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First published online July 5, 2006
doi: 10.1242/10.1242/jcs.03029

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Myogenic potential of adipose-tissue-derived cells

¹ Laboratorio di Biologia Vascolare e Terapia Genica, Centro Cardiologico Fondazione Monzino, Via Parea 4, 20138 Milan, Italy
² Laboratorio di Patologia Vascolare, Istituto Dermopatico dell'Immacolata, Via dei Monti di Creta 104, 00167 Rome, Italy
³ Istituto di Cardiologia, Università Cattolica del Sacro Cuore, Largo Agostino Gemelli 8, 00168 Rome, Italy

View larger version (59K): [in a new window]   Fig. 1. Direct co-culture of AT-SVF cells with primary myoblasts. Fresh inguinal AT-SVF cells from GFP mice were co-cultured with wild-type primary myoblasts for three days in GM and then switched to DM. Myogenic differentiation was revealed after one week by staining with an anti Troponin-T antibody (TnT). Several GFP cells (green) that have been incorporated into multinucleated myotubes expressing TnT (red) are visible. The arrow indicates a double GFP/TnT-positive mononucleated cell, shown in the inset at larger magnification, suggesting that AT-derived cells can differentiate into skeletal muscle in the absence of cell fusion with skeletal myotubes. (A) Fluorescence image of GFP positive cells, (B) fluorescence image of TnT-positive cells, (C) overlay of A and B; Hoechst staining was used to visualize nuclei (blue). Magnification, 20x. Bar, 100 µm.

View larger version (116K): [in a new window]   Fig. 2. Cell-autonomous myogenic differentiation of AT-SVF cells. Fresh inguinal AT-SVF cells were plated on fibronectin-coated transwell filters floating over a layer of primary myoblasts (A-C) or simply on a fibronectin-coated tissue-culture dish (D,E). Cultures were maintained for three days in proliferation medium and then switched to differentiation medium. Myogenic differentiation was revealed after 1 week by staining with an anti TnT antibody (red). (A,B) The image shows one of the several clusters of skeletal myotubes found on a transwell filter in a typical experiment. (C) RTPCR analysis for the indicated skeletal muscle markers of inguinal AT-SFV cells. AT, fresh, uncultured AT-SVF cells; AT+PM, AT-SVF cells cultured on transwell filters in the presence of primary myoblasts. Differentiating primary myoblasts (PM) were used as positive controls. (D,E) Spontaneous myogenic differentiation of ATSVF cells. (A,D) Fluorescence image of TnT positive cells; (B,E) merge with Hoechst to visualize nuclei (blue). (A,B) Magnification, 10x. Bar, 200 µm. (D,E) Magnification, 20x. Bar, 100 µm.

View larger version (105K): [in a new window]   Fig. 3. Myogenic conversion of AT-MSCs induced by primary myoblasts requires cell contact with myogenic cells. GFP-positive AT-MSCs were either seeded in the same dish together with primary myoblasts (top panels) or plated on 0.4 µm porous transwell filters floating on a layer of primary myoblasts (bottom panels). Myogenic differentiation was revealed by TnT staining (red) after 1 week in DM. Hoechst was used to visualize nuclei (blue). Double GFP/TnT-positive cells indicating myogenic differentiation of ATMSCs are observed only when contact between the two cell types is allowed (A-C). (A,D) Fluorescence image of GFPpositive cells; (B,E) fluorescence image of TnT-positive cells; (C,F) overlay of GFP, TnT and Hoechst staining. Magnification, 20x. Bars, 100 µm.

View larger version (50K): [in a new window]   Fig. 4. Spontaneously differentiating AT-SVF myogenic cells express satellite-cell-specific markers. (A) Transmission light image of a typical AT-derived group of proliferating myogenic cells, 5 days after plating in GM. (B) The same group of cells shown in A after 24 hours in DM, when myotubes start to form (magnification, 20x; Bar, 50 µm). (C,D) Immunofluorescence staining with an anti-Pax7 antibody of proliferating myogenic cells. To highlight the specificity of the Pax7 staining, we chose an image field where myogenic cells groups were close to morphologically different, non-myogenic cells. Small nuclei of myogenic cells express Pax7 while bigger nuclei of adjacent, nonmyogenic cells, are negative for Pax7. (C) Fluorescence image of Pax7-positive cells (red). (D) merge with Hoechst (blue) to visualize nuclei. Magnification, 40x. Bar, 25 µm. (E) RT-PCR analysis for the indicated markers of isolated myogenic clones. pPM, proliferating, skeletal-muscle-derived, primary myoblasts; pATM, proliferating AT-derived myogenic clones; AT, fresh, uncultured AT-SVF cells.

View larger version (82K): [in a new window]   Fig. 5. AT-SVF cells participate in skeletal muscle regeneration. ATSVF cells from GFP mice were injected into the adductor muscle of GFP-negative syngenic mice were ischemia was induced by femoral artery removal. Engrafted GFP-expressing cells in the injected muscle were visualized by an anti-GFP antibody (green) 7 days after injection. A wide region of GFP positive fibers are present in muscle sections from treated hind limbs (B) while no GFP staining is observed in control sections from PBS-injected limbs (A). Nuclei are visualized by Hoechst (blue). Magnification, 40x. Bar, 50 µm.

View larger version (54K): [in a new window]   Fig. 6. Transplantation of wild-type AT-SVF cells rescues dystrophin expression in mdx mice. Immunostaining of adductor muscles transverse sections with an antibody against the C-terminal portion of dystrophin. Fresh AT-SVF cells were injected into the left adductor muscle of mdx mice. PBS injected age-matched mdx mice were used as controls. Staining for dystrophin (green) was performed 21 days after injection. Nuclei are visualized by Hoechst (blue). Dystrophin expression is totally absent in PBS-injected muscles while clusters of dystrophin-positive fibers are with both centrally and peripherally located nuclei in mice injected with AT-SVF cells. (A) Wild-type uninjected; (B) mdx injected with PBS; (C) mdx injected with AT-SVF cells. Magnification, 40x. Bar, 50 µm.