Generation of muscle progenitors from human-induced pluripotent stem cells

Abstract

BackgroundSmall molecules have a role in the differentiation of human-induced pluripotent stem cells (hiPSCs) into different cell linages. The aim of this study was to evaluate the differentiation of hiPSCs into cardiac or skeletal myogenic progenitors with a single small molecule.MethodshiPSCs were treated with three different small molecules such as Isoxazole-9, Danazol and Givinostat in serum-free medium for 7 days. Cell viability, qRT-PCR, western blots, and immunostaining were assessed after treatment of hiPSCs with small molecules.ResultsHigher hiPSC viability was observed in hiPSCs treated with Isoxazole-9 (25 µM), Danazol (25 µM) and Givinostat (150 nM) versus control (P < 0.05). Givinostat had dual effect by generating both skeletal and cardiac progenitor cells versus Isoxazole-9 and Danazol after 7 days. Givinostat treatment induced upregulation of skeletal myogenic genes and their protein expression levels on day 4 and further increased on day 8 (P < 0.05) versus control. Furthermore,positive stained cells for Pax3, Myf5, MyoD1, dystrophin, desmin, myogenin, and β-catenin at 1 month. Givinostat increased upregulation of cardiac gene expression levels versus control after day 4 (P < 0.05), with positive stained cells for Nkx2.5, GATA4, TnT, TnI, connexin 43 and α-sarcomeric actinin at 1 month.ConclusionsPretreatment of hiPSCs with Givinostat represents a viable strategy for producing both cardiac/skeletal myogenic progenitors in vitro for cell therapies against myocardial infarction and Duchenne muscular dystrophy.