13mm glass coverslips (Fisher Scientific, Pittsburg, PA, USA) pre-coated with polyornithine (0.1mg/mL in water) and Matrigel (0.67mg/mL in DMEM/F12) (BD Biosciences, Bedford, MA, USA) coated at a density of 100,000 cells per coverslip. Cells were allowed to adhere to coverslips for 24 hours, and then cultured in neural differentiation media supplemented with 10ng/mL brain derived neurotrophic factor (BDNF) (Peprotech, Rocky Hill, NJ, USA), 10ng/mL glial derived neurotrophic factor (GDNF) (Peprotech), 10ng/mL insulin-like growth factor (IGF) (Peprotech), and 1µg/mL laminin (Sigma-Aldrich) to allow for the differentiation of neural cells. Half of the media was changed 3 times per week, and neural cells were cultured for 12 weeks or more.
