NSCs were obtained from C57/Bl6 mouse embryos at E14. The whole embryonic brain was dissected and dissociated as described previously . Briefly, the brain was cut into small pieces, incubated with 0.05% trypsin EDTA for 30 minutes at room temperature, and mechanically triturated through a 26-gauge needle. Cells were cultured in serum-free Neurobasal medium (Invitrogen, Breda, The Netherlands) supplemented with human recombinant epidermal growth factor (20 ng/ml, Invitrogen), basic fibroblast growth factor (20 ng/ml, Invitrogen), B27 (Invitrogen), penicillin-streptomycin 1% (Sigma-Aldrich, Zwijndrecht, The Netherlands), L-glutamine 1%, and glutamax 1% in T25 (Nunc, Roskilda, Denmark) culture flasks in a humidified 5% CO2/95% air incubator at 37°C. Within 5–7 days, the cells grew as free-floating neurospheres and were passaged after mechanical dissociation through a fire-polished Pasteur pipette after 3 days. After two passages, neurospheres were used for the OPC differentiation induction experiments. These neurospheres were directly plated or dissociated (after a short exposure [5 minutes] to 0.1 mM ethylene glycol tetra-acetic acid) through a fire-polished Pasteur pipette and subsequently cultured in poly-L-lysin-laminin–coated chamber slides (Nunc), approximately 104 cells per well. Three differentiation conditions were evaluated. The first condition was cultured in the serum-free Neurobasal medium supplemented with B27, L-glutamine 1%, glutamax 1%, and penicillin-streptomycin 1% (Sigma-Aldrich). The second condition was cultured in the oligodendrocyte-specific Sato medium, consisting of Dulbecco's modified Eagle's medium with additives glutamax 1%, L-glutamine 1%, penicillin-streptomycin 1%, putrescine (16 mg/ml, Sigma), thyroxine (400 mg/ml, Sigma), triiodothyroxine (400 mg/ml, Sigma), progesterone (6.2 ng/ml, Sigma), sodium selenite (5 ng/ml, Sigma), bovine serum albumin (100 mg/ml, Sigma), insulin (5 mg/ml, Sigma), and transferrin (50 mg/ml, Sigma). During the first 2 days of culture, the Sato medium was supplemented with Shh (100 ng/ml; R&D Systems, Abington, UK), FGF-2 (10 ng/ml; R&D Systems), and PDGF-aa (10 ng/ml; R&D Systems). The third condition was cultured in the same Sato medium as described in the second condition, but after previous transfection of the NSCs with the Olig1 gene.