Cardiac Differentiation from Stem Cells

Cardiac differentiation was conducted as described previously by our group^{12 (link)}. Briefly, SMM18 or SMMB2 cells were suspended in serum-free differentiation medium (SFD)^{7 (link)} containing Iscove’s modified Dulbecco’s medium (Thermo Fisher Scientific) and F12 medium (Thermo Fisher Scientific), supplemented with B27 without retinoic acid (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), Glutamax, ascorbic acid (Wako), and 1-thioglycerol for 2 days. Then, cells were primed toward mesoderm by culturing with growth factors including activin-A (R&D Systems), BMP4 (R&D Systems), and VEGF (Wako) from day 2 to day 4. At day 4, cells were plated and induced to the cardiac lineage using bFGF (Wako), FGF10 (R&D Systems), and VEGF. After 7 days, beating-cells were observed. To enrich cardiomyocyte yields, fresh medium-containing puromycin was fed to the cells at day 7 and cultured for 3 days. Following antibiotic selection, more than 90% of cells expressing the cardiomyocyte marker, cardiac troponin T (cTNT), were obtained. The purified cardiomyocytes were used for further analyses. For the long-term culture, we plated the cardiomyocytes at day 10 and cultured in the SFD medium up to day 38 of differentiation.