STUDIES OF OLIGODENDROCYTE DEVELOPMENT USING A TISSUE CULTURE MODEL (SERUM-FREE, FIBROBLAST GROWTH FACTOR, EPIDERMAL)

摘要

The mechanisms controlling the generation of the oligodendrocyte are not presently understood. A tissue culture model was used to investigate oligodendrocyte development. Cell-type-specific markers were employed to identify oligodendrocytes, astrocytes and neurons by immunofluorescence. To investigate cell proliferation ('3)H-thymidine incorporation and autoradiography were used.;Oligodendrocyte precursors were found to proliferate prior to expressing galactocerebroside. The level of proliferation of galactocerebroside positive oligodendrocytes was low. Immature oligodendrocytes were extremely sensitive to irradiation by ('3)H-thymidine.;A serum-free culture medium was optimised for oligodendrocyte survival in mixed cultures. A medium supplemented with insulin, transferrin, selenium and triiodothyronine sustained oligodendrocyte growth for 3 weeks. In addition, astrocytes, neurons and glial cell precursors survived in this medium. Oligodendrocytes expressed galactocerebroside earlier in the absence of serum than in culture medium containing 2.5% serum.;For prolonged survival in serum-free medium an initial exposure of the cells to serum was necessary. To overcome this serum requirement, glycoproteins of the extracellular matrix were tested for their effect on brain cell attachment and survival. Dissociated cells, which had not been exposed to serum, attached well to uncoated plastic and to a complex basal lamina-like material (produced by corneal endothelial cells). Attachment was slightly reduced on fibronectin and type IV collagen and inhibited considerably by laminin, type I collagen and serum. On each substratum, attachment was reduced when cells were seeded in medium containing 2.5% serum. The only substratum which promoted the survival of a mixed population of cells, in the absence of serum, was the complex extracellular matrix. The effect of epidermal growth factor and fibroblast growth factor on cell survival in a totally serum-free system was investigated. Each hormone stimulated the survival and proliferation of a different population of cells.;Using combined autoradiography and immunofluorescence an increased ('3)H-thymidine incorporation by galactocerebroside positive oligodendrocytes was demonstrated after various times of exposure to fibroblast growth factor.;Epidermal growth factor stimulated the proliferation of astrocytes, but not galactocerebroside positive oligodendrocytes. However, it did play a role in oligodendrocyte generation by both direct and indirect mechanisms and the target cell was the galactocerebroside negastive oligodendrocyte precursor.