds via alternative routes. Despite evidence that visceral organs are formed via embryonic and extra-embryonic trajectories, the production of organ-specific cell types in vitro focuses on the embryonic one. Here we resolve these differentiation routes using massively parallel single-cell RNA sequencing to generate datasets from FOXA2Venus reporter mouse embryos and embryonic stem cell differentiation towards endoderm. To relate cell types in these datasets, we develop a single-parameter computational approach and identify an intermediate en route from extra-embryonic identity to embryonic endoderm, which we localize spatially in embryos at embryonic day 7.5. While there is little evidence for this cell type in embryonic stem cell differentiation, by following the extra-embryonic trajectory starting with naïve extra-embryonic endoderm stem cells we can generate embryonic gut spheroids. Exploiting developmental plasticity therefore offers alternatives to pluripotent cells and opens alternative avenues for in vitro differentiation.
Asunto(s)
Endodermo , Transcriptoma , Animales , Diferenciación Celular/genética , Células Madre Embrionarias , Femenino , Regulación del Desarrollo de la Expresión Génica , Estratos Germinativos , Ratones , EmbarazoRESUMEN
With the aim of producing ß cells for replacement therapies to treat diabetes, several protocols have been developed to differentiate human pluripotent stem cells to ß cells via pancreatic progenitors. While in vivo pancreatic progenitors expand throughout development, the in vitro protocols have been designed to make these cells progress as fast as possible to ß cells. Here, we report on a protocol enabling a long-term expansion of human pancreatic progenitors in a defined medium on fibronectin, in the absence of feeder layers. Moreover, through a screening of a polymer library we identify a polymer that can replace fibronectin. Our experiments, comparing expanded progenitors to directly differentiated progenitors, show that the expanded progenitors differentiate more efficiently into glucose-responsive ß cells and produce fewer glucagon-expressing cells. The ability to expand progenitors under defined conditions and cryopreserve them will provide flexibility in research and therapeutic production.
Asunto(s)
Células Secretoras de Insulina , Células Madre Pluripotentes , Diferenciación Celular , Fibronectinas/farmacología , Humanos , Páncreas , PolímerosRESUMEN
Shaping the animal body plan is a complex process that involves the spatial organization and patterning of the different germ layers. Recent advances in live imaging have started to unravel the cellular choreography underlying this process in mammals, however, the sequence of events transforming an unpatterned cell ensemble into structured territories is largely unknown. Here, using gastruloids -3D aggregates of mouse embryonic stem cells- we study the formation of one of the three germ layers, the endoderm. We show that the endoderm is generated from an epiblast-like homogeneous state by a three-step mechanism: (i) a loss of E-cadherin mediated contacts in parts of the aggregate leading to the appearance of islands of E-cadherin expressing cells surrounded by cells devoid of E-cadherin, (ii) a separation of these two populations with islands of E-cadherin expressing cells flowing toward the aggregate tip, and (iii) their differentiation into an endoderm population. During the flow, the islands of E-cadherin expressing cells are surrounded by cells expressing T-Brachyury, reminiscent of the process occurring at the primitive streak. Consistent with recent in vivo observations, the endoderm formation in the gastruloids does not require an epithelial-to-mesenchymal transition, but rather a maintenance of an epithelial state for a subset of cells coupled with fragmentation of E-cadherin contacts in the vicinity, and a sorting process. Our data emphasize the role of signaling and tissue flows in the establishment of the body plan.
Asunto(s)
Endodermo , Estratos Germinativos , Animales , Cadherinas , Diferenciación Celular , Movimiento Celular , Gastrulación , Mamíferos , RatonesRESUMEN
In human, endoderm is induced in two waves, with the first being the extra-embryonic primitive endoderm (PrE), otherwise known as hypoblast, induced during blastocyst development, and the second being gastrulation-stage definitive endoderm (DE). The PrE gives rise to the primary and secondary yolk sac, and has supportive functions during pregnancy for nutrient provision, with descendants of this extra-embryonic lineage also playing a role in embryonic patterning. As in DE specification, we recently found that PrE could be induced in vitro by Wnt and Nodal-related signaling, but that the critical difference was in the pluripotent starting point for differentiation. Thus, blastocyst-like naïve human pluripotent stem cells retain the unique capacity to differentiate into PrE cultures, a cell type resembling the pre-implantation hypoblast. The PrE cells could then be expanded as stable naïve extra-embryonic endoderm (nEnd) cell lines, capable of indefinite self-renewal. Here, we describe detailed protocols to differentiate naïve pluripotent stem cells into PrE and then expand the cultures as nEnd, including descriptions of morphology, passaging technique, and troubleshooting.
