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Culture
Neuronal differentiation
Seven days after outgrowth, the EPI-NCSCs were trypsinized; collected by centrifugation (1000 rpm for 5 min) and replated at a cell density of 50,000 cells/well on collagen I coated 6-well plates. After attachment of the cells, 1.5 ml neurobasal medium (consisting of DMEM, GDNF, BDNF, NT-3, 10 ng/ml each and B27 2% all Invitrogen, Canada) was added according to Anderson et al. [21]. The medium was refreshed every other day until Y-27632 grew to confluency, usually after 14 days for rodent and 17 days for human stem cells.
Glial differentiation
Glial differentiation was promoted by increasing the cellular density based on a report by Yang et al. [8]. Isolated EPI-NCSCs were kept in the same culture dish and neurobasal medium was added. Confluency was usually reached after 17 days.
Immunohistochemistry
Table 1.
Primary and secondary antibodies used to identify stem cells (nestin), neurons (TUJ1), Schwann cells (Krox-20) and oligodendrocytes (OSP)
DilutionCompany