Combination of BMP-2 and 5-AZA is advantageous in rat bone marrow-derived mesenchymal stem cells differentiation into cardiomyocytes

Abstract

Bone morphogenetic protein-2 (BMP-2) has a crucial role in the development of cardiogenesis, and is used in inducing bone marrow mesenchymal stem cells (BMMSCs) to differentiate into cardiomyocytes.We have examined a combination of BMP-2 and 5-azacytidine (5-AZA) in inducing these differentiation effects. BMMSCs were collected and purified from bone marrow of 4-week-old Sprague-Dawley (SD) rats by density-gradient centrifugation and differential attachment. The fourth passage subculture of BMMSCs, selected by cytometry for purity and identification, was divided into four groups: a control group, BMP-2 treated, 5-AZA treated, and a combination of BMP-2 and 5-AZA treatment. Expression of cardiac Troponin I (cTnI) and Connexin 43 (CX-43) in BMMSCs after induction were detected by immunofluorescence and Western blot. Flow cytometry analysis was used for differentiation rates and apoptosis of induced BMMSCs, through the expression of cardiac Troponin T (cTnT) and Annexin V-FITC&PI kit, respectively. BMP-2 can ameliorate apoptosis of BMMSCs caused by 5-AZA and promote the differentiation of BMMSCs into cardiomyocyte-like cells. Thus a combination of BMP-2 and 5-AZA can significantly improve the cardiac differentiation with fewer cell damage effects, making it a safe and effective method of induction in vitro. © 2013 International Federation for Cell Biology.