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A developmental coordinate of the spectrum of pluripotency among mice, monkeys, and humans

ABSTRACT: The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of the spectrum of pluripotent stem cells (PSCs) in vitro. To explore the ontogeny of human/primate pluripotency, we performed comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys. Here we show that after specification in the blastocysts [embryonic day (E)7], cyEPI undergoes major transcriptome changes upon implantation. Thereafter, cyEPI, while generating gastrulating cells (~E13), maintains its transcriptome relatively stably over a week, retaining a unique set of pluripotency genes while acquiring properties for “neuron differentiation.” h/cyPSCs show the highest similarity to post-implantation late cyEPI (~E17), which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI (E5.5) and epiblast-like cells (EpiLCs) in vitro. These findings not only reveal divergence/coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro.

ORGANISM(S): Mus musculus Macaca fascicularis

PROVIDER: GSE74767 | GEO | 2016/08/24

SECONDARY ACCESSION(S): PRJNA301445

REPOSITORIES: GEO

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A developmental coordinate of the spectrum of pluripotency among mice, monkeys, and humans

Project description:The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of the spectrum of pluripotent stem cells (PSCs) in vitro. To explore the ontogeny of human/primate pluripotency, we performed comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys. Here we show that after specification in the blastocysts [embryonic day (E)7], cyEPI undergoes major transcriptome changes upon implantation. Thereafter, cyEPI, while generating gastrulating cells (~E13), maintains its transcriptome relatively stably over a week, retaining a unique set of pluripotency genes while acquiring properties for âneuron differentiation.â h/cyPSCs show the highest similarity to post-implantation late cyEPI (~E17), which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI (E5.5) and epiblast-like cells (EpiLCs) in vitro. These findings not only reveal divergence/coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro. Single cell transcriptome analysis of cynomolgus monkey embryo E6-17 and mouse embryo E4.5 - E6.5 as well as of cynomogus monkey embryonic stem cells (ESCs) and mouse ESC, epiblast like cells (EpiLCs), using SC3-seq technology.

2016-08-24 | E-GEOD-74767 | biostudies-arrayexpress

Stem cell lines of the early mouse embryo

Project description:Expression profiling of stem cell lines derived from the early embryo representing the trophoblast, primitive endoderm, early epiblast (inner cell mass E3.5) and late post-implantation epiblast (E5.5). Cells were grown without feeders and harvested. Comparisons were made to provide evidence of unique gene expression between the cell lines. Specifically, pre-implantation (ICM, epiblast and primitive endoderm, and trophoblast), as well as pre-implantation and post-implantation epiblasts.

2012-05-21 | E-GEOD-34799 | biostudies-arrayexpress

Stem cell lines of the early mouse embryo

2012-05-22 | GSE34799 | GEO

De novo formation of apical-basal polarity by Rap1 regulates epiblast patterning

Project description:The epiblast is the first cell type that forms apical-basal polarity de novo as the mouse embryo implants into the maternal uterus, while the extraembryonic neighbours of the epiblast - trophectoderm and primitive endoderm - retain their pre-established polarity beyond implantation [1]; however, it is still unclear how the epiblast establishes apical-basal polarity de novo. Here, we focused on Rap1 signaling pathway, which is activated during the transition of the epiblast from the naïve to primed state of pluripotency during implantation [2]. Through the preestablished in vitro three-dimensional culture system [3], genetic knockouts and proximity-biotinylation analyses, we found that Rap1 integrates multiple signals that contribute to de novo formation of apical-basal polarity. Importantly, formation of apical-basal polarity in the epiblast is essential for its correct patterning and proper communication with the extraembryonic lineages. Altogether, these results not only dissect molecular details of de novo apical-basal polarity formation, but also have broader implications for epithelial polarity and development.

2022-06-14 | PXD028315 | Pride

Induction of Rosette-to-Lumen stage embryoids using reprogramming paradigms in ESCs

Project description:Blastocyst-derived stem cell lines were shown to self-organize into embryo-like structures in 3D cell culture environments. Here, we provide evidence that embryo-like structures can be generated solely based on transcription factor-mediated reprogramming of embryonic stem cells in a simple 3D co-culture system. Embryonic stem cells in these cultures self-organize into elongated, compartmentalized embryo-like structures reflecting aspects of the inner regions of the early post-implantation embryo. Single-cell RNA-seq reveals transcriptional profiles resembling epiblast, primitive-/visceral endoderm, and extraembryonic ectoderm of early murine embryos around E4.5–E5.5. In this stem cell-based embryo model, progression from rosette formation to lumenogenesis accompanied by progression from naïve- to primed pluripotency was observed within Epi-like cells. Additionally, lineage specification of primordial germ cells and distal/anterior visceral endoderm-like cells was observed in epiblast- or visceral endoderm-like compartments, respectively. The system presented in this study allows for fast and reproducible generation of embryo-like structures, providing an additional tool to study aspects of early embryogenesis.

