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Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI1B-dependent blood and SOX17-positive endothelium

ABSTRACT: The genetic regulatory network controlling early fate choices during human blood cell development are not well understood. We use human pluripotent stem cell reporter lines to track the development of endothelial and haematopoietic populations in an in vitro model of human yolk-sac development. We identified SOX17-CD34+CD43- endothelial cells at day 2 of blast colony development, as a haemangioblast-like branch point from which SOX17-CD34+CD43+ blood cells and SOX17+CD34+CD43- endothelium subsequently arose. Most human blood cell development was dependent on RUNX1. Deletion of RUNX1 only permitted a single wave of yolk sac-like primitive erythropoiesis, but no yolk sac myelopoiesis or aorta-gonad-mesonephros (AGM)-like haematopoiesis. Blocking GFI1/1B activity with a small molecule inhibitor abrogated all blood cell development, even in cell lines with an intact RUNX1 gene. Together, our data defines the hierarchical requirements for both RUNX1 and GFI1/1B during early human haematopoiesis arising from a yolk sac-like SOX17- haemogenic endothelial intermediate.

ORGANISM(S): Homo sapiens

PROVIDER: GSE124085 | GEO | 2020/11/11

REPOSITORIES: GEO

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Project description:Human embryonic stem cells (hESCs) are a powerful tool for modeling regenerative therapy. To search for the genes that promote hematopoietic development from human pluripotent stem cell, we overexpressed a list of hematopoietic regulator genes in human pluripotent stem cell-derived CD34+CD43- endothelial cells (ECs) enriched in hemogenic endothelium. Among genes tested, only SOX17, a gene encoding a transcription factor of the SOX family, promoted cell growth and supported expansion of CD34+CD43+CD45-/low cells expressing a hemogenic endothelial maker VE-cadherin. SOX17 was highly expressed in CD34+CD43- ECs but at a low level in CD34+CD43+CD45- pre-hematopoietic progenitor cells (pre-HPCs) and CD34+CD43+CD45+ HPCs. SOX17-overexpressing cells formed sphere-like colonies and generated few hematopoietic progenies. However, they retained hemogenic potential and gave rise to hematopoietic progenies upon inactivation of SOX17. Global gene expression analyses revealed that the CD34+CD43+CD45-/low cells expanded upon overexpression of SOX17 are hemogenic endothelium-like cells developmentally placed between ECs and pre-HPCs. Of interest, SOX17 also reprogrammed both pre-HPCs and HPCs into hemogenic endothelium-like cells. Genome-wide mapping of SOX17 revealed that SOX17 directly activates transcription of key regulator genes for vasculogenesis, hematopoiesis, and erythrocyte differentiation. Depletion of SOX17 in CD34+CD43- ECs severely compromised their hemogenic activity. These findings suggest that SOX17 plays a critical role in priming hemogenic potential in ECs, thereby regulates hematopoietic development from hESCs. This SuperSeries is composed of the SubSeries listed below. ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent, Palo Alto, Calif., USA). MEFs were subjected to ChIP assay using a Ring1B antibody. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification. Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions. Human ES cells were differentiated for 6 days in EBs, then CD34+CD43-CD45- endothelial cells were isolated, plated onto OP9 cells, and transduced with the 4OH-tamoxifen (4OHT)-inducible 3M-CM-^WFLAG-tagged Sox17-ERT retrovirus. The cells were seeded on OP9 stromal cells and cultured in the presence of 4OH-tamoxifen. At day 27 of the co-culture with OP9 cells, CD34+CD43+CD45low hemogenic endothelium-like cells overexpressing Sox17-ERT were collected by CD34 magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip analysis. ChIP on chip analysis was carried out using the Mouse Promoter ChIP-on-chip Microarray Set (G4490A, Agilent, Palo Alto, Calif., USA). MEFs were subjected to ChIP assay using a Ring1B antibody. Purified immunoprecipitated and input DNA was subjected to T7 RNA polymerase-based amplification. Labeling, hybridization and washing were carried out according to the Agilent mammalian ChIP-on-chip protocol (ver.9.0). Scanned images were quantified with Agilent Feature Extraction software under standard conditions. Human ES cells were differentiated for 6 days in EBs, then CD34+CD43-CD45- endothelial cells were isolated, plated onto OP9 cells, and transduced with the 4OH-tamoxifen (4OHT)-inducible 3M-CM-^WFLAG-tagged Sox17-ERT retrovirus. The cells were seeded on OP9 stromal cells and cultured in the presence of 4OH-tamoxifen. At day 27 of the co-culture with OP9 cells, CD34+CD43+CD45low hemogenic endothelium-like cells overexpressing Sox17-ERT were collected by CD34 magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip analysis.

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Human yolk sac-like haematopoiesis generates RUNX1-, GFI1- and/or GFI1B-dependent blood and SOX17-positive endothelium

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