Embryonic mouse forebrain-derived NSC were isolated from the forebrains of CD-1 mice at embryonic day (E) 14.5 and were cultured according to previously described methods [35,37]. Briefly, dissected forebrain tissues were mechanically homogenized in NSC media DMEM/F12 1:1 (Wisent, Quebec, Canada) containing HEPES, glutamine, antibiotic/antimycotic, glucose, recombinant human epidermal growth factor (rhEGF) (Sigma Aldrich, Oakville, Ontario, Canada, 20 ng/ml), basic fibroblast growth factor (bFGF) (Upstate (Millipore), Billerica, MA, USA, 20 ng/ml), heparin (Sigma Aldrich, Oakville, Ontario, Canada, 2 μg/ml) and hormone mix. Dissociated single cells were plated at a density of 105 cells/cm2 in NSC media. The media were refreshed every 48 h and cells were cultured under these conditions for 7 days to generate neurospheres. Primary neurospheres were gently dissociated to single cells by accutase treatment. Dissociated cells were plated on plates coated with growth factor-reduced matrigel (BD Biosciences, Mississauga, Ontario, Canada) at a density of 105 cells/cm2 in DMEM (GIBCO, Life Technologies Inc, Burlington, Ontario, Canada) and 10% Fetal Bovine Serum (Invitrogen, Life Technologies Inc, Burlington, Ontario, Canada) in the absence of rhEGF and bFGF. Cells were differentiated
