Developmental regulation of endothelial-to-hematopoietic transition from iPSCs [sci-RNA-seq]
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PROVIDER: PRJNA1144913 | ENA |
REPOSITORIES: ENA
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Project description:Hematopoietic stem cells (HSCs) arise in mid-gestation from a specialized hemogenic endothelium (HE). In this process, HE cells undergo a unique fate change termed endothelial-to-hematopoietic transition, or EHT. While induced pluripotent stem cells (iPSCs) give rise to HE with robust hemogenic potential, the generation of bona fide HSCs from iPSCs remains a challenge. Here, we map single cell dynamics of EHT during embryoid body differentiation from iPSCs and integrate it with human embryo datasets to identify key transcriptional differences between in vitro and in vivo cell states. We further map ligand-receptor interactions associated with differential expression of developmental programs in the iPSC system. We found that the expression of endothelial genes was incompletely repressed during iPSC EHT. Elevated FGF signaling by FGF23, an endothelial pathway ligand, was associated with differential gene expression between in vitro and in vivo EHT. Chemical inhibition of FGF signaling during EHT increased HSPC generation in the zebrafish system, while an FGF agonist had the opposite effect. Consistently, chemical inhibition of FGF signaling increased hemogenic output from iPSCs. In summary, we map the dynamics of EHT from iPSCs at single cell resolution and identify ligand-receptor interactions that can be modulated to improve iPSC differentiation protocols. We show, as proof of principle, that chemical inhibition of FGF signaling during EHT improves hematopoietic output in zebrafish and the iPSC system.
2024-08-23 | GSE274082 | GEO
Project description:Hematopoietic stem cells (HSCs) arise in mid-gestation from a specialized hemogenic endothelium (HE). In this process, HE cells undergo a unique fate change termed endothelial-to-hematopoietic transition, or EHT. While induced pluripotent stem cells (iPSCs) give rise to HE with robust hemogenic potential, the generation of bona fide HSCs from iPSCs remains a challenge. Here, we map single cell dynamics of EHT during embryoid body differentiation from iPSCs and integrate it with human embryo datasets to identify key transcriptional differences between in vitro and in vivo cell states. We further map ligand-receptor interactions associated with differential expression of developmental programs in the iPSC system. We found that the expression of endothelial genes was incompletely repressed during iPSC EHT. Elevated FGF signaling by FGF23, an endothelial pathway ligand, was associated with differential gene expression between in vitro and in vivo EHT. Chemical inhibition of FGF signaling during EHT increased HSPC generation in the zebrafish system, while an FGF agonist had the opposite effect. Consistently, chemical inhibition of FGF signaling increased hemogenic output from iPSCs. In summary, we map the dynamics of EHT from iPSCs at single cell resolution and identify ligand-receptor interactions that can be modulated to improve iPSC differentiation protocols. We show, as proof of principle, that chemical inhibition of FGF signaling during EHT improves hematopoietic output in zebrafish and the iPSC system.
2024-08-23 | GSE274084 | GEO
Project description:Developmental regulation of endothelial-to-hematopoietic transition from iPSCs [10X 3' sc-RNA-seq]
| PRJNA1144919 | ENA
Project description:This dataset contains single cell RNAseq of human iPSCs undergoing cytokine-mediated differentiation alongside targeted CRISPR-mediated endogenous gene activation. For this experiment we sequenced single cells at day 10 of the differentiation protocol, when the cells undergo the endothelial-to-hematopoietic transition described in the paper Petazzi et al, BiorXiv DOI 10.1101/2022.10.04.510611 The dataset contains four libraries: (1) iSAM line infected with non-targeting guide RNA, (2) iSAM line infected with non-targeting guide RNA treated with Dox, (3) iSAM line infected with targeting guide RNAs library, (4) iSAM line infected with targeting guide RNAs library treated with Dox
2024-03-21 | E-MTAB-12748 | biostudies-arrayexpress
Project description:Single-cell RNAseq of differentiating human iPSCs during endothelial-to-hematopoietic transition with targeted CRISPR activation
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Project description:Corneal endothelial cells (CECs) are critical to maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cells (PBMC)-originated induced pluripotent stem cells (iPSCs)-derived CECs. We isolated PBMC and programmed the mononuclear cells to generate iPSCs. Subsequently, the PBMC-originated iPSCs were differentiated to CECs. The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent, and CECs-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSCs-derived CECs and human corneal endothelium (CE) were examined by mass spectrometry-based proteome sequencing. The PBMC-originated iPSCs expressed pluripotent-specific markers at levels similar to expression in H9 human embryonic stem cells (hESCs). Phase contrast microscopy illustrated that iPSCs-derived CECs are tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CECs-associated markers were expressed at many orders of magnitude higher in iPSCs-derived CECs at days 13, 20, and 30 compared to their respective levels in iPSCs. Importantly, only residual expression levels of pluripotency markers were detected in iPSCs-derived CECs. Mass spectrometry-based proteome profiling identified 10,575 proteins in iPSCs-derived CECs. In parallel, we completed proteome profiling of the human CE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSCs-derived CECs suggesting a 90.82% overlap between the iPSCs-derived CECs and human CE proteomes. Importantly, cryopreservation of iPSCs-derived CECs did not affect the tight adherence of CECs, and their hexagonal-like shape while expressing high levels of CECs-associated markers. We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the human CE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSCs-derived CECs.
2018-06-06 | PXD009142 | Pride
Project description:Here, we focused on the intermediate stages of SCR by comparing the somatic cell line induced by OCT4, SOX2, and KLF4 (OSK) for 7 days with mouse embryonic fibroblasts (MEFs), iPSCs, and embryonic stem cells (ESCs). Transcriptional profiles of these four cell lines were analyzed by microarray, and we found that the transition process from day 7 to the formation of iPSCs is crucial for SCR and that the reverse expression patterns can provide more candidate markers to distinguish ESCs and somatic cells iPSC. Data confirmed that the viral infection results in defense innate immunity, DNA damage, and apoptosis in MEFs, which slows down cell proliferation and immortalization to inhibit SCR. Although SCR is initiated by OSK, the p53 signaling pathway can affect the transcriptional regulatory networks through cell cycle and genomic instability as a powerful core node. MEFs were derived from embryonic day 13.5 C57BL6 mice embryos. Cell line day7 is MEF induced by OCT4, SOX2, and KLF4 (OSK) for 7 days with mouse embryonic fibroblasts (MEFs), iPSCs, and embryonic stem cells (ESCs).
2012-10-01 | E-GEOD-37397 | biostudies-arrayexpress
Project description:Oct4 is widely considered the most important among the four Yamanaka reprogramming factors. Here we show that the combination of Sox2, Klf4, and cMyc (SKM) suffices for reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs). Simultaneous induction of Sox2 and cMyc in fibroblasts triggers immediate retroviral silencing, which explains the discrepancy with previous studies that attempted but failed to generate iPSCs without Oct4 using retroviral vectors. SKM induction could partially activate the pluripotency network even in Oct4-knockout fibroblasts. Importantly, reprogramming in the absence of exogenous Oct4 results in greatly improved developmental potential of iPSCs, determined by their ability to give rise to all-iPSC mice in the tetraploid complementation assay. Our data suggest that overexpression of Oct4 during reprogramming leads to off-target gene activation during reprogramming and epigenetic aberrations in resulting iPSCs, and thereby bear major implications for further development and application of iPSC technology.
2019-11-06 | GSE137001 | GEO