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An alternate protocol for establishment of primary caprine fetal myoblast cell culture: an in vitro model for muscle growth study

An Erratum to this article was published on 03 July 2013

Abstract

Cultured myoblasts have been used extensively as an in vitro model in understanding the underlying mechanisms of myogenesis. Various protocols for establishing a pure myoblast culture have been reported which involve the use of special procedures like flow cytometry and density gradient centrifugation. In goat, only a few protocols for establishing a myogenic cell culture are available and these protocols use adult muscle tissues which often does not yield sufficient numbers of precursor cells with adequate proliferative capacity. Considering the disadvantages of adult myoblasts, we are proposing an alternate protocol using caprine fetus which does not require any special procedures. In the present study, more than 90–95% fetal-derived cell populations had the typical spindle to polyhedral shape of myoblast cell and stained positive for desmin, hence confirming their myogenic origin. These cells attained the maximum confluency as early as 3–4 d against 3 wk by adult myoblasts indicating a better growth potential. Further, quantitative real-time PCR analysis revealed a higher expression (p < 0.01) of myogenic regulatory factors (i.e., myogenic determination factor 1, myogenic factor 5, and myogenin) and myostatin (MSTN) in the fetal as compared to the adult myoblasts. Consequently, higher proliferation and differentiation ability along with higher abundance of myogenic markers and MSTN make the fetal myoblasts a better in vitro model.

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References

  • Anderson J. E.; Weber M.; Vargas C. Deflazacort increases laminin expression and myogenic repair, and induces early persistent functional gain in mdx mouse muscular dystrophy. Cell Transplantation 9: 551–564; 2000.

    PubMed  CAS  Google Scholar 

  • Bischoff R. The satellite cell and muscle regeneration. In: Engel A. G.; Franszini-Armstrong C. (eds) Myogenesis. McGraw-Hill, New York, pp 97–118; 1994.

    Google Scholar 

  • Blanco-Bose W. E.; Blau H. M. Laminin-induced change in conformation of pre-existing alpha-7beta-1 integrin signals secondary myofiber formation. Developmental Biology 233: 148–160; 2001.

    PubMed  Article  CAS  Google Scholar 

  • Burattini S.; Ferri P.; Battistelli M.; Curci R.; Luchetti F.; Falcieri E. C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. European Journal of Histochemistry 48(3): 223–233; 2004.

    PubMed  CAS  Google Scholar 

  • Carlson M. E.; Conboy M. J.; Hsu M.; Barchas L.; Jeong J.; Agrawal A.; Mikels A. J.; Agrawal S.; Schafer D. V.; Conboy I. M. Relative roles of TGF-beta1 and Wnt in the systemic regulation and aging of satellite cell responses. Aging Cell 8: 676–689; 2009.

    PubMed  Article  CAS  Google Scholar 

  • Charge S.; Rudnicki M. Cellular and molecular regulation of muscle regeneration. Physiological Reviews 84: 209–238; 2004.

    PubMed  Article  CAS  Google Scholar 

  • Conboy I. M.; Conboy M. J.; Smythe G. M.; Rando T. A. Notch-mediated restoration of regenerative potential to aged muscle. Science 302: 1575–1577; 2003.

    PubMed  Article  CAS  Google Scholar 

  • Dai Y.; Roman M.; Naviaux R. K.; Verma I. M. Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo. Proceedings of the National Academy of Sciences of the United States of America 89: 10892–10895; 1992.

    PubMed  Article  CAS  Google Scholar 

  • Deveaux V.; Picard B.; Bouley J.; Cassar-Malek I. et al. Location of myostatin expression during bovine myogenesis in vivo and in vitro. Reproduction, Nutrition, Development 43: 527–542; 2003.

    PubMed  Article  CAS  Google Scholar 

  • Dodson M. V.; McFarland D. C.; Martin E. L.; Brarmon M. A. Isolation of satellite cells from ovinc skeletal muscles. Journal of Tissue Culture Methods 10: 233; 1986.

    Article  Google Scholar 

  • Edom-Vovard F.; Mouly V.; Barbet J. P.; Butler-Browne G. S. The four populations of myoblasts involved in human limb muscle formation are present from the onset of primary myotube formation. Journal of Cell Science 112: 191–199; 1999.

    PubMed  CAS  Google Scholar 

  • Evans H. E.; Sack W. O. Prenatal development of domestic laboratory mammals: growth curves, external features and selected references. Zentralblatt für Veterinärmedizin. Reihe C 2: 11–45; 1973.

    CAS  Google Scholar 

  • Foulstone E. J.; Huser C.; Crown A. L.; Holly J. M.; Stewart C. E. Differential signalling mechanisms predisposing primary human skeletal muscle cells to altered proliferation and differentiation: roles of IGF-I and TNF alpha. Experimental Cell Research 294: 223–235; 2004.

