Fig. 4.

GET of MYOD promotes myogenic differentiation of hESCs. (A) Scheme of testing the differentiation activity of transduced MYOD in HUES7 cells. HUES7 cells were plated onto gelatinized plastic and cultured in DMEM containing 10% (vol/vol) FCS. Cells were fed daily with DMEM containing 10% (vol/vol) FCS and P21-mR-MYOD-8R (0, 1, 5, 10, or 50 µg/mL) for 7 d. Media was then changed to DMEM containing 2% (vol/vol) horse serum (HS), human recombinant insulin, and P21-mR-MYOD-8R and fed daily for 3 d. (B–F) P21-mR-MYOD-8R drives myogenic differentiation of HUES7 cells to multinucleated Myotubes. (B) Light microscopy of HUES7 cells cultured under the myogenic regime supplemented with SIN MYOD (to overexpress MYOD) or transduced with P21-mR-MYOD-8R. Elongated fused Myotubes and single myocytes are generated with SIN-MYOD or high doses of P21-mR-MYOD-8R. (Scale bar, 100 µm.) (C) MYOD-dependent myogenic differentiation of hESCs. Relative gene expression analyses of HUES7 cultures using quantitative PCR. Cultures supplemented with SIN MYOD (to overexpress MYOD) or transduced with P21-mR-MYOD-8R have increased endogenous MYOD expression and skeletal muscle-specific ACTA1 expression. Error bars indicate SE. (D and E) P21-mR-MYOD-8R differentiated cells are multinucleated. (D) Quantitation of mean nuclei number per cell using PI staining. Error bars indicate SD. (E) Fluorescence microscopy images of HUES7 cells differentiated with P21-mR-MYOD-8R (50 µg/mL) and stained with nuclear dye DAPI. (Scale bar, 50 µm.) (F) P21-mR-MYOD-8R differentiated cells are MYOGENIN-positive. Quantitation of the percentage of MYOGENIN-positive cells using immunolabeling. Error bars indicate SD. n = 6.