Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders

Abstract

Background: Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies.

Methods: Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders.

Results: The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice.

Conclusions: Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.

Figure 1

Proliferative potential. Lifespan plots (mean population doublings; MPD) of the parental populations and the immortalized cell lines derived from them.

Figure 2

Myogenic markers and in vitro differentiation. (A) Reverse transcriptase PCR comparing primary (P) and immortalized (Im.) cell lines (control, oculopharyngeal muscular dystrophy (OPMD), Duchenne muscular dystrophy (DMD)) for myogenic markers (MyoD, N-CAM and desmin). Fibroblasts were used as a negative control. (B) Immunofluorescence was carried out using MF20, an antibody directed against sarcomeric myosin (green) after 5 days of differentiation. Specific antibody labeling was visualized using Alexa Fluor 488 secondary antibody (green). Nuclei were visualized with Hoechst (blue). Original magnification × 100.

Figure 3

Detection of human immortalized cells injected into in the tibialis anterior muscle of Rag2^-/- γC^-/- C5^-/- mice. (A) Human nuclei were visualized using an anti-lamin A/C antibody (red) and fibers expressing human proteins were visualized using an anti-human spectrin-specific antibody (green). Nuclei are counterstained with Hoechst. Original magnification × 200. (B) The immortalized cells were present preferentially as myonuclei (top panel), with a small number found in interstitial space (arrow, bottom panel), identified by human lamin expression (laminin staining in green, laminA/C and spectrin staining in red, Hoechst in blue, on the LGMD2B muscle section as an example). Original magnification × 600.

Figure 4

Antisense oligonucleotides (AONs) tested in immortalized cells for exon-skipping pre-clinical studies. (A) Validation of AO51 efficacy for exon 51 skipping on primary (Prim.; Duchenne muscular dystrophy (DMD)) and immortalized cultures of a Δ48-50 patient with DMD. (B) Evaluation of the efficacy of various AONs on control immortalized cells targeting exons 17 and 18.