    PubMed  Article  CAS  Google Scholar 

  • Freshney R. I. Culture of animal cells. A manual of basic technique. 3rd ed. Liss, New York; 1994.

    Google Scholar 

  • Goel H. L.; Dey C. S. Focal adhesion kinase tyrosine phosphorylation is associated with myogenesis and modulated by insulin. Cell Proliferation 35: 131–142; 2002.

    PubMed  Article  CAS  Google Scholar 

  • Gospodarowicz D.; Wcseman J.; Moran J. S.; Lindstrom J. Effect of fibroblast growth factor on the division and fusion of bovine myoblasts. The Journal of Cell Biology 70: 395; 1976.

    PubMed  Article  CAS  Google Scholar 

  • Gros J.; Manceau M.; Thome V.; Marcelle C. A common somitic origin for embryonic muscle progenitors and satellite cells. Nature 435: 954–958; 2005.

    PubMed  Article  CAS  Google Scholar 

  • Grounds M. D.; Garrett K. L.; Lai M. C.; Wright W. E.; Beilharz M. W. Identification of skeletal muscle precursor cells in vivo by use of MyoD1 and myogenin probes. Cell and Tissue Research 267: 99–104; 1992.

    PubMed  Article  CAS  Google Scholar 

  • Guillot P. V.; Gotherstrom C.; Chan J.; Kurata H.; Fisk N. M. Human first-trimester fetal MSC express pluripotency markers and grow faster and have longer telomeres than adult MSC. Stem Cells 25: 646–654; 2007.

    PubMed  Article  CAS  Google Scholar 

  • Gussoni E.; Pavlath G. K.; Lanctot A. M.; Sharma K. R.; Miller R. G.; Steinmsn L.; Blau H. M. Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation. Nature 356: 435–438; 1992.

    PubMed  Article  CAS  Google Scholar 

  • Harper J. M. M.; Soar J. B.; Buttery P. J. Changes in protein metabolism of ovine primary muscle cultures on treatment with growth hormone, insulin, insulin-like growth factor I or epidermal growth factor. Journal of Endocrinology 112: 87–96; 1987.

    PubMed  Article  CAS  Google Scholar 

  • Hauschka S. Clonal analysis of vertebrate myogenesis: 3. Developmental changes in the muscle-colony-forming cells of the human fetal limb. Developmental Biology 37: 345–368; 1974.

    PubMed  Article  CAS  Google Scholar 

  • Hembree J. R.; Hathaway M. R.; Dayton W. R. Isolation and culture of fetal porcine myogenic cells and the effect of insulin, IGF-1, and sera on protein turnover in porcine myotube cultures. Journal of Animal Science 69: 3241–3250; 1991.

    PubMed  CAS  Google Scholar 

  • Jones G. E.; Murphy S. J.; Watt D. J. Segregation of the myogenic cell lineage in mouse muscle development. Journal of Cell Science 97: 659–667; 1990.

    PubMed  Google Scholar 

  • Karpati G.; Ajdukovic D.; Arnold D.; Gledhill R. B.; Guttmann R.; Holland P. et al. Myoblast transfer in Duchenne muscular dystrophy. Annals of Neurology 34: 8–17; 1993.

    PubMed  Article  CAS  Google Scholar 

  • Kassar-Duchossoy L.; Gayraud-Morel B.; Gomes D.; Rocancourt D.; Buckingham M.; Shinin V.; Tajbakhsh S. Mrf4 determines skeletal muscle identity in Myf5: Myod double-mutant mice. Nature 431: 466–471; 2004.

    PubMed  Article  CAS  Google Scholar 

  • Kaufman S. J.; Foster R. F. Replicating myoblasts express a muscle-specific phenotype. Proceedings of the National Academy of Sciences of the United States of America 85: 9606–9610; 1988.

    PubMed  Article  CAS  Google Scholar 

  • Kirk S.; Oldham J.; Kambadur R.; Sharma M.; Dobbie P.; Bass J. Myostatin regulation during skeletal muscle regeneration. Journal of Cellular Physiology 184: 356–363; 2000.

    PubMed  Article  CAS  Google Scholar 

  • Law P. K.; Goodwin T. G.; Wang M. G. Normal myoblast injections provide genetic treatment for murine dystrophy. Muscle & Nerve 11: 525–533; 1988.

    Article  CAS  Google Scholar 

  • Lawson M. A.; Purslow P. P. Differentiation of myoblasts in serum-free media: effects of modified media are cell line-specific. Cells, Tissues, Organs 167: 130–137; 2000.

    PubMed  Article  CAS  Google Scholar 

  • Lewis M. P.; Tippett H. L.; Sinanan A. C.; Morgan M. J.; Hunt N. P. Gelatinase-B (matrix metalloproteinase-9; MMP-9) secretion is involved in the migratory phase of human and murine muscle cell cultures. Journal of Muscle Research and Cell Motility 21: 223–233; 2000.

