Impacts of STRA6 deletion in the in vitro hESC differentiation into CMs and SMCs. (A and B) Flow cytometry analysis showing the ratios of wild-type (WT) and STRA6-KO hESC-derived STRA6⁺ cells at Day 6 in CM differentiation. SSC, side scatter. **P < 0.01. (C) Western blotting analysis for expression of STRA6 protein with β-actin protein (a loading control) in WT and STRA6-KO cells at Days 3 and 6 in hESC-CM differentiation. (D and E) Representative images on flow cytometry analysis showing the ratios of a cell proliferation marker Ki67⁺ (left), an SHF/heart progenitor marker ISL1⁺ (middle), and a differentiated CM marker TNNT2⁺ (right) in WT (top) and STRA6-KO (bottom) cells at Days 6 (D) and 12 (E) in hESC-CM differentiation. (F) Statistical data of the ratios of %Ki67⁺ (left), %ISL1⁺ (middle), and %TNNT2⁺ (right) in (D) and (E). ^#P = not significant. (G) Flow cytometry analysis and statistical data showing the ratios of vascular SMCs (PDGFRB⁺SM22⁺) at Day 6 in mesodermal SMC (mSMC) differentiation of WT and STRA6-KO hESCs with or without treatment with retinoic acid (RA, 0.5 μM) during Days 1–6. *P < 0.05 and **P < 0.01. (H) Flow cytometry analysis and statistical data showing the ratios of vascular SMCs (PDGFRB⁺SM22⁺) at Day 14 in NCC-derived SMC differentiation of WT and STRA6-KO hESCs with or without treatment with RA (0.5 μM) during Days 6–10. **P < 0.01 and ***P < 0.001. Differences between groups were examined with one-way ANOVA followed by Tukey–Kramer post hoc test.