• 1. Qiu J, Cai D, Filbin MT. Glial inhibition of nerve regeneration in the mature mammalian CNS. Glia 2000;15: 166174.

  • 2. Sandvig A, Berry M, Barrett LB, et al. Myelin-, reactive glia-, and scar-derived CNS axon growth inhibitors: expression, receptor signaling, and correlation with axon regeneration. Glia 2004;46: 225251.

    • Search Google Scholar
    • Export Citation
  • 3. Parrinello S, Napoli I, Ribeiro S, et al. EphB signaling directs peripheral nerve regeneration through Sox2-dependent Schwann cell sorting. Cell 2010;143: 145155.

    • Search Google Scholar
    • Export Citation
  • 4. Reichardt LF, Bixby JL, Hall DE, et al. Integrins and cell adhesion molecules: neuronal receptors that regulate axon growth on extracellular matrices and cell surfaces. Dev Neurosci 1989;11: 332347.

    • Search Google Scholar
    • Export Citation
  • 5. Rothblum K, Stahl RC, Carey DJ. Constitutive release of alpha4 type V collagen N-terminal domain by Schwann cells and binding to cell surface and extracellular matrix heparan sulfate proteoglycans. J Biol Chem 2004;279: 5128251288.

    • Search Google Scholar
    • Export Citation
  • 6. Fu SY, Gordon T. The cellular and molecular basis of peripheral nerve regeneration. Mol Neurobiol 1997;14: 67116.

  • 7. Shakhbazau A, Martinez JA, Xu QG, et al. Evidence for a systemic regulation of neurotrophin synthesis in response to peripheral nerve injury. J Neurochem 2012;122: 501511.

    • Search Google Scholar
    • Export Citation
  • 8. David S, Aguayo AJ. Axonal elongation into peripheral nervous system “bridges” after central nervous system injury in adult rats. Science 1981;214: 931933.

    • Search Google Scholar
    • Export Citation
  • 9. Bunge MB. Novel combination strategies to repair the injured mammalian spinal cord. J Spinal Cord Med 2008;31: 262269.

  • 10. Fouad K, Schnell L, Bunge MB, et al. Combining Schwann cell bridges and olfactory-ensheathing glia grafts with chondroitinase promotes locomotor recovery after complete transection of the spinal cord. J Neurosci 2005;25: 11691178.

    • Search Google Scholar
    • Export Citation
  • 11. Zhou XH, Ning GZ, Feng SQ, et al. Transplantation of autologous activated Schwann cells in the treatment of spinal cord injury: six cases, more than five years of follow-up. Cell Transplant 2012; 21: S39S47.

    • Search Google Scholar
    • Export Citation
  • 12. Guest J, Santamaria AJ, Benavides FD. Clinical translation of autologous Schwann cell transplantation for the treatment of spinal cord injury. Curr Opin Organ Transplant 2013;18: 682689.

    • Search Google Scholar
    • Export Citation
  • 13. Takami T, Oudega M, Bates ML, et al. Schwann cell but not olfactory ensheathing glia transplants improve hindlimb locomotor performance in the moderately contused adult rat thoracic spinal cord. J Neurosci 2002;22: 66706681.

    • Search Google Scholar
    • Export Citation
  • 14. Pearse DD, Pereira FC, Marcillo AE, et al. cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury. Nat Med 2004;10: 610616.

    • Search Google Scholar
    • Export Citation
  • 15. Keirstead HS, Morgan SV, Wilby MJ, et al. Enhanced axonal regeneration following combined demyelination plus Schwann cell transplantation therapy in the injured adult spinal cord. Exp Neurol 1999;159: 225236.

    • Search Google Scholar
    • Export Citation
  • 16. Kalbermatten DF, Erba P, Mahay D, et al. Schwann cell strip for peripheral nerve repair. J Hand Surg Eur Vol 2008;33: 587594.

