Fig. 4.

Effective enrichment of MLC-2v-positive human early ventricular cardiomyocytes based on the EGFP selection system. a FACS sorting showing positive cardiomyocytes derived from MYL2^EGFP/w hESCs 25 days after cardiac differentiation. b Representative green fluorescence (EGFP), bright field (BFEGFP), and merged images of MYL2^EGFP/w hESC-derived cardiomyocytes before and after FACS sorting. Scale bars, 100 μm. c Immunofluorescence microscopy showing coexpression of EGFP and MLC-2v in FACS sorted MYL2^EGFP/w hESC-derived cardiomyocytes. Scale bars, 50 μm. d Double immunofluorescence staining showing coexpression of EGFP and MLC-2v in FACS sorted MYL2^EGFP/w hESC-derived cardiomyocytes (1). MLC-2v was expressed in all of the cTnT-positive MYL2^EGFP/w hESC-derived cardiomyocytes post FACS (2). MLC-2a only showed background expression level in the cTnT-positive MYL2^EGFP/w hESC-derived cardiomyocytes post FACS (3). Scale bars, 100 μm. e Vitality assessment of the FACS selected MYL2^EGFP/w-CMs by live cell staining for markers of apoptosis. Representative graph as detected by flow cytometry analysis. f Percentages of ventricular-like, atrial-like, and nodal-like cells produced from MYL2^EGFP/w-CMs before and after FACS sorting as determined by single cell patch clamp. EGFP enhanced green fluorescent protein, MLC-2v cardiac ventricular isoform of myosin light chain-2, V-like ventricular-like cells, A-like atrial-like cells, N-like nodal-like cells