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

Immortalized pathological human myoblasts: towards a universal tool for the study of... 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. Background some of these diseases has been deciphered, stimulating Muscular dystrophies constitute a heterogeneous group the development of novel gene-based (or mRNA-based) of genetic muscle diseases characterized by progressive (for example, gene therapy, exon-skipping or codon muscle weakness, wasting and degeneration, some of read-through), cell-based and pharmacological therapies these features are common to muscle aging [1,2]. Over [3], which can either target the mutation directly, or tar- the past few years, the genetics and pathophysiology of get the consequences of that mutation, such as muscle wasting, atrophy or denervation. To assess these rapidly developing therapeutic advances, there is a crucial need * Correspondence: vincent.mouly@upmc.fr to develop standardized tools to determine the cellular † Contributed equally and molecular mechanisms that trigger the physiopatho- Thérapie des maladies du muscle strié, Institut de Myologie, UM76, UPMC Université Paris 6, Paris, France logic modifications, and to assess these new therapeutic Full list of author information is available at the end of the article © 2011 Mamchaoui et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 2 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 strategies in preclinical trials. Transgenic mice have DMD is the most common childhood muscular dys- often been used to investigate the physiopathology of trophy. It is caused by mutations in the dystrophin gene muscular dystrophies [4-6]; however, the mutation encoding an essential protein of the muscle membrane remains in a murine context, and there are often major cytoskeleton [14], leading to rapid and progressive skele- differences between humans and mice; for example, a tal-muscle weakness. FSHD is a progressive muscle dis- mutation in the dystrophin gene results in a mild patho- ease caused by contractions in a 3.3 kb repeat region logical phenotype in mdx mice but in a progressive and (D4Z4) located at 4q35.2 [15], which first affects the fatal disease (Duchenne muscular dystrophy; DMD) in muscles of the face and upper limb girdle with asymme- try, and later the lower limb girdle. OPMD is a rare, humans. Furthermore, not every mutation can be cre- ated and evaluated in murine models, and mechanisms autosomal dominant, late-onset degenerative muscle dis- common to aging and dystrophies may differ between order caused by a short (GCG) triplet expansion in the mice and humans. Consequently, human primary myo- poly(A) binding protein nuclear 1 (PABPN1)gene[16], blasts isolated from dystrophic patient biopsies provide which affects the eyelid and pharyngeal muscles. the most pertinent experimental models to assess a vari- LGMD2B is a recessive muscle disease caused by muta- ety of human genetic mutations in their natural genomic tions in the dysferlin gene, a muscle membrane protein environment. Although in vitro models do not fully known to be involved in membrane repair [17] and traf- recapitulate the in vivo environment, cell-culture sys- ficking. The disease is characterized by early and slowly tems allow rapid, high-throughput screening of mole- progressive weakness and atrophy of the pelvic and cules or oligonucleotides, and new strategies can be shoulder girdle muscles in early adulthood. Finally, easily tested prior to validation in animal models, which CMD refers to a clinically and genetically heterogeneous is a costly and time-consuming process. The main draw- group of dystrophies, which result in the onset of mus- backs of using in vitro primary cultures of human cells cle weakness at birth or in childhood, and involve muta- derived from muscle biopsies are their purity, their lim- tions in several proteins such as collagen, laminin, ited proliferative capacity, and the variation in pheno- integrin, and nesprin 1 [18]. type when amplified in vitro; their phenotype will always In this study, we report for the first time that for each be confounded by modifications due to cellular senes- of thesemuscular dystrophies,wewereabletoproduce cence, which will progressively occur during cell amplifi- reliable and stable immortalized cell lines from human cation [7,8]. myoblasts isolated from biopsies, resulting in robust in The two major mechanisms responsible for this vitro models that can also be implanted in vivo.This replicative cellular senescence seen in human myo- non-exhaustive list of cellular models will provide blasts are (i) activation of the p16-mediated cellular powerful and valuable tools for the scientific community stress pathway, and (ii) the progressive erosion of telo- investigating these pathological conditions and/or their meres at each cell division until they reach a critical mechanisms. as they overcome the problem of limited length that will trigger p53 activation and cell-cycle proliferation usually present in myoblasts. These models exit [9,10]. Introduction of the telomerase catalytic should also be useful in the development of gene or cell subunit (human telomerase reverse transcriptase; therapies and pharmacological strategies for muscular hTERT) cDNA alone will result in an extension of the dystrophies, some of which might also be used to com- lifespan and even immortalization in a variety of cell bat muscle weakness in the elderly. types, including endothelial cells and fibroblasts [11,12]. However, we have shown that the expression Methods of both hTERT and cyclin-dependent kinase (CDK)-4 is Ethics approval required to successfully overcome cellular senescence Muscle biopsies (Table 1) were obtained from the BTR in human myoblasts [13]; while hTERT elongates the (Bank of Tissues for Research, a partner in the EU net- INK4a telomere, CDK-4 blocks the p16 -dependent stress work EuroBioBank) or from neurologists, in accordance pathway. with European recommendations and French legislation. In the present study, our goal was to create a large Surgical procedures were performed in accordance with collection of immortalized human myoblastsisolated the legal regulations in France and European Union from a wide range of neuromuscular disorders (DMD, ethics guidelines for animal research. facioscapulohumeral muscular dystrophy (FSHD), oculo- pharyngeal muscular dystrophy (OPMD), limb-girdle Human myoblast cultures muscular dystrophy (LGMD2B or dysferlinopathy) and Human myoblasts were isolated from biopsies and culti- congenital muscular dystrophy (CMD)), which could be vated as described previously [19] in a growth medium used as experimental tools to study these diseases and consisting of 199 medium and DMEM (Invitrogen to develop new therapeutic strategies. Carlsbad, CA) in a 1:4 ratio, supplemented with 20% Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 3 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 Table 1 Muscle biopsies obtained from various neuromuscular dystrophies. Name Disease Genetic defect Donor muscle Age CTRL None None Semitendinosus 25 years CMD Congenital muscular dystrophy 2345G > T; nesprin-1 gene Paravetrebral 16 years DMD Duchenne muscular dystrophy Deletion of exon 48-50; dystrophin gene Quadriceps 20 months FSHD Fascioscapulohumeral muscular dystrophy 2 D4Z4 contraction Subscapularis 27 years LGMD2B Limb-girdle muscular dystrophy type 2B 1448C > A and 107T > A; dysferlin gene Quadriceps 40 years OPMD Oculopharyngeal muscular dystrophy Expansion (GCG) -(GCG) ; PABPN1 gene Cricopharyngeal 60 years 9 6 FCS (Invitrogen), 2.5 ng/ml hepatocyte growth factor Reverse transcriptase PCR (Invitrogen), 0.1 μmol/l dexamethasone (Sigma-Aldrich, To analyze the expression of myogenic markers in pro- St. Louis, MO, USA) and 50 μg/ml gentamycin (Invitro- liferating primary and immortalized cell lines, 1 μg RNA gen). The myogenic purity of the populations was moni- from each cell line was used for the cDNA synthesis tored by immunocytochemistry using desmin as