Recapitulating human peri-gastrulation development via self-organization of extended pluripotent stem cells
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ABSTRACT: Stem cell models that replicate the gastrulation process in human embryos have been created, but they lack the essential extraembryonic cells needed for early embryonic development and patterning. Here, we introduce a robust and efficient method that prompts human extended pluripotent stem (EPS) cells to self-organize into embryo-like structures, called peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. These peri-gastruloids simulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and early neurulation. Single-cell RNA sequencing unveiled transcriptomic characteristics of these human peri-gastruloids, which closely resemble the primary peri-gastrulation cell types found in human and non-human primates. Our results emphasize the remarkable self-organizing ability of EPS cells to generate advanced human embryo-like structures. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.
Project description:We analyzed scRNA-seq data in human pluripotent stem cells derived peri-gastrulation trilaminar embryonic disc (PTED) human embryo models with trilaminar embryonic disc-, amnion- and yolk sac-like structures.
Project description:The cellular microenvironment shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and benefit from hypoxic culture in vitro. Yet, how different components of the hypoxia response impact stem cell transcriptional networks and lineage choices remains unclear. Here we investigated the effect of acute and prolonged hypoxia on stem cell states and differentiation efficiencies of embryonic and extraembryonic cells. We show that prolonged hypoxia enhances differentiation of embryonic stem (ES) cells towards the mesendoderm lineage by transcriptionally priming cells with a primitive streak signature including Wnt3 and T expression. Exposure to hypoxia in ES culture or during formation of gastrulation-mimicking organoids (gastruloids) moderates T expression and enhances structural complexity. Hypoxic gastruloids generated without exogenous Wnt induction can spontaneously elongate and self-organize. Direct gene regulation by Hif1a, combined with DNA demethylation and metabolic rewiring modulate the transcriptional response and phenotypic outcome. Our findings highlight the influence of the microenvironment on stem cell function and provide a rationale supportive of applying physiological conditions in synthetic embryo models.
Project description:Recent advances in synthetic mammalian embryo models have opened new avenues to understand the complex events controlling lineage specification and morphogenetic processes occurring during peri-implantation and early organogenesis. Two main strategies have been developed to build embryo-like structures (ELSs): by assembling extraembryonic and embryonic stem cells (ESCs) or by subjecting ESCs to various morphogens. Here, we show that mouse ESCs solely exposed to chemical inhibition of SUMOylation, a post-translational modification which acts as a general chromatin barrier to cell fate transitions, generates ELSs comprising both anterior neural and trunk-associated regions. HypoSUMOylation-instructed ESCs first give rise to adherent spheroids which, once in suspension, self-organize into gastrulating structures containing cell types spatially and functionally related to embryonic and extraembryonic compartments. Alternatively, spheroids cultured in an optimized droplet-microfluidic device form elongated structures that undergo axial organization reminiscent of natural embryo morphogenesis. Single-cell RNA-sequencing further revealed a variety of cellular lineages including properly positioned anterior neuronal cell types, Schwann cell precursors and paraxial mesoderm segmented into somite-like structures. Mechanistically, transient SUMOylation suppression gradually increases DNA methylation genome-wide and repressive marks deposition at Nanog, enhancing ESC plasticity. Our approach provides a proof of principle for a potential strategy to study early embryogenesis by targeting molecular roadblocks of cell fate change to shape multicellular architecture.
Project description:Gastruloids are small aggregates of murine Embryonic Stem (ES) cells stimulated to undergo gastrulation-like symmetry breaking events in vitro (van den Brink, S. C. et al. 2014, Simunovic, M. & Brivanlou, A. H 2017). We analyzed the transcriptomic profiles of gastruloids at different time-points after aggregation (AA) and compared them with those of mouse embryo at different stages, raging from e6 to e9.5. We show that gastruloids are capable of organizing a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimics that of the embryo in terms of gene expression.
Project description:Several in vitro models have been developed to recapitulate mouse embryogenesis solely from embryonic stem cells (ESCs). Despite mimicking many aspects of early development, they fail to capture the interactions between embryonic and extraembryonic tissues. To overcome this difficulty, we have developed a mouse ESC-based in vitro model that reconstitutes the pluripotent ESC lineage and the two extra-embryonic lineages of the post-implantation embryo by transcription factor-mediated induction. This unified model recapitulates developmental events from embryonic day 5.5 to 8.5, including gastrulation, and formation of the anterior-posterior axis, brain, a beating heart structure, and the development of extraembryonic tissues, including yolk sac and chorion. Comparing single-cell RNA sequencing from individual structures with time-matched natural embryos identified remarkably similar transcriptional programs across lineages, but also showed when and where the model diverges from the natural program. Our findings demonstrate an extraordinary plasticity of ESCs to self-organize and generate a whole embryo-like structure.
