Fig. 3.
Effective enrichment of MLC-2v-positive human early ventricular cardiomyocytes based on the neomycin selection system. a Compared with the wildtype control (H7-derived cardiomyocytes), MYL2Neo/w hESC-derived cardiomyocytes show normal growth following G418 selection for 8–10 days after plating, while control cells died completely. b Flow cytometry analysis shows a higher percentage of MLC-2v-positive cells derived from MYL2Neo/w hESCs post G418 selection. c Representative immunofluorescence staining images reveal that MLC-2v was expressed in almost all of the G418 selected cTnT-positive MYL2Neo/w hESC-derived cardiomyocytes, while in control cells without G418 selection only partial cells showed MLC-2v expression. Scale bars, 100 μm. d Quantification of MLC-2v/cTnT expression ratio in MYL2Neo/w hESC-derived cardiomyocytes before and post G418 selection. n = 298, ***P < 0.001 by two-tailed Student’s t test. e–g Quantitative PCR showing expression of ventricular markers MYL2 and HAND1 (e) was significantly upregulated, while atrial markers GJA5, TBX5, and MYH6 (f) and nodal markers SHOX2, TBX3, and TBX18 (g) were significantly reduced in MYL2Neo/w hESC-derived cardiomyocytes post G418 selection. Data shown as the mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ns not statistically significant by two-tailed Student’s t test. h Quantification of beating rates for MYL2Neo/w hESC-derived cardiomyocytes in response to cardiac pharmaceutical reagents before and post G418 selection. i Percentages of ventricular-like, atrial-like, and nodal-like cells produced from MYL2Neo/w hESC-derived cardiomyocytes 30 days after cardiac differentiation before and post G418 selection as determined by single cell patch clamp. MYL2 myosin light chain 2, V-like ventricular-like cells, A-like atrial-like cells, N-like nodal-like cells