Morphologically and physiologically comparable skeletal muscle cells differentiated from Control-iPStet-MyoD and DMD-iPStet-MyoD.
(a) Quantitative RT-PCR of skeletal muscle marker expression in skeletal muscle cells induced from Control-iPStet-MyoD and DMD-iPStet-MyoD, shown with black and white bars, respectively. Time course analyses were performed at days 0, 2, 4, 6 and 9. Relative mRNA levels of DMD, CKM and TPM2 were analysed. Data were normalised by setting data from day 9 = 1. n = 3. Beta-actin was used as an internal control. (b) Relative mRNA expression of DMD, CKM and TPM2 at day 9 in Control- and DMD-Myocytes. Gene expression levels were normalised to those of Hu5/E18 cells at day 5 of differentiation. Ubiquitin C was used as an internal control. (c) Immunocytochemistry of MHC, CKM and skeletal muscle actin (SMA) in skeletal muscle cells induced from Control-iPStet-MyoD and DMD-iPStet-MyoD merged with DAPI. All immunofluorescence analyses were conducted at day 9 of differentiation. Scale bar, 200 μm. (d) Western blotting analyses of dystrophin protein expression in induced myotubes detecting the rod domain and C-terminus. The intensity of each band was normalised to that of myosin heavy chain (MHC). Cells were collected at days 9–12 of differentiation. (e) Electron microscopy images of induced skeletal muscle cells from Control-iPStet-MyoD and DMD-iPStet-MyoD at day 9. Myofibrils and mitochondria are indicated with arrows and arrowheads, respectively. Scale bars, 1 μm. 1:19,000 (Control) and 1:18,200 (DMD).