Objectives: Myosatellite cells (MSCs) constitute small subpopulation (2-10 %) of mononuclear stem and progenitor cells residing in mature skeletal muscle. These cells lay in a niche between the plasma membrane of the myofiber and the basal laminal membrane. In adult skeletal muscle, MSCs are generally quiescent, but ready to be activated, self-renew and differentiate for muscle growth and regeneration. Aim of the study has been to isolate MSCs from human skeletal muscle and test their capacity to generate mature myofibers in vitro, to develop a useful model for studies of muscle differentiation and for the screening of new molecules to be employed in the treatment of sarcopenia and skeletal muscle diseases. Materials and methods: Human MSCs have been isolated from biopsies of skeletal muscles (pectoralis major, recto abdominal muscles) in healthy young adult volunteers undergoingplasticsurgery.Themincedspecimenshavebeendigested with collagenase at 37 °C for 3 h. Primary cells have been cultured in a specific growth medium (PromoCell cod.C39360) and differentiated for 14 days with appropriate differentiation medium (PromoCell cod.C-39366) for 14 days to evaluate the expression of main markers of myogenic differentiation by real-time-qPCR and immunocytochemistry. Results:MSCs express PAX-7, the main early marker of myogenic differentiation. After 4 days in culture, elongated cells resembling myotubes have appeared in culture. After 10 days in culture a significant increase in the expression of markers of myogenic differentiation has been observed (p<0.005 with respect to baseline levels). The expression of the protein myosin heavy chain, characterizing mature striated muscle fibers, was confirmed. Conclusions: MSCs derived from human skeletal muscle biopsies constitute a good model to study mechanisms of skeletal muscle differentiation and can serve to test in the future therapeutical agents for skeletal muscle diseases in a preclinical setting.
PRIMARY CULTURES OF HUMAN SKELETAL MUSCLE SATELLITE CELLS: A NOVEL MODEL TO ASSESS SKELETAL MUSCLE CELL DIFFERENTIATION / Cianferotti, L.; Romagnoli, C.; Vigna, M.; Zonefrati, R.; Mavilia, C.; Galli, G.; Innocenti, M.; Marcucci G; Tanini, A.; Brandi, M. L.. - In: OSTEOPOROSIS INTERNATIONAL. - ISSN 0937-941X. - ELETTRONICO. - 27:(2016), pp. S251-S251.
PRIMARY CULTURES OF HUMAN SKELETAL MUSCLE SATELLITE CELLS: A NOVEL MODEL TO ASSESS SKELETAL MUSCLE CELL DIFFERENTIATION
CIANFEROTTI, LUISELLA;ROMAGNOLI, CECILIA;ZONEFRATI, ROBERTO;MAVILIA, CARMELO;GALLI, GIANNA;INNOCENTI, MARCO;TANINI, ANNALISA;MARCUCCI, GEMMA;Marcucci G
2016
Abstract
Objectives: Myosatellite cells (MSCs) constitute small subpopulation (2-10 %) of mononuclear stem and progenitor cells residing in mature skeletal muscle. These cells lay in a niche between the plasma membrane of the myofiber and the basal laminal membrane. In adult skeletal muscle, MSCs are generally quiescent, but ready to be activated, self-renew and differentiate for muscle growth and regeneration. Aim of the study has been to isolate MSCs from human skeletal muscle and test their capacity to generate mature myofibers in vitro, to develop a useful model for studies of muscle differentiation and for the screening of new molecules to be employed in the treatment of sarcopenia and skeletal muscle diseases. Materials and methods: Human MSCs have been isolated from biopsies of skeletal muscles (pectoralis major, recto abdominal muscles) in healthy young adult volunteers undergoingplasticsurgery.Themincedspecimenshavebeendigested with collagenase at 37 °C for 3 h. Primary cells have been cultured in a specific growth medium (PromoCell cod.C39360) and differentiated for 14 days with appropriate differentiation medium (PromoCell cod.C-39366) for 14 days to evaluate the expression of main markers of myogenic differentiation by real-time-qPCR and immunocytochemistry. Results:MSCs express PAX-7, the main early marker of myogenic differentiation. After 4 days in culture, elongated cells resembling myotubes have appeared in culture. After 10 days in culture a significant increase in the expression of markers of myogenic differentiation has been observed (p<0.005 with respect to baseline levels). The expression of the protein myosin heavy chain, characterizing mature striated muscle fibers, was confirmed. Conclusions: MSCs derived from human skeletal muscle biopsies constitute a good model to study mechanisms of skeletal muscle differentiation and can serve to test in the future therapeutical agents for skeletal muscle diseases in a preclinical setting.
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