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Human epicardium-derived cells fuse with high efficiency with skeletal myotubes and differentiate toward the skeletal muscle phenotype: a comparison study with stromal and endothelial cells.

Gentile A, Toietta G, Pazzano V, Tsiopoulos VD, Giglio AF, Crea F, Pompilio G, Capogrossi MC, Di Rocco G - Mol. Biol. Cell (2011)

Bottom Line: Results are compared to those obtained with mesenchymal stromal cells (MSCs) and with endothelial cells, another mesodermal derivative.We additionally show that vascular cell adhesion molecule 1 (VCAM1) expression levels of nonmuscle cells are modulated by soluble factors secreted by skeletal myoblasts and that VCAM1 function is required for fusion to occur.Finally, treatment with interleukin (IL)-4 or IL-13, two cytokines released by differentiating myotubes, increases VCAM1 expression and enhances the rate of fusion of EPDCs and MSCs, but not that of endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio di Patologia Vascolare, Istituto Dermopatico dell'Immacolata-IRCCS, 00167 Rome, Italy.

ABSTRACT
Recent studies have underscored a role for the epicardium as a source of multipotent cells. Here, we investigate the myogenic potential of adult human epicardium-derived cells (EPDCs) and analyze their ability to undergo skeletal myogenesis when cultured with differentiating primary myoblasts. Results are compared to those obtained with mesenchymal stromal cells (MSCs) and with endothelial cells, another mesodermal derivative. We demonstrate that EPDCs spontaneously fuse with pre-existing myotubes with an efficiency that is significantly higher than that of other cells. Although at a low frequency, endothelial cells may also contribute to myotube formation. In all cases analyzed, after entering the myotube, nonmuscle nuclei are reprogrammed to express muscle-specific genes. The fusion competence of nonmyogenic cells in vitro parallels their ability to reconstitute dystrophin expression in mdx mice. We additionally show that vascular cell adhesion molecule 1 (VCAM1) expression levels of nonmuscle cells are modulated by soluble factors secreted by skeletal myoblasts and that VCAM1 function is required for fusion to occur. Finally, treatment with interleukin (IL)-4 or IL-13, two cytokines released by differentiating myotubes, increases VCAM1 expression and enhances the rate of fusion of EPDCs and MSCs, but not that of endothelial cells.

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Human EPDCs, MSCs, and endothelial cells are incorporated into mouse skeletal myotubes and are reprogrammed to express human skeletal muscle specific genes. Adult EPDCs were cocultured with differentiating mouse primary myoblasts. Equal numbers of MSCs and endothelial cells were used for comparison. (A–C) Combined bright-field and green fluorescent protein (GFP) fluorescent (green) images of living cells. Human cells were labeled with a lentiviral vector expressing GFP. Several GFP-labeled multinucleated myotubes, indicating that human cells are participating in myotube formation, are visible with all three cell types. The number of green myotubes is much lower in the case of endothelial cells compared to EPDCs and MSCs. (D–F) Immunofluorescence of fixed cells labeled with antibodies that recognize both mouse and human Troponin T (hTNT, green) but only human LaminA/C (red). Several hybrid myotubes containing both human (red) and mouse (Hoechst blue) nuclei are visible. G–I: Fixed cells were stained with antibodies specific for hTnT (green) and human LaminA/C (red). Hoechst (blue) was used to visualize all nuclei. Independently from the cell origin, human nuclei inside hybrid myotubes are phenotypically reprogrammed to express skeletal muscle specific proteins. ENDO: endometrium-derived endothelial cells. Magnification: 20×, Bars: 50 μm. D′–F′, G′– I′: 2× enlarged boxes of D–F and G–I, respectively.
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Figure 2: Human EPDCs, MSCs, and endothelial cells are incorporated into mouse skeletal myotubes and are reprogrammed to express human skeletal muscle specific genes. Adult EPDCs were cocultured with differentiating mouse primary myoblasts. Equal numbers of MSCs and endothelial cells were used for comparison. (A–C) Combined bright-field and green fluorescent protein (GFP) fluorescent (green) images of living cells. Human cells were labeled with a lentiviral vector expressing GFP. Several GFP-labeled multinucleated myotubes, indicating that human cells are participating in myotube formation, are visible with all three cell types. The number of green myotubes is much lower in the case of endothelial cells compared to EPDCs and MSCs. (D–F) Immunofluorescence of fixed cells labeled with antibodies that recognize both mouse and human Troponin T (hTNT, green) but only human LaminA/C (red). Several hybrid myotubes containing both human (red) and mouse (Hoechst blue) nuclei are visible. G–I: Fixed cells were stained with antibodies specific for hTnT (green) and human LaminA/C (red). Hoechst (blue) was used to visualize all nuclei. Independently from the cell origin, human nuclei inside hybrid myotubes are phenotypically reprogrammed to express skeletal muscle specific proteins. ENDO: endometrium-derived endothelial cells. Magnification: 20×, Bars: 50 μm. D′–F′, G′– I′: 2× enlarged boxes of D–F and G–I, respectively.

