Histochemistry and Cell Biology

, Volume 149, Issue 5, pp 537–543 | Cite as

Establishment of a myelinating co-culture system with a motor neuron-like cell line NSC-34 and an adult rat Schwann cell line IFRS1

  • Shizuka Takaku
  • Hideji Yako
  • Naoko Niimi
  • Tomoyo Akamine
  • Daiji Kawanami
  • Kazunori Utsunomiya
  • Kazunori Sango
Short Communication


Co-culture models of neurons and Schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained by primary culture with embryonic or neonatal animals. A spontaneously immortalized Schwann cell line IFRS1 from long-term cultures of adult Fischer rat peripheral nerves has been shown to retain fundamental ability to myelinate neurites in co-cultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed PC12 cells. Our current investigation focuses on the establishment of stable co-culture system with IFRS1 cells and NSC-34 motor neuron-like cells. NSC-34 cells were seeded at a low density (2 × 103/cm2) and maintained for 5–7 days in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (BDNF; 10 ng/mL). Upon observation of neurite outgrowth under a phase-contrast microscope, the NSC-34 cells were exposed to an anti-mitotic agent mitomycin C (1 µg/mL) for 12–16 h, then co-cultured with IFRS1 cells (2 × 104/cm2), and maintained in serum-containing medium supplemented with ascorbic acid (50 µg/mL), BDNF (10 ng/mL), and ciliary neurotrophic factor (10 ng/mL). Double immunofluorescence staining carried out at day 28 of the co-culture showed myelin protein (P0 or PMP22)-immunoreactive IFRS1 cells surrounding the βIII tubulin-immunoreactive neurites. This co-culture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g., amyotrophic lateral sclerosis, Charcot–Marie–Tooth diseases, and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them.


Motor neuron-like cells Immortalized Schwann cells Co-culture Myelination Mitomycin C 



This study was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (JSPS KAKENHI 16K07048). We would like to thank Dr. Kazuhiko Watabe for providing us NSC-34 cells, Drs. Tatsufumi Murakami, Tomoko Ishibashi and Mari Suzuki for helpful suggestions, and the late Kyoko Ajiki for her enormous contribution to the histochemical analyses.

Author contributions

ST and KS conducted cell culture. ST, HY, NN, and TA conducted immunocytochemical analysis and image presentation. KS, DK, and KU designed the experiments and KS supervised the project. ST and KS drafted the manuscript.

Compliance with ethical standards

Conflict of interest

There is no conflict of interest.


