A Neuron-Free Microfiber Assay to Assess Myelin Sheath Formation

Important advances in our understanding of oligodendrocyte precursor cell biology and differentiation have stemmed from in vitro experiments using cultures of isolated primary oligodendrocyte precursor cells. To examine the process of myelination in the final stages of oligodendrocyte development, experimental systems have previously been limited to models utilizing neurons. Recent advances in three-dimensional culture systems, however, have opened the possibility to observe myelin sheath formation with only one cell type, the oligodendrocyte precursor cell. In this chapter, such a method is described for examining oligodendrocyte myelin sheath formation with isolated oligodendrocytes in the absence of neurons. This assay is ideal for gaining mechanistic insight into oligodendrocyte-specific regulation of myelin sheath formation. Oligodendrocyte heterogeneity can be readily assessed, determining whether different oligodendrocyte sources influence myelin sheath formation. As well, the direct impact of both physical and molecular cues on oligodendrocytes can be determined in this defined system. This assay extends the capability of two-dimensional oligodendrocyte cultures, permitting post-differentiation analysis of myelinating oligodendrocytes, the number of sheaths formed by individual oligodendrocytes, as well as the lengths of myelin sheaths formed.