Directed Differentiation of Endothelial Cells

VECs were differentiated as previously described (Pan et al., 2016 (link); Wu et al., 2018 (link)). In brief, human ESC clones were seeded in matrigel coated 6-well plates in mTeSR medium at day 0. ESCs were washed with IMDM medium (Gibco) and cultured in EGM-2 medium (Lonza) supplemented with 25 ng/mL BMP4 (R&D), 3 μmol/L CHIR99021 (Selleck), 3 μmol/L IWP2 (Selleck) and 4 ng/mL FGF2 (JPC) for 3 days. At day 4, Cells were rinsed with IMDM medium and cultured in EGM-2 medium supplemented with 50 ng/mL VEGF (HumanZyme), 10 ng/mL IL6 (Peprotech) and 20 ng/mL FGF2 (JPC) for 3 days. Endothelial cells were sorted with CD201-PE (Biolegend, 351904, 1:300), CD34-FITC (BD biosciences, 555821, 1:100). IgG-FITC (BD biosciences, 555748) and IgG-PE (BD biosciences, 555749) were used as isotype controls.