Figure 3.
(A) Schematics of optimized cardimyogenic differentiation protocol to obtain in vitro CSC-derived cardiomyocytes. (B) c-kitpos (green) CSC cardiospheres express multipotent stemness markers and Wnt3a (red). Bar = 50µm. (C) Representative light microscopy images showing (top) clonal CSC-derived cardiospheres grown in suspension and (bottom) differentiated cardiospheres 14 days after the treatment with the optimized stage-specific TGF-β-Family/Wnt Inhibitor cocktail. (D) Cumulative RT-PCR data for the expression of myogenic genes in CSC-derived cardiospheres from day 0 to day 14 in the optimized stage-specific TGF-β-Family/Wnt Inhibitor cocktail. Ccna2, Cyclin A2; Tnnt2, cardiac troponin t2; Myl2, myosin light chain 2; Myh, myosin heavy chain; Actc1, cardiac Actin; Pln, phospholamban; Atp2a, ATPase Sarcoplasmic/Endoplasmic Reticulum Ca2+ Transporting 1 (also known as SERCA Ca(2+)-ATPases). *p < 0.05 vs. 0d; #p < 0.05 vs. 1d; ^p < 0.05 vs. 5d; §p < 0.05 vs. 10d. (E) Dissociated cells from CSC-derived beating cardiospheres stain positive for the cardiomyocyte lineage, exhibiting sarcomeric structures. MHC, myosin heavy chain; MLC2A, myosin light chain 2A; cTnI, cardiac troponin I. DAPI (blue staining) depicts cell nuclei. Bar = 20 µm..(adapted from Vicinanza et al. 2017 [25]).