    PubMed  Article  CAS  Google Scholar 

  • Li Y.; Huard J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. American Journal of Pathology 161: 895–907; 2002.

    PubMed  Article  Google Scholar 

  • Lindon C.; Montarras D.; Pinset C. Cell cycle-regulated expression of the muscle determination factor myf-5 in proliferating myoblasts. The Journal of Cell Biology 140: 111–118; 1998.

    PubMed  Article  CAS  Google Scholar 

  • Linge C.; Green M. R.; Brooks R. F. A method for removal fibroblasts from human tissue culture systems. Experimental Cell Research 85: 519–528; 1989.

    Article  Google Scholar 

  • Livak K. J.; Schmittgen T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-[delta][delta] CT method. Methods 25: 402–408; 2001.

    PubMed  Article  CAS  Google Scholar 

  • McCroskery S.; Thomas M.; Maxwell L.; Sharma M.; Kambadur R. Myostatin negatively regulates satellite cell activation and self-renewal. The Journal of Cell Biology 162: 1135–1147; 2003.

    PubMed  Article  CAS  Google Scholar 

  • McFarlane C.; Langley B.; Thomas M.; Hennebry A.; Plummer E.; Nicholas G.; McMahon C.; Sharma M.; Kambadur R. Proteolytic processing of myostatin is auto-regulated during myogenesis. Developmental Biology 283: 58–69; 2005.

    PubMed  Article  CAS  Google Scholar 

  • McFarland D. C. Cell culture as a tool for the study of poultry skeletal muscle development. Conference: In vitro approaches to understanding growth and development. 818–829; 1992.

  • McKinnell I. W.; Parise G.; Rudnicki M. A. Muscle stem cells and regenerative myogenesis. Current Topics in Developmental Biology 71: 113–130; 2005.

    PubMed  Article  CAS  Google Scholar 

  • Megeney L. A.; Rudnicki M. A. Determination versus differentiation and the MyoD family of transcription factors. Biochemical Cell Biology 73(9–10): 723–732; 1995.

    Article  CAS  Google Scholar 

  • Merkulova T.; Keller A.; Oliviero P.; Marotte F.; Samuel J. L.; Rappaport L.; Lamand N.; Lucas M. Thyroid hormones differentially modulate enolase isozymes during skeletal and cardiac muscle development. American Journal of Physiology, Endocrinology and Metabolism 278: E330–E339; 2000.

    CAS  Google Scholar 

  • Morgan J. E. Myogenicity in vitro and in vivo of mouse muscle cells separated on discontinuous Percoll gradients. Journal of the Neurological Sciences 85: 197–207; 1988.

    PubMed  Article  CAS  Google Scholar 

  • Morgan J. E.; Hoffman E. P.; Partridge T. A. Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse. The Journal of Cell Biology 111: 2437–2449; 1990.

    PubMed  Article  CAS  Google Scholar 

  • Oldham J. M.; Martyn J. A.; Sharma M.; Jeanplong F.; Kambadur R.; Bass J. J. Molecular expression of myostatin and MyoD is greater in double-muscled than normal-muscled cattle fetuses. American Journal of Physiology 280: R1488–R1493; 2001.

    PubMed  CAS  Google Scholar 

  • Pappa K.; Anagnou N. Novel sources of fetal stem cells: where do they fit on the developmental continuum? Regenerative Medicine 4: 423–433; 2009.

    PubMed  Article  Google Scholar 

  • Péault B.; Rudnicki M.; Torrente Y.; Cossu G.; Tremblay J. P.; Partridge T.; Gussoni E.; Kunkel L. M.; Huard J. Stem and progenitor cells in skeletal muscle development, maintenance, and therapy. American Society of Genetic Therapy; 2007. doi:10.1038/mt.sj.6300145.

    Google Scholar 

  • Perry R. L. S.; Rudnicki M. A. Molecular mechanisms regulating myogenic determination and differentiation. Frontiers in Bioscience 5: d750–d767; 2000.

    PubMed  Article  CAS  Google Scholar 

  • Qu Z.; Balkir L.; Van Deutekom J. C. T. et al. Development of approaches to improve cell survival in myoblast transfer therapy. The Journal of Cell Biology 142: 1257–1267; 1998.

    PubMed  Article  CAS  Google Scholar 

  • Quinn L. S.; Ong L. D.; Roeder R. A. Paracrine control of myoblast proliferation and differentiation by fibroblasts. Developmental Biology 140(1): 8–19; 1990.

    PubMed  Article  CAS  Google Scholar 

  • Rando T. A.; Blau H. M. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. The Journal of Cell Biology 125(6): 275–1287; 1994.

