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Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation

  • Mainak Das
  • John W. Rumsey
  • Neelima Bhargava
  • Cassie Gregory
  • Lisa Riedel
  • Jung Fong Kang
  • James J. Hickman
Article

Abstract

This work describes the step-by-step development of a novel, serum-free, in vitro cell culture system resulting in the formation of robust, contracting, multinucleate myotubes from dissociated skeletal muscle cells obtained from the hind limbs of fetal rats. This defined system consisted of a serum-free medium formulation developed by the systematic addition of different growth factors as well as a nonbiological cell growth promoting substrate, N-1[3-(trimethoxysilyl) propyl] diethylenetriamine. Each growth factor in the medium was experimentally evaluated for its effect on myotube formation. The resulting myotubes were evaluated immunocytochemically using embryonic skeletal muscle, specifically the myosin heavy chain antibody. Based upon this analysis, we propose a new skeletal muscle differentiation protocol that reflects the roles of the various growth factors which promote robust myotube formation. Further observation noted that the proposed skeletal muscle differentiation technique also supported muscle–nerve coculture. Immunocytochemical evidence of nerve–muscle coculture has also been documented. Applications for this novel culture system include biocompatibility and skeletal muscle differentiation studies, understanding myopathies, neuromuscular disorders, and skeletal muscle tissue engineering.

Keywords

Muscle differentiation Muscle-nerve coculture Myotube Serum-free medium Synthetic substrate 

Notes

Acknowledgments

The F1.652 monoclonal antibody developed by Helan Blau was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA. This work was supported by DARPA grant DARPA-ITO N65236-01-1-7400 and NIH grant number 5R01 NS 050452. The initial experiments for this work were performed in the Bioengineering Department at Clemson University, Clemson, SC.

