neurospheres were dissociated into single cells by pipetting and plated at a density of 200 000 cells per well on coverslips coated with poly-l-ornithine (Sigma-Aldrich) and fibronectin (Sigma-Aldrich) into 24-well plates. To induce neuronal differentiation, the cells were further cultured for 10 days in the neural culture medium (without human leukemia inhibitory factor or basic fibroblast growth factor) supplemented with 2% (v/v) B27 (Life Technologies), 10 ng ml−1 brain-derived neurotrophic factor (R&D Systems, Minneapolis, MN, USA), 10 ng ml−1 glial-derived neurotrophic factor (R&D Systems), 200 μm ascorbic acid (Sigma-Aldrich) and 1 mm dibutyryl-cAMP (Sigma-Aldrich).
