ROCK suppression promotes the differentiation and expansion of ECs from Flk1⁺ MPCs. (A) Protocol for differentiation of ECs. ESCs were cultured without leukemia inhibitory factor for 4.5 days for mesodermal induction. Flk1⁺ MPCs were then sorted by MACS and cultured with VEGF-A (50 ng/mL) for 2 days otherwise indicated in the presence of each indicated inhibitor, and their differentiation status was analyzed on day 6.5. (B) Immunofluorescence image showing CD144⁺ EC colonies, α-SMA⁺ MCs, and DAPI⁺ nuclei in differentiating Flk1⁺ MPCs. Scale bar represents 100 μm. (C) Representative FACS analysis of CD31⁺/CD144⁺ ECs incubated with or without VEGF-A(50 ng/mL). (D) Relative population of CD31⁺/CD144⁺ ECs grown with different inhibitors. Population of CD31⁺/CD144⁺ ECs grown with only VEGF-A (Control) was regarded as 100%. Each group, n = 3. *P < .01 versus Control. (E) Representative FACS analysis of CD31⁺/CD144⁺ ECs incubated with PBS (Control) or Y27632 (10μM). (F) Percentage of CD31⁺/CD144⁺ EC population incubated with various concentrations of Y27632. Each group, n = 3. *P < .01 versus 0. (G) Relative cell number of CD31⁺/CD144⁺ ECs incubated with various concentrations of Y27632. Each group, n = 3. *P < .01 versus 0. (H) Immunofluorescence images showing CD144⁺ EC colonies, α-SMA⁺ MCs, and DAPI⁺ nuclei in differentiating Flk1⁺ MPCs incubated with PBS (Control) or Y27632 (10μM). Scale bars represent 100 μm. (I-J) Comparisons of EC colony formation in a given area (cm²) and cell number in each EC colony at 36 hours after the endothelial induction. Each group, n = 4. *P < .01 versus Control. (K) Percentage of CD31⁺/CD144⁺ EC population incubated with PBS (Control) or Y27632 (10μM) in Flk1⁺ MPCs derived from different kinds of ESCs (E14tg2a and R1) and iPSC. Each group, n = 3. *P < .01 versus Control. (L) Percentage of CD31⁺/CD144⁺ EC population incubated with various ROCK inhibitors. Each group, n = 3. *P < .01 versus Control. (M) Immunoblotting for RhoA, ROCK1, and ROCK2 in different cell types: ESC (E14tg2a), Flk1⁺ MPCs, ECs, and MCs. (N) Percentage of CD31⁺/CD144⁺ EC population incubated with nonspecific control siRNA (Control), siRNA for ROCK1 (siROCK1), ROCK2 (siROCK2), or both (siROCK1 and siROCK2). Each group, n = 4. *P < .01 versus Control.