Generation of Brain Organoids from hiPSCs

To generate brain organoids, hiPSC cultures were dissociated into a single cell suspension using Accutase (Innovative Cell Technologies, Inc., #AT-104) for 20 minutes at 37 °C, diluted with PBS, transferred to a 15 mL conical tube, and then centrifuged for 5 minutes at 300 x g. The supernatant was removed and the hiPSCs were resuspended in warm mTeSR Plus with 10 μM Rock inhibitor (StemCell Technologies #72304) and counted on an automated cell counter (Bio-Rad, #1450102). Approximately 3 × 10⁶ hiPSCs were seeded per well of a 24-well AggreWell-800 plate (StemCell Technologies, #34815) which was centrifuged at 100 x g for 3 minutes to form spheroid aggregates (Day 0). After 24–48 hours, the spheroids were dislodged from the microwells and transferred to an Ultra-Low Attachment 6-well plate (Costar, #3471) using a wide bore P1000 pipette tip. The spheroids were maintained in suspension culture on an orbital shaker (~95 rpm) and guided via directed differentiation into brain organoids using previously described methods (S2 Table) [46 (link)].