marker. (Superscript III; Invitrogen) using random hexamer pri- Enrichment of myogenic cells was performed using an mers. cDNA (1 μl) was used as a template for PCR using N-CAM, MyoD and desmin specific primers. The immunomagnetic cell sorting system (MACS; Miltenyi primer sequences and detailed PCR protocols used are Biotec, Paris, France) according to the manufacturer’s available on request. instructions. Briefly, cells were labeled with anti-CD56 (a specific marker of myoblasts) microbeads, and then separated in a MACS column placed in a magnetic field. Induction of host muscle regeneration and implantation Purification was checked by immunochemistry using a of human cells -/- -/- -/- desmin marker. Differentiation was induced at conflu- Immunodeficient Rag2 gC C5 mice aged 2 to 3 ence by replacing the growth medium with DMEM sup- months were anesthetized with an intraperitoneal injec- plemented with 100 μg/ml transferrin, 10 μg/ml insulin tion of ketamine hydrochloride (80 mg/kg) and xylasin and 50 μg/ml of gentamycin (Sigma-Aldrich). (10 mg/kg) (Sigma-Aldrich). To induce severe muscle damage and trigger regeneration, the recipient tibialis Cell transduction anterior (TA) muscles were exposed to cryodamage, and hTERT and Cdk4 cDNA were cloned into different a single injection of immortalized human cells (15 μlof 5 5 pBABE retroviral vectors containing puromycin and cell suspension containing 2.5 × 10 or 5 × 10 cells in neomycin selection markers, respectively. Infection was PBS) was administered as described previously [23]. carried out as described previously [20]. Transduced cell Four weeks after transplantation, the recipient TA mus- cultures were selected with puromycin (0.2 μg/ml) and/ cles were dissected, mounted in gum tragacanth, and or neomycin (0.3 mg/ml) for 8 days. The infected cells frozen in liquid nitrogen-cooled isopentane for later were purified as described previously if necessary, and analysis. were then seeded at clonal density. Selected individual myogenic clones were isolated from each population, Immunofluorescence using glass cylinders, and their proliferation and differ- In vitro and in vivo characterizations were performed by immunolabeling as described previously [23-25]. Antibo- entiation capacities were characterized. dies used were directed against myosin isoforms (MF20, mouse IgG2b, 1:20 dilution; Developmental Studies Telomere length analysis Hybridoma Bank, DSHB, Iowa City, IA), lamin A/C Genomic DNA was extracted from each proliferating (clone JOL2, mouse IgG1, 1:300; AbCam, Cambridge, cell line using a salting-out procedure. Telomere length was determined by using a quantitative (q)PCR method, Cambridgeshire, UK), lamin A/C (NCL-LAM A/C, clone as previously described [21,22]. PCR amplification was 636, mouse IgG2b, 1:400, Novocastra, Newcastle-upon- achieved using telomere (T) and single-copy gene 36B4 Tyne, Tyne and Wear, UK), spectrin (NCL-Spec1, clone (acidic ribosomal phosphoprotein P0) (S) primers. The RBC2/3D5, mouse IgG2b, 1:50; Novocastra), and lami- mean telomere length was calculated as the ratio of telo- nin (rabbit polyclonal, Z 0097, 1:400; Dako, Trappes, mere repeats to 36B4 copies, represented as the T:S France). The secondary antibodies used were Alexa ratio. Each sample was run in triplicate, using 20 ng of Fluor 488-conjugated goat anti-mouse IgG2b (Molecular DNA per replicate, and three independent runs were Probes, Montluçon, France), Alexa Fluor 647-conjugated analyzed. The primer sequences and detailed PCR pro- goat anti-rabbit (Molecular Probes), and Cy3-conjugated tocols used are available on request. goat anti-mouse IgG1 (Jackson Immunoresearch, West Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 4 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 Grove, PA, USA). Images were visualized using a micro- reaching senescence, but remained within the range of scope (Olympus Corp., Tokyo, Japan), and digitized that seen both in myoblasts and in other stem cells (12 using a charge-coupled device (CCD) camera (Olympus to 20 kb). Corp., Tokyo, Japan). In vitro characterization of immortalized cells Antisense oligonucleotides transfection and reverse To confirm that the immortalized cell lines maintained transcriptase PCR their myogenic signature, we compared the expression Cells were seeded in six-well plates and grown in of several markers in proliferating primary and immor- growth medium. Transfection of antisense oligonucleo- talized cell lines from control, OPMD and DMD biop- tides (AONs) was performed using 1 μl of transfection sies. In all of them, we confirmed the expression of the reagent (Lipofectamin 2000; Invitrogen) per μg of AONs myogenic markers desmin, neural cell adhesion mole- for 4 hours. The chemistry used for AONs was 2’-O- cule (N-CAM) and MyoD (Figure 2A). methyl-phosphorothioates. All transfections were per- In addition, we also tested their ability to differentiate formed with at least two independent duplicates. Cells into myotubes, using immunostaining with MF20 anti- were changed to differentiation medium before transfec- body, which recognizes all skeletal-muscle myosin heavy tion. Typically 24 to 48 hours after transfection, RNA chains (MyHCs). After 5 days in differentiation condi- was extracted from the cells using Trizol or Qiagen col- tions, all of the immortalized cell lines were able to fuse umn kit (Qiagen Inc., Valencia, CA, USA). 1 μgRNA into myotubes expressing MyHCs (Figure 2B). was used for the cDNA synthesis (Superscript III; Invi- trogen) with DMD exon-specific primers. cDNA (2 μl) Induction of host muscle regeneration and implantation was used as a template for a first PCR reaction. From of human cells this first reaction of 25 cycles, 1 μl of the product was To investigate the in vivo behavior of these immorta- removed and used as a template for a second nested lized cells, cells were grafted into damaged TA muscles -/- -/- -/- PCR of 35 cycles. PCR products were analyzed on 1.5 to of Rag2 gC C5 mice; injected muscles were ana- 2% agarose gels. The primer sequences and detailed lyzed 4 weeks after transplantation, which corresponds PCR protocols used are available on request. to a complete fiber regeneration process, using antibo- dies specific for human lamin A/C (expressed in all Results human nuclei) and human spectrin (expressed in differ- Immortalized myoblast lines generated from dystrophic entiated fibers). For each injected clone (control, DMD, muscles FSHD, OPMD, CMD or LGMD2B), mature muscle Primary cultures from distinct muscular dystrophies fibers containing human spectrin protein and human (DMD, FSHD, OPMD, CMD and LGMD2B, Table 1) lamin A/C+ nuclei were seen (Figure 3A). No tumors were co-transduced with two retroviral vectors expres- were ever observed in these immunodeficient mice. sing hTERT and CDK-4 cDNA. Co-transduced cells Using antibodies specific for the basal lamina protein were selected by neomycin and puromycin and then laminin, lamin A/C (human nuclei) and spectrin (speci- purified using magnetic beads coupled to antibodies fic to the human protein) to identify fiber sarcolemma, directed against the myogenic marker CD56. Following we investigated if these cell lines could replenish the culture at clonal density, individual myogenic clones muscle stem-cell niche (allowing self-renewal), at the with extended proliferative lifespans, as compared to the periphery of the muscle fiber and beneath the basal untranduced cells, were isolated from each population. lamina. Whereas the vast majority of the lamin A/C- In contrast to the parental populations, which stopped positive nuclei (97%) were found as myonuclei (upper proliferating at various stages of the culture, depending panel, Figure 3B), we observed the unexpected finding on the type of dystrophy, the selected immortalized that all the human cells outside the muscle fibers were clones were still able to proliferate after prolonged present in the interstitial space, separated from the amplification in vitro under the same culture conditions fibers by a basal lamina (lower panel, Figure 3B), and (Figure 1). All immortalized clones were cultivated until not in the satellite-cell niche, suggesting that the they had achieved at least twice as many divisions as the immortalized cells were engaged preferentially in the parental population. differentiation pathway and not in the self-renewal Telomere length was measured in each clone (Table process. 