Cobalt Chloride Pretreatment Promotes Cardiac Differentiation of Human Embryonic Stem Cells Under Atmospheric Oxygen Level

Our previous study demonstrated the direct involvement of the HIF-1 alpha subunit in the promotion of cardiac differentiation of murine embryonic stem cells (ESCs). We report the use of cobalt chloride to induce HIF-1 alpha stabilization in human ESCs to promote cardiac differentiation. Treatment of undifferentiated hES2 human ESCs with 50 mu M cobalt chloride markedly increased protein levels of the HIF-1 alpha subunit, and was associated with increased expression of early cardiac specific transcription factors and cardiotrophic factors including NK2.5, vascular endothelial growth factor, and cardiotrophin-1. When pretreated cells were subjected to cardiac differentiation, a notable increase in the occurrence of beating embryoid bodies and sarcomeric actinin-positive cells was observed, along with increased expression of the cardiac-specific markers, MHC-A, MHC-B, and MLC2V. Electrophysiological study revealed increased atrial- and nodal-like cells in the cobalt chloride-pretreated group. Confocal calcium imaging analysis indicated that the maximum upstroke and decay velocities were significantly increased in both noncaffeine and caffeine-induced calcium transient in cardiomyocytes derived from the cobalt chloride-pretreated cells, suggesting these cells were functionally more mature. In conclusion, our study demonstrated that cobalt chloride pretreatment of hES2 human ESCs promotes cardiac differentiation and the maturation of calcium homeostasis of cardiomyocytes derived from ESCs.