Mentions: To test whether EPDCs can be directed toward a skeletal muscle phenotype, we performed experiments in which EPDCs, previously infected with a lentiviral vector harboring a green fluorescent protein (GFP) expression cassette, were cocultured with equal amounts of unlabeled differentiating mouse primary myoblasts in an 80% confluent plate. Surprisingly, after 48 h in reduced-serum differentiation medium (DM) many GFP+ contractile multinucleated cells were detected, indicating that EPDCs had been recruited to form skeletal myotubes (Figure 2A). The number of GFP+ myotubes corresponded approximately to 12% (12 ± 2%) of total myotubes, as calculated from three different experiments. When the GFP-labeled AT-MSCs were used, numerous GFP+ myotubes were also observed reaching a maximum of 5%. When GFP-labeled, endometrium-derived primary endothelial cells were used in the coculture, however, the number of GFP+ myotubes was markedly reduced compared to the other two cell types, and their number never exceeded 0.2% of total myotubes. To more precisely determine and compare the number of human nuclei incorporated into skeletal myotubes in the three cell types, coculture experiments with mouse myoblasts were performed with unlabeled human cells and by plating at lower (30%) density. Cells were then stained with an antibody against Troponin T (TnT) to identify myotubes and with an antibody specific for human laminA/C, to identify human nuclei (Figure 2, D–F). In all three cases it was possible to detect skeletal myotubes containing from 1 to 3 human nuclei together with several mouse nuclei. The fusion index, calculated as the ratio between the number of human nuclei inside myotubes and the total number of myotubes, was different, however, for the three cell types and was 11.12% ± 4.23% for EPDCs, 5.06% ± 3.22% for AT-MSCs, and 0.20% ± 0.04% for endothelial cells.


Human epicardium-derived cells fuse with high efficiency with skeletal myotubes and differentiate toward the skeletal muscle phenotype: a comparison study with stromal and endothelial cells.

Gentile A, Toietta G, Pazzano V, Tsiopoulos VD, Giglio AF, Crea F, Pompilio G, Capogrossi MC, Di Rocco G - Mol. Biol. Cell (2011)