  1. Acquarone M, de Melo TM, Meireles F, Brito-Moreira J, Oliveira G, Ferreira ST, Castro NG, Tovar-Moll F, Houzel JC, Rehen SK (2015) Mitomycin-treated undifferentiated embryonic stem cells as a safe and effective therapeutic strategy in a mouse model of Parkinson’s disease. Front Cell Neurosci 9:97CrossRefPubMedPubMedCentralGoogle Scholar
  2. Cashman NR, Durham HD, Blusztajn JK, Oda K, Tabira T, Shaw IT, Dahrouge S, Antel JP (1992) Neuroblastoma × spinal cord (NSC) hybrid cell lines resemble developing motor neurons. Dev Dyn 194:209–221CrossRefPubMedGoogle Scholar
  3. Chan JR, Cosgaya JM, Wu YJ, Shooter EM (2001) Neurotrophins are key mediators of the myelination program in the peripheral nervous system. Proc Natl Acad Sci USA 98:14661–14668CrossRefPubMedPubMedCentralGoogle Scholar
  4. Domagala W, Woźniak L, Lasota J, Weber K, Osborn M (1990) Vimentin is preferentially expressed in high-grade ductal and medullary, but not in lobular breast carcinomas. Am J Pathol 137:1059–1064PubMedPubMedCentralGoogle Scholar
  5. Eggett CJ, Crosier S, Manning P, Cookson MR, Menzies FM, McNeil CJ, Shaw PJ (2000) Development and characterisation of a glutamate-sensitive motor neurone cell line. J Neurochem 74:1895–1902CrossRefPubMedGoogle Scholar
  6. Eldridge CF, Bunge MB, Bunge RP, Wood PM (1987) Differentiation of axon-related Schwann cells in vitro. I. Ascorbic acid regulates basal lamina assembly and myelin formation. J Cell Biol 105:1023–1034CrossRefPubMedGoogle Scholar
  7. Gingras M, Beaulieu MM, Gagnon V, Durham HD, Berthod F (2008) In vitro study of axonal migration and myelination of motor neurons in a three-dimensional tissue-engineered model. Glia 56:354–364CrossRefPubMedGoogle Scholar
  8. Greene LA, Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci USA 73:2424–2428CrossRefPubMedPubMedCentralGoogle Scholar
  9. Hyung S, Yoon Lee B, Park JC, Kim J, Hur EM, Francis Suh JK (2015) Coculture of primary motor neurons and schwann cells as a model for in vitro myelination. Sci Rep 5:15122CrossRefPubMedPubMedCentralGoogle Scholar
  10. Ishii T, Kawakami E, Endo K, Misawa H, Watabe K (2017) Myelinating cocultures of rodent stem cell line-derived neurons and immortalized Schwann cells. Neuropathology 37:475–481CrossRefPubMedGoogle Scholar
  11. La Marca R, Cerri F, Horiuchi K, Bachi A, Feltri ML, Wrabetz L, Blobel CP, Quattrini A, Salzer JL, Taveggia C (2011) TACE (ADAM17) inhibits Schwann cell myelination. Nat Neurosci 14:857–865CrossRefPubMedGoogle Scholar
  12. Lang EM, Schlegel N, Reiners K, Hofmann GO, Sendtner M, Asan E (2008) Single-dose application of CNTF and BDNF improves remyelination of regenerating nerve fibers after C7 ventral root avulsion and replantation. J Neurotrauma 25:384–400CrossRefPubMedGoogle Scholar
  13. Li X, Huang J, May JM (2003) Ascorbic acid spares alpha-tocopherol and decreases lipid peroxidation in neuronal cells. Biochem Biophys Res Commun 305:656–661CrossRefPubMedGoogle Scholar
  14. Lobsiger CS, Smith PM, Buchstaller J, Schweitzer B, Franklin RJ, Suter U, Taylor V (2001) a conditionally immortalized Schwann cell precursor line that generates myelin. Glia 36:31–47CrossRefPubMedGoogle Scholar
  15. Matusica D, Fenech MP, Rogers ML, Rush RA (2008) Characterization and use of the NSC-34 cell line for study of neurotrophin receptor trafficking. J Neurosci Res 86:553–565CrossRefPubMedGoogle Scholar
  16. Miles GB, Yohn DC, Wichterle H, Jessell TM, Rafuse VF, Brownstone RM (2004) Functional properties of motoneurons derived from mouse embryonic stem cells. J Neurosci 24:7848–7858CrossRefPubMedGoogle Scholar
  17. Mitani K, Sekiguchi F, Maeda T, Tanaka Y, Yoshida S, Kawabata A (2016) The prostaglandin E2/EP4 receptor/cyclic AMP/T-type Ca(2+) channel pathway mediates neuritogenesis in sensory neuron-like ND7/23 cells. J Pharmacol Sci 130:177–180CrossRefPubMedGoogle Scholar
  18. Murakami T, Saitoh I, Inada E, Kurosawa M, Iwase Y, Noguchi H, Terao Y, Yamasaki Y, Hayasaki H, Sato M (2013) STO feeder cells are useful for propagation of primarily cultured human deciduous dental pulp cells by eliminating contaminating bacteria and promoting cellular outgrowth. Cell Med 6:75–81CrossRefPubMedPubMedCentralGoogle Scholar
