Endothelial Differentiation of iPSCs

Endothelial differentiation was performed as described previously with some modifications^{24 (link)}. The iPSCs were cultured in mTeSR1 medium (Stemcell Technologies) on dishes coated with iMatrix511 (Nippi; (0.5 µg/cm²). After the colony size was grown up to 750–1000 μm in diameter, the medium was changed to Essential8 medium (Thermo Fisher Scientific) containing 4 μM of CHIR99021 (Wako), 80 ng/mL of BMP4 (HumanZyme), and 80 ng/mL of VEGF (Thermo Fisher Scientific). Then 48 to 51 h later, cells were detached using TrypLE Express (Thermo Fisher Scientific) and seeded onto dishes coated with LM411-E8 (Nippi; 0.4 μg/cm²) with a density of 2500 ~ 10,000/cm² in Stempro-34 SFM (Thermo Fisher Scientific) containing 80 ng/ml of VEGF. Four to six days later, cells were detached using TrypLE Express and sorted using Alexa Fluor 647 mouse anti-human CD31 antibody (1 μg/1 × 10⁶ cells; BD Biosciences) and PE mouse anti-human CD144 antibody (0.5 μg/1 × 10⁶ cells; BD Biosciences) with a FACSAria II flow cytometer (BD Biosciences).