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 percentage of CD31-positive ECs was about 91% and PDGFRβ-positive pericytes about 7% (Figure 1F). The PDGFRβ+/CD31− pericytes were purified by fluorescence-activated cell sorting (FACS) and positive for α-smooth muscle actin (αSMA), SM22α, NG2, and CALPONIN (Figure 1G). We examined the function of these ECs by using the tube formation assay. The ECs generated vascular-like structures on Matrigel. Gel immunostaining showed that these structures consisted of CD31-positive ECs attached to αSMA-positive pericytes (Figure 1H).