2) and ranged from 10.3 kb to 24.8 kb with no differ- ence between the clones and control immortalized myo- Immortalized cell lines as a useful tool for therapeutic blasts (17.6 kb). The length of the telomeres in all of the preclinical studies immortalized myogenic clones was always well above To show that these cell lines could be powerful tools to the 6 to 7 kb limit usually seen in control cells that are develop therapeutic strategies, we used them to evaluate Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 5 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 Figure 1 Proliferative potential. Lifespan plots (mean population doublings; MPD) of the parental populations and the immortalized cell lines derived from them. the efficiency of AONs in an exon-skipping strategy for and this AON is currently being used in a phase I/II DMD. The first one tested was AO51, which resulted in clinical trial. Using immortalized control cells, we were efficient skipping of exon 51 using both the primary and able to screen a range of AONs targeting exons 17, 18, immortalized DMD cell lines (Δ48 to 50; Figure 4A) 21, 22, 43, 44 and 45 of the dystrophin gene. RT-PCR Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 6 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 Table 2 Mean telomere length of control and immortalized cell lines. Name Clone Number of Telomere length, kb, mean divisions ± SEM CTRL C25Cl48 127 17.6 ± 0.3 CMD CMDCl12 42 20.8 ± 1.7 DMD DMDCl2 57.9 10.3 ± 0.1 FSHD FSHDCl17 37.9 24.8 ± 1.6 LGMD2B LGMD2Cl11 27.4 17.2 ± 3.0 OPMD OPMDCl2 47.6 20.0 ± 0.5 CMD, congenital muscular dystrophy; DMD, Duchenne muscular dystrophy; FSHD, fascioscapulohumeral muscular dystrophy; LGMD2B, limb-girdle muscular dystrophy type 2B; OPMD, oculopharyngeal muscular dystrophy identified efficient skipping for two of them (Figure 4B, and data not shown). Discussion Immortal human cell lines, as long as they retain their capacity to express a specific program, are essential to study cellular and molecular mechanisms (as exempli- fied by the number of studies conducted on the mouse cell line C2C12), and responses to potential therapeutic strategies. The relatively short proliferative lifespan of human myoblasts, reduced even more in dystrophic conditions by successive cycles of degeneration/regen- eration in vivo prior to isolation and the modification in their myogenic potential as they approach senescence [26-28], limits their potential use. As a consequence, any assessment of pathological mechanisms or of thera- peutic strategies will be biased by the presence of senes- cent cells, which will modify the behavior of the population. This is even more crucial for high-through- Figure 2 Myogenic markers and in vitro differentiation. (A) Reverse transcriptase PCR comparing primary (P) and immortalized put screening of molecules, for which large numbers of (Im.) cell lines (control, oculopharyngeal muscular dystrophy cells are required. Immortalization can solve this pro- (OPMD), Duchenne muscular dystrophy (DMD)) for myogenic blem, as long as the cell lines are stable and retain most markers (MyoD, N-CAM and desmin). Fibroblasts were used as a of the characteristics of the unmodified parental popula- negative control. (B) Immunofluorescence was carried out using tion. This has been shown to be a problem with the MF20, an antibody directed against sarcomeric myosin (green) after 5 days of differentiation. Specific antibody labeling was visualized C2C12 cell line, as the phenotype drifts and therefore using Alexa Fluor 488 secondary antibody (green). Nuclei were can vary both within and between different laboratories. visualized with Hoechst (blue). Original magnification × 100. Immortal cell lines have been generated from human skeletal muscle, such as those derived from rhabdomyo- sarcomas, a rare form of skeletal-muscle tumor. How- ever, these lines often have impaired fusion downregulates both the p16 and p19Arf tumor suppres- characteristics and perturbed myogenic programs sor genes, encoded by the Ink4 locus. These cells had [29,30]. Other approaches have used transduction of the an extended lifespan with no chromosomal rearrange- large T antigen from SV40, which binds Rb and p53, ment, but the differentiation potential of control myo- but does not stop telomere shortening. Although these blasts was found to be impaired [33]. This year, a report cells do have an extended lifespan, they are not immor- described the same approach as we have used in the tal, and extensive telomeric erosion results in an present study (introduction of hTERT and CDK4) using increased frequency of chromosomal rearrangement [31] muscle cells isolated from patients affected with FSHD and a defective differentiation program [32]. More [34]. The FSHD mutation causes a defect within myo- recently, cell lines were generated by the transduction of genic cells, thus the establishment of a clonal myogenic primary myoblasts with both hTERT and Bmi-1, which cell line permit reproducible study of the consequences Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 7 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 myogenicity during successive passages of primary cul- tures of muscle cells isolated from patients with OPMD [35]; many muscle diseases are subject to a similar decrease in myogenicity. In this report, we describe the isolation of immortalized human myoblast lines from a wide range of neuromuscular diseases, using a combina- tion of hTERT and CDK-4. This wide range of diseases paves the way to searching for common mechanisms between distinct dystrophies, and even those shared with muscle aging, as opposed to those mechanisms specific for each disease. We found that these cell lines have extended proliferative lifespans, and maintain their capacity to differentiate both in vitro and in vivo after transplantation into the regenerating muscles of immu- nodeficient mice. We found that these human myoblast cell lines expressing myogenic markers could colonize the host muscles and form mature fibers, thus providing an ideal model to assess therapeutic strategies in vivo, which is closer to bona fide differentiation of muscle stem cells than the converted fibroblasts described pre- viously [36]. We also found that these cells do not replenish the muscle stem-cell niche as primary human myoblasts do under the same conditions of implantation [23], but are engaged primarily in the differentiation pathway. The process of immortalization involves over- expression of both hTERT and CDK-4, and further investigations will be needed to analyze how this may influence the balance between self-renewal and differentiation. Conclusions These human myoblast lines represent a powerful tool to assess signaling and/or functional deregulations in neuromuscular diseases, particularly those in which Figure 3 Detection of human immortalized cells injected into -/- -/- -/- these mechanisms have not yet been clearly elucidated gC C5 mice. (A) in the tibialis anterior muscle of Rag2 (FSHD, OPMD) or those with features common to mus- Human nuclei were visualized using an anti-lamin A/C antibody (red) and fibers expressing human proteins were visualized using an cle aging, such as atrophy or muscle wasting. We have anti-human spectrin-specific antibody (green). Nuclei are described only a subset of the cell lines produced; we counterstained with Hoechst. Original magnification × 200. (B) The have now generated more than 35 cell lines with various immortalized cells were present preferentially as myonuclei (top mutations covering a range of 14 different pathologies, panel), with a small number found in interstitial space (arrow, as well as cell