days, the culture medium was replaced with RPMI + B27 medium with 100 ng/mL VEGF165 (Wako) and 1 mM 8-bromoadenosine-3′,5′-cyclic monophosphate sodium salt (8-bromo-cAMP) (Nacalai Tesque) for 1 day. Primitive mesoderm cells were purified with autoMACS (Miltenyi Biotec) using anti-KDR antibody and anti-APC MicroBeads (Miltenyi Biotec) and recultured at a density of 60,000 cells/cm2 with RPMI + B27 medium, 100 ng/mL VEGF165, and 1 mM 8-bromo-cAMP. One day after MACS sorting, the culture medium was replaced with RPMI + B27 medium and 100 ng/mL VEGF165. Then ECs were cultured with human endothelial serum-free medium (SFM) (Life Technologies) supplemented with 20 ng/mL bFGF (Wako), 10 ng/mL EGF (Life Technologies), and 10 μg/mL human plasma fibronectin (Life Technologies), and refreshed every 3 days.
