For differentiation, PSCs were passaged and grown as floating aggregates (Fig. 1b) in low adherent plates. The passaged cells were transferred to a 15 ml conical tube and centrifuged at 1000 rpm for 1 min, followed by careful removal of the supernatant. The cell pellet was resuspended in KSR medium (20% knockout serum replacement, DMEM, 2 mM l-Glutamine, 10 µM β-mercaptoethanol and penicillin-streptomycin) with ROCK inhibitor (Y-27632, 5 µM) and gently triturated using a P1000 pipette to obtain a homogeneous cell suspension. About 2.5 × 106 cells were added to each well of a 6-well low adherent plate with 3 ml of KSR medium in each well. High cell density per well optimized the survival of cells. Rock inhibitor (Y-27632, 5 µM) was added on the first day of differentiation to prevent dissociation-induced death of hPSCs [25]. For neural induction, cells were treated with LDN193189 (100 nM) from day 0 to day 14 and with SB431542 (10 µM) from day 0 to day 7 [26].