References

  1. Alterio J.; Courtois Y.; Robelin J.; Bechet D.; Martelly I. Acidic and basic fibroblast growth factor mRNAs are expressed by skeletal muscle satellite cells. Biochem. Biophys. Res. Commun 1663: 1205–1512; 1990. doi: 10.1016/0006-291X(90)90994-X.PubMedCrossRefGoogle Scholar
  2. Anderson J. E.; Liu L.; Kardami E. Distinctive patterns of basic fibroblast growth factor (bFGF) distribution in degenerating and regenerating areas of dystrophic (mdx) striated muscles. Dev. Biol 1471: 96–109; 1991. doi: 10.1016/S0012-1606(05)80010-7.PubMedCrossRefGoogle Scholar
  3. Arnold H. H.; Winter B. Muscle differentiation: more complexity to the network of myogenic regulators. Curr. Opin. Genet. Dev 85: 539–544; 1998. doi: 10.1016/S0959-437X(98)80008-7.PubMedCrossRefGoogle Scholar
  4. Biesecker G. The complement SC5b-9 complex mediates cell adhesion through a vitronectin receptor. J. Immunol 1451: 209–214; 1990.PubMedGoogle Scholar
  5. Bordet T.; Lesbordes J. C.; Rouhani S.; Castelnau-Ptakhine L.; Schmalbruch H.; Haase G.; Kahn A. Protective effects of cardiotrophin-1 adenoviral gene transfer on neuromuscular degeneration in transgenic ALS mice. Hum. Mol. Genet 1018: 1925–1933; 2001. doi: 10.1093/hmg/10.18.1925.PubMedCrossRefGoogle Scholar
  6. Brand-Saberi B. Genetic and epigenetic control of skeletal muscle development. Ann. Anat 1873: 199–207; 2005. doi: 10.1016/j.aanat.2004.12.018.PubMedCrossRefGoogle Scholar
  7. Brand-Saberi B.; Christ B. Genetic and epigenetic control of muscle development in vertebrates. Cell Tissue Res 2961: 199–212; 1999. doi: 10.1007/s004410051281.PubMedCrossRefGoogle Scholar
  8. Brand T.; Butler-Browne G.; Fuchtbauer E. M.; Renkawitz-Pohl R.; Brand-Saberi B. EMBO Workshop Report: Molecular genetics of muscle development and neuromuscular diseases Kloster Irsee, Germany, September 26–October 1, 1999. Embo J 199: 1935–1941; 2000. doi: 10.1093/emboj/19.9.1935.PubMedCrossRefGoogle Scholar
  9. Burgess W. H.; Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu. Rev. Biochem 58: 575–606; 1989. doi: 10.1146/annurev.bi.58.070189.003043.PubMedCrossRefGoogle Scholar
  10. Carrasco D. I.; English A. W. Neurotrophin 4/5 is required for the normal development of the slow muscle fiber phenotype in the rat soleus. J. Exp. Biol 206Pt 13: 2191–2200; 2003. doi: 10.1242/jeb.00412.PubMedCrossRefGoogle Scholar
  11. Chen J.; von Bartheld C. S. Role of exogenous and endogenous trophic factors in the regulation of extraocular muscle strength during development. Invest. Ophthalmol. Vis. Sci 4510: 3538–3545; 2004. doi: 10.1167/iovs.04-0393.PubMedCrossRefGoogle Scholar
  12. Choi-Lundberg D. L.; Bohn M. C. Ontogeny and distribution of glial cell line-derived neurotrophic factor (GDNF) mRNA in rat. Brain Res. Dev. Brain Res 851: 80–88; 1995. doi: 10.1016/0165-3806(94)00197-8.PubMedCrossRefGoogle Scholar
  13. Christ B.; Brand-Saberi B. Limb muscle development. Int. J. Dev. Biol 467: 905–914; 2002.PubMedGoogle Scholar
  14. Clegg C. H.; Linkhart T. A.; Olwin B. B.; Hauschka S. D. Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor. J. Cell Biol 1052: 949–956; 1987. doi: 10.1083/jcb.105.2.949.PubMedCrossRefGoogle Scholar
  15. Das M.; Gregory C. A.; Molnar P.; Riedel L. M.; Wilson K.; Hickman J. J. A defined system to allow skeletal muscle differentiation and subsequent integration with silicon microstructures. Biomaterials 2724: 4374–4380; 2006. doi: 10.1016/j.biomaterials.2006.03.046.PubMedCrossRefGoogle Scholar
  16. Das M.; Molnar P.; Devaraj H.; Poeta M.; Hickman J. J. Electrophysiological and morphological characterization of rat embryonic motoneurons in a defined system. Biotechnol. Prog 196: 1756–1761; 2003. doi: 10.1021/bp034076l.PubMedCrossRefGoogle Scholar