lines from control subjects of various bottom panel), identified by human lamin expression (laminin staining in green, laminA/C and spectrin staining in red, Hoechst in ages. The in vivo implantation of these cells offers the blue, on the LGMD2B muscle section as an example). Original possibility to investigate the consequences of defined magnification × 600. mutations on cellular behavior in vivo, particularly with regard to their regenerative capacity. Finally, the devel- opment of therapeutic strategies, whether these strate- of this mutation within myoblasts without the contami- gies imply gene, cell or pharmacological therapy nation of non-myogenic cells. The decrease in myogeni- involving high-throughput screening, is facilitated using city in the primary culture, and consequently the these tools before assessment in animal models. As an enrichment of non-myogenic cells during amplification, example, we have described a rapid screen of a range of is a considerable problem in the study of dystrophy dis- AONs in an exon-skipping strategy for DMD. In conclu- eases. For example, in 2006, we described rapid loss of sion, the co-transduction of hTERT and CDK4 Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 8 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 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. List of abbreviations generates human myoblasts immortal in culture that AONs: antisense oligonucleotides; CDK-4: cyclin-dependent kinase 4; CMD: maintain myogenic potential both in vitro and in vivo, congenital muscular dystrophy; DMD: Duchenne muscular dystrophy; DMEM: offering new cell paradigms for pathophysiological stu- Dulbecco’s modified Eagle’s medium; FCS: fetal calf serum; FSHD: facioscapulohumeral muscular dystrophy; hTERT: human telomerase reverse dies and novel therapeutic strategies. transcriptase; LGMD2B: limb-girdle muscular dystrophy type 2b; N-CAM: Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 9 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 neural cell adhesion molecule; OPMD: oculopharyngeal muscular dystrophy; 6. Allamand V, Campbell KP: Animal models for muscular dystrophy: PBS: phosphate-buffered serum; PCR: polymerase chain reaction. valuable tools for the development of therapies. Hum Mol Genet 2000, 9:2459-2467. Acknowledgements 7. Mouly V, Aamiri A, Perie S, Mamchaoui K, Barani A, Bigot A, Bouazza B, We thank all the patients who provided the biopsies to establish the Francois V, Furling D, Jacquemin V, et al: Myoblast transfer therapy: is primary cultures. We also thank the MSG study group, particularly D. Furling there any light at the end of the tunnel? Acta Myol 2005, 24:128-133. for fruitful discussions and L. Dollé, S. Sandal, M. Oloko, S. Vasseur and M. 8. Webster C, Blau HM: Accelerated age-related decline in replicative life- Chapart for technical assistance. We thank Genosafe for help with the span of Duchenne muscular dystrophy myoblasts: implications for cell transductions and Steve Wilton for providing the antisense oligonucleotides. and gene therapy. Somat Cell Mol Genet 1990, 16:557-565. This work was supported by the MYORES Network of Excellence (contract 9. Renault V, Thornell LE, Eriksson PO, Butler-Browne G, Mouly V: Regenerative 511978) and TREAT-NMD (contract LSHM-CT-2006-036825) from the potential of human skeletal muscle during aging. Aging Cell 2002, European Commission 6th FP, MYOAGE (contract HEALTH-F2-2009-223576) 1:132-139. from the Seventh FP, the ANR Genopath-INAFIB, the ANR MICRORNAS, 10. Wright WE, Shay JW: Historical claims and current interpretations of MyoGrad (GK1631, German Research Foundation), the Duchenne Parent replicative aging. Nat Biotechnol 2002, 20:682-688. Project Netherlands, CNRS, INSERM, University Pierre and Marie Curie, AFM 11. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, (Association Française contre les Myopathies) (including network grant Shay JW, Lichtsteiner S, Wright WE: Extension of life-span by introduction #15123), the Jain Foundation, Parents Project of Monaco, and the European of telomerase into normal human cells. Science 1998, 279:349-352. Parent Project. 12. 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Nowak KJ, Davies KE: Duchenne muscular dystrophy and dystrophin: of Child Health, University College, London, UK. Muscle Research Unit, pathogenesis and opportunities for treatment. EMBO Rep 2004, 5:872-876. Experimental and Clinical Research Center, Charité University Hospital and 15. Tawil R: Facioscapulohumeral muscular dystrophy. Neurotherapeutics 2008, Max Delbrück Center for Molecular Medicine, Berlin, Germany. Faculté de 5:601-606. Médecine de Marseille, Université de la Méditerranée, Inserm UMRS 910 16. Brais B, Bouchard JP, Xie YG, Rochefort DL, Chretien N, Tome FM, Génétique Médicale et Génomique Fonctionnelle, Marseille, France. Lafreniere RG, Rommens JM, Uyama E, Nohira O, et al: Short GCG Department of Neurology, Hillel Yaffe Medical Center, PO Box 169, Hadera, expansions in the PABP2 gene cause oculopharyngeal muscular 38100, Israel. UT Southwestern Medical Center, Department of Cell Biology, dystrophy. Nat Genet 1998, 18:164-167. Dallas, TX 75390, USA. Laboratoire LBCM, Departement de Biologie, Faculté 17. Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, Williamson R, McNeil PL, des Sciences, Agadir, Maroc. Campbell KP: Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature 2003, 423:168-172. Authors’ contributions 18. Reed UC: Congenital muscular dystrophy. Part I: a review of KM, CT, AB, EN, and SC designed and performed the in vitro and in vivo phenotypical and diagnostic aspects. Arq Neuropsiquiatr 2009, 67:144-168. experiments, analyzed data, and wrote the manuscript. AW, PKK, SM, JK, and 19. Bigot A, Klein AF, Gasnier E, Jacquemin V, Ravassard P, Butler-Browne G, -/- AA provided technical support. JDS provided the immunodeficient Rag2 Mouly V, Furling D: Large CTG repeats trigger p16-dependent premature -/- -/- γC C5 mice for the in vivo experiments. JLSG, FM, SP, SS, NL, SB, and TV senescence in myotonic dystrophy type 1 muscle precursor cells. Am J provided biopsies, and WEW provided DNA constructs. TV and AA discussed Pathol 2009, 174:1435-1442. the results and gave expert advice. GBB and VM provided conceptual input 20. Di Donna S, Mamchaoui K, Cooper RN, Seigneurin-Venin S, Tremblay J, and supervision. and wrote the manuscript. All authors read and approved Butler-Browne GS, Mouly V: Telomerase can extend the proliferative the final manuscript. capacity of human myoblasts, but does not lead to their immortalization. Mol Cancer Res 2003, 1:643-653. Competing interests 21. Cawthon RM: Telomere measurement by quantitative PCR. Nucleic Acid The authors declare that they have no competing interests. Research 2002, 30:e47. 22. Cawthon RM: Telomere length measurement by a novel monochrome Received: 20 June 2011 Accepted: 1 November 2011 multiplex quantitative PCR method. Nucleic Acid Research 2009, 37:e21. Published: 1 November 2011 23. Negroni E, Riederer I, Chaouch S, Belicchi M, Razini P, Di Santo J, Torrente Y, Butler-Browne GS, Mouly V: In vivo myogenic potential of human CD133+ muscle-derived stem cells: a quantitative study. Mol Ther 2009, References 17:1771-1778. 1. McNally EM, Pytel P: Muscle diseases: the muscular dystrophies. Annu Rev 24. Cooper RN, Irintchev A, Di Santo JP, Zweyer M, Morgan JE, Partridge TA, Pathol 2007, 2:87-109. Butler-Browne GS, Mouly V, Wernig A: A new immunodeficient mouse 2. Ruegg MA, Glass DJ: Molecular mechanisms and treatment options for model for human myoblast transplantation. Hum Gene Ther 2001, muscle wasting diseases. Annu Rev Pharmacol Toxicol 2011, 51:373-395. 12:823-831. 