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Bone marrow mesenchymal stem cells (MSCs) can differentiate into smooth muscle cells (SMCs) and have tremendous potential for cell therapy and tissue engineering. In this study, to understand the effects of TGF-beta3 on rat bone marrow-derived MSCs and the underlying molecular mechanism of this differentiation process, we investigated that the changes of myocardin-related transcription factors (MRTFs) at the transcriptional level after rat MSCs were treated with TGF-beta3. The results showed that TGF-beta3 increased the expression of contractile genes, such as SM22, smooth muscle-myosin heavy chain (SM-MHC), SM-alpha-actin in MSCs. When TGF-beta3 induced MSCs differentiation into SMCs, myocardin and MRTF-A were activated. The data indicated that TGF-betai3 induced rat bone marrow-derived MSCs differentiation into SMCs by activating mypcardin and MRTF-A.