(EGF) (PeproTech), and 2 µg/ml heparin (Sigma). Attached neuroepithelial cells were fed every other day for 7 days. Neural rosette structures appeared by around day 15, were lifted off the plate, and grown in suspension in Neural Progenitor Medium to form neurospheres. For neuronal differentiation, neurospheres were dissociated with Accutase and plated on culture plates coated with 100 µg/ml poly-l-ornithine (Sigma) and 10 µg/ml laminin (Sigma). Neuronal cultures were maintained in Neural Differentiation Medium consisting of Neural Progenitor Medium, brain-derived neurotrophic factor (10 ng/ml; PeproTech), and glial cell–derived growth factor (10 ng/ml; PeproTech) without bFGF, EGF, or heparin. Neurons were observed after several days and were further differentiated for 30 days or longer. Most of the experiments were performed on the neuronal culture differentiated beyond 8 weeks.
