For the induction of neurospheres, hiPSCs were incubated with TrypLE Select (Life Technologies, Carlsbad, CA, USA) for 10 min at 37 °C. The digestion was quenched with 0.02% w/v trypsin inhibitor (Sigma-Aldrich, St Louis, MO, USA) in phosphate-buffered saline (PBS). The hiPSCs were dissociated into single cells by pipetting and plated at a density of 10 000 cells ml−1 in an uncoated T75 flask containing the neural culture medium20 supplemented with human leukemia inhibitory factor (Merck Millipore, Darmstadt, Germany) and with basic fibroblast growth factor (PeproTech, Rocky Hill, NJ, USA). The cells were cultured in an atmosphere containing 4% O2 and 5% CO2 for 14 days. The neurospheres were passaged repeatedly by culture in the same manner. The neurospheres in passages four to seven were used for analysis. The neurospheres were then collected for neural differentiation. For neural differentiation, the 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
