Organoid Formation from Mouse ESCs

The protocol for organoid formation was based on the cortical differentiation protocol above. Briefly, 2000 cells were seeded per well of a round bottom, ultralow attachment, 96-well plate in mESC basal media with LIF, SB431542, and dorsomorphin. Forty-eight hours later, the spent medium was removed and replaced with NMM⁺ SB431542, dorsomorphin, I-BET151, and retinoic acid. The next day, this medium was removed, and NMM with SB431542 and Dorsomorphin was added. Forty-eight hours later, half the volume of the culture medium was replaced. At days 6 and 8, half of the medium was replaced with basal media (NMM). On day 10, the organoids were split evenly and 24 organoids were placed into 6-cm ultralow attachment dishes. On day 16, organoids were embedded in undiluted Matrigel (Corning, 356234, lot 229001) in the same medium and kept on a shaker at 16 rpm for at least 21 days.