  17. Das M.; Molnar P.; Gregory C.; Riedel L.; Jamshidi A.; Hickman J. J. Long-term culture of embryonic rat cardiomyocytes on an organosilane surface in a serum-free medium. Biomaterials 2525: 5643–5647; 2004. doi: 10.1016/j.biomaterials.2004.01.020.PubMedCrossRefGoogle Scholar
  18. Das M.; Rumsey J. W.; Gregory C. A.; Bhargava N.; Kang J. F.; Molnar P.; Riedel L.; Guo X.; Hickman J. J. Embryonic motoneuron–skeletal muscle co-culture in a defined system. Neuroscience 1462: 481–488; 2007a. doi: 10.1016/j.neuroscience.2007.01.068.PubMedCrossRefGoogle Scholar
  19. Das M.; Wilson K.; Molnar P.; Hickman J. J. Differentiation of skeletal muscle and integration of myotubes with silicon microstructures using serum-free medium and a synthetic silane substrate. Nat. Protoc 27: 1795–1801; 2007b. doi: 10.1038/nprot.2007.229.PubMedCrossRefGoogle Scholar
  20. Dolcet X.; Soler R. M.; Gould T. W.; Egea J.; Oppenheim R. W.; Comella J. X. Cytokines promote motoneuron survival through the Janus kinase-dependent activation of the phosphatidylinositol 3-kinase pathway. Mol. Cell Neurosci 186: 619–631; 2001. doi: 10.1006/mcne.2001.1058.PubMedCrossRefGoogle Scholar
  21. Donovan M. J.; Hahn R.; Tessarollo L.; Hempstead B. L. Identification of an essential nonneuronal function of neurotrophin 3 in mammalian cardiac development. Nat. Genet 142: 210–213; 1996. doi: 10.1038/ng1096-210.PubMedCrossRefGoogle Scholar
  22. Dutton E. K.; Uhm C. S.; Samuelsson S. J.; Schaffner A. E.; Fitzgerald S. C.; Daniels M. P. Acetylcholine receptor aggregation at nerve-muscle contacts in mammalian cultures: induction by ventral spinal cord neurons is specific to axons. J. Neurosci 1511: 7401–7416; 1995.PubMedGoogle Scholar
  23. Golden J. P.; DeMaro J. A.; Osborne P. A.; Milbrandt J.; Johnson E. M. Jr. Expression of neurturin, GDNF, and GDNF family-receptor mRNA in the developing and mature mouse. Exp. Neurol 1582: 504–528; 1999. doi: 10.1006/exnr.1999.7127.PubMedCrossRefGoogle Scholar
  24. Gonzalez A. M.; Buscaglia M.; Ong M.; Baird A. Distribution of basic fibroblast growth factor in the 18-day rat fetus: localization in the basement membranes of diverse tissues. J. Cell Biol 1103: 753–765; 1990. doi: 10.1083/jcb.110.3.753.PubMedCrossRefGoogle Scholar
  25. Gullberg D.; Sjoberg G.; Velling T.; Sejersen T. Analysis of fibronectin and vitronectin receptors on human fetal skeletal muscle cells upon differentiation. Exp. Cell Res 2201: 112–123; 1995. doi: 10.1006/excr.1995.1297.PubMedCrossRefGoogle Scholar
  26. Hannon K.; Kudla A. J.; McAvoy M. J.; Clase K. L.; Olwin B. B. Differentially expressed fibroblast growth factors regulate skeletal muscle development through autocrine and paracrine mechanisms. J. Cell. Biol 1326: 1151–1159; 1996. doi: 10.1083/jcb.132.6.1151.PubMedCrossRefGoogle Scholar
  27. Heinrich G. A novel BDNF gene promoter directs expression to skeletal muscle. BMC Neurosci 4: 11; 2003. doi: 10.1186/1471-2202-4-11.PubMedCrossRefGoogle Scholar
  28. Henderson C. E.; Phillips H. S.; Pollock R. A.; Davies A. M.; Lemeulle C.; Armanini M.; Simmons L.; Moffet B.; Vandlen R. A.; Simpson L. C. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science 2665187: 1062–1064; 1994. doi: 10.1126/science.7973664.PubMedCrossRefGoogle Scholar
  29. Hickman J. J.; Bhatia S. K.; Quong J. N.; Schoen P.; Stenger D. A.; Pike C. J.; Cotman C. W. Rational pattern design for in-vitro cellular networks using surface photochemistry. J. Vac. Sci. Technol. A 123: 607–616; 1994. doi: 10.1116/1.578844.CrossRefGoogle Scholar
