Abstract

One of the promising approaches for the treatment of cardiac disease is stem cell therapy. In this study, we compared the cardiomyogenic differentiation rate, from human adipose-derived stem cells (hADSCs) in a three-dimensional (3D) hanging drop (HD) spheroid culture system, versus a two-dimensional (2D) culture condition at different concentrations of 5-azacytidine (5-Aza). 5-Azaytidine (5-Aza) is a pyrimidine nucleoside analogue of cytidine that initiates cell differentiation programs through DNA demethylation. The hADSCs were isolated and cultured both in 2D and 3D HD conditions, with either 10 or 50 μM concentrations of 5-Aza. Then DNA content, gene expression, and protein content were analyzed. 3D HD culture resulted in a higher percentage of cells in G0/G1 and S phase in the cell division cycle, whereas 2D culture led to a greater percentage of cells in the G2/M phase. A significantly higher gene expression rate of HAND1, HAND2, cTnI, Cx43, βMHC, GATA4, NKX2.5, and MLC2V was observed in HD treated with 50 μM 5-Aza. This was confirmed by immunocytochemistry. These findings suggest that 50 μM concentration of 5-Aza can induce hADSCs to differentiate into cardiomyocytes. The differentiation rate was significantly higher when accompanied by the 3D HD culture system. This work provides a new culture system for cell differentiation for cardiovascular tissue engineering.