hPSC colonies were first treated with Accutase (Sigma-Aldrich) for 10 min at 37°C. Cells were rinsed briefly with phosphate-buffered saline (PBS) before being collected, centrifuged, and resuspended in mTeSR1 containing the ROCK inhibitor Y27632 (10 μM; Tocris). Singly dissociated hPSCs were plated onto coverslips at an initial cell seeding density of 50 × 103 cells cm−2 and cultured overnight. For Gel-2D and Gel-3D cultures, the coverslip was precoated with the Geltrex gel bed, whereas for Glass-2D and Glass-3D conditions, glass coverslips were precoated with 1% Geltrex solution for 1 hour at room temperature. On the following day (day 1), culture medium was switched to fresh N2B27-based neural induction medium (see below). For Gel-3D and Glass-3D conditions, this neural induction medium contained 2% (v/v) Geltrex. Thereafter, fresh neural induction medium with or without 2% (v/v) Geltrex supplement was exchanged daily.

N2B27-based neural induction medium comprised Advance DMEM/F12 (Gibco):Neurobasal medium (1:1; Gibco), 0.5× N2 (Gibco), 0.5× B27 (Gibco), 1× nonessential amino acids (Gibco), 2 mM l-glutamine (Gibco), and 0.1 mM β-mercaptoethanol (Sigma-Aldrich). N2B27-based neural induction medium further contained the TGF-β pathway inhibitor SB (10 μM; STEMCELL Technologies) and the BMP inhibitor LDN (0.1 μM; STEMCELL Technologies).

For dorsalization of NE cysts, 3 μM CHIR (STEMCELL Technologies) was supplemented into neural induction medium from day 4 to day 9. For ventralization of NE cysts, all-trans RA (1 μM; STEMCELL Technologies), recombinant human SHH (10 or 100 nM; PeproTech), and/or SAG (1 μM; STEMCELL Technologies) were supplemented into neural induction medium from day 4. For MN induction, the following chemicals were added to neural induction medium from day 9 or day 12: BDNF (10 ng ml−1; R&D Systems), GDNF (10 ng ml−1; PeproTech), CNTF (10 ng ml−1; PeproTech), IGF-1 (10 ng ml−1; PeproTech), cAMP (1 μM; Sigma-Aldrich), and AA (0.2 μg ml−1; Sigma-Aldrich).

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