3. Trollet C, Athanasopoulos T, Popplewell L, Malerba A, Dickson G: Gene 25. Cooper RN, Thiesson D, Furling D, Di Santo JP, Butler-Browne GS, Mouly V: therapy for muscular dystrophy: current progress and future prospects. Extended amplification in vitro and replicative senescence: key factors Expert Opin Biol Ther 2009, 9:849-866. implicated in the success of human myoblast transplantation. Hum Gene 4. Trollet C, Anvar SY, Venema A, Hargreaves IP, Foster K, Vignaud A, Ferry A, Ther 2003, 14:1169-1179. Negroni E, Hourde C, Baraibar MA, et al: Molecular and phenotypic 26. Decary S, Mouly V, Hamida CB, Sautet A, Barbet JP, Butler-Browne GS: characterization of a mouse model of oculopharyngeal muscular Replicative potential and telomere length in human skeletal muscle: dystrophy reveals severe muscular atrophy restricted to fast glycolytic implications for satellite cell-mediated gene therapy. Hum Gene Ther fibres. Hum Mol Genet 2010. 1997, 8:1429-1438. 5. Vignaud A, Ferry A, Huguet A, Baraibar M, Trollet C, Hyzewicz J, Butler- 27. Renault V, Piron-Hamelin G, Forestier C, DiDonna S, Decary S, Hentati F, Browne G, Puymirat J, Gourdon G, Furling D: Progressive skeletal muscle Saillant G, Butler-Browne GS, Mouly V: Skeletal muscle regeneration and weakness in transgenic mice expressing CTG expansions is associated the mitotic clock. Exp Gerontol 2000, 35:711-719. with the activation of the ubiquitin-proteasome pathway. Neuromuscul Disord 2010, 20:319-325. Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 10 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 28. Bigot A, Jacquemin V, Debacq-Chainiaux F, Butler-Browne GS, Toussaint O, Furling D, Mouly V: Replicative aging down-regulates the myogenic regulatory factors in human myoblasts. Biol Cell 2008, 100:189-199. 29. Rossi S, Poliani PL, Cominelli M, Bozzato A, Vescovi R, Monti E, Fanzani A: Caveolin 1 is a marker of poor differentiation in Rhabdomyosarcoma. Eur J Cancer 2010. 30. Wang S, Guo L, Dong L, Li S, Zhang J, Sun M: TGF-beta1 signal pathway may contribute to rhabdomyosarcoma development by inhibiting differentiation. Cancer Sci 2010, 101:1108-1116. 31. Stewart N, Bacchetti S: Expression of SV40 large T antigen, but not small t antigen, is required for the induction of chromosomal aberrations in transformed human cells. Virology 1991, 180:49-57. 32. Mouly V, Edom F, Decary S, Vicart P, Barbert JP, Butler-Browne GS: SV40 large T antigen interferes with adult myosin heavy chain expression, but not with differentiation of human satellite cells. Exp Cell Res 1996, 225:268-276. 33. Cudre-Mauroux C, Occhiodoro T, Konig S, Salmon P, Bernheim L, Trono D: Lentivector-mediated transfer of Bmi-1 and telomerase in muscle satellite cells yields a duchenne myoblast cell line with long-term genotypic and phenotypic stability. Hum Gene Ther 2003, 14:1525-1533. 34. Stadler G, Chen JC, Wagner K, Robin JD, Shay JW, Emerson CP Jr, Wright WE: Establishment of clonal myogenic cell lines from severely affected dystrophic muscles - CDK4 maintains the myogenic population. Skelet Muscle 2011, 1:12. 35. Perie S, Mamchaoui K, Mouly V, Blot S, Bouazza B, Thornell LE, St Guily JL, Butler-Browne G: Premature proliferative arrest of cricopharyngeal myoblasts in oculo-pharyngeal muscular dystrophy: therapeutic perspectives of autologous myoblast transplantation. Neuromuscul Disord 2006, 16:770-781. 36. Chaouch S, Mouly V, Goyenvalle A, Vulin A, Mamchaoui K, Negroni E, Di Santo J, Butler-Browne G, Torrente Y, Garcia L, Furling D: Immortalized skin fibroblasts expressing conditional MyoD as a renewable and reliable source of converted human muscle cells to assess therapeutic strategies for muscular dystrophies: validation of an exon-skipping approach to restore dystrophin in Duchenne muscular dystrophy cells. Hum Gene Ther 2009, 20:784-790. doi:10.1186/2044-5040-1-34 Cite this article as: Mamchaoui et al.: Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders. Skeletal Muscle 2011 1:34. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

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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. Background some of these diseases has been deciphered, stimulating Muscular dystrophies constitute a heterogeneous group the development of novel gene-based (or mRNA-based) of genetic muscle diseases characterized by progressive (for example, gene therapy, exon-skipping or codon muscle weakness, wasting and degeneration, some of read-through), cell-based and pharmacological therapies these features are common to muscle aging [1,2]. Over [3], which can either target the mutation directly, or tar- the past few years, the genetics and pathophysiology of get the consequences of that mutation, such as muscle wasting, atrophy or denervation. To assess these rapidly developing therapeutic advances, there is a crucial need * Correspondence: vincent.mouly@upmc.fr to develop standardized tools to determine the cellular † Contributed equally and molecular mechanisms that trigger the physiopatho- Thérapie des maladies du muscle strié, Institut de Myologie, UM76, UPMC Université Paris 6, Paris, France logic modifications, and to assess these new therapeutic Full list of author information is available at the end of the article © 2011 Mamchaoui et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 2 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 strategies in preclinical trials. Transgenic mice have DMD is the most common childhood muscular dys- often been used to investigate the physiopathology of trophy. It is caused by mutations in the dystrophin gene muscular dystrophies [4-6]; however, the mutation encoding an essential protein of the muscle membrane remains in a murine context, and there are often major cytoskeleton [14], leading to rapid and progressive skele- differences between humans and mice; for example, a tal-muscle weakness. FSHD is a progressive muscle dis- mutation in the dystrophin gene results in a mild patho- ease caused by contractions in a 3.3 kb repeat region logical phenotype in mdx mice but in a progressive and (D4Z4) located at 4q35.2 [15], which first affects the fatal disease (Duchenne muscular dystrophy; DMD) in muscles of the face and upper limb girdle with asymme- try, and later the lower limb girdle. OPMD is a rare, humans. Furthermore, not every mutation can be cre- ated and evaluated in murine models, and mechanisms autosomal dominant, late-onset degenerative muscle dis- common to aging and dystrophies may differ between order caused by a short (GCG) triplet expansion in the mice and humans. Consequently, human primary myo- poly(A) binding protein nuclear 1 (PABPN1)gene[16], blasts isolated from dystrophic patient biopsies provide which affects the eyelid and pharyngeal muscles. the most pertinent experimental models to assess a vari- LGMD2B is a recessive muscle disease caused by muta- ety of human genetic mutations in their natural genomic tions in the dysferlin gene, a muscle membrane protein environment. Although in vitro models do not fully known to be involved in membrane repair [17] and traf- recapitulate the in vivo environment, cell-culture sys- ficking. The disease is characterized by early and slowly tems allow rapid, high-throughput screening of mole- progressive weakness and atrophy of the pelvic and cules or oligonucleotides, and new strategies can be shoulder girdle muscles in early adulthood. Finally, easily tested prior to validation in animal models, which CMD refers to a clinically and genetically heterogeneous is a costly and time-consuming process. The main draw- group of dystrophies, which result in the onset of mus- backs of using in vitro primary cultures of human cells cle weakness at birth or in childhood, and involve muta- derived from muscle biopsies are their purity, their lim- tions in several proteins such as collagen, laminin, ited proliferative capacity, and the variation in pheno- integrin, and nesprin 1 [18]. type when amplified in vitro; their phenotype will always In this study, we report for the first time that for each be confounded by modifications due to cellular senes- of thesemuscular dystrophies,wewereabletoproduce cence, which will progressively occur during cell amplifi- reliable and stable immortalized cell lines from human cation [7,8]. myoblasts isolated from biopsies, resulting in robust in The two major mechanisms responsible for this vitro models that can also be implanted