Different methods of inducing differentiation of rat skeletal muscle satellite cells into insulin-producing cells: a comparative study

Abstract

Objective To investigate the effects of different methods of inducing differentiation of rat skeletal muscle satellite cells (MDSCs) into insulin-producing cells (IPCs) , and to provide a experimental and theoretical basis for stem cell therapy of type 1 diabetes. Methods The rat MDSCs were isolated and cultured by twice-enzyme digestion and differential adherent culture, and differentiated into IPCs by different induction methods. The morphology of MDSCs-IPCs was identified by dithizone staining. Immunohistochemistry was used to detect the expression of Q-Desmin, α-Sarcomeric Actinin, MyoD1, Myf5, PAX7 genes and identify MDSCs. The expression levels of C-peptide and insulin in MDSCs-IPCs were determined by Q-PCR and Western blotting. The biological function of MDSCs-IPCs was examined by the insulin release test. Results At 4 h after inoculation, some adherent MDSCs developed small protrusions. At 48 h, most of the adherent cells showed the shape of spindle with abundant cytoplasm and high refractive index. The fusiform shape of the cells became more apparent and developed rapidly along with the prolonging time of culture. The findings of immunohistochemistry showed that Desmin, α-Sarcomeric Actinin, MyoD1, Myf5 and PAX7 were positive in MDSCs. Co-culture induction, two-step induction and five-step induction could induce MDSCs into dithizone-positive IPCs in varying degrees. Q-PCR results showed that the expression of C-peptide and Insulin in MDSCs-IPCs induced by five-step induction was significantly higher than that induced by co-culture and two-step induction (P<0.01) . Insulin release assay showed that the insulin secretion of β-cells stimulated by 5.6 mmol/L and 16.7 nmlol/L glucose for 2 hours was (20.3+4.2) mU/L and 60.5+9.3) mU/L, respectively. The insulin secretion of MDSCs-IPCs induced by five-step induction was (16.1+3.7) mU/L and (40.9+7.3) mU/L, which were significantly higher than that of co-culture[ (10.9+2.7) mU/L and (30.2+6.7) mU/L]and two-step induction[ (6.2+2.1) mU/L and (12.9+3.3) mU/L], respectively. Conclusion MDSCs are easy to isolate, culture and proliferate. The five-step induction method is more suitable for inducing IPCs in vitro. Key words: Skeletal muscle-derived satellite cells; Insulin-producing cells; Induction method; Function