VYI-ECs have neovasculogenic capabilities in vitro and in vivo. (A) Diagram of VYI-EC preparation for analyses of neovasculogenic activities. VYI-ECs were applied for tube-forming assay, scratch wound healing assay, microfluidic angiogenesis assay using VEGF-A (50 ng/mL; Control) or VEGF-A (50 ng/mL) plus Y27632 (10μM; Y72632) as supplemental materials, and in vivo Matrigel plug implantation assay. (B) Representative phase-contrast images showing network and branch formations of VYI-ECs at 12 hours. Scale bars represent 100 μm. (C) Number of branch points in a given area (cm²). Branch point was defined as the contact point of 3 or more endothelial tubes. Each group, n = 3. *P < .05 versus Control. (D) Representative phase-contrast images showing invasion of VYI-ECs (white dotted lines) into wound regions at 6 hours. White solid lines indicate the boundaries of wounding. Scale bars represent 100 μm. (E) Percentage of invasions. Area of wound at 0 hours is regarded as 100%. Each group, n = 3. *P < .05 versus Control. (F left): Representative phase-contrast and immunofluorescence images showing the sprouting of CD144⁺/FITC-lectin⁺ ECs into ECM scaffold at 48 hours after cell seeding. Upper ECM scaffold provides a negative gradient of Y27632 (0-10μM; Control), whereas lower ECM scaffold provides a constant concentration of Y27632 (10μM; Y27632). Nuclei were stained with DAPI. Scale bars represent 100 μm. (Right): Average length of sprouting ECs. *P < .05 versus Control. (G) Diagram of in vivo Matrigel plug assay. VYI-ECs derived from iPSCs were mixed with Matrigel supplemented with VEGF-A (500 ng/mL) and implanted into the dorsal flank of Tie2-GFP mouse. Implanted Matrigel was immunostained for CD31⁺ blood vessels and stained for DAPI⁺ nuclei. Donor-derived CD31⁺/GFP⁻ blood vessels are formed in the gel, whereas invasion of recipient-derived CD31⁺/GFP⁺ blood vessels into the gel is detected. White arrows indicate the implanted VYI-ECs that have been integrated into recipient vessel. Scale bars represent 50 μm.