Asunto(s)
Endodermo , Células Madre Pluripotentes , Diferenciación Celular , Línea Celular , Estratos Germinativos , HumanosRESUMEN
The cellular cytoskeleton provides the cell with a mechanical rigidity that allows mechanical interaction between cells and the extracellular environment. The actin structure plays a key role in mechanical events such as motility or the establishment of cell polarity. From the earliest stages of development, as represented by the ex vivo expansion of naïve embryonic stem cells (ESCs), the critical mechanical role of the actin structure is becoming recognized as a vital cue for correct segregation and lineage control of cells and as a regulatory structure that controls several transcription factors. Naïve ESCs have a characteristic morphology, and the ultrastructure that underlies this condition remains to be further investigated. Here, we investigate the 3D actin cytoskeleton of naïve mouse ESCs using super-resolution optical reconstruction microscopy (STORM). We investigate the morphological, cytoskeletal, and mechanical changes in cells cultured in 2i or Serum/LIF media reflecting, respectively, a homogeneous preimplantation cell state and a state that is closer to embarking on differentiation. STORM imaging showed that the peripheral actin structure undergoes a dramatic change between the two culturing conditions. We also detected micro-rheological differences in the cell periphery between the cells cultured in these two media correlating well with the observed nano-architecture of the ESCs in the two different culture conditions. These results pave the way for linking physical properties and cytoskeletal architecture to cell morphology during early development.
Asunto(s)
Actinas/metabolismo , Forma de la Célula , Células Madre Embrionarias de Ratones/citología , Citoesqueleto de Actina/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Elasticidad , Imagenología Tridimensional , Ratones , Nanopartículas/química , ViscosidadRESUMEN
The metazoan-specific acetyltransferase p300/CBP is involved in activating signal-induced, enhancer-mediated transcription of cell-type-specific genes. However, the global kinetics and mechanisms of p300/CBP activity-dependent transcription activation remain poorly understood. We performed genome-wide, time-resolved analyses to show that enhancers and super-enhancers are dynamically activated through p300/CBP-catalyzed acetylation, deactivated by the opposing deacetylase activity, and kinetic acetylation directly contributes to maintaining cell identity at very rapid (minutes) timescales. The acetyltransferase activity is dispensable for the recruitment of p300/CBP and transcription factors but essential for promoting the recruitment of TFIID and RNAPII at virtually all enhancers and enhancer-regulated genes. This identifies pre-initiation complex assembly as a dynamically controlled step in the transcription cycle and reveals p300/CBP-catalyzed acetylation as the signal that specifically promotes transcription initiation at enhancer-regulated genes. We propose that p300/CBP activity uses a "recruit-and-release" mechanism to simultaneously promote RNAPII recruitment and pause release and thereby enables kinetic activation of enhancer-mediated transcription.
Asunto(s)
Elementos de Facilitación Genéticos , ARN Polimerasa II/metabolismo , Iniciación de la Transcripción Genética , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Animales , Biocatálisis , Cromatina/metabolismo , Regulación hacia Abajo/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Ratones , Modelos Biológicos , Proteínas Nucleares/metabolismo , Unión Proteica , Factor de Transcripción TFIID/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Embryonic stem cells (ESCs) are derived from the pre-implantation mammalian blastocyst. At this point in time, the newly formed embryo is concerned with the generation and expansion of both the embryonic lineages required to build the embryo and the extra-embryonic lineages that support development. When used in grafting experiments, embryonic cells from early developmental stages can contribute to both embryonic and extra-embryonic lineages, but it is generally accepted that ESCs can give rise to only embryonic lineages. As a result, they are referred to as pluripotent, rather than totipotent. Here, we consider the experimental potential of various ESC populations and a number of recently identified in vitro culture systems producing states beyond pluripotency and reminiscent of those observed during pre-implantation development. We also consider the nature of totipotency and the extent to which cell populations in these culture systems exhibit this property.
Asunto(s)
Blastocisto/metabolismo , Linaje de la Célula , Células Madre Embrionarias Humanas/metabolismo , Células Madre Totipotentes/metabolismo , Animales , Blastocisto/citología , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Totipotentes/citologíaRESUMEN
Johnny Thunders once wrote, "You can't put your arms around a memory " and the song continues, "don't try." Yet, when complex morphogenetic movements accompany differentiation, the cellular memory of signaling becomes essential. In this issue of Cell Stem Cell, Gunne-Braden et al. (2020) report a network wrapping its arms around the memory of BMP signaling, a phenomenon known as hysteresis.
