449. IGF-1 Treatment Enhances the Myogenic Potential of Human Skeletal Muscle-Derived Stem Cells

Human muscle-derived stem cells (hMDSCs) have been shown to promote the regeneration of a variety of tissues, including damaged heart, peripheral nerve, bone, articular cartilage and skeletal muscle1xSee all References, 2xSee all References, 3xSee all References; however, without appropriate stimulation their differentiation potential remains limited.4xSee all References In this study we examined whether insulin-like growth factor 1 (IGF-1) treatment could also enhance the myogenic potential of hMDSC, since it has been shown that IGF-1 treatment increased the myogenic potential of bone marrow-derived stem cells (BMSCs).5xSee all References Methods: HMDSCs were isolated from human adult muscle biopsies via a modified preplate technique, as previously described.6xSee all References Young female hMDSCs were plated in proliferation medium at a density of 1.58 × 104 cells/cm2. After 24h in proliferation medium, the cells were cultured for an additional 7 days in myogenic medium containing various levels of IGF-1: 0ng/mL, 50 ng/mL, 100 ng/mL, or 200 ng/mL. To evaluate the extent of myogenesis, cells were fixed in MeOH and stained for fast myosin heavy chain (fMHC). Activity levels of creatine kinase, which is highly expressed in tissues like skeletal muscle that rapidly consume ATP, were measured according to the company's protocol. Additionally, RNA was extracted from cells, and cDNA was synthesized. Semi-quantitative PCR and qRT-PCR analyses were performed for myogenin, a late myogenic differentiation marker. Results: (1) fMHC staining indicated that IGF-1 treatment increased myotube formation (Fig. 1AFig. 1A). However, the mortality rates of the cells in all IGF-1 treatment groups were increased compared to the non-treated group. (2) hMDSCs treated with 50 ng/ml IGF-1 had significantly higher CK activity compared to all other groups when normalized to total protein. Furthermore, cells treated with 200 ng/ml IGF-1 also had significantly more CK activity than the untreated cells. This suggests that IGF-1 may enhance CK activity at low levels. At higher IGF-1 concentrations, CK activity may be reduced due to increased cell death (Fig. 1BFig. 1B). (3) Semi-quantitative analysis showed that IGF-1 treatment, at all concentrations, increased myogenin expression. The 100ng/mL concentration appeared to have the greatest effect. These results were corroborated by qRT-PCR analysis, which indicated that, compared to the non treated group, the 50ng/mL, 100ng/mL, and 200ng/mL groups had a 16.1 fold, 30.1 fold, and 14.3 fold increase in myogenin expression, respectively. Conclusion: These results indicate that IGF-1 enhances the myogenic potential of hMDSCs. However, we observed more cell death in IGF-1 treatment groups, which may be a result of overgrown cells during differentiation. In the future, we will determine whether IGF-1 treated hMDSCs display a higher regenerative potential in skeletal muscle, after injury and disease, than non-treated hMDSCs.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide