NBN defined medium supports the development of O4+/O1- immunopanned pro-oligodendroglia.

Maintenance of immunopanned cells in culture medium in the absence of serum or pre-conditioning by other neural cell types such as astrocytes can be problematic. Here we report the novel use of a chemically defined medium, which we refer to as NBN since it contains N-2 supplement, B-27 supplement, and N-acetyl-L-cysteine, for maintaining O4+/O1- immunopanned pro-oligodendroglia. Since we had previously characterized O4+/O1- immunopanned pro-oligodendroglia in astrocyte-conditioned basal defined medium (BDM; [24]), we compared their proliferation and differentiation in NBN medium or in NBN medium containing 40% NBN medium pre-conditioned by astrocytes. At 4 DIC in NBN, 23% of O4+ cells were BrdU+ while in conditioned NBN medium, 91% of O4+ cells were BrdU+. At 7 DIC in either medium, less than 25% of O4+ cells were BrdU+. O4+/O1- immunopanned pro-oligodendroglia cultured in NBN medium developed extensive processes and membranous expansions characteristic of mature oligodendroglia. At 4 DIC in NBN medium, approximately 100% of cells were O4+, 80% were O1+, and 54% were MBP+. By contrast, at 4 DIC in conditioned NBN, 87% of cells were O4+, 12% were O1+, and 2% were MBP+. At 7 DIC, there were no differences in the percentages of cells that expressed O4, O1, or MBP in either NBN or conditioned NBN. These results indicate that NBN defined medium supports the development of O4+/O1- immunopanned pro-oligodendroglia, and promotes more rapid maturation than conditioned NBN. The ability to maintain cells of the oligodendroglial lineage immunopanned at specific developmental stages in NBN defined medium should facilitate studies designed to identify effects of growth factors or toxins on oligodendroglia.

Development of O4+/O1- immunopanned pro-oligodendroglia in vitro.

In this study, O4+/O1- pro-oligodendroglia isolated by immunopanning from cerebral hemispheres of P3-P5 rats were evaluated during their maturation in culture. Immunopanning yielded 3-4 x 10(5) cells/cerebrum, with 98% O4+ and 6% O1+. There was heterogeneity in the morphologies of immunopanned cells ranging from simple bipolar cells to more complex multipolar cells. As a first step in determining potential differentiative responses of mature oligodendroglia, we examined glial fibrillary acidic protein (GFAP) expression in response to fetal bovine serum (FBS) by cultures established from O4+/O1- immunopanned cells grown for 1, 14, or 21 days, exposed to 20% FBS for 6-7 days and fixed and immunostained on days 7, 21 or 28 in culture (DIC). When immunopanned cells were exposed to FBS following 1 day in serum-free medium, 88% expressed GFAP and when immunopanned cells were cultured for 14 days prior to FBS exposure, 78% expressed GFAP. By contrast, when cells were cultured for 21 days prior to FBS exposure (when a majority of the cells expressed O1 and myelin basic protein (MBP)), only 19% of the cells expressed GFAP (p < 0.001). Cells that were O4+/GFAP- even in the presence of FBS often exhibited a mature oligodendroglial morphology. Among immunopanned cells that responded to FBS by expression of GFAP, both process-bearing (similar to type 2 astroglia) and flattened, polygonal (similar to type 1 astroglia) GFAP+ cells were observed. These results confirm the utility of immunopanning for the isolation of pro-oligodendroglia and demonstrate that oligodendroglia that develop in vitro from O4+/O1- immunopanned cells become resistant to GFAP induction by FBS.