Modulation of Stem Cells Differentiation and Myostatin as an approach to Counteract fibrosis in Muscle Dystrophy and Regeneration after Injury

摘要

We have completed in Year 3 the in vitro characterization of the three muscle derived stem cells (MDSC) isolated from the wild type (WT), myostatin knock out (Mst KO), and mdx mice. We had shown in Years 1 and 2 that the in vitro myogenic differentiation and myotube formation by MDSC was refractory to modulation by myostatin, follistatin, myostatin antibodies and shRNA, hormones, and nitric oxide, and specifically that the Mst KO and mdx MDSC were totally unable to form myotubes.. We have now found out: a) that this inability of the Mst KO and mdx MDSC to form myotubes in vitro occurs despite they can differentiate into myofibroblasts and neural and smooth muscle cells, albeit not into adipocytes, b) is not overcome even in dual paracrine and juxtacrine cultures with WT MDSC and with C2C12 myoblasts, and c) is not accompanied by the silencing of stem cell genes, like Sca 1 and Oct-4. MDSC from a myostatin+/dystrophin+ transgenic mouse expressing gfp under the Oct-4 promoter, morphologically resemble very small embryonic-like (VSEL) stem cells. Although the three MDSC cultures showed similar levels of expression for 98% of the 260 stem cell and myogenic genes assayed, the genetic inactivation of myostatin or dystrophin was associated with a dramatic down-regulation of Actc1, Acta1, Spp1, MyoD, and notch 2, critical genes for the onset of myogenesis. We also have defined novel antifibrotic strategies to apply concurrently with MDSC implantation into mdx mice during Year 4. The in vivo studies to determine the capacity of WT MDSC alone to repair notexin-injured diaphragm and gastrocnemius in old mdx mice are now ongoing.