Molecular mechanisms involved in TGF-β1-induced Muscle-derived stem cells differentiation to smooth muscle cells.

We investigated the molecular mechanisms involved in transforming growth factor beta 1 (TGF-β1)-induced myogenic stem cell differentiation to smooth muscle cells. We isolated muscle-derived stem cells (MDSCs) from gastrocnemius muscles following their identification by immunohistochemistry analysis of desmin and flow cytometry analysis of SCA-1, CD34, and CD45. MDSCs at passage 3 (PP3) were cultured in vitro to examine the effects of MDSC induction. Gene ontology and KEGG pathway analyses were performed to analyze these differentially expressed genes. Reduced representation bisulfite sequencing was performed in TGF-β1-treated and untreated cells to evaluate differences in the methylation status and analyze the chromosomal distribution of differentially methylated sites (DMSs). Significant morphological changes to cells were observed at PP3, and most PP3 cells were positive for desmin and SCA-1, and were confirmed to be MDSCs. Results of western blot and immunohistochemistry analyses suggested that expressions of a-SMA and CNN1 significantly increased after treatment with TGF-β1. Global transcriptome analysis identified 1996 differentially expressed genes (MSC_TGFβ1/MSC_NC). Results of methylome analysis indicated that there were more hypermethylation sites in the untreated group than in the TGF-β1-treated group. Most DMSs were hypermethylated, whereas a small portion was hypomethylated. The chromosomal distribution of DMSs indicated that chromosome 1 had the highest proportion of DMSs, whereas the Y chromosome had the fewest DMSs. Sud2, Pcdh19, and Nat14 are potential core genes involved in cell differentiation. These results may explain the mechanisms of cell differentiation and provide useful information regarding diseases such as pelvic organ prolapse.