For neural differentiations, NPCs were dissociated with Accutase and plated in neural differentiation media (DMEM/F12, 1x N2, 1X B27-RA, 20 ng/ml BDNF (Peprotech), 20 ng/ml GDNF (Peprotech), 1 mm dibutyrl-cyclicAMP (Sigma), 200 nm ascorbic acid (Sigma) onto PORN/Laminin-coated plates. Density is critical and the following guidelines were used: two-well permanox slide, 80–100,000 cells/well; 24-well, 40–60,000 cells/well; six-well, 200,000 cells/well. hiPSC derived-neurons were differentiated for 1–3 months. Notably, synapse maturation occurs most robustly in vitro when hiPSC neurons are cocultured with wildtype human cerebellar astrocytes (Sciencell). 0.5% FBS was supplemented into neural differentiation media for all astrocyte coculture experiments.
