Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein

•MyoD was engineered to contain protein transduction domain and endosome-disruptive INF7 peptide.•The engineered MyoD-IT showed efficient nuclear targeting through an endosomal escape by INF7 peptide.•By applying MyoD-IT, human adipose-derived stem cells (hASCs) were differentiated into myogenic cells.•hASCs differentiated by applying MyoD-IT fused to myotubes through co-culturing with mouse myoblasts.•Myogenic differentiation using MyoD-IT is a safe method without the concern of altering the genome. Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method.