  • 17. Hood B, Levene HB, Levi AD. Transplantation of autologous Schwann cells for the repair of segmental peripheral nerve defects. Neurosurg Focus 2009; 26: E4.

    • Search Google Scholar
    • Export Citation
  • 18. Daly W, Yao L, Zeugolis D, et al. A biomaterials approach to peripheral nerve regeneration: bridging the peripheral nerve gap and enhancing functional recovery. J R Soc Interface 2012;9: 202221.

    • Search Google Scholar
    • Export Citation
  • 19. Lehmann HC, Höke A. Schwann cells as a therapeutic target for peripheral neuropathies. CNS Neurol Disord Drug Targets 2010;9: 801806.

    • Search Google Scholar
    • Export Citation
  • 20. Ansselin AD, Corbeil SD, Davey DF. Culture of Schwann cells from adult animals. In Vitro Cell Dev Biol Anim 1995;31: 253254.

  • 21. Haastert-Talimi K. Culture and proliferation of highly purified adult Schwann cells from rat, dog, and man. Methods Mol Biol 2012;846: 189200.

    • Search Google Scholar
    • Export Citation
  • 22. Mosahebi A, Woodward B, Wiberg M, et al. Retroviral labeling of Schwann cells: in vitro characterization and in vivo transplantation to improve peripheral nerve regeneration. Glia 2001;34: 817.

    • Search Google Scholar
    • Export Citation
  • 23. Bunge RP. The cell of Schwann. In: Asbury AK, McKhann GM, McDonald WI, eds. Diseases of the nervous system. Philadelphia: Saunders, 1986; 153162.

    • Search Google Scholar
    • Export Citation
  • 24. Bunge MB, Wood PM, Tynan LB, et al. Perineurium originates from fibroblasts: demonstration in vitro with a retroviral marker. Science 1989;243: 229231.

    • Search Google Scholar
    • Export Citation
  • 25. Morrissey TK. Kleitman N, Bunge RP. Isolation and functional characterization of Schwann cells derived from adult peripheral nerve. J Neurosci 1991;11: 24332442.

    • Search Google Scholar
    • Export Citation
  • 26. Li R. Culture methods for selective growth of normal rat and human Schwann cells. Methods Cell Biol 1998;57: 167186.

  • 27. Haastert K, Seef P, Stein VM, et al. A new cell culture protocol for enrichment and genetic modification of adult canine Schwann cells suitable for peripheral nerve tissue engineering. Res Vet Sci 2009;87: 140142.

    • Search Google Scholar
    • Export Citation
  • 28. Techangamsuwan S, Imbschweiler I, Kreutzer R, et al. Similar behaviour and primate-like properties of adult canine Schwann cells and olfactory ensheathing cells in long-term culture. Brain Res 2008;1240: 3138.

    • Search Google Scholar
    • Export Citation
  • 29. Gautron M, Jazat F, Ratinahirana H, et al. Alterations in myelinated fibres in the sciatic nerve of rats after constriction: possible relationships between the presence of abnormal small myelinated fibres and pain-related behaviour. Neurosci Lett 1990;111: 2833.

    • Search Google Scholar
    • Export Citation
  • 30. Haastert K, Mauritz C, Chaturvedi S, et al. Human and rat adult Schwann cell cultures: fast and efficient enrichment and highly effective non-viral transfection protocol. Nat Protoc 2007;2: 99104.

    • Search Google Scholar
    • Export Citation
  • 31. Lim JH, Boozer L, Mariani CL, et al. Generation and characterization of neurospheres from canine adipose tissue-derived stromal cells. Cell Reprogram 2010;12: 417425.

    • Search Google Scholar
    • Export Citation
  • 32. Bolin LM, Iismaa TP, Shooter EM. Isolation of activated adult Schwann cells and a spontaneously immortal Schwann cell clone. J Neurosci Res 1992;33: 231238.

