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Single-Cell RNA-seq Defines the Three Cell Lineages of the Human Blastocyst

ABSTRACT: Here we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with those that are human-specific. Importantly, we validate our RNA-sequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast including the transcription factor KLF17. Key components of the TGF-β signaling pathway including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1 are also enriched in the human epiblast. Intriguingly, inhibition of TGF-β signaling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although key trophectoderm factors Id2, Elf5, and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression dynamics including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparisons of the human epiblast to existing embryonic stem cells (hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared to mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE66507 | GEO | 2015/08/26

SECONDARY ACCESSION(S): PRJNA277181

REPOSITORIES: GEO

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Single-Cell RNA-seq Defines the Three Cell Lineages of the Human Blastocyst

Project description:Despite morphological similarities, relatively little is known about conserved developmental processes in human and mouse pre-implantation embryos. Here we provide the first comprehensive single-cell RNA-sequencing comparison of human and mouse embryos from the zygotic to the blastocyst stage. We establish a robust computational pipeline allowing us to elucidate human-specific transcriptional Programs. Importantly, we validate our RNA-sequencing findings by Immunofluorescence analysis, which further reveals differences in human and mouseembryo gene expression. For example, although key trophectoderm factors Id2, Elf5, Eomes and Tcfap2c/Ap2? are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we identify several genes exclusively expressed in the human pluripotent epiblast including the transcription factor KLF17 and key components of the TGF-? signaling pathway LEFTY1, LEFTY2, NODAL and ACVRL1/ALK1 whose expression is absent from the mouse inner cell mass. Conversely, we also identify genes with conserved expression dynamics including Foxa2/FOXA2, which we show for the first time is restricted to the primitive endoderm in both human and mouse embryos. Our analysis highlights significant differences in human pre-implantation development compared to mouse and provides a molecular blueprint to understand human embryogenesis. Single-Cell RNA-seq

2015-08-26 | E-GEOD-66507 | biostudies-arrayexpress

Reversion to naïve human pluripotency creates a new methylation landscape devoid of blastocyst or germline memory

Project description:Human embryonic stem cells (hESCs) typically exhibit "primed" pluripotency, analogous to stem cells derived from the mouse post-implantation epiblast. This has led to a search for growth conditions that support self-renewal of hESCs akin to hypomethylated naïve epiblast cells in human pre-implantation embryos. We have discovered that reverting primed hESCs to a hypomethylated naïve state or deriving a new hESC line under naïve conditions results in the establishment of Stage Specific Embryonic Antigen 4 (SSEA4) negative hESC lines with a transcriptional program resembling the human pre-implantation epiblast. In contrast, we discovered that the methylome of naïve hESCs in vitro is distinct from the human epiblast in vivo with loss of DNA methylation at primary imprints and a lost "memory" of the methylation state of the human oocyte. This failure to recover the naïve epiblast methylation landscape appears to be a consistent feature of self-renewing hypomethylated naïve hESCs in vitro.

2016-02-04 | GSE76970 | GEO

RNA-seq of hESCs undergoing primed to naive-like resetting via exogenous KLF17 overexpression

Project description:RNA-seq of H9 human embryonic stem cells cultured under naive pluripotency-supportive PXGL conditions either with (Dox) or without (UI) exogenous KLF17 expression for five days. As well as a day 0 control sample (hESCs 24hrs after plating in mTeSR), both UI and +Dox samples were collected at days 1, 2 and 5 after switching to naive-permissive PXGL culture conditions. Only Dox-induced hESCs survive after the first passage and form stable lines in PXGL with naive-like morphology and transcriptome, which could be maintained until naive passage 5 (p5).

2021-09-30 | E-MTAB-10915 | biostudies-arrayexpress

Novel culture conditions facilitating the induction of naïve pluripotency in human embryonic stem cells

Project description:It is now well recognized that human embryonic stem cells (hESCs)1 closely resemble mouse epiblast stem cells (mEpiSCs)2-5 exhibiting primed pluripotency unlike mouse ESCs (mESCs) which acquire a naïve pluripotent state4-8. Efforts have been made to trigger naïve pluripotency in hESCs9-11 for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines12. We report here a novel culture medium facilitating rapid induction of naïve pluripotency in established hESCs. Our medium also allows derivation of naïve mESCs from blastocyst stage which has not been shown earlier. The established naïve hESCs could survive long-term single cell passaging, maintain a normal karyotype, exhibit upregulation of naïve pluripotency genes and were dependent on signaling pathways similar to naïve mESCs. Also, they undergo global DNA demethylation, cluster together with previously described naïve hESCs13 and show a distinctive long non-coding RNA profile. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs which is fast, reproducible, can be employed to derive naïve mESCs and can induce efficient differentiation. Three primed and matching naive human embryonic stem cell lines were profiled in duplo.