    Article  Google Scholar 

  • Renault V.; Piron-Hamelin G.; Forestier C.; DiDonna S.; Decary S.; Hentati F.; Saillant G.; Butler-Browne G. S.; Mouly V. Skeletal muscle regeneration and the mitotic clock. Experimental Gerontology 35: 711–719; 2000.

    PubMed  Article  CAS  Google Scholar 

  • Roe J. A.; Harper J. M. M.; Buttery P. J. Protein metabolism in ovine primary muscle culture derived from satellite cells—effects of selected peptide hormones and growth factors. Journal of Endocrinology 122: 565; 1989.

    PubMed  Article  CAS  Google Scholar 

  • Schienda J.; Engleka K. A.; Jun S.; Hansen M. S.; Epstein J. A.; Tabin C. J.; Kunkel L. M.; Kardon G. Somitic origin of limb muscle satellite and side population cells. Proceedings of the National Academy of Sciences of the United States of America 103: 945–950; 2006.

    PubMed  Article  CAS  Google Scholar 

  • Shefer G.; Van de Mark D. P.; Richardson J. B.; Yablonka-Reuveni Z. Satellite-cell pool size does matter: defining the myogenic potency of aging skeletal muscle. Developmental Biology 294: 50–66; 2006.

    PubMed  Article  CAS  Google Scholar 

  • Springer M. L.; Rando T.; Blau H. M. Gene delivery to muscle. In: Boyle A. L. (ed) Current protocols in human genetics. Unit 13.4. Wiley, New York; 1997.

    Google Scholar 

  • Stockdale F.; Holtzer H. DNA synthesis and myogenesis. Experimental Cell Research 24: 508; 1961.

    PubMed  Article  CAS  Google Scholar 

  • Takashima A. Establisment of fibroblast cultures. Current Protocols in Cell Biology; 2001. doi:10.1002/0471143030.cb0201s00.

    PubMed  Google Scholar 

  • Tripathi A. K.; Ramani U. V.; Ahir V. B.; Rank D. N.; Joshi C. G. A modified enrichment protocol for adult caprine skeletal muscle stem cell. Cytotechnology 62: 483–488; 2010.

    PubMed  Article  Google Scholar 

  • Webster C.; Pavlath G.; Parks D.; Walsh F.; Blau H. Isolation of human myoblasts with the fluorescence-activated cell sorter. Experimental Cell Research 174: 252–265; 1988.

    PubMed  Article  CAS  Google Scholar 

  • Weintraub H.; Tapscott S. J.; Davis R.; Thayer M. J.; Adam M. A.; Lassar A. B.; Dusty Miller A. Activation of muscle-specific genes in pigment, nerve, fat, liver and fibroblast cell lines by forced expression of MyoD. Proceedings of the National Academy of Sciences of the United States of America 86: 5434–5438; 1989.

    PubMed  Article  CAS  Google Scholar 

  • Yablonka-Reuveni Z.; Anderson S. K.; Bowen–Pope D. F.; Nameroff M. Biochemical and morphological differences between fibroblasts and myoblasts from embryonic chicken skeletal muscle. Cell and Tissue Research 252: 339–348; 1988.

    PubMed  Article  CAS  Google Scholar 

  • Yamanouchi K.; Hosoyama T.; Murakami Y.; Nakano S.; Nishihara M. Satellite cell differentiation in goat skeletal muscle single fiber culture. Journal of Reproduction and Development 55: 252–255; 2009.

    PubMed  Article  CAS  Google Scholar 

  • Yamanouchi K.; Hosoyana T.; Murokami Y.; Nishihara M. Myogenic and adipogenic properties of goat skeletal muscle stem cell. Journal of Reproduction and Development 53: 51–58; 2007.

    PubMed  Article  CAS  Google Scholar 

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Acknowledgments

This work was carried out under the project supported by Competitive Research Grant (C2132) under National Agriculture Innovation Project (NAIP, Component 4), Indian Council of Agricultural Research (ICAR), New Delhi. The financial help in the form of Institute Scholarship to SPS during his Ph.D. study is also acknowledged. Authors are grateful to Vishakh Walia, Senior Research Fellow, Genome Analysis Lab, AG division, IVRI, Izatnagar, Bareilly, India for critical reading of the manuscript.

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Correspondence to Abhijit Mitra.

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Editor: T. Okamoto

Satyendra Pal Singh, Rohit Kumar, and Priya Kumari contributed equally to this work.

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Singh, S.P., Kumar, R., Kumari, P. et al. An alternate protocol for establishment of primary caprine fetal myoblast cell culture: an in vitro model for muscle growth study. In Vitro Cell.Dev.Biol.-Animal 49, 589–597 (2013). https://doi.org/10.1007/s11626-013-9642-0

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Keywords

  • Fetal myoblast
  • Cell culture
  • Desmin staining
  • Goat
  • Myostatin
  • Myogenic regulatory factors
  • Gene expression