Restoration of Dystrophin in Duchenne Muscular Dystrophy by Human iPS Cells Derived Skeletal Muscle Progenitor Cells

Background and Objective Duchenne muscular dystrophy (DMD) is caused by mutations of the gene that encodes the protein dystrophin. Loss of dystrophin leads to severe and progressive muscle‐wasting in both skeletal and heart muscles. Human induced pluripotent stem cells (hiPSCs) offer an important opportunity to treat a number of diseases. Here, we investigated whether givinostat, a histone deacetylase inhibitor (HDACi), could reprogram hiPSCs into muscle progenitor cells (MPCs) for DMD treatment. Methods and Results hiPSCs were treated with CHIR99021(10 μM) for two days and followed by givinostat (100 nM) for 5 days in E6 medium (Fig. 1). After culture in E6 medium for additional seven days, cells were analyzed for myogenic makers (Pax7, desmin) expression. Reprogrammed cells (MPCs) from 4 human iPS cell lines expressed Pax7 and desmin. MPCs were differentiated into myotubes in N2 or DMEM medium with 2% horse serum expressing MF20. Additionally, we also generated MPCs from hiPSCs using CHIR99021 and FGF according to a previous study (Fig. 2) and referred these cells as control‐MPCs. We observed that givinostat induced MPCs exhibited superior proliferation and migration capacity using CCK‐8 assay, colony assay and migration assay compared to control‐MPCs. Moreover, we transplanted human myoblasts (1×10 5 ) and MPCs (1×10 5 ) labeled with GFP into tibialis anterior (TA) muscles of Mdx/scid mice, after injury with cardiotoxin (CTX) for one day prior to cell transplantation. One month after transplantation, dystrophin and GFP double positive fibers were analyzed. Givinostat induced MPCs showed higher engraftment capacity and restoration of dystrophin than treatment with control‐MPCs and human myoblasts. In addition, givinostat induced MPCs treated muscle contained significantly less inflammatory cells and showed limited muscle necrosis and fibrosis. Conclusion We successfully generated highly expandable and integration free MPCs from multiple hiPS cell lines using CHIR99021 and givinostat. Givinostat induced MPCs showed marked and impressive regeneration and restored dystrophin in injured tibialis muscle compared to control MPCs. It is concluded that hiPSCs pharmacologically reprogrammed into MPCs with a small molecule, givinostat might be effective cellular source for treatment of muscle injury and restoration of dystrophin in DMD. Support or Funding Information National Institutes of Health grants R01 HL134354 & R01 AR070029 (M Ashraf, Y Tang, NL Weintraub)Schematic outline of generation of MPCs from hiPSCs using CHIR99021 and givinostat (Givi) small molecule combo.Schematic outline generation of control MPCs from hiPSC.