Optimal population of embryonic stem cells in hanging drop culture for in-vitro differentiation to cardiac myocytes

Pluripotent embryonic stem (ES) cells differentiate to cardiac myocytes in vitro by many other previous reports demonstrated "hanging-drop" method. In this study, the number of ES cells in each hanging-drop plays an important role in the cultivation of cardiac myocytes. We examined the optimal hanging-drop size to obtain embryonic stem cell-derived cardiac cells (ESCMs) in vitro using specific labeled mouse ES cells (hCGP7) which were stably transfected with the enhanced green fluorescent protein (GFP) under transcriptional control of the cardiac myocyte-specific promoter with fluorescence-activated cell sorting (FACS) analysis. GFP-positive cells were sorted efficiently in the hanging-drop cell number of approximately 800-1600 cells/drop. The present data will be an important element to establish the optimal methods of ES cellular differentiation in vitro.