in vivo.This replicative cellular senescence seen in human myo- non-exhaustive list of cellular models will provide blasts are (i) activation of the p16-mediated cellular powerful and valuable tools for the scientific community stress pathway, and (ii) the progressive erosion of telo- investigating these pathological conditions and/or their meres at each cell division until they reach a critical mechanisms. as they overcome the problem of limited length that will trigger p53 activation and cell-cycle proliferation usually present in myoblasts. These models exit [9,10]. Introduction of the telomerase catalytic should also be useful in the development of gene or cell subunit (human telomerase reverse transcriptase; therapies and pharmacological strategies for muscular hTERT) cDNA alone will result in an extension of the dystrophies, some of which might also be used to com- lifespan and even immortalization in a variety of cell bat muscle weakness in the elderly. types, including endothelial cells and fibroblasts [11,12]. However, we have shown that the expression Methods of both hTERT and cyclin-dependent kinase (CDK)-4 is Ethics approval required to successfully overcome cellular senescence Muscle biopsies (Table 1) were obtained from the BTR in human myoblasts [13]; while hTERT elongates the (Bank of Tissues for Research, a partner in the EU net- INK4a telomere, CDK-4 blocks the p16 -dependent stress work EuroBioBank) or from neurologists, in accordance pathway. with European recommendations and French legislation. In the present study, our goal was to create a large Surgical procedures were performed in accordance with collection of immortalized human myoblastsisolated the legal regulations in France and European Union from a wide range of neuromuscular disorders (DMD, ethics guidelines for animal research. facioscapulohumeral muscular dystrophy (FSHD), oculo- pharyngeal muscular dystrophy (OPMD), limb-girdle Human myoblast cultures muscular dystrophy (LGMD2B or dysferlinopathy) and Human myoblasts were isolated from biopsies and culti- congenital muscular dystrophy (CMD)), which could be vated as described previously [19] in a growth medium used as experimental tools to study these diseases and consisting of 199 medium and DMEM (Invitrogen to develop new therapeutic strategies. Carlsbad, CA) in a 1:4 ratio, supplemented with 20% Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 3 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 Table 1 Muscle biopsies obtained from various neuromuscular dystrophies. Name Disease Genetic defect Donor muscle Age CTRL None None Semitendinosus 25 years CMD Congenital muscular dystrophy 2345G > T; nesprin-1 gene Paravetrebral 16 years DMD Duchenne muscular dystrophy Deletion of exon 48-50; dystrophin gene Quadriceps 20 months FSHD Fascioscapulohumeral muscular dystrophy 2 D4Z4 contraction Subscapularis 27 years LGMD2B Limb-girdle muscular dystrophy type 2B 1448C > A and 107T > A; dysferlin gene Quadriceps 40 years OPMD Oculopharyngeal muscular dystrophy Expansion (GCG) -(GCG) ; PABPN1 gene Cricopharyngeal 60 years 9 6 FCS (Invitrogen), 2.5 ng/ml hepatocyte growth factor Reverse transcriptase PCR (Invitrogen), 0.1 μmol/l dexamethasone (Sigma-Aldrich, To analyze the expression of myogenic markers in pro- St. Louis, MO, USA) and 50 μg/ml gentamycin (Invitro- liferating primary and immortalized cell lines, 1 μg RNA gen). The myogenic purity of the populations was moni- from each cell line was used for the cDNA synthesis tored by immunocytochemistry using desmin as marker. (Superscript III; Invitrogen) using random hexamer pri- Enrichment of myogenic cells was performed using an mers. cDNA (1 μl) was used as a template for PCR using N-CAM, MyoD and desmin specific primers. The immunomagnetic cell sorting system (MACS; Miltenyi primer sequences and detailed PCR protocols used are Biotec, Paris, France) according to the manufacturer’s available on request. instructions. Briefly, cells were labeled with anti-CD56 (a specific marker of myoblasts) microbeads, and then separated in a MACS column placed in a magnetic field. Induction of host muscle regeneration and implantation Purification was checked by immunochemistry using a of human cells -/- -/- -/- desmin marker. Differentiation was induced at conflu- Immunodeficient Rag2 gC C5 mice aged 2 to 3 ence by replacing the growth medium with DMEM sup- months were anesthetized with an intraperitoneal injec- plemented with 100 μg/ml transferrin, 10 μg/ml insulin tion of ketamine hydrochloride (80 mg/kg) and xylasin and 50 μg/ml of gentamycin (Sigma-Aldrich). (10 mg/kg) (Sigma-Aldrich). To induce severe muscle damage and trigger regeneration, the recipient tibialis Cell transduction anterior (TA) muscles were exposed to cryodamage, and hTERT and Cdk4 cDNA were cloned into different a single injection of immortalized human cells (15 μlof 5 5 pBABE retroviral vectors containing puromycin and cell suspension containing 2.5 × 10 or 5 × 10 cells in neomycin selection markers, respectively. Infection was PBS) was administered as described previously [23]. carried out as described previously [20]. Transduced cell Four weeks after transplantation, the recipient TA mus- cultures were selected with puromycin (0.2 μg/ml) and/ cles were dissected, mounted in gum tragacanth, and or neomycin (0.3 mg/ml) for 8 days. The infected cells frozen in liquid nitrogen-cooled isopentane for later were purified as described previously if necessary, and analysis. were then seeded at clonal density. Selected individual myogenic clones were isolated from each population, Immunofluorescence using glass cylinders, and their proliferation and differ- In vitro and in vivo characterizations were performed by immunolabeling as described previously [23-25]. Antibo- entiation capacities were characterized. dies used were directed against myosin isoforms (MF20, mouse IgG2b, 1:20 dilution; Developmental Studies Telomere length analysis Hybridoma Bank, DSHB, Iowa City, IA), lamin A/C Genomic DNA was extracted from each proliferating (clone JOL2, mouse IgG1, 1:300; AbCam, Cambridge, cell line using a salting-out procedure. Telomere length was determined by using a quantitative (q)PCR method, Cambridgeshire, UK), lamin A/C (NCL-LAM A/C, clone as previously described [21,22]. PCR amplification was 636, mouse IgG2b, 1:400, Novocastra, Newcastle-upon- achieved using telomere (T) and single-copy gene 36B4 Tyne, Tyne and Wear, UK), spectrin (NCL-Spec1, clone (acidic ribosomal phosphoprotein P0) (S) primers. The RBC2/3D5, mouse IgG2b, 1:50; Novocastra), and lami- mean telomere length was calculated as the ratio of telo- nin (rabbit polyclonal, Z 0097, 1:400; Dako, Trappes, mere repeats to 36B4 copies, represented as the T:S France). The secondary antibodies used were Alexa ratio. Each sample was run in triplicate, using 20 ng of Fluor 488-conjugated goat anti-mouse IgG2b (Molecular DNA per replicate, and three independent runs were Probes, Montluçon, France), Alexa Fluor 647-conjugated analyzed. The primer sequences and detailed PCR pro- goat anti-rabbit (Molecular Probes), and Cy3-conjugated tocols used are available on request. goat anti-mouse IgG1 (Jackson Immunoresearch, West Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 4 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 Grove, PA, USA). Images were visualized using a micro- reaching senescence, but remained within the range of scope (Olympus Corp., Tokyo, Japan), and digitized that seen both in myoblasts and in other stem cells (12 using a charge-coupled device (CCD) camera (Olympus to 20 kb). Corp., Tokyo, Japan). In vitro characterization of immortalized cells Antisense oligonucleotides transfection and reverse To confirm that the immortalized cell lines maintained transcriptase PCR their myogenic signature, we compared the expression Cells were seeded in six-well plates and grown in of several markers in proliferating primary and immor- growth medium. Transfection of antisense oligonucleo- talized cell lines from control, OPMD and DMD biop- tides (AONs) was performed using 1 μl of transfection sies. In all of them, we confirmed the expression of the reagent (Lipofectamin 2000; Invitrogen) per μg of AONs myogenic markers desmin, neural cell adhesion mole- for 4 hours. The chemistry used for AONs was 2’-O- cule (N-CAM) and MyoD (Figure 2A). methyl-phosphorothioates. All transfections were per- In addition, we also tested their ability to differentiate formed with at least two independent duplicates. Cells into myotubes, using immunostaining with MF20 anti- were changed to differentiation medium before transfec- body, which recognizes all skeletal-muscle myosin heavy tion. Typically 24 to 48 hours after transfection, RNA chains (MyHCs). After 5 days in differentiation condi- was extracted from the cells using Trizol or Qiagen col- tions, all of the immortalized cell lines were able to fuse umn kit (Qiagen Inc., Valencia, CA, USA). 