Asunto(s)
Células Madre Embrionarias Humanas , Proteína Morfogenética Ósea 4 , Diferenciación Celular , Factor de Transcripción GATA3 , Humanos , Transducción de SeñalRESUMEN
The role of the homeobox transcriptional repressor HESX1 in embryonic stem cells (ESCs) remains mostly unknown. Here, we show that Hesx1 is expressed in the preimplantation mouse embryo, where it is required during developmental diapause. Absence of Hesx1 leads to reduced expression of epiblast and primitive endoderm determinants and failure of diapaused embryos to resume embryonic development after implantation. Genetic deletion of Hesx1 impairs self-renewal and promotes differentiation toward epiblast by reducing the expression of pluripotency factors and decreasing the activity of LIF/STAT3 signaling. We reveal that Hesx1-deficient ESCs show elevated ERK pathway activation, resulting in accelerated differentiation toward primitive endoderm, which can be prevented by overexpression of Hesx1. Together, our data provide evidence for a novel role of Hesx1 in the control of self-renewal and maintenance of the undifferentiated state in ESCs and mouse embryos.
Asunto(s)
Diferenciación Celular/genética , Autorrenovación de las Células/genética , Diapausa/genética , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Eliminación de Gen , Proteínas Represoras/deficiencia , Animales , Biomarcadores , Desarrollo Embrionario , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Proteínas de Homeodominio , Factor Inhibidor de Leucemia/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Modelos Biológicos , Fenotipo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Transducción de SeñalRESUMEN
Central to understanding cellular behaviour in multi-cellular organisms is the question of how a cell exits one transcriptional state to adopt and eventually become committed to another. Fibroblast growth factor-extracellular signal-regulated kinase (FGF -ERK) signalling drives differentiation of mouse embryonic stem cells (ES cells) and pre-implantation embryos towards primitive endoderm, and inhibiting ERK supports ES cell self-renewal1. Paracrine FGF-ERK signalling induces heterogeneity, whereby cells reversibly progress from pluripotency towards primitive endoderm while retaining their capacity to re-enter self-renewal2. Here we find that ERK reversibly regulates transcription in ES cells by directly affecting enhancer activity without requiring a change in transcription factor binding. ERK triggers the reversible association and disassociation of RNA polymerase II and associated co-factors from genes and enhancers with the mediator component MED24 having an essential role in ERK-dependent transcriptional regulation. Though the binding of mediator components responds directly to signalling, the persistent binding of pluripotency factors to both induced and repressed genes marks them for activation and/or reactivation in response to fluctuations in ERK activity. Among the repressed genes are several core components of the pluripotency network that act to drive their own expression and maintain the ES cell state; if their binding is lost, the ability to reactivate transcription is compromised. Thus, as long as transcription factor occupancy is maintained, so is plasticity, enabling cells to distinguish between transient and sustained signals. If ERK signalling persists, pluripotency transcription factor levels are reduced by protein turnover and irreversible gene silencing and commitment can occur.
Asunto(s)
Linaje de la Célula , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Animales , Quinasas MAP Reguladas por Señal Extracelular/genética , Complejo Mediador/deficiencia , Complejo Mediador/metabolismo , Ratones , Unión Proteica , Transcripción GenéticaRESUMEN
Embryonic stem cells (ESCs) exist in at least two states that transcriptionally resemble different stages of embryonic development. Naïve ESCs resemble peri-implantation stages and primed ESCs the pre-gastrulation epiblast. In mouse, primed ESCs give rise to definitive endoderm in response to the pathways downstream of Nodal and Wnt signalling. However, when these pathways are activated in naïve ESCs, they differentiate to a cell type resembling early primitive endoderm (PrE), the blastocyst-stage progenitor of the extra-embryonic endoderm. Here, we apply this context dependency to human ESCs, showing that activation of Nodal and Wnt signalling drives the differentiation of naïve pluripotent cells toward extra-embryonic PrE, or hypoblast, and these can be expanded as an in vitro model for naïve extra-embryonic endoderm (nEnd). Consistent with observations made in mouse, human PrE differentiation is dependent on FGF signalling in vitro, and we show that, by inhibiting FGF receptor signalling, we can simplify naïve pluripotent culture conditions, such that the inhibitor requirements closer resemble those used in mouse. The expandable nEnd cultures reported here represent stable extra-embryonic endoderm, or human hypoblast, cell lines.This article has an associated 'The people behind the papers' interview.