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DNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library in haploid human embryonic stem cells (hESCs) differentiated into three germ layers

ABSTRACT: Pluripotent stem cells can differentiate into all embryonic germ layers, a process that is orchestrated by the dissolution of pluripotency network and activation of pro-differentiation pathways. Yet, the genes essential for these cell fate transitions in human are still elusive. Here, we used a genome-wide loss-of-function library established in haploid hPSCs and differentiated these cells into into the three germ layers. We performed a genetic screening of the differentited library and identified genes that are essential for the differentiation of human pluripotent stem cells (hPSCs) towards ectoderm, mesoderm and endoderm.

ORGANISM(S): Homo sapiens

PROVIDER: GSE143369 | GEO | 2020/07/30

REPOSITORIES: GEO

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RNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library in haploid human embryonic stem cells (hESCs) differentiated into three germ layers

Project description:Pluripotent stem cells can differentiate into all embryonic germ layers, a process that is orchestrated by the dissolution of pluripotency network and activation of pro-differentiation pathways. Yet, the genes essential for these cell fate transitions in human are still elusive. Here, we used a genome-wide loss-of-function library established in haploid hPSCs and differentiated these cells into into the three germ layers. We analyzed the transcriptome of the differentiated mutant library cells to assess their differentiation status, as well as to determine lineage-specific expression of essential genes for the differentiation of hESCs into each germ layer.

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Transcriptome analysis of differentiated human embryonic stem cells (hESCs) upon CRISPR/Cas9-mediated mutagenesis of lineage-specific and common essential genes

Project description:Pluripotent stem cells can differentiate into all embryonic germ layers, a process that is orchestrated by the dissolution of pluripotency network and activation of pro-differentiation pathways. We established a genome-wide loss-of-function library in haploid hPSCs and differentiated these cells into the three germ layers. The analyses of our high-throughput genetic screening revealed germ layer-specific essential transcription factors, as well as common essential genes for the transition from pluripotency into the three embryonic germ layer fates. Here, we have generated individual knockouts of seven lineage-specific and four common essential genes in human embryonic stem cells by CRISPR/Cas9 mutagenesis and analyzed the transcriptomes of the mutants upon either directed or spontaneous differentiation.

2020-07-30 | GSE143371 | GEO

DNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library established in haploid human embryonic stem cells (hESCs)

Project description:The maintenance of pluripotency requires coordinated expression of a set of essential genes. Using our recently established haploid human pluripotent stem cells (hPSCs), we generated a genome-wide loss-of-function library targeting 18,166 protein-coding genes to define the essential genes in hPSCs. We could allude to an intrinsic bias of essentiality across cellular-compartments, uncover two opposing roles for tumor-suppressor genes, and link autosomal-recessive disorders with growth-retardation phenotypes to early embryogenesis. hPSC-enriched essential genes mainly encode transcription factors and proteins related to cell-cycle and DNA-repair, revealing that a quarter of the nuclear-factors are essential for normal growth. Our screen also led to the identification of growth-restricting genes whose loss-of-function provides growth advantage to hPSCs, highlighting the role of the P53-mTOR pathway in this context. Overall, we have constructed an atlas of essential and growth-restricting genes in hPSCs, revealing key aspects of cellular essentiality and providing a reference for future studies on human pluripotency.

2018-04-30 | GSE111309 | GEO

Secreted footprint of pluripotency in Man-13 HeSCs

Project description:With a view to developing a much-needed non-invasive method for monitoring the healthy pluripotent state of human stem cells in culture, we undertook proteomic analysis of the waste medium from cultured induced (Rebl.PAT) human pluripotent stem cells (hPSCs). Cells were grown in E8 medium to maintain pluripotency, and then transferred to FGF2 and TGFβ deficient E6 media for 48 hours to replicate an early, undirected dissolution of pluripotency. We identified a distinct proteomic footprint associated with early loss of pluripotency in both hPSC lines, and a strong correlation with changes in the transcriptome. We demonstrate that multiplexing of four E8- against four E6- enriched secretome biomarkers provides a robust, diagnostic metric for pluripotent state. These biomarkers were further confirmed by Western blotting which demonstrated consistent correlation with the pluripotent state across cell lines, and in response to a recovery assay.

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DNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library in haploid human embryonic stem cells (hESCs) differentiated into three germ layers

Project description:DNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library in haploid human embryonic stem cells (hESCs) differentiated into three germ layers

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Expression data of E7.5 primary germ layers

Project description:Expression data of E7.5 primary germ layers

| PRJNA438676 | ENA

DNA methylation profiling of embryonic stem cell differentiation into the three germ layers

Project description:This SuperSeries is composed of the following subset Series: GSE32080: DNA methylation profiling of embryonic stem cell differentiation into the three germ layers [MeDIP analysis] GSE32081: DNA methylation profiling of embryonic stem cell differentiation into the three germ layers [Expression analysis] Refer to individual Series

2012-02-15 | E-GEOD-32082 | biostudies-arrayexpress

RNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library in haploid human embryonic stem cells (hESCs) differentiated into three germ layers

Project description:RNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library in haploid human embryonic stem cells (hESCs) differentiated into three germ layers

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Expression data of E7.5 primary germ layers [microarray]

Project description:Expression data of E7.5 primary germ layers [microarray]

| PRJNA438680 | ENA

High-throughput screen in post-implantation haploid epiblast stem cells reveals Hs3st3b1 as a modulator for reprogramming

Project description:Epiblast stem cells (EpiSCs) derived from post-implantation epiblast are pluripotent stem cells, epigenetically distinct from embryonic stem cells (ESCs), which are widely used in reprogramming studies. Recent achieved haploid cell lines in mammalian open a new era for high-throughput genetic screening, due to their homozygous phenotypes. Here, we report the generation of mouse haploid EpiSCs (haEpiSCs) from post-implantation embryos at embryonic day 6.5 (E6.5). These cells maintain one set of chromosomes, express pluripotent and EpiSC specific genes, and have potentials to differentiate into three germ layers. We also develop a massive mutagenesis protocol with haEpiSCs, and subsequently perform reprogramming genetic screening using this mutation library. Multiple mutants related to various pathway modules are implicated. The validation experiment proves that Knock-out (KO) of Hst3st3b1 (One of the candidates) can promote reprogramming of EpiSCs to ground state efficiently. Our results open the possibility of utilizing haEpiSCs to uncover fundamental biological processes including cell fate alternations.

2021-01-06 | GSE164250 | GEO

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