Advertisement

Schwann Cells pp 213-232 | Cite as

Scalable Differentiation and Dedifferentiation Assays Using Neuron-Free Schwann Cell Cultures

  • Paula V. MonjeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1739)

Abstract

This chapter describes protocols to establish simplified in vitro assays of Schwann cell (SC) differentiation in the absence of neurons. The assays are based on the capacity of isolated primary SCs to increase or decrease the expression of myelination-associated genes in response to the presence or absence of cell permeable analogs of cyclic adenosine monophosphate (cAMP). No special conditions of media or substrates beyond the administration or removal of cAMP analogs are required to obtain a synchronous response on differentiation and dedifferentiation. The assays are cost-effective and far easier to implement than traditional myelinating SC-neuron cultures. They are scalable to a variety of plate formats suited for downstream experimentation and analysis. These cell-based assays can be used as drug discovery platforms for the evaluation of novel agents controlling the onset, maintenance, and reversal of the differentiated state using any typical adherent SC population.

Key words

Primary cultures cAMP In vitro assays Myelin markers Cell morphology Krox-20 c-Jun O1 MAG 

Notes

Acknowledgments

The author appreciates the guidance provided by Dr. Patrick Wood and the assistance provided by Drs. Jennifer Soto and Ketty Bacallao in the development of these protocols. James Guest, Kristine Ravelo, and Gonzalo Piñero are acknowledged for performing critical review of the manuscript. The work presented in this chapter was generously supported by the NIH-NINDS (NS084326), The Craig Neilsen Foundation (339576), The Miami Project to Cure Paralysis, and The Buoniconti Fund. The author declares no conflicts of interest with the contents of this article.

References

  1. 1.
    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(2):1023–1034CrossRefPubMedGoogle Scholar
  2. 2.
    Sobue G, Pleasure D (1984) Schwann cell galactocerebroside induced by derivatives of adenosine 3′,5′-monophosphate. Science 224(4644):72–74CrossRefPubMedGoogle Scholar
  3. 3.
    Jessen KR, Mirsky R, Morgan L (1991) Role of cyclic AMP and proliferation controls in Schwann cell differentiation. Ann N Y Acad Sci 633:78–89CrossRefPubMedGoogle Scholar
  4. 4.
    Glenn TD, Talbot WS (2013) Analysis of Gpr126 function defines distinct mechanisms controlling the initiation and maturation of myelin. Development 140(15):3167–3175CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Monk KR, Oshima K, Jors S, Heller S, Talbot WS (2011) Gpr126 is essential for peripheral nerve development and myelination in mammals. Development 138(13):2673–2680CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Bacallao K, Monje PV (2015) Requirement of cAMP signaling for Schwann cell differentiation restricts the onset of myelination. PLoS One 10(2):e0116948.  https://doi.org/10.1371/journal.pone.0116948 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Monje PV, Rendon S, Athauda G, Bates M, Wood PM, Bunge MB (2009) Non-antagonistic relationship between mitogenic factors and cAMP in adult Schwann cell re-differentiation. Glia 57(9):947–961CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Bacallao K, Monje PV (2013) Opposing roles of PKA and EPAC in the cAMP-dependent regulation of schwann cell proliferation and differentiation [corrected]. PLoS One 8(12):e82354.  https://doi.org/10.1371/journal.pone.0082354 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Monje PV, Soto J, Bacallao K, Wood PM (2010) Schwann cell dedifferentiation is independent of mitogenic signaling and uncoupled to proliferation: role of cAMP and JNK in the maintenance of the differentiated state. J Biol Chem 285(40):31024–31036CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Pinero G, Berg R, Andersen ND, Setton-Avruj P, Monje PV (2016) Lithium reversibly inhibits Schwann cell proliferation and differentiation without inducing myelin loss. Mol Neurobiol.  https://doi.org/10.1007/s12035-016-0262-z
  11. 11.
    Soto J, Monje PV (2017) Axon contact-driven Schwann cell dedifferentiation. Glia.  https://doi.org/10.1002/glia.23131
  12. 12.
    Morrissey TK, Kleitman N, Bunge RP (1991) Isolation and functional characterization of Schwann cells derived from adult peripheral nerve. J Neurosci 11(8):2433–2442PubMedGoogle Scholar
  13. 13.
    Andersen ND, Srinivas S, Pinero G, Monje PV (2016) A rapid and versatile method for the isolation, purification and cryogenic storage of Schwann cells from adult rodent nerves. Sci Rep 6:31781.  https://doi.org/10.1038/srep31781 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Maldonado EN, Alderson NL, Monje PV, Wood PM, Hama H (2008) FA2H is responsible for the formation of 2-hydroxy galactolipids in peripheral nervous system myelin. J Lipid Res 49(1):153–161.  https://doi.org/10.1194/jlr.M700400-JLR200 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.The Miami Project to Cure Paralysis, Department of Neurological SurgeryUniversity of Miami Miller School of MedicineMiamiUSA

Personalised recommendations