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

The genetic regulatory network controlling early fate choices during human blood cell development are not well understood. We used 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 aortagonad-mesonephros (AGM)-like haematopoiesis. Blocking GFI1 and/or GFI1B activity with a small molecule inhibitor abrogated all blood cell development, even in cell lines with an intact RUNX1 gene. Together, our data define the hierarchical requirements for RUNX1, GFI1 and/or GFI1B during early human haematopoiesis arising from a yolk sac-like SOX17-negative haemogenic endothelial intermediate.