Myogenic Differentiation from MYOGENIN-Mutated Human iPS Cells by CRISPR/Cas9

Figure 2

Generation of hMYOGENIN-mutated hiPS cells. A schematic model for the induction of myogenic cells derived from hiPS cells by overexpression of MYOD1 marked with mCherry (red) after administrating Dox (a). A flowchart of the time course for the identification of MYOG-mutated hiPS cells (b). FACS analyses to isolate hiPS cells after the introduction of the pX458-hMYOG+189 vector (c). Genomic sequence data around the region targeted by pX458-hMYOG+189. 5 bp of deletion in clone number 28 and 1 bp of insertion in clone number C3 (dashed lines, (d)). Established hiPS cells were immunostained with undifferentiated pluripotent cell markers, anti-SSEA4 (green in left panel), anti-OCT3/4 (red in left panel), anti-TRA1-80 (green in right panel), and anti-NANOG (red in right panel) antibodies. Nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 100 μm (e). Differentiated myogenic cells after Dox treatment for 7 days were immunostained with anti-MYOG N-terminus (N-term, red) and C-terminus (C-term, green) antibodies. Nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 100 μm (upper in (f)). Putative MYOG protein structures both in wild-type (MYOG WT) and mutated cells (MYOG mut) (lower in (f)).

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