A low dose of BCNU decreases division and promotes differentiation of O-2A/OPCs. Cells grown at clonal density were exposed 1 day after plating to low-dose BCNU (2.5 μM for 1 h), a dosage that did not cause significant killing (< 5%) of O-2A/OPCs in mass culture. The number of undifferentiated O-2A/OPCs and differentiated cells (oligodendrocytes) was determined in each individual clone from a total number of 50 clones in each condition by morphological examination and by immunostaining with A2B5 and anti-GalC (galactocerebroside) antibodies (to label O-2A/OPCs and oligodendrocytes, respectively). (a) Schematic diagram of the differentiation potential of O-2A/OPCs. Bipolar O-2A/OPCs can undergo continued cell division(s) to form new precursor cells (red), and can differentiate into multipolar postmitotic oligodendrocytes (green). Alternatively, an O-2A/OPC can differentiate directly into an oligodendrocyte without further cell divisions. (b) An example of one clone in culture. Immunostaining with A2B5 (red) and anti-GalC (green) identifies six O-2A/OPCs and two oligodendrocytes. Cell nuclei stained in blue (DAPI). Scale bar represents 20 μm. (c) Composition of progenitors and oligodendrocytes in a representative experiment of control cultures analyzed 8 days after plating optic nerve-derived O-2A/OPCs at clonal density. Multiple clones with three or more O-2A/OPCs were seen. (d) In parallel BCNU-treated cultures, analyzed 8 days after plating at clonal density (7 days after BCNU exposure), no clones contained more than two O-2A/OPCs. Experiments were performed in triplicate in at least two independent experiments. In the experiments represented in (c) and (d) the proliferation and differentiation of O-2A/OPCs were followed over a time course of up to 10 days after BCNU treatment. Results are presented as representative three-dimensional graphs. The number of progenitors per clone is shown on the x (horizontal) axis, the number of oligodendrocytes on the z (orthogonal) axis and the number of clones with any particular composition on the y (vertical) axis.