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Skeletal muscle characteristics are preserved in hTERT/cdk4 human myogenic cell lines.

ABSTRACT: BACKGROUND:hTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research, being used as therapeutically pertinent models of various neuromuscular disorders and in numerous fundamental studies of muscle cell function. However, the cell cycle is linked to other cellular processes such as integrin regulation, the PI3K/Akt pathway, and microtubule stability, raising the question as to whether genetic modification related to the cell cycle results in secondary effects that could undermine the validity of these cell models. RESULTS:Here we subjected five healthy and disease muscle cell isolates to transcriptomic analysis, comparing immortalized lines with their parent primary populations in both differentiated and undifferentiated states, and testing their myogenic character by comparison with non-myogenic (CD56-negative) cells. Principal component analysis of global gene expression showed tight clustering of immortalized myoblasts to their parent primary populations, with clean separation from the non-myogenic reference. Comparison was made to publicly available transcriptomic data from studies of muscle human pathology, cell, and animal models, including to derive a consensus set of genes previously shown to have altered regulation during myoblast differentiation. Hierarchical clustering of samples based on gene expression of this consensus set showed that immortalized lines retained the myogenic expression patterns of their parent primary populations. Of 2784 canonical pathways and gene ontology terms tested by gene set enrichment analysis, none were significantly enriched in immortalized compared to primary cell populations. We observed, at the whole transcriptome level, a strong signature of cell cycle shutdown associated with senescence in one primary myoblast population, whereas its immortalized clone was protected. CONCLUSIONS:Immortalization had no observed effect on the myogenic cascade or on any other cellular processes, and it was protective against the systems level effects of senescence that are observed at higher division counts of primary cells.

SUBMITTER: Thorley M

PROVIDER: S-EPMC5146814 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Skeletal muscle characteristics are preserved in hTERT/cdk4 human myogenic cell lines.

Thorley Matthew M Duguez Stéphanie S Mazza Emilia Maria Cristina EMC Valsoni Sara S Bigot Anne A Mamchaoui Kamel K Harmon Brennan B Voit Thomas T Mouly Vincent V Duddy William W

Skeletal muscle 20161208 1

<h4>Background</h4>hTERT/cdk4 immortalized myogenic human cell lines represent an important tool for skeletal muscle research, being used as therapeutically pertinent models of various neuromuscular disorders and in numerous fundamental studies of muscle cell function. However, the cell cycle is linked to other cellular processes such as integrin regulation, the PI3K/Akt pathway, and microtubule stability, raising the question as to whether genetic modification related to the cell cycle results ...[more]

PMID: 27931240

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Project description:Muscle fibre denervation and declining numbers of muscle stem (satellite) cells are defining characteristics of ageing skeletal muscle. The aim of this study was to investigate the potential for lifelong recreational exercise to offset muscle fibre denervation and compromised satellite cell content and function, both at rest and under challenged conditions. Sixteen elderly lifelong recreational exercisers (LLEX) were studied alongside groups of age-matched sedentary (SED) and young subjects. Lean body mass and maximal voluntary contraction were assessed, and a strength training bout was performed. From muscle biopsies, tissue and primary myogenic cell cultures were analysed by immunofluorescence and RT-qPCR to assess myofibre denervation and satellite cell quantity and function. LLEX demonstrated superior muscle function under challenged conditions. When compared with SED, the muscle of LLEX was found to contain a greater content of satellite cells associated with type II myofibres specifically, along with higher mRNA levels of the beta and gamma acetylcholine receptors (AChR). No difference was observed between LLEX and SED for the proportion of denervated fibres or satellite cell function, as assessed in vitro by myogenic cell differentiation and fusion index assays. When compared with inactive counterparts, the skeletal muscle of lifelong exercisers is characterised by greater fatigue resistance under challenged conditions in vivo, together with a more youthful tissue satellite cell and AChR profile. Our data suggest a little recreational level exercise goes a long way in protecting against the emergence of classic phenotypic traits associated with the aged muscle. KEY POINTS: The detrimental effects of ageing can be partially offset by lifelong self-organized recreational exercise, as evidence by preserved type II myofibre-associated satellite cells, a beneficial muscle innervation status and greater fatigue resistance under challenged conditions. Satellite cell function (in vitro), muscle fibre size and muscle fibre denervation determined by immunofluorescence were not affected by recreational exercise. Individuals that are recreationally active are far more abundant than master athletes, which sharply increases the translational perspective of the present study. Future studies should further investigate recreational activity in relation to muscle health, while also including female participants.

Project description:ObjectivesAccording to myosatellite cell lines (MSCs) established in vitro from diploid and triploid flounder, we compared the characters of growth and differentiation of their MSCs. The results would be useful for learning the muscle development mechanism in teleosts.Materials and methodsThe skeletal muscle cells from the diploid and triploid olive flounder Paralichthys olivaceus were isolated and cultured in vitro, respectively, and the cells were characterized at the morphology and molecular level; meanwhile, the performance of these cells' proliferation and differentiation were analyzed.ResultsTwo new skeletal muscle cell lines (POMSCS(2n) and POMSCS(3n)) from diploid and triploid flounder have been respectively subcultured for 67 times and 66 times. The cultured cells were mostly spindle-like mononuclear cells. They have normal flounder diploid karyotype (2n=48t) and triploid karyotype (3n=72t), respectively. Muscle satellite cell gene marker (pax7b) and myogenic cell protein marker (Desmin) were all expressed in cells of two cell lines. Both of the cells could differentiate into the large polynucleated muscle fibre cells, and immunofluorescence reactions of myosin heavy chain (MyHC) were positive. There were more cells of POMSCS(3n) to differentiate into the muscle fibre cells than that of POMSCS(2n). However, POMSCS(2n) cells proliferated more rapidly than those of POMSCS(3n) (P < 0.05). The significant fluorescent signals were observed in both POMSCS(2n) and POMSCS(3n) cells after transfected with pEGFP-N3 reporter plasmid.ConclusionsThe two cell lines have been established and characterized as MSCs. We suppose that it might be the differentiation capacity, rather than the proliferation activity of MSCs to play a key role in the better growth of triploid ones than diploid. Both cell lines will become the ideal tools to learn the mechanism of fish MSCs proliferation, differentiation and regeneration during muscle development in the future.

Dataset Information

Skeletal muscle characteristics are preserved in hTERT/cdk4 human myogenic cell lines.

Publications

Skeletal muscle characteristics are preserved in hTERT/cdk4 human myogenic cell lines.

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