title: In Vitro Differentiation of Mature Myofibers for Live Imaging.
abstract: Skeletal muscles are composed of myofibers, the biggest cells in the mammalian body and one of the few syncytia. How the complex and evolutionarily conserved structures that compose it are assembled remains under investigation. Their size and physiological features often constrain manipulation and imaging applications. The culture of immortalized cell lines is widely used, but it can only replicate the early steps of differentiation. Here, we describe a protocol that enables easy genetic manipulation of myofibers originating from primary mouse myoblasts. After one week of differentiation, the myofibers display contractility, aligned sarcomeres and triads, as well as peripheral nuclei. The entire differentiation process can be followed by live imaging or immunofluorescence. This system combines the advantages of the existing ex vivo and in vitro protocols. The possibility of easy and efficient transfection as well as the ease of access to all differentiation stages broadens the potential applications. Myofibers can subsequently be used not only to address relevant developmental and cell biology questions, but also to reproduce muscle disease phenotypes for clinical applications.
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa.
authors.forename: Mafalda R
authors.initials: MR
authors.lastname: Pimentel
authors.affiliation: Myology Research Center, UM76-INSERM U974-CNRS FRE 361, Sorbonne University, UPMC University of Paris 6.
authors.forename: Sestina
authors.initials: S
authors.lastname: Falcone
authors.affiliation: Myology Research Center, UM76-INSERM U974-CNRS FRE 361, Sorbonne University, UPMC University of Paris 6.
authors.forename: Bruno
authors.initials: B
authors.lastname: Cadot
authors.affiliation: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa; Myology Research Center, UM76-INSERM U974-CNRS FRE 361, Sorbonne University, UPMC University of Paris 6; edgargomes@medicina.ulisboa.pt.
authors.forename: Edgar R
authors.initials: ER
authors.lastname: Gomes
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique / Q000295:instrumentation / Q000379:methods*; D006614:Hindlimb; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D018482:Muscle, Skeletal / Q000166:cytology*; D032446:Myoblasts / Q000166:cytology; D014162:Transfection / Q000295:instrumentation / Q000379:methods