Muscular dystrophies constitute a heterogeneous group of genetic muscle diseases characterized by progressive muscle weakness, wasting and degeneration, some of these features are common to muscle aging [1, 2]. Over the past few years, the genetics and pathophysiology of some of these diseases has been deciphered, stimulating the development of novel gene-based (or mRNA-based) (for example, gene therapy, exon-skipping or codon read-through), cell-based and pharmacological therapies , which can either target the mutation directly, or target the consequences of that mutation, such as muscle wasting, atrophy or denervation. To assess these rapidly developing therapeutic advances, there is a crucial need to develop standardized tools to determine the cellular and molecular mechanisms that trigger the physiopathologic modifications, and to assess these new therapeutic strategies in preclinical trials. Transgenic mice have often been used to investigate the physiopathology of muscular dystrophies [4–6]; however, the mutation remains in a murine context, and there are often major differences between humans and mice; for example, a mutation in the dystrophin gene results in a mild pathological phenotype in mdx mice but in a progressive and fatal disease (Duchenne muscular dystrophy; DMD) in humans. Furthermore, not every mutation can be created and evaluated in murine models, and mechanisms common to aging and dystrophies may differ between mice and humans. Consequently, human primary myoblasts isolated from dystrophic patient biopsies provide the most pertinent experimental models to assess a variety of human genetic mutations in their natural genomic environment. Although in vitro models do not fully recapitulate the in vivo environment, cell-culture systems allow rapid, high-throughput screening of molecules or oligonucleotides, and new strategies can be easily tested prior to validation in animal models, which is a costly and time-consuming process. The main drawbacks of using in vitro primary cultures of human cells derived from muscle biopsies are their purity, their limited proliferative capacity, and the variation in phenotype when amplified in vitro; their phenotype will always be confounded by modifications due to cellular senescence, which will progressively occur during cell amplification [7, 8].
The two major mechanisms responsible for this replicative cellular senescence seen in human myoblasts are (i) activation of the p16-mediated cellular stress pathway, and (ii) the progressive erosion of telomeres at each cell division until they reach a critical length that will trigger p53 activation and cell-cycle exit [9, 10]. Introduction of the telomerase catalytic subunit (human telomerase reverse transcriptase; hTERT) cDNA alone will result in an extension of the lifespan and even immortalization in a variety of cell types, including endothelial cells and fibroblasts [11, 12]. However, we have shown that the expression of both hTERT and cyclin-dependent kinase (CDK)-4 is required to successfully overcome cellular senescence in human myoblasts ; while hTERT elongates the telomere, CDK-4 blocks the p16INK4a-dependent stress pathway.
In the present study, our goal was to create a large collection of immortalized human myoblasts isolated from a wide range of neuromuscular disorders (DMD, facioscapulohumeral muscular dystrophy (FSHD), oculopharyngeal muscular dystrophy (OPMD), limb-girdle muscular dystrophy (LGMD2B or dysferlinopathy) and congenital muscular dystrophy (CMD)), which could be used as experimental tools to study these diseases and to develop new therapeutic strategies.
DMD is the most common childhood muscular dystrophy. It is caused by mutations in the dystrophin gene encoding an essential protein of the muscle membrane cytoskeleton , leading to rapid and progressive skeletal-muscle weakness. FSHD is a progressive muscle disease caused by contractions in a 3.3 kb repeat region (D4Z4) located at 4q35.2 , which first affects the muscles of the face and upper limb girdle with asymmetry, and later the lower limb girdle. OPMD is a rare, autosomal dominant, late-onset degenerative muscle disorder caused by a short (GCG)n triplet expansion in the poly(A) binding protein nuclear 1 (PABPN1) gene , which affects the eyelid and pharyngeal muscles. LGMD2B is a recessive muscle disease caused by mutations in the dysferlin gene, a muscle membrane protein known to be involved in membrane repair  and trafficking. The disease is characterized by early and slowly progressive weakness and atrophy of the pelvic and shoulder girdle muscles in early adulthood. Finally, CMD refers to a clinically and genetically heterogeneous group of dystrophies, which result in the onset of muscle weakness at birth or in childhood, and involve mutations in several proteins such as collagen, laminin, integrin, and nesprin 1 .
In this study, we report for the first time that for each of these muscular dystrophies, we were able to produce reliable and stable immortalized cell lines from human myoblasts isolated from biopsies, resulting in robust in vitro models that can also be implanted in vivo. This non-exhaustive list of cellular models will provide powerful and valuable tools for the scientific community investigating these pathological conditions and/or their mechanisms. as they overcome the problem of limited proliferation usually present in myoblasts. These models should also be useful in the development of gene or cell therapies and pharmacological strategies for muscular dystrophies, some of which might also be used to combat muscle weakness in the elderly.