Characterization of a subset of oligodendrocytes separated on the basis of selective adherence properties

Abstract

A subset of oligodendrocytes (B_3,f) was isolated by taking advantage of selective cell–substratum interaction. B_3,f cells were characterized morphologically, biochemically, and immunocytochemically. Oligodendrocytes were isolated from 4-to-6-month-old lamb brains by a modified version of our published procedure [Szuchet et al, J Neurosci Methods 3:7–19, 1980]. Freshly isolated cells from band III were plated on plastic culture plates at a concentration of 2 × 10⁶ cells/ml. Approximately 40% of the cells attached to the plate under these conditions. The remaining cells formed small floating clusters. We refer to the latter as B_3,f oligodendrocytes. After 4 to 5 days, the supernatant containing B_3,f cells was removed and centrifuged, and the pellet was resuspended in culture medium and replated on polylysine-coated petri dishes. B_3,f oligodendrocytes attached to this surface and extended an intricate network of processes. The purity of the cultures, judged by the number of cells staining with a monoclonal antibody against galactocerebroside was 98–99%. This high degree of cell homogeneity was maintained throughout the life of the cultures. B_3,f cells appeared to be highly differentiated and remained so in vitro. This is surmised by the expression of oligodendrocytic characteristic functions such as high levels of CNPase activity typically, 5 μM/min/mgP; high incorporation of H₂ ³⁵SO₄ into sulfatides, an overall lipid metabolism that mimics events associated with myelinogenesis [Szuchet et al, PNAS 80:7019–7023, 1983]; the presence, detected immunocytochemically, of myelin-associated glycoprotein and myelin basic proteins. It is concluded that this culture system offers an opportunity for studying the biology of interfascicular oligodendrocytes and their interaction with neurons and/or astrocytes. It also should open up a way of examining the relevance of oligodendrocyte polymorphism.