Abstract
Myelination cell culture systems are useful tools for studying myelin biology and myelin-related disorders. Compared to a number of established protocols for dissociated pure oligodendrocyte (OL) culture, methods for myelination culture are limited. We recently developed a mixed neuron-glia coculture system that generates robust and efficient myelination. By optimizing cell culture conditions, dissociated neural progenitor cells from embryonic rat spinal cords develop into neurons and glial cells including profiles of oligodendrocyte (OL) lineage. Within 4 weeks, OL progenitor cells (OPC) proliferate, differentiate into mature OLs, and myelinate axons. The formation of compact myelin sheath is confirmed by electron microscopy. For morphological analysis by light microscopy, cells grown on glass coverslips are fixed and immunostained for various myelin-related proteins, including those embedded within the myelin sheath and those clustered at the node of Ranvier. Myelinated axons can be quantified readily by either manual counting or ImageJ software. The culture system may also be used for electron microscopic analysis by slightly modifying the cell culture procedure.
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References
Bhatt A, Fan LW, Pang Y (2014) Strategies for myelin regeneration: lessons learned from development. Neural Regen Res 9(14):1347–1350. https://doi.org/10.4103/1673-5374.137586
Fan LW, Bhatt A, Tien LT, Zheng B, Simpson KL, Lin RC, Cai Z, Kumar P, Pang Y (2015) Exposure to serotonin adversely affects oligodendrocyte development and myelination in vitro. J Neurochem 133(4):532–543. https://doi.org/10.1111/jnc.12988
McCarthy KD, de Vellis J (1980) Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol 85(3):890–902
Pang Y, Cai Z, Rhodes PG (2000) Effects of lipopolysaccharide on oligodendrocyte progenitor cells are mediated by astrocytes and microglia. J Neurosci Res 62(4):510–520. https://doi.org/10.1002/1097-4547(20001115)62:4<510::AID-JNR5>3.0.CO;2-F
Pang Y, Cai Z, Rhodes PG (2005) Effect of tumor necrosis factor-alpha on developing optic nerve oligodendrocytes in culture. J Neurosci Res 80(2):226–234. https://doi.org/10.1002/jnr.20450
Pang Y, Campbell L, Zheng B, Fan L, Cai Z, Rhodes P (2010) Lipopolysaccharide-activated microglia induce death of oligodendrocyte progenitor cells and impede their development. Neuroscience 166(2):464–475. https://doi.org/10.1016/j.neuroscience.2009.12.040
Pang Y, Zheng B, Fan LW, Rhodes PG, Cai Z (2007) IGF-1 protects oligodendrocyte progenitors against TNFalpha-induced damage by activation of PI3K/Akt and interruption of the mitochondrial apoptotic pathway. Glia 55(11):1099–1107. https://doi.org/10.1002/glia.20530
Pang Y, Zheng B, Kimberly SL, Cai Z, Rhodes PG, Lin RC (2012) Neuron-oligodendrocyte myelination co-culture derived from embryonic rat spinal cord and cerebral cortex. Brain Behav 2(1):53–67. https://doi.org/10.1002/brb3.33
Acknowledgments
This work was supported by NIH grants 2R56NS054278, MH084194, and by funds from the Department of Pediatrics, University of Mississippi Medical Center. We thank Glenn Hoskins for his excellent technical support in EM study.
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Pang, Y., Simpson, K., Javier Miguel-Hidalgo, J., Savich, R. (2018). Neuron/Oligodendrocyte Myelination Coculture. In: Woodhoo, A. (eds) Myelin. Methods in Molecular Biology, vol 1791. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7862-5_10
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DOI: https://doi.org/10.1007/978-1-4939-7862-5_10
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7861-8
Online ISBN: 978-1-4939-7862-5
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