a, Schematic of experimental timeline (2D expansion for 5 passages with p38i followed by 3D culture). GM: 3D growth media, Diff: 3D differentiation media, G4 day 4 in growth media, D4,7, days 4,7 in differentiation media). b, Representative images of Pax7 and Ki67 staining in 2D-expanded passage-5 CD56+ myoblasts and D7 myobundles with myofibers stained with F-actin (DAPI-stained nuclei are blue). c-e, Representative images of D7 myobundles showing expression of transcription factors Pax7, MyoG, and MyoD, and cell adhesion molecule M-cadherin (M-cad). f, Gene expression in cells isolated from passage 5 expanded 2D cultures (2D) or myobundles (G4, D4, D7) normalized to RPL13 and shown relative to 2D group (N = 1 donor, n = 3–17 samples per group, 1 sample = cells from 4 to 8 myobundles or separate 2D cultures). Data: mean ± SEM. *p < .05; **p < .01; ***p < .001.