The Fc receptor CD32 identifies primed human hemogenic endothelial cells [scRNA-seq]

During embryonic development, blood cells emerge from a subset of specialized endothelial cells, named hemogenic endothelial cells (HECs), via a process known as endothelial-to-hematopoietic transition (EHT). A thorough characterization of HECs and their EHT is essential to guide the efforts to derive this population from human pluripotent stem cells (hPSCs), a critical step to generate therapeutic blood products in vitro. However, current known markers used to isolate HECs are insufficient as they also enrich for arterial endothelial cells that are associated with HECs. To identify specific human HEC markers, we performed transcriptomic analysis of 28-32-day human embryos, a developmental stage characterized by active EHT. We observed that the expression of FCGR2B, encoding for the Fc receptor CD32 previously associated with other specialized endothelia, is highly enriched in the ACE+CD34+ endothelial cell population that contains HECs. Functional ex vivo analyses confirmed that multilineage hematopoietic potential is highly enriched in CD32+ endothelial cells isolated from the aorta-gonad-mesonephros region and yolk sac of human embryos. In addition, CD32 emerged as selective marker for hPSC-derived HECs across different hematopoietic programs. Remarkably, our analyses showed that CD32 expression identifies HECs that are irreversibly bound to undergo EHT. As such, CD32 expression enriches for cells with hemogenic potential with a higher specificity for hPSC-derived HECs than other known HEC markers. These findings provide a simple method for isolating HECs from human embryos and hPSC cultures, allowing the efficient generation of hematopoietic cells in vitro. Overall design: H1 human embryonic stem cells were expanded in serum-free conditions and differentiated towards endothelial and hematopoietic fate as reported in Ditadi et al., 2015 (doi.org/10.1038/ncb3161); Ditadi et al, 2016 (doi.org/10.1016/j.ymeth.2015.10.001). At day8 of Wnt-dependent hematopoietic cultures, CD34+CD43-CXCR4-CD73- cells (referred to as HE) were isolated by FACS sorting and used for RNA extraction, library preparation and single cell RNA sequencing.