Purification of O-2A progenitor cells from 7 day old rats was as described previously.17 The O-2A progenitor cells were purified by immunopanning using antibody-coated dishes. Negative selection with Ran-2 antibody43 was used to eliminate type-1 astrocytes, followed by anti-galactocerebroside (GalC) antibody44 panning to remove oligodendrocytes. The O-2 A progenitors were further purified from the remaining cell suspension by capture on a tissue culture dish coated with the A2B5 antibody.45
Platelet-derived growth factor (PDGFAA, R&D Systems) and basic fibroblast growth factor (&FGF; Pepro Tech) were added daily, each at 10ng/ml, both to increase the number of progenitor cells and also to maintain them in an undifferentiated state.46
Increasing numbers of O-2A progenitors were obtained by repeated passage on to new plates. Immature and mature oligodendrocytes were derived from the same pool of cells as the O-2A progenitors following passage of O-2A progenitors on to new dishes. Immature oligodendrocytes were allowed to partially differentiate in the presence of bFGF, 10ng/ml added daily. O-2A progenitors were allowed to spontaneously differentiate into mature oligodendrocytes by withdrawal of growth factors (PDFGAA and bFGF), following passage on to new dishes. The mature oligodendrocytes were then cultured under two different conditions for at least five days prior to harvest. One set of cultures were supplemented with CNTF (cilliary neurotrophic factor - recombinant rat CNTF; Precision Research Biochemicals) at a concentration of 5ng/ml daily, while the other set of oligodendrocytes were cultured in DMEM-BS alone.17
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