The combination of angiotensin II and 5-azacytidine promotes cardiomyocyte differentiation of rat bone marrow mesenchymal stem cells

Abstract

Bone marrow mesenchymal stem cells (BMMSCs) are ideal seed cells for tissue engineering and regenerative medicine. Many studies have shown that 5-azacytidine (5-aza) can induce BMMSCs to differentiate into cardiomyogenic cells, but some issues still remain to be resolved. In this study, we investigated the effects of angiotensin II (Ang II) on the proliferation and differentiation of BMMSCs induced by 5-aza in vitro. BMMSCs were isolated from the bone marrow of Sprague-Dawley rats by density gradient centrifugation. The third-passage cells were divided into four groups: the Ang II group (0. 1 μmol/l) (group A), the 5-aza group (10 μmol/l) (group B), the Ang II combined with 5-aza group (0.1 and 10 μmol/l) (group C), and the untreated group as control. After 24 h of induction, the medium was changed to the complete culture medium without any inductor, and the cells were cultured for 3 weeks. Morphological changes were observed under a phase contrast microscope. The effect of Ang II and 5-aza on BMMSC proliferation was evaluated by the methyl thiazolyl tetrazolium (MTT) assay. Cardiomyogenic cells were identified through immunofluorescence staining, and the induction ratio was examined by flow cytometry. The level of cardiac troponin I (cTnI) was examined by western blotting, and the ultrastructures of the induced cells were viewed with a transmission electron microscope. The MTT assay showed that the cell proliferation in group C outweighed that in either group A or group B, but no significant difference existed between group A and group B. The expression of specific proteins, namely, cTnI and sarcomeric α-actin in induced BMMSCs was verified as positive. Flow cytometry showed that the induction ratio in group C was higher than that in group A or group B. The protein levels of cTnI in groups A, B, and C were significantly higher than those in the control group. Transmission electron microscopy showed that the induced cells had myofilaments, z line-like substances, desmosomes, and gap junctions. Angiotensin II and 5-azacytidine can promote the proliferation and differentiation of BMMSCs into cardiomyocyte-like cells.