We first addressed the creation of an efficient EC and pericyte differentiation system derived from hiPSCs. We previously reported that the cAMP/protein kinase A/CREB pathway triggers the initiation of endothelial and hematopoietic cell differentiation via Etv2 induction (Yamamizu et al., 2009, Yamamizu et al., 2012a, Yamamizu et al., 2012b). Therefore, we treated cells from primitive mesoderm stage with cAMP and vascular endothelial growth factor (VEGF) based on our cardiomyocyte differentiation method (Masumoto et al., 2014) (Figure 1A). VEGF receptor type 2 (KDR)-positive and platelet-derived growth factor receptor α (PDGFRα)-positive cells appeared in approximately 70% of the cells at 4 days after differentiation. KDR single-positive cells and KDR/PDGFRα double-positive cells appeared after treatment with cAMP and VEGF, and KDR-positive cells were purified by auto MACS at day 5 after the differentiation and then recultured (Figure 1B). At day 9 after differentiation, the percentage of CD31/VE-CADHERIN double-positive ECs was nearly 99%, and these cells showed spindle-like structures (Figures 1C and 1D). All ECs took up low-density lipoprotein (LDL) into the cytoplasm, indicating functional ECs (Figure 1E). At day 12 after differentiation, the
