Fig. 3.

Human DEC lines of both, normal (MB^N1/MB^N2) and normal and DMD donor (MB^N/MB^DMD) origin, maintain dystrophin expression, proliferation properties and undergo myogenic differentiation in culture. (a) Representative immunofluorescence images of dystrophin expression (magenta) in MB^N1/MB^N2 and MB^N/MB^DMD DEC in vitro up to 21 days after fusion (n = 4, magnification 400X, scale bar 10 µm). (b) Dystrophin expression in cultured MB^N1/MB^N2 and MB^N/MB^DMD DEC up to 21 days after fusion quantified by Taqman PCR (n = 3, mean ± SD). (c) Flow cytometry analysis confirming proliferation of MB^N1/MB^N2 (upper row) and MB^N/MB^DMD (lower row) DEC up to day 21 post-fusion. Decrease in the intensity of eFluor 670 Proliferative Dye correlated with decrease in the intensity of double PKH67/PKH26 staining used to confirm DEC fusion. (d) Immunofluorescence images of MB^N1/MB^N2 and MB^N/MB^DMD DEC expressing sarcolemmal glycoproteins, alpha-sarcoglycan (yellow), beta-sarcoglycan (violet) and dystrophin (red) co-expressed with the motor protein skeletal myosin heavy chain (SMHC, green), and fusion protein desmin (green) after 7 days of stimulation in the myogenic differentiation media. Upper panel: Immunofluorescence images of normal (MB^N) and DMD-affected (MB^DMD) undifferentiated myoblast controls before fusion confirming lack of dystrophin expression in MB^DMD and a diffused distribution of SMHC in both MB^N and MB^DMD. Lower panel: Immunofluorescence images of MB^N, MB^DMD, MB^N1/MB^N2 and MB^N/MB^DMD DEC after 7-day stimulation in the myogenic differentiation medium confirming expression of dystrophin–glycoprotein complex (DGC) in MB^N/MB^DMD DEC after fusion suggesting restoration of functional DGC. Uniform distribution of alpha-sarcoglycan and beta-sarcoglycan was observed after 7 days of induced differentiation further confirming progression of myogenic maturation. Nuclei were counterstained with DAPI (blue). Scale bar 20 µm