1 μgRNA into myotubes expressing MyHCs (Figure 2B). was used for the cDNA synthesis (Superscript III; Invi- trogen) with DMD exon-specific primers. cDNA (2 μl) Induction of host muscle regeneration and implantation was used as a template for a first PCR reaction. From of human cells this first reaction of 25 cycles, 1 μl of the product was To investigate the in vivo behavior of these immorta- removed and used as a template for a second nested lized cells, cells were grafted into damaged TA muscles -/- -/- -/- PCR of 35 cycles. PCR products were analyzed on 1.5 to of Rag2 gC C5 mice; injected muscles were ana- 2% agarose gels. The primer sequences and detailed lyzed 4 weeks after transplantation, which corresponds PCR protocols used are available on request. to a complete fiber regeneration process, using antibo- dies specific for human lamin A/C (expressed in all Results human nuclei) and human spectrin (expressed in differ- Immortalized myoblast lines generated from dystrophic entiated fibers). For each injected clone (control, DMD, muscles FSHD, OPMD, CMD or LGMD2B), mature muscle Primary cultures from distinct muscular dystrophies fibers containing human spectrin protein and human (DMD, FSHD, OPMD, CMD and LGMD2B, Table 1) lamin A/C+ nuclei were seen (Figure 3A). No tumors were co-transduced with two retroviral vectors expres- were ever observed in these immunodeficient mice. sing hTERT and CDK-4 cDNA. Co-transduced cells Using antibodies specific for the basal lamina protein were selected by neomycin and puromycin and then laminin, lamin A/C (human nuclei) and spectrin (speci- purified using magnetic beads coupled to antibodies fic to the human protein) to identify fiber sarcolemma, directed against the myogenic marker CD56. Following we investigated if these cell lines could replenish the culture at clonal density, individual myogenic clones muscle stem-cell niche (allowing self-renewal), at the with extended proliferative lifespans, as compared to the periphery of the muscle fiber and beneath the basal untranduced cells, were isolated from each population. lamina. Whereas the vast majority of the lamin A/C- In contrast to the parental populations, which stopped positive nuclei (97%) were found as myonuclei (upper proliferating at various stages of the culture, depending panel, Figure 3B), we observed the unexpected finding on the type of dystrophy, the selected immortalized that all the human cells outside the muscle fibers were clones were still able to proliferate after prolonged present in the interstitial space, separated from the amplification in vitro under the same culture conditions fibers by a basal lamina (lower panel, Figure 3B), and (Figure 1). All immortalized clones were cultivated until not in the satellite-cell niche, suggesting that the they had achieved at least twice as many divisions as the immortalized cells were engaged preferentially in the parental population. differentiation pathway and not in the self-renewal Telomere length was measured in each clone (Table process. 2) and ranged from 10.3 kb to 24.8 kb with no differ- ence between the clones and control immortalized myo- Immortalized cell lines as a useful tool for therapeutic blasts (17.6 kb). The length of the telomeres in all of the preclinical studies immortalized myogenic clones was always well above To show that these cell lines could be powerful tools to the 6 to 7 kb limit usually seen in control cells that are develop therapeutic strategies, we used them to evaluate Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 5 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 Figure 1 Proliferative potential. Lifespan plots (mean population doublings; MPD) of the parental populations and the immortalized cell lines derived from them. the efficiency of AONs in an exon-skipping strategy for and this AON is currently being used in a phase I/II DMD. The first one tested was AO51, which resulted in clinical trial. Using immortalized control cells, we were efficient skipping of exon 51 using both the primary and able to screen a range of AONs targeting exons 17, 18, immortalized DMD cell lines (Δ48 to 50; Figure 4A) 21, 22, 43, 44 and 45 of the dystrophin gene. RT-PCR Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 6 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 Table 2 Mean telomere length of control and immortalized cell lines. Name Clone Number of Telomere length, kb, mean divisions ± SEM CTRL C25Cl48 127 17.6 ± 0.3 CMD CMDCl12 42 20.8 ± 1.7 DMD DMDCl2 57.9 10.3 ± 0.1 FSHD FSHDCl17 37.9 24.8 ± 1.6 LGMD2B LGMD2Cl11 27.4 17.2 ± 3.0 OPMD OPMDCl2 47.6 20.0 ± 0.5 CMD, congenital muscular dystrophy; DMD, Duchenne muscular dystrophy; FSHD, fascioscapulohumeral muscular dystrophy; LGMD2B, limb-girdle muscular dystrophy type 2B; OPMD, oculopharyngeal muscular dystrophy identified efficient skipping for two of them (Figure 4B, and data not shown). Discussion Immortal human cell lines, as long as they retain their capacity to express a specific program, are essential to study cellular and molecular mechanisms (as exempli- fied by the number of studies conducted on the mouse cell line C2C12), and responses to potential therapeutic strategies. The relatively short proliferative lifespan of human myoblasts, reduced even more in dystrophic conditions by successive cycles of degeneration/regen- eration in vivo prior to isolation and the modification in their myogenic potential as they approach senescence [26-28], limits their potential use. As a consequence, any assessment of pathological mechanisms or of thera- peutic strategies will be biased by the presence of senes- cent cells, which will modify the behavior of the population. This is even more crucial for high-through- Figure 2 Myogenic markers and in vitro differentiation. (A) Reverse transcriptase PCR comparing primary (P) and immortalized put screening of molecules, for which large numbers of (Im.) cell lines (control, oculopharyngeal muscular dystrophy cells are required. Immortalization can solve this pro- (OPMD), Duchenne muscular dystrophy (DMD)) for myogenic blem, as long as the cell lines are stable and retain most markers (MyoD, N-CAM and desmin). Fibroblasts were used as a of the characteristics of the unmodified parental popula- negative control. (B) Immunofluorescence was carried out using tion. This has been shown to be a problem with the MF20, an antibody directed against sarcomeric myosin (green) after 5 days of differentiation. Specific antibody labeling was visualized C2C12 cell line, as the phenotype drifts and therefore using Alexa Fluor 488 secondary antibody (green). Nuclei were can vary both within and between different laboratories. visualized with Hoechst (blue). Original magnification × 100. Immortal cell lines have been generated from human skeletal muscle, such as those derived from rhabdomyo- sarcomas, a rare form of skeletal-muscle tumor. How- ever, these lines often have impaired fusion downregulates both the p16 and p19Arf tumor suppres- characteristics and perturbed myogenic programs sor genes, encoded by the Ink4 locus. These cells had [29,30]. Other approaches have used transduction of the an