N2 neuroplex

iPSCs were differentiated into NPCs as previously described [45 (link), 46 (link)]. Briefly, iPSCs colonies were cultured for 2 days in the presence of DMEM/F12 1:1 with 1x Glutamax (Life Technologies), 1x N2 NeuroPlex (N2; Gemini Bio-products), 1x penicillin-streptomycin (PS; Life Technologies), 10-μm SB431542 (SB; StemGent), and 1-μm dorsomorphin (Dorso; Tocris Biosciences). The colonies were lifted off and kept in suspension, under rotation (95 rpm) for 7 days to form embryoid bodies (EB). EBs were gently disrupted, plated onto Matrigel-coated plates, and cultured in DMEM/F12 1:1 with 1x HEPES, 1x Glutamax, 1x PS, 0.5x N2, 0.5x Gem21 NeuroPlex (Gem21; Gemini Bio-products), supplemented with 20-ng/mL basic fibroblast growth factor (bFGF; Life Technologies) for 7 days. Next, neural rosettes were manually collected, dissociated with Accutase (Thermo Fisher), and NPCs were plated onto poly-L-ornithine/laminin-coated plates. NPCs were expanded in the presence of bFGF and fed every other day. Neural differentiation was promoted by bFGF withdrawn from the medium; ROCK inhibitor (Y-27632; Calbiochem, Sigma-Aldrich) was added at 5 μm for the first 2 days. Medium was changed twice a week, and cells were allowed to differentiate for as long as needed.

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.

All iPSC were provided by the Muotri lab. Cortical NPCs were differentiated from hiPSC as described previously (Griesi-Oliveira et al., 2015 (link)). To differentiate NPCs to cortical neurons, NPCs were dissociated with Accutase (StemCell Technologies), plated on a 10-cm tissue culture dishes coated with 10 μg/ml poly-L-ornithine (PLO; Sigma, P3655) and 2.5 μg/ml mouse laminin (1 mg, Invitrogen 23017-015), and then cultured for 2 weeks in NPC medium (DMEM/F12 supplemented with 1% penicillin-streptomycin, N2 NeuroPlex (Gemini Bio-products), NeuroCult SM1 (Stem-Cell technologies), and 20 ng/mL basic fibroblast growth factor (bFGF; Life Technologies). When NPCs reached 90% confluency, NPCs were differentiated into cortical neurons by bFGF withdrawal. Differentiating cells were fed with NPC medium every 3 days. hiPSC-derived cultures were differentiated for 4–5 weeks to allow growth of healthy neurons, extension of long processes, and formation of densely interconnected networks. Neurons were then dissociated and replated on 35-mm imaging plates with glass bottoms, as described below, and kept differentiating for a further 3–4 weeks with excellent cell survival, cell recovery and connectivity re-growth.