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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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Expression of fusion-related genes in human nonmuscle cells. Human cells were cultured for 48 h in DM in the absence or presence (+) of IL-4. mRNA expression was evaluated by semiquantitative RT-PCR analysis. Human myoblasts (MYO) were used as positive control. ADAM12: disintegrin and metalloproteinase domain-containing protein 12; MRC1: mannose receptor isoform 1; NTN3: Netrin3.
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Figure 9: Expression of fusion-related genes in human nonmuscle cells. Human cells were cultured for 48 h in DM in the absence or presence (+) of IL-4. mRNA expression was evaluated by semiquantitative RT-PCR analysis. Human myoblasts (MYO) were used as positive control. ADAM12: disintegrin and metalloproteinase domain-containing protein 12; MRC1: mannose receptor isoform 1; NTN3: Netrin3.

Mentions: One possibility for the extremely low fusion competence of endothelial cells could be that they fail to express some fusion-related genes when compared to fusion-competent cells such as EPDCs and MSCs. To verify such a hypothesis, we conducted a mini-screening by RT-PCR for the expression of various genes known to be involved in the fusion process and at the same time tested whether they are modulated by IL-4. Those genes include the metalloprotease ADAM12, the mannose receptor (MRC1), Netrin3 (NTN3), the integrin chains α9 (ITGα9) and the isoform A and D of integrin β1 (ITGβ1A and ITGβ1D) (Schwander et al., 2003; Kang et al., 2004; Jansen and Pavlath, 2006, 2008; Quach et al., 2009). In addition, we included integrin α7 (ITGα7), which, although it has never been directly reported to be involved in fusion, represents the tissue-specific partner subunit of ITGβ1D (Mayer, 2003). We excluded from the screening molecules 1) those molecules that could act nonautonomously, such as soluble factors, because in a coculture system they could be provided by muscle cells, as well as 2) fusion molecules already known to be expressed in endothelial cells. Interestingly, with the exception of VCAM1, all the tested molecules resulted to be expressed at comparable levels in EPDCs and AT-MSCs (Figure 9). The expression of ITGβ1D and ITGα7, however, was practically undetectable in endothelial cells both in the presence and absence of IL-4. Moreover, apart from a mild inductive effect on MRC1 of approximately 20%, confirmed by normalized densitometry data (unpublished data), none of the analyzed genes seemed to be affected by treatment with IL-4. Thus, the inability to express some fusion-related genes may effectively explain the reduced fusion propensity of 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)

Expression of fusion-related genes in human nonmuscle cells. Human cells were cultured for 48 h in DM in the absence or presence (+) of IL-4. mRNA expression was evaluated by semiquantitative RT-PCR analysis. Human myoblasts (MYO) were used as positive control. ADAM12: disintegrin and metalloproteinase domain-containing protein 12; MRC1: mannose receptor isoform 1; NTN3: Netrin3.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046056&req=5

Figure 9: Expression of fusion-related genes in human nonmuscle cells. Human cells were cultured for 48 h in DM in the absence or presence (+) of IL-4. mRNA expression was evaluated by semiquantitative RT-PCR analysis. Human myoblasts (MYO) were used as positive control. ADAM12: disintegrin and metalloproteinase domain-containing protein 12; MRC1: mannose receptor isoform 1; NTN3: Netrin3.
Mentions: One possibility for the extremely low fusion competence of endothelial cells could be that they fail to express some fusion-related genes when compared to fusion-competent cells such as EPDCs and MSCs. To verify such a hypothesis, we conducted a mini-screening by RT-PCR for the expression of various genes known to be involved in the fusion process and at the same time tested whether they are modulated by IL-4. Those genes include the metalloprotease ADAM12, the mannose receptor (MRC1), Netrin3 (NTN3), the integrin chains α9 (ITGα9) and the isoform A and D of integrin β1 (ITGβ1A and ITGβ1D) (Schwander et al., 2003; Kang et al., 2004; Jansen and Pavlath, 2006, 2008; Quach et al., 2009). In addition, we included integrin α7 (ITGα7), which, although it has never been directly reported to be involved in fusion, represents the tissue-specific partner subunit of ITGβ1D (Mayer, 2003). We excluded from the screening molecules 1) those molecules that could act nonautonomously, such as soluble factors, because in a coculture system they could be provided by muscle cells, as well as 2) fusion molecules already known to be expressed in endothelial cells. Interestingly, with the exception of VCAM1, all the tested molecules resulted to be expressed at comparable levels in EPDCs and AT-MSCs (Figure 9). The expression of ITGβ1D and ITGα7, however, was practically undetectable in endothelial cells both in the presence and absence of IL-4. Moreover, apart from a mild inductive effect on MRC1 of approximately 20%, confirmed by normalized densitometry data (unpublished data), none of the analyzed genes seemed to be affected by treatment with IL-4. Thus, the inability to express some fusion-related genes may effectively explain the reduced fusion propensity of 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