Regeneration of TA muscle injury by MYOD transduced hAFS cells. Left TA muscle of immunodeficient BALB/cSlc-nu mice was injured with cardiotoxin (0.4 nM/mouse), and EGFP positive hAFS cells transduced with EV and MYOD viruses. (a) H&E staining of TA muscles at 7 and 21 days after injection of EV- or MYOD-hAFS cells (black dashed line: injured site). (b) The area of muscle tissue was measured using the i-solution image program at 7 and 21 days after injection (n = 9; three per mouse). The muscle area was significantly increased in the MYOD-hAFS cell-injected group compared to the group injected with EV-hAFS cells or the control (*P <0.0002 and **P <0.002). (c) The size of muscle fibers of MYOD-hAFS cell-transplanted TA muscle was larger than that of CTX only or EV-hAFS cell injected TA muscles. Using the i-solution program, the average size of centrally nucleated myofibers and whole myofibers was calculated (n = 48; 16 per mouse) (*P < .004 and **P <0.03). The percentage of centrally nucleated fibers over total fibers was also calculated (n = 48; 16 per mouse) (*P <0.02). (d) Injected hAFS cells were visualized with eGFP signal (green) and DAPI with confocal microscopy (white dashed line: regenerating muscle fiber). (e) TA muscles injected with CTX only (control), EV- or MYOD-hAFS cells were stained with α-BTX at 21 days after injection. The area of α-BTX binding was measured using the i-solution image program (n = 32; 16 per mouse) (*P <0.03 and **P <0.02) (Scale bar; a = 500 μm, 7 days of c = 10 μm and others = 20 μm). α-BTX, α-bungarotoxin; CTX, cardiotoxin; DAPI, 4',6-diamidino-2-phenylindole; EV, empty vector; hAFS, human amniotic fluid stem; TA, tibialis anterior.