RESUMO
The human extraembryonic mesoderm (EXM) is an important tissue in the postimplantation embryo which is specified before gastrulation in primates but not in rodents. EXM is mesenchymal and plays an important role in embryogenesis, including early erythropoiesis, and provides mechanical support to the developing embryo. Recently, it has been shown that self-renewing extraembryonic mesoderm cells (EXMCs) can be modeled in vitro by using human naive pluripotent stem cells. Here, we present a detailed step-by-step protocol to induce EXMCs from naive pluripotent stem cells in vitro.
Assuntos
Mesoderma , Células-Tronco Pluripotentes , Animais , Humanos , Embrião de Mamíferos , Desenvolvimento Embrionário , Primatas , Diferenciação CelularRESUMO
A hallmark of primate postimplantation embryogenesis is the specification of extraembryonic mesoderm (EXM) before gastrulation, in contrast to rodents where this tissue is formed only after gastrulation. Here, we discover that naive human pluripotent stem cells (hPSCs) are competent to differentiate into EXM cells (EXMCs). EXMCs are specified by inhibition of Nodal signaling and GSK3B, are maintained by mTOR and BMP4 signaling activity, and their transcriptome and epigenome closely resemble that of human and monkey embryo EXM. EXMCs are mesenchymal, can arise from an epiblast intermediate, and are capable of self-renewal. Thus, EXMCs arising via primate-specific specification between implantation and gastrulation can be modeled in vitro. We also find that most of the rare off-target cells within human blastoids formed by triple inhibition (Kagawa et al., 2021) correspond to EXMCs. Our study impacts our ability to model and study the molecular mechanisms of early human embryogenesis and related defects.
Assuntos
Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Embrião de Mamíferos , Camadas Germinativas , Humanos , Mesoderma , PrimatasRESUMO
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.
Assuntos
Células-Tronco Pluripotentes , Complexo Repressor Polycomb 2 , Diferenciação Celular/genética , Cromatina/genética , Histonas/genética , Humanos , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Trofoblastos/metabolismoRESUMO
Dosage compensation between the sexes results in one X chromosome being inactivated during female mammalian development. Chromosome-wide transcriptional silencing from the inactive X chromosome (Xi) in mammalian cells is erased in a process termed X-chromosome reactivation (XCR), which has emerged as a paradigm for studying the reversal of chromatin silencing. XCR is linked with germline development and induction of naive pluripotency in the epiblast, and also takes place upon reprogramming somatic cells to induced pluripotency. XCR depends on silencing of the long non-coding RNA (lncRNA) X inactive specific transcript (Xist) and is linked with the erasure of chromatin silencing. Over the past years, the advent of transcriptomics and epigenomics has provided new insights into the transcriptional and chromatin dynamics with which XCR takes place. However, multiple questions remain unanswered about how chromatin and transcription related processes enable XCR. Here, we review recent work on establishing the transcriptional and chromatin kinetics of XCR, as well as discuss a model by which transcription factors mediate XCR not only via Xist repression, but also by direct targeting of X-linked genes.