  30. Hornik C.; Brand-Saberi B.; Rudloff S.; Christ B.; Fuchtbauer E. M. Twist is an integrator of SHH, FGF, and BMP signaling. Anat. Embryol. (Berl) 2091: 31–39; 2004. doi: 10.1007/s00429-004-0412-3.CrossRefGoogle Scholar
  31. Lesbordes J. C.; Bordet T.; Haase G.; Castelnau-Ptakhine L.; Rouhani S.; Gilgenkrantz H.; Kahn A. In vivo electrotransfer of the cardiotrophin-1 gene into skeletal muscle slows down progression of motor neuron degeneration in pmn mice. Hum. Mol. Genet 1114: 1615–1625; 2002. doi: 10.1093/hmg/11.14.1615.PubMedCrossRefGoogle Scholar
  32. Li L.; Olson E. N. Regulation of muscle cell growth and differentiation by the MyoD family of helix-loop-helix proteins. Adv. Cancer Res 58: 95–119; 1992. doi: 10.1016/S0065-230X(08)60292-4.PubMedCrossRefGoogle Scholar
  33. Lin L. F.; Doherty D. H.; Lile J. D.; Bektesh S.; Collins F. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science 2605111: 1130–1132; 1993. doi: 10.1126/science.8493557.PubMedCrossRefGoogle Scholar
  34. Liu J.; Rumsey J. W.; Das M.; Molnar P.; Gregory C.; Riedel L.; Hickman J. J. Electrophysiological and immunocytochemical characterization of DRG neurons on an organosilane surface in serum-free medium. In Vitro Cell. Dev. Biol. Anim 445–6: 162–168; 2008. doi: 10.1007/s11626-008-9097-x.PubMedCrossRefGoogle Scholar
  35. Mitsumoto H.; Klinkosz B.; Pioro E. P.; Tsuzaka K.; Ishiyama T.; O’Leary R. M.; Pennica D. Effects of cardiotrophin-1 (CT-1) in a mouse motor neuron disease. Muscle Nerve 246: 769–777; 2001. doi: 10.1002/mus.1068.PubMedCrossRefGoogle Scholar
  36. Moore J. W.; Dionne C.; Jaye M.; Swain J. L. The mRNAs encoding acidic FGF, basic FGF and FGF receptor are coordinately downregulated during myogenic differentiation. Development 1113: 741–748; 1991.PubMedGoogle Scholar
  37. Morrow N. G.; Kraus W. E.; Moore J. W.; Williams R. S.; Swain J. L. Increased expression of fibroblast growth factors in a rabbit skeletal muscle model of exercise conditioning. J. Clin. Invest 856: 1816–1820; 1990. doi: 10.1172/JCI114640.PubMedCrossRefGoogle Scholar
  38. Mousavi K.; Parry D. J.; Jasmin B. J. BDNF rescues myosin heavy chain IIB muscle fibers after neonatal nerve injury. Am. J. Physiol. Cell. Physiol 2871: C22–C29; 2004. doi: 10.1152/ajpcell.00583.2003.PubMedCrossRefGoogle Scholar
  39. Nishikawa J.; Sakuma K.; Sorimachi Y.; Yoshimoto K.; Yasuhara M. Increase of Cardiotrophin-1 immunoreactivity in regenerating and overloaded but not denervated muscles of rats. Neuropathology 251: 54–65; 2005. doi: 10.1111/j.1440-1789.2004.00587.x.PubMedCrossRefGoogle Scholar
  40. Nugent M. A.; Iozzo R. V. Fibroblast growth factor-2. Int. J. Biochem. Cell. Biol 322: 115–120; 2000. doi: 10.1016/S1357-2725(99)00123-5.PubMedCrossRefGoogle Scholar
  41. Oakley R. A.; Lefcort F. B.; Clary D. O.; Reichardt L. F.; Prevette D.; Oppenheim R. W.; Frank E. Neurotrophin-3 promotes the differentiation of muscle spindle afferents in the absence of peripheral targets. J. Neurosci 1711: 4262–4274; 1997.PubMedGoogle Scholar
  42. Ohuchi H.; Noji S. Fibroblast-growth-factor-induced additional limbs in the study of initiation of limb formation, limb identity, myogenesis, and innervation. Cell Tissue Res 2961: 45–56; 1999. doi: 10.1007/s004410051265.PubMedCrossRefGoogle Scholar
  43. Olson E. Activation of muscle-specific transcription by myogenic helix-loop-helix proteins. Symp. Soc. Exp. Biol 46: 331–341; 1992a.PubMedGoogle Scholar
  44. Olson E. N. Interplay between proliferation and differentiation within the myogenic lineage. Dev. Biol 1542: 261–272; 1992b. doi: 10.1016/0012-1606(92)90066-P.PubMedCrossRefGoogle Scholar
  45. Olson E. N.; Perry W. M. MyoD and the paradoxes of myogenesis. Curr. Biol 21: 35–37; 1992. doi: 10.1016/0960-9822(92)90429-E.PubMedCrossRefGoogle Scholar
