Effects of Fluid Dynamic Forces Created by Rotary Orbital Suspension Culture on Cardiomyogenic Differentiation of Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the potential to become an effective resource for regenerative medicine in heart disease. The efficiency of cardiomyocyte differentiation changes drastically depending on the quality of hESC aggregates known as embryoid bodies (EBs). This study compares the production of EBs in rotary orbital suspension culture at various speeds with that in a simple static suspension culture. None of the outgrowths of EBs formed in static suspension culture developed beating. In contrast, outgrowths from EBs formed at 100 rpm had the highest rate of beating, 70%, and increased the expression of Nkx2.5, a master gene of cardiomyocyte differentiation. Outgrowths from EBs developed at slower rotational speeds showed more endoderm gene expression. At faster speeds, the expression of ectoderm markers increased. A computational hydrodynamic simulation showed that the liquid medium shear stress on the bottom was restricted to the periphery at 30 and 55 rpm and the center at 120 rpm. The analysis showed that at 100 rpm, the shear stress was uniformly distributed in the dish. Our results suggest that shear stress by fluid dynamic forces induces the differentiation of specific cell phenotypes from hESCs depending on rotational speed. For cardiomyocyte differentiation, 100 rpm was the most effective speed.