AIM To investigate the function of Wnt/β-catenin pathway in inducing rat bone marrow mesenchymal stem cells (BMMSCs) to cardiomyocytes.
METHODS Third generation BMMSCs were divided into Ang II combined with 5-aza group (angiotensin II, final concentration 0.1 μmol/L + 5-azacytidine, final concentration 10 μmol/L) and control group. After induction for 24 hours, the induction medium was abandoned and the cells were cultured in complete medium for 4 weeks. Morphological changes, proliferation ability, induction differentiation rate, expression of α-actin and ultrastructure were respectively detected by inverted phase contrast microscopy, MTT, flow cytometry, immunofluorescence staining and transmission electron microscopy. The expression level of Wnt and β-catenin was detected by Western blot.
RESULTS BMMSCs showed various morphologies in primary culture. After passage, BMMSCs grew in long shuttle shape and showed uniform growth after induction. MTT assay showed that the growth rate of Ang II combined with 5-aza group was faster than that in control group. Flow cytometry showed that the induction rate of cardiomyocytes in Ang II combined with 5-aza group was (31.2 ± 1.7)% and that in control group was (1.1 ± 0.2)%. Immunofluorescence staining showed that after induction BMMSCs expressed α-actin positively in Ang II combined with 5-aza group. Myofilament and gap junction were seen by transmission electron microscopy. Western blot results showed that the expression levels of Wnt and β-catenin in Ang II combined with 5-aza group were significantly higher than those in control group (P < 0.05).
CONCLUSION Ang II combined with 5-aza can promote the differentiation of BMMSCs into cardiomyocytes, which may be related to the activation of Wnt/β-catenin signaling pathway.
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