title: Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts.
abstract: Primary human skeletal muscle cells (hSkMCs) are invaluable tools for deciphering the basic molecular mechanisms of muscle-related biological processes and pathological alterations. Nevertheless, their use is quite restricted due to poor availability, short life span and variable purity of the cells during in vitro culture. Here, we evaluate a recently published method of hSkMCs immortalization, relying on ectopic expression of cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4) and telomerase (TERT) in myoblasts from healthy donors (n=3) and myotonic dystrophy type 1 (DM1) patients (n=2). The efficacy to maintain the myogenic and non-transformed phenotype, as well as the main pathogenetic hallmarks of DM1, has been assessed. Combined expression of the three genes i) maintained the CD56(NCAM)-positive myoblast population and differentiation potential; ii) preserved the non-transformed phenotype and iii) maintained the CTG repeat length, amount of nuclear foci and aberrant alternative splicing in immortal muscle cells. Moreover, immortal hSkMCs displayed attractive additional features such as structural maturation of sarcomeres, persistence of Pax7-positive cells during differentiation and complete disappearance of nuclear foci following (CAG)7 antisense oligonucleotide (ASO) treatment. Overall, the CCND1, CDK4 and TERT immortalization yields versatile, reliable and extremely useful human muscle cell models to investigate the basic molecular features of human muscle cell biology, to elucidate the molecular pathogenetic mechanisms and to test new therapeutic approaches for DM1 in vitro.
authors.affiliation: Department of Neurosciences, University of Padua, Italy. Electronic address: boris.pantic@yahoo.it.
authors.forename: Boris
authors.initials: B
authors.lastname: Pantic
authors.affiliation: Department of Neurosciences, University of Padua, Italy. Electronic address: doriana89b@hotmail.it.
authors.forename: Doriana
authors.initials: D
authors.lastname: Borgia
authors.affiliation: Department of Surgery, Oncology and Gastroenterology, Section of Oncology and Immunology, University of Padua, Padua, Italy. Electronic address: silvia.giunco@unipd.it.
authors.forename: Silvia
authors.initials: S
authors.lastname: Giunco
authors.affiliation: Department of Neurosciences, University of Padua, Italy. Electronic address: adriana.malena@unipd.it.
authors.forename: Adriana
authors.initials: A
authors.lastname: Malena
authors.affiliation: Division of Carcinogenesis and Cancer Prevention, National Cancer Center Research Institute, Tokyo, Japan. Electronic address: tkiyono@ncc.go.jp.
authors.forename: Tohru
authors.initials: T
authors.lastname: Kiyono
authors.affiliation: Department of Biomedical Sciences, University of Padua, Italy. Electronic address: patgen08@mail.bio.unipd.it.
authors.forename: Sergio
authors.initials: S
authors.lastname: Salvatori
authors.affiliation: Department of Surgery, Oncology and Gastroenterology, Section of Oncology and Immunology, University of Padua, Padua, Italy; Unit of Viral Oncology, Istituto Oncologico Veneto (IOV)-IRCCS, Padua, Italy. Electronic address: anita.derossi@unipd.it.
authors.forename: Anita
authors.initials: A
authors.lastname: De Rossi
authors.affiliation: Department of Biomedicine and Prevention, Tor Vergata University of Rome, Italy; Molecular Genetics Laboratory UILDM, Santa Lucia Foundation, Rome, Italy. Electronic address: giardina@med.uniroma2.it.
authors.forename: Emiliano
authors.initials: E
authors.lastname: Giardina
authors.affiliation: Department of Biomedicine and Prevention, Tor Vergata University of Rome, Italy. Electronic address: sangiuolo@med.uniroma2.it.
authors.forename: Federica
authors.initials: F
authors.lastname: Sangiuolo
authors.affiliation: Department of Neurosciences, University of Padua, Italy. Electronic address: elena.pegoraro@unipd.it.
authors.forename: Elena
authors.initials: E
authors.lastname: Pegoraro
authors.affiliation: Department of Neurosciences, University of Padua, Italy. Electronic address: lodovica.vergani@unipd.it.
authors.forename: Lodovica
authors.initials: L
authors.lastname: Vergani
authors.affiliation: Department of Biomedicine and Prevention, Tor Vergata University of Rome, Italy. Electronic address: botta@med.uniroma2.it.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Botta
mesh_terms: D017398:Alternative Splicing; D002454:Cell Differentiation; D002478:Cells, Cultured; D006801:Humans; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D032446:Myoblasts / Q000502:physiology*; D009223:Myotonic Dystrophy / Q000473:pathology*; D061251:Primary Cell Culture; D012518:Sarcomeres / Q000378:metabolism; D019680:Trinucleotide Repeat Expansion
keywords: ASOs; CDK4; Cyclin D1; Human muscle cell models; Immortalization; Myotonic dystrophy type 1; TERT