  19. Niimi N, Yako H, Tsukamoto M, Takaku S, Yamauchi J, Kawakami E, Yanagisawa H, Watabe K, Utsunomiya K, Sango K (2016) Involvement of oxidative stress and impaired lysosomal degradation in amiodarone-induced schwannopathy. Eur J Neurosci 44:1723–1733CrossRefPubMedGoogle Scholar
  20. Nishimoto S, Tanaka H, Okamoto M, Okada K, Murase T, Yoshikawa H (2015) Methylcobalamin promotes the differentiation of Schwann cells and remyelination in lysophosphatidylcholine-induced demyelination of the rat sciatic nerve. Front Cell Neurosci 9:298CrossRefPubMedPubMedCentralGoogle Scholar
  21. Roa BB, Dyck PJ, Marks HG, Chance PF, Lupski JR (1993) Dejerine–Sottas syndrome associated with point mutation in the peripheral myelin protein 22 (PMP22) gene. Nat Genet 5:269–273CrossRefPubMedGoogle Scholar
  22. Rossner M, Yamada KM (2004) What’s in a picture? The temptation of image manipulation. J Cell Biol 166:11–15CrossRefPubMedPubMedCentralGoogle Scholar
  23. Saavedra JT, Wolterman RA, Baas F, ten Asbroek AL (2008) Myelination competent conditionally immortalized mouse Schwann cells. J Neurosci Methods 174:25–30CrossRefPubMedGoogle Scholar
  24. Sabitha KR, Sanjay D, Savita B, Raju TR, Laxmi TR (2016) Electrophysiological characterization of Nsc-34 cell line using Microelectrode Array. J Neurol Sci 370:134–139CrossRefPubMedGoogle Scholar
  25. Sango K, Yamauchi J (2014) Schwann cell development and pathology. Springer, TokyoCrossRefGoogle Scholar
  26. Sango K, Yanagisawa H, Kawakami E, Takaku S, Ajiki K, Watabe K (2011) Spontaneously immortalized Schwann cells from adult Fischer rat as a valuable tool for exploring neuron–Schwann cell interactions. J Neurosci Res 89:898–908CrossRefPubMedGoogle Scholar
  27. Sango K, Kawakami E, Yanagisawa H, Takaku S, Tsukamoto M, Utsunomiya K, Watabe K (2012) Myelination in coculture of established neuronal and Schwann cell lines. Histochem Cell Biol 137:829–839CrossRefPubMedGoogle Scholar
  28. Stankoff B, Aigrot MS, Noël F, Wattilliaux A, Zalc B, Lubetzki C (2002) Ciliary neurotrophic factor (CNTF) enhances myelin formation: a novel role for CNTF and CNTF-related molecules. J Neurosci 22:9221–9227CrossRefPubMedGoogle Scholar
  29. Stonecypher MS, Chaudhury AR, Byer SJ, Carroll SL (2006) Neuregulin growth factors and their ErbB receptors form a potential signaling network for schwannoma tumorigenesis. J Neuropathol Exp Neurol 65:162–175CrossRefPubMedGoogle Scholar
  30. Syed N, Reddy K, Yang DP, Taveggia C, Salzer JL, Maurel P, Kim HA (2010) Soluble neuregulin-1 has bifunctional, concentration-dependent effects on Schwann cell myelination. J Neurosci 30:6122–6131CrossRefPubMedPubMedCentralGoogle Scholar
  31. Tep C, Kim ML, Opincariu LI, Limpert AS, Chan JR, Appel B, Carter BD, Yoon SO (2012) Brain-derived neurotrophic factor (BDNF) induces polarized signaling of small GTPase (Rac1) protein at the onset of Schwann cell myelination through partitioning-defective 3 (Par3) protein. J Biol Chem 287:1600–1608CrossRefPubMedGoogle Scholar
  32. Usuki S, Cashman NR, Miyatake T (1999) GM2 promotes ciliary neurotrophic factor-dependent rescue of immortalized motor neuron-like cell (NSC-34). Neurochem Res 24:281–286CrossRefPubMedGoogle Scholar
  33. Vent J, Wyatt TA, Smith DD, Banerjee A, Ludueña RF, Sisson JH, Hallworth R (2005) Direct involvement of the isotype-specific C-terminus of beta tubulin in ciliary beating. J Cell Sci 118:4333–4341CrossRefPubMedPubMedCentralGoogle Scholar
  34. Yang L, Zhang B, Toku K, Maeda N, Sakanaka M, Tanaka J (2000) Improvement of the viability of cultured rat neurons by the non-essential amino acids l-serine and glycine that upregulates expression of the anti-apoptotic gene product Bcl-w. Neurosci Lett 295:97–100CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Diabetic Neuropathy Project, Department of Sensory and Motor SystemsTokyo Metropolitan Institute of Medical ScienceTokyoJapan
  2. 2.Division of Diabetes, Metabolism and Endocrinology, Department of Internal MedicineJikei University School of MedicineTokyoJapan

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