by FGF2 withdrawal for a further 2 weeks. At this stage neurospheres‐containing OLIG2+ cells were further expanded for additional 2 weeks in the presence of FGF2 (10 ng/ml), PDGFα (Platelet‐derived growth factor alpha, 20 ng/ml, PeproTech), purmorphamine (1 μM), and SAG (1 μM, Calbiochem), IGF‐1 (10 ng/ml, PeproTech), T3 (60 ng/ml, Sigma), and 1 × ITS (Gibco, NY, USA) before initiating oligodendrocyte differentiation. It was possible to maintain OPC‐containing spheres in this media for up to 8 weeks. Terminal differentiation of oligodendrocytes was achieved by mitogen withdrawal (except for T3 and IGF‐1) following single‐cell dissociation using papain (Worthington Biochemical, NJ, USA) and plating on Matrigel (SLS, 354230; 1 in 100 dilution), fibronectin (20 μg/ml, F2006, Sigma‐Aldrich), laminin (10 μg/ml, L2020, Sigma‐Aldrich) coated coverslips (or plates) at a density of 20,000‐30,000 cells per 0.3 cm2. For biochemical studies, cultures were dissociated with accutase (Life technologies), and separation of O4+‐oligodendrocytes was achieved by magnetic‐activated cell sorting (MACS) using anti‐O4 MicroBeads (MACS 130‐096‐670) and MACS LS columns (MACS 130‐042‐401) following the manufacturer's instructions.
