ROCK suppression continuously promotes the expansion of ECs. (A) ICAM2⁺ ECs were further incubated with PBS (Control), Y27632 (10μM), VEGF-A (50 ng/mL; VA), or Y27632 (10μM) plus VEGF-A (50 ng/mL) for 2 days, and analyses were performed at day 8.5. (B) Immunofluorescence images showing CD144⁺ EC colonies, α-SMA⁺ MCs, and DAPI⁺ nuclei. Scale bars represent 100 μm. (C) Representative FACS analysis of CD31⁺/CD144⁺ ECs. (D) Percentage of CD31⁺/CD144⁺ EC population. Each group, n = 3. *P < .01 versus Control. (E) Relative cell number of ICAM2⁺ ECs. Each group, n = 3. *P < .01 versus Control. #P < .01 versus VEGF-A. (F) Relative population of CD31⁺/CD144⁺ ECs. Population of CD31⁺/CD144⁺ ECs grown without VEGF-A and Y27632 was regarded as 100%. Each group, n = 3. *P < .01 versus Control. #P < .01 versus VEGF-A. (G) Percentage of adherent ICAM2⁺ ECs onto plate at 6 hours. Each group, n = 3. *P < .05 versus VEGF-A. (H) Representative FACS analysis showing cell cycle in ICAM2⁺ ECs at 12 hours. (I) Percentage of ICAM2⁺ ECs under each phase of cell cycle at 12 hours. Each group, n = 3. *P < .01 versus Control. #P < .01 versus VEGF-A. (J) Representative FACS analysis of annexin V⁺/PI⁻ apoptotic ECs. (K) Percentage of annexin V⁺/PI⁻ ECs. Each group, n = 3. *P < .01 versus Control. (L) Estimation and comparison of number of ECs generated from one ESC grown with indicated agents in the 2D Matrigel system at day 6.5 and 8.5. Estimation was calculated from 5 independent incubations.