Human EPDCs, MSCs, and endothelial cells are incorporated into mouse skeletal myotubes and are reprogrammed to express human skeletal muscle specific genes. Adult EPDCs were cocultured with differentiating mouse primary myoblasts. Equal numbers of MSCs and endothelial cells were used for comparison. (A–C) Combined bright-field and green fluorescent protein (GFP) fluorescent (green) images of living cells. Human cells were labeled with a lentiviral vector expressing GFP. Several GFP-labeled multinucleated myotubes, indicating that human cells are participating in myotube formation, are visible with all three cell types. The number of green myotubes is much lower in the case of endothelial cells compared to EPDCs and MSCs. (D–F) Immunofluorescence of fixed cells labeled with antibodies that recognize both mouse and human Troponin T (hTNT, green) but only human LaminA/C (red). Several hybrid myotubes containing both human (red) and mouse (Hoechst blue) nuclei are visible. G–I: Fixed cells were stained with antibodies specific for hTnT (green) and human LaminA/C (red). Hoechst (blue) was used to visualize all nuclei. Independently from the cell origin, human nuclei inside hybrid myotubes are phenotypically reprogrammed to express skeletal muscle specific proteins. ENDO: endometrium-derived endothelial cells. Magnification: 20×, Bars: 50 μm. D′–F′, G′– I′: 2× enlarged boxes of D–F and G–I, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Human EPDCs, MSCs, and endothelial cells are incorporated into mouse skeletal myotubes and are reprogrammed to express human skeletal muscle specific genes. Adult EPDCs were cocultured with differentiating mouse primary myoblasts. Equal numbers of MSCs and endothelial cells were used for comparison. (A–C) Combined bright-field and green fluorescent protein (GFP) fluorescent (green) images of living cells. Human cells were labeled with a lentiviral vector expressing GFP. Several GFP-labeled multinucleated myotubes, indicating that human cells are participating in myotube formation, are visible with all three cell types. The number of green myotubes is much lower in the case of endothelial cells compared to EPDCs and MSCs. (D–F) Immunofluorescence of fixed cells labeled with antibodies that recognize both mouse and human Troponin T (hTNT, green) but only human LaminA/C (red). Several hybrid myotubes containing both human (red) and mouse (Hoechst blue) nuclei are visible. G–I: Fixed cells were stained with antibodies specific for hTnT (green) and human LaminA/C (red). Hoechst (blue) was used to visualize all nuclei. Independently from the cell origin, human nuclei inside hybrid myotubes are phenotypically reprogrammed to express skeletal muscle specific proteins. ENDO: endometrium-derived endothelial cells. Magnification: 20×, Bars: 50 μm. D′–F′, G′– I′: 2× enlarged boxes of D–F and G–I, respectively.
Mentions: To test whether EPDCs can be directed toward a skeletal muscle phenotype, we performed experiments in which EPDCs, previously infected with a lentiviral vector harboring a green fluorescent protein (GFP) expression cassette, were cocultured with equal amounts of unlabeled differentiating mouse primary myoblasts in an 80% confluent plate. Surprisingly, after 48 h in reduced-serum differentiation medium (DM) many GFP+ contractile multinucleated cells were detected, indicating that EPDCs had been recruited to form skeletal myotubes (Figure 2A). The number of GFP+ myotubes corresponded approximately to 12% (12 ± 2%) of total myotubes, as calculated from three different experiments. When the GFP-labeled AT-MSCs were used, numerous GFP+ myotubes were also observed reaching a maximum of 5%. When GFP-labeled, endometrium-derived primary endothelial cells were used in the coculture, however, the number of GFP+ myotubes was markedly reduced compared to the other two cell types, and their number never exceeded 0.2% of total myotubes. To more precisely determine and compare the number of human nuclei incorporated into skeletal myotubes in the three cell types, coculture experiments with mouse myoblasts were performed with unlabeled human cells and by plating at lower (30%) density. Cells were then stained with an antibody against Troponin T (TnT) to identify myotubes and with an antibody specific for human laminA/C, to identify human nuclei (Figure 2, D–F). In all three cases it was possible to detect skeletal myotubes containing from 1 to 3 human nuclei together with several mouse nuclei. The fusion index, calculated as the ratio between the number of human nuclei inside myotubes and the total number of myotubes, was different, however, for the three cell types and was 11.12% ± 4.23% for EPDCs, 5.06% ± 3.22% for AT-MSCs, and 0.20% ± 0.04% for endothelial cells.

Bottom Line: Results are compared to those obtained with mesenchymal stromal cells (MSCs) and with endothelial cells, another mesodermal derivative.We additionally show that vascular cell adhesion molecule 1 (VCAM1) expression levels of nonmuscle cells are modulated by soluble factors secreted by skeletal myoblasts and that VCAM1 function is required for fusion to occur.Finally, treatment with interleukin (IL)-4 or IL-13, two cytokines released by differentiating myotubes, increases VCAM1 expression and enhances the rate of fusion of EPDCs and MSCs, but not that of endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio di Patologia Vascolare, Istituto Dermopatico dell'Immacolata-IRCCS, 00167 Rome, Italy.

ABSTRACT
Recent studies have underscored a role for the epicardium as a source of multipotent cells. Here, we investigate the myogenic potential of adult human epicardium-derived cells (EPDCs) and analyze their ability to undergo skeletal myogenesis when cultured with differentiating primary myoblasts. Results are compared to those obtained with mesenchymal stromal cells (MSCs) and with endothelial cells, another mesodermal derivative. We demonstrate that EPDCs spontaneously fuse with pre-existing myotubes with an efficiency that is significantly higher than that of other cells. Although at a low frequency, endothelial cells may also contribute to myotube formation. In all cases analyzed, after entering the myotube, nonmuscle nuclei are reprogrammed to express muscle-specific genes. The fusion competence of nonmyogenic cells in vitro parallels their ability to reconstitute dystrophin expression in mdx mice. We additionally show that vascular cell adhesion molecule 1 (VCAM1) expression levels of nonmuscle cells are modulated by soluble factors secreted by skeletal myoblasts and that VCAM1 function is required for fusion to occur. Finally, treatment with interleukin (IL)-4 or IL-13, two cytokines released by differentiating myotubes, increases VCAM1 expression and enhances the rate of fusion of EPDCs and MSCs, but not that of endothelial cells.

Show MeSH
Related in: MedlinePlus