Fractionation and enrichment of oligodendrocytes from developing human brain

Enriched cultures of human oligodendrocytes were obtained from fetal brain specimens between 16 and 21 gestational weeks. Brain cells were separated over a Percoll density gradient and collected as two fractions with initial relative densities of approximately 1.035 g/ml and 1.102 g/ml, for fractions 1 and 2, respectively. After separation, 58.3 and 67.7% of the cells in fractions 1 and 2, respectively, were labeled by the antibody O4 that recognizes immature oligodendrocytes. A total of 15.5 and 29.4% of the cells in fractions 1 and 2, respectively, were positive for tubulin-beta(III), a marker for neurons but none of the freshly isolated cells were positive for glial fibrillary acidic protein (GFAP), a protein associated with astrocytes in the central nervous system. When the fractionated cells were cultured on poly-ornithine coated coverslips for 3 days and processed for immunocytochemistry, the percentage of O4+ oligodendrocytes decreased to less than 4% whereas GFAP+ cells increased to 1.8 and 12.4% for fractions 1 and 2 respectively. The percentage of tubulin-betaIII+ cells increased to 46 and 61% in cultures from the two Percoll fractions. This increase is probably due to the decrease in the number of oligodendrocytes. To avoid the loss of oligodendrocytes, cells were cultured as free-floating aggregates in the presence of 20 ng/ml of fibroblast growth factor-2 for 2 weeks. The resultant cultures became enriched for oligodendrocytes as demonstrated by cellular morphology and by positive O4 labeling. The method described here provides a means of obtaining enriched cultures of immature human oligodendrocytes for developmental and transplantation studies.