Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Ham, Richard G.; Clair, Judy A. St.; Webster, Cecelia; Blau, Helen M.

doi:10.1007/bf02623656

Cited by 102 publications

(44 citation statements)

References 38 publications

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“…RT-PCR-5 g of total cellular RNA was reverse transcribed using 100 units of Superscript reverse transcriptase (Life Technologies, Inc., Eggenstein, Germany) and oligo(dT) [12][13][14][15][16][17] as a primer. Human ␣NAC and skNAC fragments were amplified by PCR.…”

Section: Methodsmentioning

confidence: 99%

Cloning of Novel Injury-regulated Genes

Munz

Lochmüller²,

Werner³

1999

Journal of Biological Chemistry

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To gain insight into the molecular mechanisms underlying the wound repair process, we searched for genes that are regulated by skin injury. Using the differential display reverse transcription-polymerase chain reaction technique, we identified a gene that was strongly induced as early as 12 h after wounding. Sequence analysis revealed the identity of the corresponding protein with skeletal muscle nascent polypeptide-associated complex (skNAC), a recently identified muscle-specific transcription factor. By in situ hybridization and immunohistochemistry, we demonstrated the specific expression of skNAC in skeletal muscle cells of the panniculus carnosus at the wound edge. Furthermore, in vitro studies with cultured myoblasts revealed expression of skNAC in differentiating and differentiated, but not in proliferating, nondifferentiated cells. Differentiation of cultured myoblasts was accompanied by simultaneous expression of skNAC and the muscle-specific transcription factor myogenin. Our results provide the first evidence for a role of skNAC in muscle repair processes. Furthermore, they demonstrate the usefulness of our approach in identifying new players in wound repair.

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Section: Methodsmentioning

confidence: 99%

Cloning of Novel Injury-regulated Genes

Munz

Lochmüller²,

Werner³

1999

Journal of Biological Chemistry

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“…Human myoblasts were obtained from a postmortem biopsy. The procedure of myoblast cloning was previously described (29). Briefly, the clones are obtained by limiting dilution.…”

Section: Methodsmentioning

confidence: 99%

High efficiency of muscle regeneration after human myoblast clone transplantation in SCID mice.

Huard¹,

Verreault²,

Roy³

et al. 1994

J. Clin. Invest.

152

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SCID mouse tibialis anterior muscles were first irradiated to prevent regeneration by host myoblasts and injected with notexin to damage the muscle fibers and trigger regeneration. The muscles were then injected with roughly 5 million human myoblasts. 1 mo later, 16-33% of the normal number of muscle fibers were present in the injected muscle, because of incomplete regeneration. However, > 90% of these muscle fibers contained human dystrophin. Some newly formed muscle fibers had an accumulation of human dystrophin and desmin on a part of their membrane. Such accumulations have been demonstrated at neuromuscular junctions before suggesting that the new muscle fibers are innervated and functional. The same pool of clones of human myoblasts produced only < 4% of muscle fibers containing human dystrophin when injected in nude mice muscles.Several of the human myoblasts did not fuse and remained in interstial space or tightly associated with muscle fibers suggesting that some of them have formed satellite cells. Moreover, cultures of 98% pure human myoblasts were obtained from transplanted SCID muscles. In some mice where the muscle regeneration was not complete, the muscle fibers containing human dystrophin also expressed uniformly HLA class 1, confirming that the fibers are of human origin. The presence of hybrid muscle fibers containing human dystrophin and mouse MHC was also demonstrated following transplantation.These results establish that in absence of an immune reaction, transplanted human myoblasts participate to the muscle regeneration with a high degree of efficacy even if the animals were killed only 1 mo after the transplantation. (J. Clin. Invest.

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“…The skeletal muscle growth medium (GM) is a modification of that of Ham et al (1988): MCDB-131 with -glutamine, 50 IU/ml penicillin, 5 µg/ml streptomycin, 2·5 µg/ml amphotericin B, 0·4 µg/ml dexamethasone, 0·5 mg/ml bovine serum albumin (BSA), 0·5 mg/ml fetuin, and 10% fetal calf serum (FCS) (GIBCO, Paisley, UK). Initially, 50 µg/ml insulin (Novo Nordisk, Crawley, West Sussex, UK) was included in the GM, as suggested by Ham et al (1988); we subsequently found that cells grew equally well in the absence of insulin. Skeletal muscle fusion medium (FM) consisted of minimum essential medium (MEM)-alpha modification with 50 IU/ml penicillin, 5 µg/ml streptomycin, 1% -glutamine and 2% FCS (GIBCO).…”

Section: Mediamentioning

confidence: 99%

Characterisation of the IGF system in a primary adult human skeletal muscle cell model, and comparison of the effects of insulin and IGF-I on protein metabolism

Crown

He²,

Holly³

et al. 2000

Journal of Endocrinology

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In an attempt to address the complex and clinically challenging question of the causes of muscle wasting in patients with cachexia, we have developed a primary adult human skeletal muscle cell model. The cultured cells were characterised by immunocytochemistry using antibodies to the myofibrillar protein constituents desmin and titin. Myotube formation was confirmed biochemically by a fourfold increase in the activity of the muscle-specific enzyme creatinine kinase, and myoblast withdrawal from the cell cycle, which is essential for terminal differentiation, was associated with progressive retinoblastoma protein dephosphorylation. Having successfully confirmed the phenotype of these adult human muscle cells, we assessed their interaction with the insulin-like growth factor (IGF) system. IGF-I is known to stimulate myoblast survival, proliferation and differentiation in cell lines, and, like insulin, is a potent anabolic agent in the regulation of protein metabolism. We have shown that IGF-I stimulated both replication and differentiation of myoblasts, whilst fibroblast growth factor-2 stimulated replication but inhibited differentiation. Examining the IGF system during the process of terminal differentiation, we found that both myoblasts and myotubes expressed insulin, IGF-I and insulin-IGF-I hybrid receptors, with the levels of all three receptor types increasing on differentiation. The cells also produced a wide range of IGF binding proteins (IGFBPs) including IGFBP-2, IGFBP-4 and abundant IGFBP-3, which has not been shown to be produced by any other skeletal muscle cell line examined to date. Both insulin and IGF-I had anabolic effects on myotube protein metabolism at physiological concentrations. Insulin was more potent than IGF-I: use of the IGF analogue long R 3 IGF-I demonstrated that the effects of exogenous IGF-I on protein metabolism were not affected by the high levels of endogenous IGFBP production. In summary, we have developed and characterised a clinically relevant in vitro model with which to address the aetiology of muscle wasting associated with chronic catabolic conditions, and we anticipate that future work will enable the development of novel, effective therapeutic interventions.

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Improved media for normal human muscle satellite cells: Serum-free clonal growth and enhanced growth with low serum

Cited by 102 publications

References 38 publications

Cloning of Novel Injury-regulated Genes

Cloning of Novel Injury-regulated Genes

High efficiency of muscle regeneration after human myoblast clone transplantation in SCID mice.

Characterisation of the IGF system in a primary adult human skeletal muscle cell model, and comparison of the effects of insulin and IGF-I on protein metabolism

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