hNES cells from passage 1 to 4 were used for differentiation, induced in neural induction (NI) medium [20] with SHH (400 ng/ml; Peprotech) for 4 days, and Valproic Acid (VPA; 10 μM; Sigma) for 3 days. After induction with SHH and VPA (day 8), cells were passaged with accutase (Millipore) and split at 1:2 or 1:3 (depending on the density) on PLO/ laminin pre-coated plates. Until day 17–19, cells were kept in the same well at high-density in Neurobasal medium (Neurobasal medium (Gibco, 21103–049), 1X B-27 (50X, Gibco, 17504–044), HEPES 18 mM (Gibco, 15630–056), 0.25X Glutamax (100X, Gibco, 35050–038), 100 U/ ml Pen/strep (100X, Sigma, P0781)) with BDNF (20 ng/ ml), GDNF (10 ng/ ml), IGF1 (10 ng/ ml; all from Peprotech) and cAMP (10 μM; Sigma). At this point, they were gently dissociated using Accutase. Clumps of (proliferating) cells were discarded. The single cell suspension of day 17–19 neurons was then frozen or directly plated at 62.5k cells/ 3.8 cm2 (one well of 12WP) in direct contact or indirect contact mode in the presence of astrocytes for maturation. To
