Figure 1.
Effects of MSCs on myoblast cell growth and differentiation.
Time lapse videoimaging of C2C12 cells in single (A) and co-culture with Dil (red)-labeled MSCs (B). Quantitative analyses of C2C12 cell proliferation in the different experimental conditions (C). Note that the presence of MSCs greatly enhances myoblast proliferation. D) Confocal immunofluorescence to detect myogenin (green) expression in C2C12 cells in single and co-culture with Dil-labeled MSCs; the quantitative analysis is reported in the corresponding histogram. E) Phase contrast microscopy showing myotube formation in single and co-cultured C2C12 cells; the quantitative analysis is reported in the corresponding histogram. The results of these experiments clearly show that myogenin is up-regulated in the co-cultured C2C12 cells and that myoblasts are the only cell type to contain myogenin+ nuclei. Also their tendency to fuse into multinucleated myotube is greater in the presence of MSCs. Data represent the results of at least three independent experiments with similar results. C: *p<0.05 versus T0; °p<0.05 versus the earlier time points; #p<0.05 versus single culture. D, E: *p<0.05.
Figure 2.
Assessment of myoblast cell proliferation by cyclin A expression.
C2C12 cells in single and co-culture with Dil(red)- or GFP(green)-labeled MSCs for 24 h were incubated with specific antibodies against cyclin A (green, A,B; red, D-G) and observed by confocal microscopy. Notably, the cells with the higher immunofluorescence intensity are those located in close contact with MSCs. C) Quantitative analysis of the number of cells positive for cyclin A. Data represent the results of at least three independent experiments with similar results. *p<0.05.
Figure 3.
Notch-1 patway in differentiating C2C12 myoblasts.
A,B) mRNA and protein expression of Notch-1 receptor and its ligand, Jagged 1, evaluated by RT-PCR (A) and Western Blotting (B) in C2C12 cells in single culture; densitometric analysis of the bands normalized to β-actin is reported in the histograms. C,D) Hes-1 expression in cells treated with 5 µM DAPT, a pharmacological inhibitor of Notch-1 activation. Note that the inhibition of Notch-1 expression (C) is capable of reducing the expression of Hes-1 at the mRNA (D) and protein levels (C). Data represent the results of at least three independent experiments with similar results. A, B, *p<0.05 versus 2 h; #p<0.05 versus 24 h; °p<0.05 versus 48 h.C,D, *p<0.05.
Figure 4.
MSCs induce Notch-1 and Hes-1 expression in C2C12 myoblasts.
A) Flow cytometric analysis of CD34 and CD73 antigen expression in MSCs and C2C12 cells in single cultures and in C2C12 myoblasts after isolation from MSCs in the co-culture by immunomagnetical separation using anti CD34 and anti CD73 antibodies (CD34+ C2C12; CD73- C2C12). B,C) CD34+ C2C12 and CD73- C2C12 cells in co-culture immunomagnetically isolated from MSCs were analyzed for the expression of Notch-1 and Hes-1 by RT-PCR (B) and Western Blotting (C). Note that Notch-1 and Hes-1 expression is robustly induced in the myoblastic cells after co-culturing with MSCs as compared with the single culture. Densitometric analysis of the bands normalized to β-actin is reported in the corresponding histograms. Data represent the results of at least three independent experiments with similar results. *p<0.05.
Figure 5.
Notch-1 signaling is activated in C2C12 myoblasts upon co-culture with MSCs.
Confocal immunofluorescence analysis of Notch-1 (A-C) and Hes-1 (D-F) expression in C2C12 cells in single and co-culture with Dil(red)- or GFP(green)-labeled MSCs for 24 h. After the co-culture, C2C12 cells reveal a stronger reactivity for the activated Notch-intracellular domain (Notch-ICD) and for Hes-1, which is visible inside the nucleus. As shown in the inserts, Notch-1 is preferentially located at the cell membrane (arrows) in the single cultured C2C12 cells, whereas it is found within the cytoplasm (white arrowheads) and nucleus (grey arrowheads) in the co-cultured cells. E) C2C12 myoblast were treated with 5 µM DAPT to inhibit Notch-1 activation and assayed for Hes-1 expression. The corresponding quantitative analysis is reported in the histograms (G,H). Data represent the results of at least three independent experiments with similar results. *p<0.05.
Figure 6.
MSCs influence C2C12 myoblast proliferation through paracrine mechanisms.
C2C12 cells were grown in single culture (C2C12) or exposed to MSC-derived CM (C2C12/MSC-CM) and their proliferative activity assessed by time lapse videomicroscopy (A-C), EdU (green) incorporation (D, E), Notch-1 and Hes-1 expression by RT-PCR (F), Western blotting (G) and confocal immunofluorescence (H). Quantitative analyses of the results shown are reported in the histograms. Data represent the results of at least three independent experiments with similar results. * p<0.05 versus T0; ° p<0.05 versus the earlier time points; § p<0.05 vs 9 h; # p<0.05 versus single culture. D,E: * p<0.05.
Figure 7.
MSCs influence C2C12 myoblast proliferation through the release of VEGF.
A) Cytokine and growth factor secretion profiles by MSCs grown in C2C12 differentiation medium (MSC-CM). B) Western Blotting analysis of VEGFR2 expression in C2C12 cells in single culture (C2C12) or exposed to MSC-CM (C2C12/MSC-CM), in the presence or absence of VEGFR2 inhibitor, KRN633. C) Superimposed DIC and fluorescence image showing cellular localization of VEGFR2 in C2C12 cells; the staining (green) is mainly localized at the cell surface. D) VEGFR2 phosphorylation in C2C12 cells in the noted experimental conditions, assayed by Western Blotting analysis performed on the immunoprecipitated VEGFR2 protein. Note that VEGFR2 expression and phosphorylation levels increase in the cells exposed to MSC-CM as compared with control. E) Superimposed DIC and fluorescence image showing nuclear EdU (green) staining and corresponding quantitative analysis. (F,G) Notch-1 expression by (F) Western blotting and (G) confocal immunofluorescence in C2C12 cells in the indicated experimental conditions. The quantitative analyses are reported in the histograms. Note that EdU staining and Notch-1 expression are significantly affected by treatment with the VEGFR2 inhibitor, KRN633. Data represent the results of at least three independent experiments with similar results. * p<0.05.
Figure 8.
Effects of soluble VEGF on C2C12 myoblast proliferation and differentiation.
C2C12 cells in single culture were treated with different concentrations of soluble VEGF and assayed for Notch-1 and Hes-1 expression by RT-PCR (A), myogenin expression (green) by confocal immunofluorescence (B), and for myotube formation by phase contrast microscopy (C). The quantitative analyses are reported in the histograms. Data represent the results of at least three independent experiments with similar results.* p<0.05.