2021-11-15 | GSE188394 | GEO

Ex utero Mouse Embryogenesis from Pre-Gastrulation to Late-Organogenesis

Project description:Establishment of the mammalian body plan occurs shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. While methods for in vitro culture of pre- and peri-implantation mouse embryos are routinely utilized, approaches for robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. We develop herein highly stable ex utero post-implantation mouse embryo culture platforms, that enable appropriate development of embryos before gastrulation (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in 3D rotating bottles settings, while extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of novel static and rotating bottle culture protocols. Histological, molecular, and single cell RNA-seq analysis validate that the ex utero developed embryos recapitulate precisely in utero development. This culture system is amenable to introducing a variety of embryonic perturbations and micro-manipulations that can be followed ex utero for up to 6 days. Establishment of a system to robustly grow normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool to investigate post-implantation embryogenesis, eliminating the uterine barrier to mechanistically interrogate morphogenesis and tissue specification in mammals.

2021-02-15 | GSE149372 | GEO

The chemically-defined induction of a primitive endoderm and epiblast-like niche supports post-implantation progression from blastoids.

Project description:The early mammalian conceptus (blastocyst) contains two supporting extraembryonic tissues - the trophectoderm and the primitive endoderm (PrE) - that encase and guide the epiblast (Epi) to eventually form the all body. Modifications of the conceptus exposed key genes regulating these tissues co-development. However, the combination of signalling pathways at play remains elusive. Stem cell-based models including embryoid bodies and blastoids can be generated in large numbers and subjected to high-content screens. Here, we use combinatorial screens of proteins, GPCR ligands and small molecules to rapidly (<96 hours) and efficiently (80%) guide embryoid bodies to form a three dimensional PrE-/Epiblast-like niche in chemically-defined conditions (gel-free, serum-free). This bipotent niche spontaneously progresses, without growth factors, to form a pro-amniotic cavity surrounded by a polarized Epi covered by parietal and visceral endoderm-like cells. In blastoids, these molecules enhance the ratio and number of Gata6+/Nanog+ cells and promote the survival, expansion and morphogenesis of a post-implantation-like Epi in vitro. Altogether, modelling early development in chemically-defined conditions delineates the pathways sufficient to form a functional PrE/Epiblast niche that fuels post-implantation development.

2021-04-01 | GSE129655 | GEO

H3K4me3 Epigenomic Landscape derived from ChIP-Seq of 1,000 mouse early embryonic cells

Project description:Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) is a powerful tool to dissect global epigenetic landscapes of cells. However, this method usually consumes millions of cells. Here we develop a robust technique for performing ChIP-Seq using as low as 1,000 cells. This method combines a semi-automatic nanoliter ChIP reaction with a carrier-based sequencing library preparation strategy without pre-amplifying the ChIP product. We used this method to investigate the pattern of trimethylation of histone 3 lysine 4 (H3K4me3) of mouse post-implantation epiblast cells at embryonic day 6.5 (E6.5) and showed that it is more similar to that of mouse Epi-Stem cells (mEpiSCs) than that of mouse Embryonic Stem cells (mESCs). Together with the high similarity between the transcriptomes of EpiSCs and E6.5 epiblast cells, this suggests that mEpiSCs is a reliable in vitro model for post-implantation epiblast cells in vivo. Use 1 million mEpiSCs and 1million mESCs as the positive control, 1000 mEpiSC and 1000 mESCs samples are used to validate the protocol. 1000 mEpiblasts have two biological replicates

2015-07-24 | E-GEOD-59738 | biostudies-arrayexpress

Program of monkey embryogenesis and germ cell formation in vitro [RNA-seq]

Project description:Embryo development of post-implantation and gastrulation is a milestone of embryogenesis. However, the fundamental mechanisms underlying this process remain elusive as technological and ethical limitations. Here, we we improved an in vitro culture system that cynomolgus monkey embryos could develop to 20 days without any maternal tissues, which clearly reveal lineage specification, bi-laminar disc generation, amniotic and yolk sac formation, and primordial germ cell like cells (PGCLC). By applying single cell-omics analysis, unsupervised dimensionality reduction and clustering algorithm data show clear, distinct, stepwise development routes for the epiblast, primitive endoderm, trophectoderm and extra-embryonic mesenchyme lineage. We also found development path of PGCLCs, suggesting origin of PGLCs in amnion instead of EPI. Furthermore, signal pathways analysis showed existence of intimate interactions among lineages. By sequencing (scATAC-seq ) assay for transposase-accessible chromatin, we identify transcription factor grammar specifying of each cell type (EPI, PE and TE) and infer potential master transcriptional regulators. Collectively, the results provide the new insight into understanding molecular and cellular dynamics that underlie monkey post-implantation development, especially beyond E13. Our study provides a basic knowledge about regulation of primate pluripotency in vitro for cell replacement therapy.

2020-05-07 | GSE150004 | GEO

Program of monkey embryogenesis and germ cell formation in vitro [Bulk-seq]

2020-05-07 | GSE149974 | GEO

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