DMPE-PEG scaffold binding with TGF-β1 receptor enhances cardiomyogenic differentiation of adipose-derived stem cells

Figures

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  Fig. 1. The characteristics of cultured adipose-derived mesenchymal stem cells (ADSCs) at day 3, day 7, and day 14. ADSCs were isolated and cultured from human adipocyte tissue. By day 3, most of the cells were ovoid (a); by day 7, fibroblast-like cells were observed (b); and by day 14 (c), the ADSCs grew into clusters with a long spindle-shaped morphology. ADSCs were cultured in a lipogenic medium, and accumulation of lipids into intracellular vesicles was observed by Oil Red O staining (d). ADSCs were cultured in osteogenic medium and stained with Alizarin Red S to visualize calcium deposition and mineralization in the monolayer (e). Flow cytometry was used to detect the expression of ADSC markers (f–i) (scale bar, 100 μm)

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  Fig. 2. The effect of different concentrations (0, 0.75, 1.5, or 3 μg) of DMPE-PEG-FITC on ADSCs was analyzed by confocal microscope (a). b Fluorescence intensity as demonstrated by FACS analysis of TGF-β1 RI expression on the membranes of modified ADSCs. c Western blot analysis for the expression intensity of TGF-β1 RI protein on modified ADSCs. d–f Light microscopy analysis of the effect of DMPE-PEG on ADSCs adhesion. g Growth curve of modified and mock modified ADSCs (scale bar, 100 μm; *p < 0.05, **p < 0.01, ***p < 0.001)

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  Fig. 3. Confocal microscopy was performed to analyze (A, B) TGF-β1 binding to rTGF-β1 RI on ADSC cell membranes and to visualize (C, D) the colocalization of rTGF-β1 RI (DMPE-PEG-TGF-β1 RI-FITC) and TGF-β1 (Cy3-TGF-β1) on the surface of ADSCs at × 64 oil immersion (scale bar, 100 μm; *p < 0.05, **p < 0.01, ***p < 0.001)

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  Fig. 4. Following 1 or 2-week treatments with TGF-β1, ADSC cell morphology was compared in the mock, D-P, and D-P-TGF-β1 RI groups with a phase contrast microscope (A, B, a–c). ADSCs were immunostained with anti-TGF-β1 RI (green),anti-α-SMA (red), and cTnT (red). Nuclei were counterstained with DAPI (blue) (A, B, d–i). C, D The number of cells expressing TGF-β1 RI /α-SMA and TGF-β1 RI /cTnT were compared. E Western blot was performed to exam the protein expression of TGF-β1 RI, α-SMA, cTnT, p-SMad2/3, and SMad 2/3 in the mock, D-P, and D-P-TGF-β1 RI groups. G, F Comparison of relative mRNA and protein expression levels of TGF-β1 R1, α-SMA, and cTnT in the mock, D-P, and D-P-TGF-β1 RI groups (scale bar, 100 μm; values are expressed as mean ± S.D; *p < 0.05, **p < 0.01, ***p < 0.001)

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  Fig. 5. In vivo differentiation studies and immunofluorescence analysis of D-P and D-P-TGF-β1 RI samples (× 40 magnification) after 1 week of transplantation. a, b Modified ADSCs were labeled with the cell track reagent CMFDA (green) and immunostained with anti-α-SMA(red) or anti-cTnT (red). Nuclei were counterstained with DAPI (blue). c, d The number of modified ADSCs expressing α-SMA and cTnT were compared (scale bar, 100 μm; values are expressed as mean ± S.D; *p < 0.05, **p < 0.01, ***p < 0.001)