Dorso

Cortical organoids were generated as previously described (Trujillo et al, 2019). Briefly, PSCs cultured for approximately seven days were dissociated with 1:1 Accutase (Life Technologies):PBS, and cells were plated into a 6‐well plate (4 × 10⁶ cells/well) in mTeSR1 supplemented with 10 µM SB (Stemgent), 1 μM Dorso (R&D Systems), and 5 µM Y‐27632 (EMD‐Millipore, Burlington, MA, USA) and cultured hereafter in shaker suspension (95 rpm at 37°C). Formed spheres were fed mTeSR1 (with 10 μM SB and 1 μM Dorso) for three days followed by Media1 [Neurobasal (Life Technologies), 1× Glutamax (Life Technologies), 2% Gem21‐NeuroPlex (Gemini Bio‐Products, Sacramento, CA, USA), 1% N2‐NeuroPlex (Gemini Bio‐Products), 1% non‐essential amino acids (NEAA; Life Technologies), 1% P/S (Life Technologies), 10 μM SB, and 1 μM Dorso] for six days, every other day; Media2 (Neurobasal, 1× Glutamax, 2% Gem21, 1% NEAA, and 1% P/S) with 20 ng/ml FGF‐2 (Life Technologies) for seven days, daily; Media2 with 20 ng/ml each of FGF‐2 and EGF (PeproTech, Rocky Hill, NJ, USA) for 6 days, every other day; and Media2 with 10 ng/ml each of BDNF, GDNF, and NT‐3 (all PeproTech), 200 μM L‐ascorbic acid (Sigma‐Aldrich, St. Louis, MO, USA), and 1 mM dibutyryl‐cAMP (Sigma‐Aldrich) for 6 days, every other day. Cortical organoids were subsequently maintained indefinitely in Media2 without supplementation.