    • Search Google Scholar
    • Export Citation
  • 33. Kohama I, Lankford KL, Preiningerova J, et al. Transplantation of cryopreserved adult human Schwann cells enhances axonal conduction in demyelinated spinal cord. J Neurosci 2001;21: 944950.

    • Search Google Scholar
    • Export Citation
  • 34. Haastert K, Mauritz C, Matthies C, et al. Autologous adult human Schwann cells genetically modified to provide alternative cellular transplants in peripheral nerve regeneration. J Neurosurg 2006;104: 778786.

    • Search Google Scholar
    • Export Citation
  • 35. Kitchell RL, Evans H. The spinal nerves. In: Evans Miller's anatomy of the dog. 3rd ed. Philadelphia: WB Saunders Co, 834838.

  • 36. Sharp NJ, Wheeler SJ. Procedures: hemilaminectomy. In: Small animal spinal disorders: diagnosis and surgery. 2nd ed. St Louis: Elsevier Health Sciences, 2005; 266270.

    • Search Google Scholar
    • Export Citation
  • 37. Höke A, Redett R, Hameed H, et al. Schwann cells express motor and sensory phenotypes that regulate axon regeneration. J Neurosci 2006;26: 96469655.

    • Search Google Scholar
    • Export Citation
  • 38. Mirsky R, Woodhoo A, Parkinson DB, et al. Novel signals controlling embryonic Schwann cell development, myelination and dedifferentiation. J Peripher Nerv Syst 2008;13: 122135.

    • Search Google Scholar
    • Export Citation
  • 39. Honkanen H, Lahti O, Nissinen M, et al. Isolation, purification and expansion of myelination-competent, neonatal mouse Schwann cells. Eur J Neurosci 2007;26: 953964.

    • Search Google Scholar
    • Export Citation
  • 40. Scarpini E, Kreider BQ, Lisak RP, et al. Establishment of Schwann cell cultures from adult rat peripheral nerves. Exp Neurol 1988;102: 167176.

    • Search Google Scholar
    • Export Citation
  • 41. Askanas V, Engel WK, Dalakas MC, et al. Human Schwann cells in tissue culture: histochemical and ultrastructural studies. Arch Neurol 1980;37: 329337.

    • Search Google Scholar
    • Export Citation
  • 42. Casella GT, Bunge RP, Wood PM. Improved method for harvesting human Schwann cells from mature peripheral nerve and expansion in vitro. Glia 1996;17: 327338.

    • Search Google Scholar
    • Export Citation
  • 43. Morrissey TK, Bunge RP, Kleitman N. Human Schwann cells in vitro. I. Failure to differentiate and support neuronal health under co-culture conditions that promote full function of rodent cells. J Neurobiol 1995;28: 171189.

    • Search Google Scholar
    • Export Citation
  • 44. Komiyama T, Nakao Y, Toyama Y, et al. A novel technique to isolate adult Schwann cells for an artificial nerve conduit. J Neurosci Methods 2003;122: 195200.

    • Search Google Scholar
    • Export Citation
  • 45. Spiegel I, Peles E. A novel method for isolating Schwann cells using the extracellular domain of Necl1. J Neurosci Res 2009;87: 32883296.

    • Search Google Scholar
    • Export Citation
  • 46. Basu S, Campbell HM, Dittel BN, et al. Purification of specific cell population by fluorescence activated cell sorting. J Vis Exp 2010;41: 1546.

    • Search Google Scholar
    • Export Citation
  • 47. Schmitte R, Tipold A, Stein VM, et al. Genetically modified canine Schwann cells: in vitro and in vivo evaluation of their suitability for peripheral nerve tissue engineering. J Neurosci Methods 2010;186: 202208.

    • Search Google Scholar
    • Export Citation
  • 48. Calderón-Martínez D, Garavito Z, Spinel C, et al. Schwann cell-enriched cultures from adult human peripheral nerve: a technique combining short enzymatic dissociation and treatment with cytosine arabinoside (Ara-C). J Neurosci Methods 2002;114: 18.