2015-06-08 | E-GEOD-57271 | biostudies-arrayexpress

Wnt/TCF7L1 transcriptional repressor axis drives primitive endoderm formation by antagonizing naive and formative pluripotency

Project description:Early during preimplantation development and in heterogeneous mouse embryonic stem cells (mESC) culture, pluripotent cells are specified towards either the primed epiblast or the primitive endoderm (PE) lineage. Canonical Wnt signaling is crucial for safeguarding naive pluripotency and embryo implantation, yet the role and relevance of canonical Wnt inhibition during early mammalian development remains unknown. Here, we demonstrate that transcriptional repression exerted by Wnt/TCF7L1 promotes PE differentiation of mESCs and in preimplantation inner cell mass. Time-series RNA sequencing and promoter occupancy data reveal that TCF7L1 binds and represses genes encoding essential naive pluripotency factors and indispensable regulators of the formative pluripotency program, including Otx2 and Lef1. Consequently, TCF7L1 promotes pluripotency exit and suppresses epiblast lineage formation, thereby driving cells into PE specification. Conversely, TCF7L1 is required for PE specification as deletion of Tcf7l1 abrogates PE differentiation without restraining epiblast priming. Taken together, our study underscores the importance of transcriptional Wnt inhibition in regulating lineage specification in ESCs and preimplantation embryo development as well as identifies TCF7L1 as key regulator of this process.

2023-02-08 | GSE171204 | GEO

Novel culture conditions facilitating the induction of naïve pluripotency in human embryonic stem cells

2015-06-08 | GSE57271 | GEO

Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening [WGBS]

Project description:Background: Naïve human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional “primed” hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Methods:Here we performed high-throughput screening using a library of >3,000 well-annotated compounds to identify essential signaling requirements for naïve human pluripotency. Results:We report that MEK1/2 inhibitors can be replaced during maintenance of naïve human pluripotency by inhibitors targeting either upstream (FGFR, RAF1) or downstream (ERK1/2) kinases. Naïve hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naïve resetting in combination with PKC, ROCK, and TNKS inhibitors and Activin A. Conclusions: This work demonstrates that induction and maintenance of naïve human pluripotency are governed by distinct signaling requirements.

2021-06-15 | GSE153213 | GEO

Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening [RNA-seq]

2021-06-15 | GSE153212 | GEO

OCT4 IP-MS in Naive and Primed Human ESCs

Project description:Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes.

2021-09-12 | PXD026556 | Pride

NANOG-OCT4-SOX2 Regulatory Module in Human Embryonic Stem Cells (dataset 4)

Project description:The transcription factors Nanog, Oct4 and Sox2 are the master regulators of pluripotency in mouse embryonic stem cells (mESCs), however, their functions in human ESCs (hESCs) have not been rigorously defined. Here we show that the requirements for NANOG, OCT4 and SOX2 in hESCs differ from those in mESCs. Both NANOG and OCT4 are required for self-renewal and repress differentiation. OCT4 controls both extraembryonic and epiblast-derived cell fates in a BMP4-dependent manner. OCT4-depleted hESCs commit to trophectoderm and primitive endoderm in the presence of BMP4, but undergo neuroectoderm differentiation in the absence of BMP4. NANOG represses neuroectoderm and neural crest commitment, but has little or no effect on the other lineages. We find that SOX2 is not required for self-renewal because it is redundant with SOX3, which is induced in SOX2-depleted hESCs. Simultaneous depletion of both SOX2 and SOX3 induces differentiation into the primitive streak. Unexpectedly, we identify significant variability in the usage of pluripotency factors by individual hESC lines, suggesting that the pluripotency network is remodelled to support a continuum of developmental states. Our study revises the general view of how NANOG, OCT4 and SOX2 orchestrate self-renewal in hESCs. Total RNA obtained from EF1a-control-, OE-NANOG-, OE-OCT4- or OE-SOX2-transduced hESCs.

2012-04-05 | E-GEOD-34920 | biostudies-arrayexpress

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