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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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.
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.
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.
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.
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.
Charge S.; Rudnicki M. Cellular and molecular regulation of muscle regeneration. Physiological Reviews 84: 209–238; 2004.
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.
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.
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.
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.
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.
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.
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.
Freshney R. I. Culture of animal cells. A manual of basic technique. 3rd ed. Liss, New York; 1994.
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.
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.
Gros J.; Manceau M.; Thome V.; Marcelle C. A common somitic origin for embryonic muscle progenitors and satellite cells. Nature 435: 954–958; 2005.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Law P. K.; Goodwin T. G.; Wang M. G. Normal myoblast injections provide genetic treatment for murine dystrophy. Muscle & Nerve 11: 525–533; 1988.
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.
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.
Li Y.; Huard J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. American Journal of Pathology 161: 895–907; 2002.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Pappa K.; Anagnou N. Novel sources of fetal stem cells: where do they fit on the developmental continuum? Regenerative Medicine 4: 423–433; 2009.
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.
Perry R. L. S.; Rudnicki M. A. Molecular mechanisms regulating myogenic determination and differentiation. Frontiers in Bioscience 5: d750–d767; 2000.
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.
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.
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.
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.
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.
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.
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.
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.
Stockdale F.; Holtzer H. DNA synthesis and myogenesis. Experimental Cell Research 24: 508; 1961.
Takashima A. Establisment of fibroblast cultures. Current Protocols in Cell Biology; 2001. doi:10.1002/0471143030.cb0201s00.
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.
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.
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.
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.
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.
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.
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.
Satyendra Pal Singh, Rohit Kumar, and Priya Kumari contributed equally to this work.
Editor: T. Okamoto
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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
- Fetal myoblast
- Cell culture
- Desmin staining
- Myogenic regulatory factors
- Gene expression