(A) MHCPim-wt and (B) NTG CSCs differentiated on fixed NRCMs for 7 days, fixed and immunolabeled for Pim (green), α-sarcomeric actinin (red) and Topro (blue). MHCPim-wt differentiated CSCs express more Pim protein. (C) MHCPim-wt and (D) NTG CSCs differentiated on fixed NRCMs for 7 days, fixed and immunolabeled for GFP (green), α-sarcomeric actinin (red) and Topro (blue). MHCPim-wt differentiated CSCs express GFP. (E) MHCAkt-nuc and (F) NTG CSCs differentiated on fixed NRCMs for 7 days, fixed and immunolabeled for myc-tag (green), α-sarcomeric actinin (red) and Topro (blue). MHCAkt-nuc differentiated CSCs express increased nuclear-localized myc-tag. Scale bar is 40 μm. (G) RT-qPCR analysis of NTG CSCs postdifferentiation on live NRCMs shows an increase in relative mRNA levels of troponin T (left), tropomyosin (middle) and α-smooth muscle actin (right) compared with undifferentiated CSCs.
CSC: Cardiac stem cell; GFP: Green fluorescent protein; MHC: Myosin heavy chain; NRCM: Neonatal rat cardiomyocyte; NTG: Nontransgenic.