Project description:Gastrulation is the highly coordinated process by which the early embryo breaks symmetry, establishes germ layers and a body plan, and sets the stage for organogenesis. As early mammalian development is challenging to study in vivo, stem cell-derived models have emerged as powerful surrogates, e.g. human and mouse gastruloids. However, although single cell RNA-seq (scRNA-seq) and high-resolution imaging have been extensively applied to characterize such in vitro embryo models, a paucity of measurements of protein dynamics and regulation leaves a major gap in our understanding. Here we sought to address this by applying quantitative proteomics to human and mouse gastruloids at four key stages of their differentiation (naïve ESCs, primed ESCs, early gastruloids, late gastruloids). To the resulting data, we perform network analysis to map the dynamics of expression of macromolecular protein complexes and biochemical pathways, including identifying cooperative proteins that associate with them. With matched RNA-seq and phosphosite data from these same stages, we investigate pathway-, stage- and species-specific aspects of translational and post-translational regulation, e.g. finding peri-gastrulation stages of human vs. mice to be discordant with respect to the mitochondrial transcriptome vs. proteome, and nominating novel kinase-substrate relationships based on phosphosite dynamics. Finally, we leverage correlated dynamics to identify conserved protein networks centered around congenital disease genes. Altogether, our study showcases the potential of intersecting proteomics and in vitro embryo models to advance our understanding of early mammalian development in ways that may not be possible through transcriptomics alone.
Project description:Among all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their unrestricted developmental potential, able to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic (ExEm) tissues, in particular, those giving rise to the placenta in vivo. To date, it remains unknown whether stem cells with both embryonic and extraembryonic developmental potency can be captured and maintained in vitro. Here, we identify a new chemical cocktail that allows for the generation of stem cells with extended developmental potency from mouse and human, designated as extended pluripotent stem (EPS) cells, which is capable of chimerizing both embryonic and extraembryonic tissues. Importantly, a single mouse EPS (mEPS) cell shows widespread contribution to both embryonic and extraembryonic lineages in chimeric mouse conceptuses at late-gestation stages, and permits generation of high-grade germline competent chimeras as well as single EPS-derived viable mice by tetraploid complementation. Furthermore, human EPS (hEPS) cells contribute to embryonic and extraembryonic tissues in interspecies chimeric mouse conceptuses. Compared to known PSCs, EPS cells show unique gene modules that upregulate in embryonic cells from early preimplantation development. Further analysis shows that PARP1 inhibition is required for maintaining EPS potency. Our findings constitute a first step towards capturing pluripotent stem cells with extraembryonic developmental potentials in culture, and open new avenues for generating mammalian PSCs with robust chimeric competency for basic and translational research.
Project description:We used circular micropattern cultures to differentiate human embryonic stem cells (H1 hESCs) into microcolonies termed ‘gastruloids’, comprising germ layer and extraembryonic cells. Differentiated gastruloids were dissociated and reseeded onto micropatterns in single cells solution to study cell sorting behaviors. We applied single-cell RNA sequencing to examine transcriptomes of gastruloids and reseeded cells.
Project description:Gastrulation is a critical stage of embryonic development during which the three germ layers are established. Deciphering the molecular mechanisms underlying this process from a protein perspective remains a significant challenge. To address this, we employed a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho)protein expression during differentiation of ESCs towards gastruloids – an in vitro model of gastrulation-stage embryogenesis. Our findings revealed that many proteins exhibited temporal expression during gastrulation with unique expression profiles corresponding to the three germ layers, which we also validated using an ultra sensitive single cell proteomics approachtechnology. Additionally, we profiled enhancer interaction landscapes in ESCs and gastruloids using p300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron based perturbations combined with scRNA-seq revealed a critical role for Zeb2 in regulating mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.
Project description:Gastruloids are small aggregates of murine Embryonic Stem (ES) cells stimulated to undergo gastrulation-like symmetry breaking events in vitro (van den Brink, S. C. et al. 2014, Simunovic, M. & Brivanlou, A. H 2017). We analyzed the transcriptomic profiles of gastruloids at different time-points after aggregation (AA), showing that they are capable of organizing a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimics that of the embryo in terms of gene expression.