Current Progress and Challenges for Skeletal Muscle Differentiation from Human Pluripotent Stem Cells Using Transgene-Free Approaches

Figure 2

Derivation of skeletal myocytes and matured myotubes from human iPSCs using a transgene-free protocol. Human iPSCs can be sufficiently differentiated into myogenic progenitors and myotubes in a defined culture without genetic modification using free-floating spheres (EZ spheres) [59, 67]. (a) Human iPSC-derived myotubes were labeled with multiple myogenic proteins Pax3, Pax7, MyoD, myogenin (MyoG), and myosin heavy chain (MHC), demonstrating colocalization of those proteins in the same field. Some MyoD⁺ nuclei were overlapped with MyoG⁺ nuclei and fused on MHC⁺ myotubes (double arrow: MyoD⁺/MyoG⁺). The other nuclei were not overlapping with MHC but expressed either MyoD or MyoG (arrow: MyoD⁺/MyoG⁻; or arrow head: MyoD⁻/MyoG⁺) (C). Neither Pax3⁺ nuclei (A) nor Pax7⁺ nuclei (B) showed any localization with MyoG⁺ nuclei, which mostly fused on MHC⁺ myotubes. (b) Sarcomere formation in iPSC-derived myotubes. Titin staining revealed that striated patterns were clearly visible in the myotubes at 12 weeks MHC staining in the same cell preparations used for titin labeling. (c) Ultrastructures of iPSC-derived myotubes. After 12 weeks of terminal differentiation, mature sarcomeres were observed to be assembled into myofibrils. Morphological hallmarks, including I-band of actin filaments and A-band with distinct M line across myosin filaments, were clearly visible. Sarcomere Z lines appeared to be reasonably aligned and gave rise to a striated pattern. This figure is reproduced from Jiwlawat et al. [67] (under the Creative Commons Attribution license/public domain).

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