Project description:We did bulk and single cell RNA sequencing of blastocysts, blastoids, trophoblast stem cells (TSC) and embryonic stem cells (ESC). The goal of these experiment is to describe the transformations of the transcriptome occurring within cells (TSC, ESC) upon formation of a blastoid. E3.25 and E3.5 blastocysts are used as controls. To this end, we first did RNA sequencing of intact structures (E3.25 and E3.5 blastocysts, blastoids, and parental cell lines). In a different series of experiments, we micro-dissected blastocyst, blastoid or trophosphere structures into single cells, and sequenced their mRNAs, to infer cell identity and transcriptome variations.
Project description:Embryonic stem cells upon extrinsic induction could self-assemble into blastocyst-like structures. However, the intrinsic regulation of such blastoid forming potential remain to be addressed. We discover that the activity of nuclear receptor subfamily 1, group H, member 2 (Nr1h2) in expanded potential stem cell (EPSC) positively correlates with blastoid efficiency and quality. In addition, Nr1h2 agonist, T0901317, improves natural blastocyst development. Surprisingly, Nr1h2-activated ESC (NrESC) is rewired towards a distinct pluripotency state that is capable of self-organizing into blastoids and contribute to embryonic and extraembryonic lineage. We aim at characterizing the NrESC-derived blastoids by single-cell RNA-seq and show that blastoid comprise of epiblast-like, trophectoderm-like and primitive endoderm-like populations, which is the same as blastocyst composition and shows respective gene marker expression for each cluster. Thus, NrESC has attained a unique expanded pluripotency state driven by Nr1h2 activation.
Project description:The first lineage decisions during mouse development lead to establishment of embryonic and extraembryonic tissues. The transcription factor Cdx2 plays a central role by repressing pluripotency genes, such as Oct4 and promoting trophoblast fate at the blastocyst stage. Here we show that the transcription factor Gata3 is coexpressed with Cdx2 in the blastocyst and that overexpression of Gata3 in embryonic stem cells is sufficient to induce expression of trophoblast genes. Gata3 expression in the blastocyst does not depend on Cdx2, nor do Gata3 overexpressing cell lines require Cdx2 for expression of a subset of trophoblast genes. In the embryo, expression of Gata3, like Cdx2, depends on Tead4, and expression of both factors becomes restricted to nascent trophoblast by an Oct4-independent mechanism. These observations place Tead4 at the top of a trophoblast hierarchy, with Gata3 and Cdx2 acting downstream to induce expression of common and independent targets in this lineage. This SuperSeries is composed of the following subset Series: GSE12985: Differentiation time course of trophoblast stem cells GSE12986: Expression of Cdx2 or Gata3 in R1 mouse embryonic stem cells
Project description:Preimplantation embryo development is a precisely regulated process organized by maternally inherited and newly synthesized proteins. Recently, some studies have reported that blastocyst-like structures, named blastoids, can be generated from mouse ESCs (embryonic stem cells) or EPSCs (extended pluripotent stem cells). In this study, to explore the dynamic expression characteristics of proteins and their PTMs in mouse EPS blastoids, we revealed the protein expression profile of EPS-blastoids and metabolite characteristics by TMT-based quantitative mass spectrometry (MS) strategy. Furthermore, the protein phosphorylation sites were identified to show the phosphoproteomic analysis in blastoids compared with mouse early embryos. Above all, our study revealed the protein expression profile of EPS blastoids compared with mouse embryos during preimplantation development and indicated that glucose metabolism is key to blastoid formation.
Project description:Trophoblast stem cells represent the stem cell population of the extra-embryonic lineage and arise as a result of the first cell fate decision. From blastocyst stage onwards, a distinct epigenetic lineage barrier strictly separates mouse embryonic and extra-embryonic lineages. Recently, it has been shown that this epigenetic barrier cannot be fully overcome as the expression of TS-determining factors in embryonic stem cells lead to incomplete transdifferentiation. Here, we demonstrate that transient expression of Tfap2c, Gata3, Eomes and Ets2 in fibroblasts suffices to generate cells which are almost identical to trophoblast stem cells based on morphology, expression and methylation pattern. Further, these induced trophoblast stem cells display transgene independent self-renewal, differentiate along the extra-embryonic lineage and chimerize the placenta upon blastocyst injection. Our findings provide insights into the transcription factor networks governing trophoblast stem cell identity and offer a new tool for studying the hierarchy of those factors.
Project description:Naïve human pluripotent stem cells have the remarkable ability to self-organize into blastocyst-like structures (“blastoids”) that model lineage segregation in the pre-implantation embryo. However, the extent to which blastoids can recapitulate defining features of human post-implantation development remains unexplored. Here, we report that blastoids cultured on thick 3D extracellular matrices capture hallmarks of early post-implantation development, including epiblast lumenogenesis, rapid expansion and diversification of trophoblast lineages, and robust invasion of extravillous trophoblast cells by day 14. Extended blastoid culture results in the localized activation of primitive streak marker TBXT and the emergence of embryonic germ layers by day 21. We also show that modulation of WNT signaling alters the balance between epiblast and trophoblast fates in post-implantation blastoids. This work demonstrates that 3D-cultured blastoids offer a continuous and integrated in vitro model system of human embryonic and extraembryonic development from pre-implantation to early gastrulation stages.
Project description:Shortly after fertilization, human embryos implant into the uterus. This requires the formation of a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Appropriate stem cells can form a blastocyst model, which we termed blastoid. Here we show that naive human pluripotent stem cells (hPSCs) triply inhibited for the Hippo, TGF-β, and ERK pathways consistently and efficiently (>70%) form blastoids that generate transcriptional pre-implantation analogs of the three founding lineages (trophoblast, epiblast, primitive endoderm; >97%) according to the sequence and pace of blastocyst development. Blastoids spontaneously form an axis marked by the maturation of the polar region, which acquires the potential to specifically attach to hormonally stimulated endometrial cells, as during in utero implantation. Such human blastoids are scalable, versatile, and ethical models to explore human implantation and development.
Project description: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 apply multi-omics to comprehensively define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Integrating the chromatin-bound proteome and histone modification data sets reveals differences in the relative abundance and activities of distinct chromatin modules, identifying a strong enrichment of Polycomb Repressive Complex 2 (PRC2)-associated H3K27me3 in naive pluripotent stem cell chromatin. Single-cell approaches and human blastoid models reveal that PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, and inhibiting PRC2 promotes trophoblast fate induction and cavity formation. 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.
Project description:Embryonic stem cells upon extrinsic induction could self-assemble into blastocyst-like structures. However, the intrinsic regulation of such blastoid forming potential remain to be addressed. We discover that the activity of nuclear receptor subfamily 1, group H, member 2 (Nr1h2) in expanded potential stem cell (EPSC) positively correlates with blastoid efficiency and quality. In addition, Nr1h2 agonist, T0901317, improves natural blastocyst development. Surprisingly, Nr1h2-activated ESC (NrESC) is rewired towards a distinct pluripotency state that is capable of self-organizing into blastoids and contribute to embryonic and extraembryonic lineage. We hypothesize that Nr1h2 activation broadly affects the DNA methylation to rewire ESC state. Indeed, from WGBS data, we observe that genome-wide DNA methylation differences in NrESC or EPSC after treated with T09 . In summary, our study demonstrates a novel Nr1h2-centric regulation of expanded pluripotency.