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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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Recruitment of EPDCs and MSCs inside myotubes is stimulated by IL-4 and IL-13 while it is inhibited by VCAM1 blocking antibody. Human cells were cocultured with differentiating primary mouse myoblasts in presence of the indicated molecules for 2 d in DM. hIL-4: human recombinant IL-4; mIL-4: mouse recombinant IL-4; mhIL-4: mouse plus human IL-4; VCMab: specific anti–human VCAM1 antibody. The percentage of human nuclei inside myotubes was determined as the ratio between human lamin A/C-labeled nuclei inside TnT-labeled myotubes and the total number of human nuclei. Values are the mean ± SEM of three separate experiments. *p < 0.05; **p < 0.005.
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Figure 7: Recruitment of EPDCs and MSCs inside myotubes is stimulated by IL-4 and IL-13 while it is inhibited by VCAM1 blocking antibody. Human cells were cocultured with differentiating primary mouse myoblasts in presence of the indicated molecules for 2 d in DM. hIL-4: human recombinant IL-4; mIL-4: mouse recombinant IL-4; mhIL-4: mouse plus human IL-4; VCMab: specific anti–human VCAM1 antibody. The percentage of human nuclei inside myotubes was determined as the ratio between human lamin A/C-labeled nuclei inside TnT-labeled myotubes and the total number of human nuclei. Values are the mean ± SEM of three separate experiments. *p < 0.05; **p < 0.005.

Mentions: It has been reported that fusion of mouse BM-MSCs to differentiating skeletal muscle cells is increased in the presence of exogenously added IL-4 (Schulze et al., 2005). To test whether the same applies to human cells, we performed coculture experiments in the presence of IL-4 or IL-13. The addition of either one of the two cytokines did not significantly affect the fusion with myotubes of endothelial cells (unpublished data). In contrast, treatment with human IL-4 or with the cross-species reactive mouse IL-13, determined an increment of the number of fusion events for both EPDCs and AT-MSCs (Figure 7A). In particular, IL-4 increased the percentage of fusion by approximately 35% and 15% in EPDCs and AT-MSCs, respectively, whereas IL-13 raised the percentage by 20% in EPDCs and 30% in AT-MSCs (Figure 7A). Interestingly, treatment with mouse recombinant IL-4 also significantly enhanced the fusion rate to similar (in the case of EPDCs) or even higher (in the case of AT-MSCs) levels when compared to the human protein. Moreover, if the two proteins (human and mouse IL-4) are added together, their effect is additive. Because the mouse protein may only act on mouse cells, its effect on human cell recruitment has to be indirect. These results suggest that additional mechanisms, probably acting also on recruiting myotubes and not only directly on recruited cells, may mediate the effects of IL-4 on myotube growth.


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)

Recruitment of EPDCs and MSCs inside myotubes is stimulated by IL-4 and IL-13 while it is inhibited by VCAM1 blocking antibody. Human cells were cocultured with differentiating primary mouse myoblasts in presence of the indicated molecules for 2 d in DM. hIL-4: human recombinant IL-4; mIL-4: mouse recombinant IL-4; mhIL-4: mouse plus human IL-4; VCMab: specific anti–human VCAM1 antibody. The percentage of human nuclei inside myotubes was determined as the ratio between human lamin A/C-labeled nuclei inside TnT-labeled myotubes and the total number of human nuclei. Values are the mean ± SEM of three separate experiments. *p < 0.05; **p < 0.005.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046056&req=5

Figure 7: Recruitment of EPDCs and MSCs inside myotubes is stimulated by IL-4 and IL-13 while it is inhibited by VCAM1 blocking antibody. Human cells were cocultured with differentiating primary mouse myoblasts in presence of the indicated molecules for 2 d in DM. hIL-4: human recombinant IL-4; mIL-4: mouse recombinant IL-4; mhIL-4: mouse plus human IL-4; VCMab: specific anti–human VCAM1 antibody. The percentage of human nuclei inside myotubes was determined as the ratio between human lamin A/C-labeled nuclei inside TnT-labeled myotubes and the total number of human nuclei. Values are the mean ± SEM of three separate experiments. *p < 0.05; **p < 0.005.
Mentions: It has been reported that fusion of mouse BM-MSCs to differentiating skeletal muscle cells is increased in the presence of exogenously added IL-4 (Schulze et al., 2005). To test whether the same applies to human cells, we performed coculture experiments in the presence of IL-4 or IL-13. The addition of either one of the two cytokines did not significantly affect the fusion with myotubes of endothelial cells (unpublished data). In contrast, treatment with human IL-4 or with the cross-species reactive mouse IL-13, determined an increment of the number of fusion events for both EPDCs and AT-MSCs (Figure 7A). In particular, IL-4 increased the percentage of fusion by approximately 35% and 15% in EPDCs and AT-MSCs, respectively, whereas IL-13 raised the percentage by 20% in EPDCs and 30% in AT-MSCs (Figure 7A). Interestingly, treatment with mouse recombinant IL-4 also significantly enhanced the fusion rate to similar (in the case of EPDCs) or even higher (in the case of AT-MSCs) levels when compared to the human protein. Moreover, if the two proteins (human and mouse IL-4) are added together, their effect is additive. Because the mouse protein may only act on mouse cells, its effect on human cell recruitment has to be indirect. These results suggest that additional mechanisms, probably acting also on recruiting myotubes and not only directly on recruited cells, may mediate the effects of IL-4 on myotube growth.

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