Effect of bone morphogenetic protein-4 (BMP-4) on cardiomyocyte differentiation from mouse embryonic stem cell.

The present study was designed to evaluate the effect of BMP-4 on mouse embryonic stem cells (ESCs)-derived cardiomyocyte. Cardiac differentiation of the mouse ESCs was initiated by embryoid bodies (EBs) formation in hanging drops, transfer of EBs to the suspension culture and then plating onto gelatin-coated tissue culture plates. BMP-4 was added to culture medium throughout the suspension period. Cultures were observed daily with an inverted microscope for the appearance of contracting clusters. At the early, intermediate and terminal stages of differentiation, the choronotropic responses of cardiomyocytes to cardioactive drugs were assessed, and the cardiomyocytes immunostained for cardiac troponin I, desmin, alpha-actinin and nebulin. The contracting clusters were isolated for ultrastructural evaluation, at day 14 after plating. Moreover, total RNA extracted from contracting EBs of early and terminal stages of differentiation were examined for oct-4, alpha- and beta-myosin heavy chain, myosin light chain-2V and atrial natriuretic factor expression. The BMP-4 treatment resulted in a decrease in the percent of beating EBs and the percent of developing cardiomyocytes per EBs. As a whole, the chronotropic responses of beating cardiac clusters to cardioactive drugs in control group were better than BMP-4 treated group. The cardiomyocytes of both groups were positive immunostained for applied antibodies except for nebulin. Moreover, in the BMP-4 treated group, the ultrastructural characteristics and cardiac-specific genes expression were all retarded in the terminal stage of cardiomyocytes development. In conclusion, BMP-4 had an inhibitory effect on cardiomyocyte differentiation from the mouse ESCs in terms of ultrastructural characteristics, genes expression and functional properties.