  46. Oppenheim R. W.; Wiese S.; Prevette D.; Armanini M.; Wang S.; Houenou L. J.; Holtmann B.; Gotz R.; Pennica D.; Sendtner M. Cardiotrophin-1, a muscle-derived cytokine, is required for the survival of subpopulations of developing motoneurons. J. Neurosci 214: 1283–1291; 2001.PubMedGoogle Scholar
  47. Peroulakis M. E.; Forger N. G. Ciliary neurotrophic factor increases muscle fiber number in the developing levator ani muscle of female rats. Neurosci. Lett 2962–3: 73–76; 2000. doi: 10.1016/S0304-3940(00)01649-9.PubMedCrossRefGoogle Scholar
  48. Ravenscroft M. S.; Bateman K. E.; Shaffer K. M.; Schessler H. M.; Jung D. R.; Schneider T. W.; Montgomery C. B.; Custer T. L.; Schaffner A. E.; Liu Q. Y.; Li Y. X.; Barker J. L.; Hickman J. J. Developmental neurobiology implications from fabrication and analysis of hippocampal neuronal networks on patterned silane-modified surfaces. J. Am. Chem. Soc 12047: 12169–12177; 1998. doi: 10.1021/ja973669n.CrossRefGoogle Scholar
  49. Scaal M.; Bonafede A.; Dathe V.; Sachs M.; Cann G.; Christ B.; Brand-Saberi B. SF/HGF is a mediator between limb patterning and muscle development. Development 12621: 4885–4893; 1999.PubMedGoogle Scholar
  50. Schaffner A. E.; Barker J. L.; Stenger D. A.; Hickman J. J. Investigation of the factors necessary for growth of hippocampal neurons in a defined system. Journal of Neuroscience Methods 621–2: 111–119; 1995. doi: 10.1016/0165-0270(95)00063-1.PubMedCrossRefGoogle Scholar
  51. Schwarz J. J.; Chakraborty T.; Martin J.; Zhou J. M.; Olson E. N. The basic region of myogenin cooperates with two transcription activation domains to induce muscle-specific transcription. Mol. Cell. Biol 121: 266–275; 1992.PubMedGoogle Scholar
  52. Sheng Z.; Pennica D.; Wood W. I.; Chien K. R. Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival. Development 1222: 419–428; 1996.PubMedGoogle Scholar
  53. Simon M.; Porter R.; Brown R.; Coulton G. R.; Terenghi G. Effect of NT-4 and BDNF delivery to damaged sciatic nerves on phenotypic recovery of fast and slow muscles fibres. Eur. J. Neurosci 189: 2460–2466; 2003. doi: 10.1046/j.1460-9568.2003.02978.x.PubMedCrossRefGoogle Scholar
  54. Spargo B. J.; Testoff M. A.; Nielsen T. B.; Stenger D. A.; Hickman J. J.; Rudolph A. S. Spatially controlled adhesion, spreading, and differentiation of endothelial-cells on self-assembled molecular monolayers. Proc. Natl. Acad. Sci. U. S. A 9123: 11070–11074; 1994. doi: 10.1073/pnas.91.23.11070.PubMedCrossRefGoogle Scholar
  55. Stenger D. A.; Georger J. H.; Dulcey C. S.; Hickman J. J.; Rudolph A. S.; Nielsen T. B.; McCort S. M.; Calvert J. M. Coplanar molecular assemblies of aminoalkylsilane and perfluorinated alkylsilane—characterization and geometric definition of mammalian-cell adhesion and growth. J. Am. Chem. Soc 11422: 8435–8442; 1992. doi: 10.1021/ja00048a013.CrossRefGoogle Scholar
  56. Yang L. X.; Nelson P. G. Glia cell line-derived neurotrophic factor regulates the distribution of acetylcholine receptors in mouse primary skeletal muscle cells. Neuroscience 1283: 497–509; 2004. doi: 10.1016/j.neuroscience.2004.06.067.PubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2009

Authors and Affiliations

  • Mainak Das
    • 1
    • 2
  • John W. Rumsey
    • 1
  • Neelima Bhargava
    • 1
  • Cassie Gregory
    • 2
  • Lisa Riedel
    • 1
    • 2
  • Jung Fong Kang
    • 1
    • 2
  • James J. Hickman
    • 1
    • 2
  1. 1.NanoScience Technology CenterUniversity of Central FloridaOrlandoUSA
  2. 2.Department of BioengineeringClemson UniversityClemsonUSA

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