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Genome-scale CRISPR Screen Reveals Essential Biological Process Determining Human Lineage Specification

ABSTRACT: Human pluripotent stem cells (hPSCs) have an unlimited capacity to self-renewal and generate any somatic cell types in the human body, offer a promising cell resource for disease modeling and transplantation. However, the systematic mechanism leading the conversion of hPSCs into lineage specification, which is the earliest cellular event during human embryonic development, is elusive. Here, combining a lineage-specific reporter with inducible CRISPR/Cas9 system, we conducted a genome-scale screening in hESCs for overall drawing the track of cell fate decision. We defined eight functional modules containing embryonic development, mRNA processing, metabolism process, epigenetic regulation, cell cycle and others, that participate in full to the lineage commitment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE132309 | GEO | 2020/11/27

REPOSITORIES: GEO

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SMAD2/3 interactome in human pluripotent stem cells

Project description:This experiment was designed to determine the interactome of SMAD2/3 in human pluripotent stem cells (hPSCs). hPSCs were cultured in standard pluripotency-promoting conditions, or induced to differentiate towards the definitive endoderm lineage for 36h. Endogenous SMAD2/3 was immunoprecipitated from nuclear extracts in these two conditions using a specific antibody. Non-immune IgG immunoprecipitations were performed as negative controls. Three biological replicates per conditions were analyzed by quantitative label-free mass spectrometry.

2018-01-04 | PXD005285 | Pride

Notch-HES1 signaling axis controls hemato-endothelial fate decisions of human embyronic and induced pluripotent cells

Project description:Notch signaling regulates several cellular processes including cell fate decisions and proliferation in both invertebrates and mice. However, comparatively less is known about the role of Notch during early human development. Here, we examined the function of Notch signaling during hematopoietic lineage specification from human pluripotent stem cells (hPSCs) of both embryonic and adult fibroblast origin. Using immobilized Notch ligands and siRNA to Notch receptors we have demonstrated that Notch1, but not Notch2 activation, induced HES1 expression and generation of committed hematopoietic progenitors. Using gain and loss of function approaches, this was shown to be attributed to Notch signaling regulation through HES1, that dictated cell fate decisions from bipotent precursors either to the endothelial or hematopoietic lineages at the clonal level. Our study reveals a previously unappreciated role for the Notch pathway during early human hematopoiesis, whereby Notch signaling via HES1 represents a toggle switch of hematopoietic vs. endothelial fate specification. Human pluripotent stem cells (hPSCs) have differentiation potential into three embryonic germ layers including blood. Notch signaling is one of important signaling pathways involved in blood differentiation of hPSCs. Thus, in order to examine the effect of Notch signaling pathways during hematopoietic differentiation of hPSCs, embryoid bodies (EBs) were formed and cultured for 10 days in the combination of cytokines and growth factors (Chadwick, Blood, 2003; 300 ng/ml of SCF, 300 ng/ml of Flt-3L, 10 ng/ml of IL-3, 10 ng/ml of IL-6, and 50 ng/ml of G-CSF) to induce differentiation into blood. Additionally, CD31+CD45- bipotent hemogenic precursors were isolated from day10 hematopoietic EBs (Wang et al., Immunity, 2004)

2013-06-01 | E-GEOD-47466 | biostudies-arrayexpress

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Project description:Graphene has been selected as a candidate for synthetic feeder-free culture substrate guiding human/mouse multipotent stem cell lineage specification, and culturing pluripotent stem cells in a number of studies. However, conventional graphene is not an ideal biomaterial to maintain the pluripotency of human pluripotent stem cells (hPSC) including hESCs/hiPSCs due to its intrinsic hydrophobicity and relatively flat surface topography. Here, we applied morphology-controlled nanocrystalline graphene (NG) coating onto the culture substrates via diffusion-associated synthesis (DAS) process and cultivated hPSCs. It is found that enhanced hydrophilicity and controlled surface roughness of DAS-NG enabled tight focal adhesion of hPSCs onto the DAS-NG coated culture substrate and retained pluripotency for over 2 weeks. It is also found hPSCs grown on DAS-NG shared comparable global gene expression profile with hPSCs grown on mouse embryonic fibroblast (MEF). Importantly, the similarities in cell adhesion gene expression between hPSCs grown on DAS-NG and hPSCs on MEF suggest DAS-NG may provide comparable physical cues with MEF for sustaining pluripotency. Taken together, our findings show a new reliable method for culturing hPSCs in feeder-free condition using DAS-graphene. Human iPSCs and human ESC H9 were seeded onto DAS-NG coated glass, ITO or QU, CVD-grown graphene coated glass, uncoated glass and mitomycin-C treated CF1. Human pluripotent stem cells seeded on each culture substrate were cultured in human embryonic stem cell medium composed of knockout DMEM (GIBCO-10829) supplemented with 20% knockout serum replacement (GIBCO-10828), 1mM L-glutamine, 1% penicillin/streptomycin (PAA-P11-010), 1% MEM-non essential amino acid (PAA-M11-003), 0.1mM beta-mercaptoethanol, and 5ng/ml human basic fibroblast growth factor.

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Genome-scale CRISPR Screen Reveals Essential Biological Process Determining Human Lineage Specification

Project description:Genome-scale CRISPR Screen Reveals Essential Biological Process Determining Human Lineage Specification

| PRJNA547581 | ENA

Expression data from human iPS cells treated by a small molecule KY02111

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2016-10-17 | GSE81074 | GEO

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros

2016-10-17 | GSE81080 | GEO

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2022-04-12 | GSE158887 | GEO

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