CD32 allows capturing blood cells emergence in slow motion during human embryonic development

During development, in the embryo proper 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) driven by time-specific Notch signaling activation1. HECs represent an elusive cell population as they are rare and transient, rapidly generating blood cells, and specific markers are lacking. Therefore, it remains unclear how and when the hematopoietic fate is specified and how blood cell emergence is molecularly regulated. Notably, thorough characterization of this process is essential to guide the generation of therapeutic blood products in vitro from human pluripotent stem cells (hPSCs). To identify specific human HEC markers, we performed transcriptomic analysis of 28-32-day human embryos, a developmental stage characterized by active hematopoiesis. We observed that the expression of FCGR2B, encoding for the Fc receptor CD32, 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 human embryos. In addition, clonal analysis revealed that 90% of CD32+ hPSC-derived endothelial cells are bona fide HECs. We leveraged this specificity to study how HECs commit to the blood fate. Remarkably, our analyses indicated that HECs progress through different states culminating with the one identified by CD32 expression. Indeed CD32+ HECs no longer require Notch to generate hematopoietic progeny and display full commitment to hematopoiesis even before the expression of hematopoietic markers. These findings provide a precise method for isolating HECs primed to the blood fate from human embryos and hPSC cultures, thus allowing the efficient generation of hematopoietic cells in vitro.