extended lifespan with no chromosomal rearrange- large T antigen from SV40, which binds Rb and p53, ment, but the differentiation potential of control myo- but does not stop telomere shortening. Although these blasts was found to be impaired [33]. This year, a report cells do have an extended lifespan, they are not immor- described the same approach as we have used in the tal, and extensive telomeric erosion results in an present study (introduction of hTERT and CDK4) using increased frequency of chromosomal rearrangement [31] muscle cells isolated from patients affected with FSHD and a defective differentiation program [32]. More [34]. The FSHD mutation causes a defect within myo- recently, cell lines were generated by the transduction of genic cells, thus the establishment of a clonal myogenic primary myoblasts with both hTERT and Bmi-1, which cell line permit reproducible study of the consequences Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 7 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 myogenicity during successive passages of primary cul- tures of muscle cells isolated from patients with OPMD [35]; many muscle diseases are subject to a similar decrease in myogenicity. In this report, we describe the isolation of immortalized human myoblast lines from a wide range of neuromuscular diseases, using a combina- tion of hTERT and CDK-4. This wide range of diseases paves the way to searching for common mechanisms between distinct dystrophies, and even those shared with muscle aging, as opposed to those mechanisms specific for each disease. We found that these cell lines have extended proliferative lifespans, and maintain their capacity to differentiate both in vitro and in vivo after transplantation into the regenerating muscles of immu- nodeficient mice. We found that these human myoblast cell lines expressing myogenic markers could colonize the host muscles and form mature fibers, thus providing an ideal model to assess therapeutic strategies in vivo, which is closer to bona fide differentiation of muscle stem cells than the converted fibroblasts described pre- viously [36]. We also found that these cells do not replenish the muscle stem-cell niche as primary human myoblasts do under the same conditions of implantation [23], but are engaged primarily in the differentiation pathway. The process of immortalization involves over- expression of both hTERT and CDK-4, and further investigations will be needed to analyze how this may influence the balance between self-renewal and differentiation. Conclusions These human myoblast lines represent a powerful tool to assess signaling and/or functional deregulations in neuromuscular diseases, particularly those in which Figure 3 Detection of human immortalized cells injected into -/- -/- -/- these mechanisms have not yet been clearly elucidated gC C5 mice. (A) in the tibialis anterior muscle of Rag2 (FSHD, OPMD) or those with features common to mus- Human nuclei were visualized using an anti-lamin A/C antibody (red) and fibers expressing human proteins were visualized using an cle aging, such as atrophy or muscle wasting. We have anti-human spectrin-specific antibody (green). Nuclei are described only a subset of the cell lines produced; we counterstained with Hoechst. Original magnification × 200. (B) The have now generated more than 35 cell lines with various immortalized cells were present preferentially as myonuclei (top mutations covering a range of 14 different pathologies, panel), with a small number found in interstitial space (arrow, as well as cell lines from control subjects of various bottom panel), identified by human lamin expression (laminin staining in green, laminA/C and spectrin staining in red, Hoechst in ages. The in vivo implantation of these cells offers the blue, on the LGMD2B muscle section as an example). Original possibility to investigate the consequences of defined magnification × 600. mutations on cellular behavior in vivo, particularly with regard to their regenerative capacity. Finally, the devel- opment of therapeutic strategies, whether these strate- of this mutation within myoblasts without the contami- gies imply gene, cell or pharmacological therapy nation of non-myogenic cells. The decrease in myogeni- involving high-throughput screening, is facilitated using city in the primary culture, and consequently the these tools before assessment in animal models. As an enrichment of non-myogenic cells during amplification, example, we have described a rapid screen of a range of is a considerable problem in the study of dystrophy dis- AONs in an exon-skipping strategy for DMD. In conclu- eases. For example, in 2006, we described rapid loss of sion, the co-transduction of hTERT and CDK4 Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 8 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 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. List of abbreviations generates human myoblasts immortal in culture that AONs: antisense oligonucleotides; CDK-4: cyclin-dependent kinase 4; CMD: maintain myogenic potential both in vitro and in vivo, congenital muscular dystrophy; DMD: Duchenne muscular dystrophy; DMEM: offering new cell paradigms for pathophysiological stu- Dulbecco’s modified Eagle’s medium; FCS: fetal calf serum; FSHD: facioscapulohumeral muscular dystrophy; hTERT: human telomerase reverse dies and novel therapeutic strategies. transcriptase; LGMD2B: limb-girdle muscular dystrophy type 2b; N-CAM: Mamchaoui et al. Skeletal Muscle 2011, 1:34 Page 9 of 10 http://www.skeletalmusclejournal.com/content/1/1/34 neural cell adhesion molecule; OPMD: oculopharyngeal muscular dystrophy; 6. Allamand V, Campbell KP: Animal models for muscular dystrophy: PBS: phosphate-buffered serum; PCR: polymerase chain reaction. valuable tools for the development of therapies. Hum Mol Genet 2000, 9:2459-2467. Acknowledgements 7. Mouly V, Aamiri A, Perie S, Mamchaoui K, Barani A, Bigot A, Bouazza B, We thank all the patients who provided the biopsies to establish the Francois V, Furling D, Jacquemin V, et al: Myoblast transfer therapy: is primary cultures. We also thank the MSG study group, particularly D. Furling there any light at the end of the tunnel? Acta Myol 2005, 24:128-133. for fruitful discussions and L. Dollé, S. Sandal, M. Oloko, S. Vasseur and M. 8. Webster C, Blau HM: Accelerated age-related decline in replicative life- Chapart for technical assistance. We thank Genosafe for help with the span of Duchenne muscular dystrophy myoblasts: implications for cell transductions and Steve Wilton for providing the antisense oligonucleotides. and gene therapy. Somat Cell Mol Genet 1990, 16:557-565. This work was supported by the MYORES Network of Excellence (contract 9. Renault V, Thornell LE, Eriksson PO, Butler-Browne G, Mouly V: Regenerative 511978) and TREAT-NMD (contract LSHM-CT-2006-036825) from the potential of human skeletal muscle during aging. Aging Cell 2002, European Commission 6th FP, MYOAGE (contract HEALTH-F2-2009-223576) 1:132-139. from the Seventh FP, the ANR Genopath-INAFIB, the ANR MICRORNAS, 10. Wright WE, Shay JW: Historical claims and current interpretations of MyoGrad (GK1631, German Research Foundation), the Duchenne Parent replicative aging. Nat Biotechnol 2002, 20:682-688. Project Netherlands, CNRS, INSERM, University Pierre and Marie Curie, AFM 11. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, (Association Française contre les Myopathies) (including network grant Shay JW, Lichtsteiner S, Wright WE: Extension of life-span by introduction #15123), the Jain Foundation, Parents Project of Monaco, and the European of telomerase into normal human cells. Science 1998, 279:349-352. Parent Project. 12. 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Hum Gene Ther 2009, 20:784-790. doi:10.1186/2044-5040-1-34 Cite this article as: Mamchaoui et al.: Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders. Skeletal Muscle 2011 1:34. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: Nov 1, 2011

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