    • Search Google Scholar
    • Export Citation
  • 49. Chen LE, Liu K, Seaber AV, et al. Recombinant human glial growth factor 2 (rhGGF2) improves functional recovery of crushed peripheral nerve (a double-blind study). Neurochem Int 1998;33: 341351.

    • Search Google Scholar
    • Export Citation
  • 50. Mahanthappa NK, Anton ES, Matthew WD. Glial growth factor 2, a soluble neuregulin, directly increases Schwann cell motility and indirectly promotes neurite outgrowth. J Neurosci 1996;16: 46734683.

    • Search Google Scholar
    • Export Citation
  • 51. Chuah MI, Cossins J, Woodhall E, et al. Glial growth factor 2 induces proliferation and structural changes in ensheathing cells. Brain Res 2000;857: 265274.

    • Search Google Scholar
    • Export Citation
  • 52. Cannella B, Hoban CJ, Gao YL, et al. The neuregulin, glial growth factor 2, diminishes autoimmune demyelination and enhances remyelination in a chronic relapsing model for multiple sclerosis. Proc Natl Acad Sci U S A 1998;95: 1010010105.

    • Search Google Scholar
    • Export Citation
  • 53. Minghetti L, Goodearl AD, Mistry K, et al. Glial growth factors I–III are specific mitogens for glial cells. J Neurosci Res 1996;43: 684693.

    • Search Google Scholar
    • Export Citation

Advertisement

Generation of pure cultures of autologous Schwann cells by use of biopsy specimens of the dorsal cutaneous branches of the cervical nerves of young adult dogs

Ji-Hey Lim DVM, PhD1 and Natasha J. Olby VET MB, PhD2
View More View Less
  • 1 Department of Clinical Sciences, College of Veterinary Medicine, and the Comparative Medicine Institute, North Carolina State University, Raleigh, NC 27606.
  • | 2 Department of Clinical Sciences, College of Veterinary Medicine, and the Comparative Medicine Institute, North Carolina State University, Raleigh, NC 27606.

Abstract

OBJECTIVE To identify an optimal technique for isolation, purification, and amplification of Schwann cells (SCs) from biopsy specimens of the dorsal cutaneous branches of the cervical nerves of dogs.

SAMPLE Biopsy specimens of dorsal cervical cutaneous nerves from the cadavers of three 1- to 2-year-old dogs.

PROCEDURES Nerve specimens were dissected, predegenerated, and dissociated to isolate single cells. After culture to enhance SC growth, cells were immunopurified by use of magnetic beads. Cell purity was evaluated by assessing expression of cell surface antigens p75 (to detect SCs) and CD90 (to detect fibroblasts). Effects of various concentrations of recombinant human glial growth factor 2 (rhGGF2) on SC proliferation were tested. Cell doubling time was assessed in SC cultures with selected concentrations of rhGGF2.

RESULTS Mean ± SD wet weight of nerve fascicles obtained from the biopsy specimens was 16.8 ± 2.8 mg. A mean predegeneration period of 8.6 days yielded approximately 6,000 cells/mg of nerve tissue, and primary culture yielded 43,000 cells/mg of nerve tissue in a mean of 11 days, of which 39.9 ± 9.1% expressed p75. Immunopurification with magnetic beads yielded a mean of 85.4 ± 1.9% p75-positive cells. Two passages of subculture with 10μM cytosine arabinoside further enhanced SC purity to a mean of 97.8 ± 1.2% p75-positive cells. Finally, rhGGF2 supplementation at a range of 40 to 100 ng/mL increased the SC proliferation rate up to 3-fold.

CONCLUSIONS AND CLINICAL RELEVANCE SCs could be cultured from biopsy specimens of dorsal cervical cutaneous nerves and purified and expanded to generate adequate numbers for autologous transplants to treat dogs with spinal cord and peripheral nerve injuries.

Contributor Notes

Address correspondence to Dr. Olby (Natasha_olby@ncsu.edu).