Human iPSC-Derived Skeletal Muscle Myoblasts

ERBB3⁺/NGFR⁺ Population of Human iPSC-Derived Myoblasts Exhibits Rapid Differentiation to Myotubes

Neurovascular cells play important roles in skeletal muscle biology, including myogenesis, maturation, and regeneration. While in vitro studies have examined motor neurons and endothelial cells independently, their combined effects on muscle development remain unclear. Here, Das et al. developed a triculture system using human iPSC-derived skeletal myocytes, iPSC-derived motor neurons, and primary human endothelial cells, maintained under controlled media conditions.

The human iPSC line Penn123i-SV20 was differentiated into a mixture of myocytes, myotubes, and skeletal muscle progenitor cells (SMPCs) as described. Cells were enriched using antibodies against ERBB3 and NGFR, markers of PAX7+ myogenic progenitors, then expanded and cryopreserved as SMPCs for later myotube formation. ERBB3+NGFR+ cells efficiently formed myotubes within 4 days after initiating differentiation (Fig. 1).

Quantitative PCR showed increased expression of myogenic transcription factors (PAX7, MYOD1, MYOG), structural proteins (DMD, TNNT1, TTN), and myosin heavy chain genes (MYH1, MYH3, MYH6, MYH7, MYH8) (Fig. 1B), consistent with prior reports. Myogenic identity was confirmed by uniform expression of skeletal myosin heavy chain (MYHC) and embryonic MYH3 (Fig. 1C). Based on these findings, ERBB3+/NGFR+ myoblasts were used as the starting cell source for all subsequent experiments.