Figure 2. Functional human hemogenic endothelium can be phenotypically identified and sorted from hESC-RUNX1c-tdTomato cells.

(A) Representative flow cytometry plots of differentiating hESC-RUNX1c-tdTomato cells at Day 11 expressing typical endothelial surface antigens (CD144⁺CD31⁺), but absent for early hematopoietic surface antigens (CD144⁺CD41a⁻, CD144⁺CD43⁻). Phenotypic endothelial cells were gated (top panel) and assessed for tdTomato expression (bottom panel). A fraction of endothelial cells were tdTomato^dim (red histogram) as compared to control hESCs lacking the GFP-RUNX1c-tdTomato reporter (blue histogram), consistent with the identification of hemogenic endothelium. n=2 independent, biological replicates. (B) Schema of FACS sorting hESC-RUNX1c-tdTomato cells into hemogenic endothelium (HE), endothelial cells that lack hematopoietic potential (non-HE), and early hematopoietic progenitor cells (HP). (C) Sorted HE cells seeded on fibronectin-coated wells with EGM-2 media for 5 days and subsequently immunofluorescently imaged for CD31 (left column). HE stained positive for CD31 (purple), GFP (green), and were tdTomato^dim (red) while retaining a stereotypical cobblestone morphology of traditional endothelial cells. Untransfected human umbilical vein endothelial cells (HUVECs) are shown as an endogenous control (right column). (D) Presorted hESC-RUNX1c-tdTomato-derived adherent cells, sorted HE, and sorted non-HE were plated in Stage II hemato-endothelial differentiation media for two additional days to promote hematopoiesis. Both Presort and HE populations robustly generated non-adherent GFP⁺tdTomato⁺ hematopoietic progenitor cells, while non-HE did not adequately support hematopoietic development. Underlying non-HE cells further retain their endothelial morphology. Bar=100μm.