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J Neurosci Res. 1999 Aug 1;57(3):304-14.

Fractionation and enrichment of oligodendrocytes from developing human brain.

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1
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI 53706, USA.

Abstract

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.

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