Neural induction

Neural induction

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 Geltrex gel bed at an initial cell seedingdensity of 50 × 10³ cells cm⁻² and cultured overnight. On the following day (day 1), culture medium was switched to fresh N2B27-based neural induction medium (see below). For Gel-3D conditions, this neural induction medium was supplemented with 2% (v/v) Geltrex.

N2B27 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). For neural induction, N2B27 medium was supplemented with the TGF-β pathway inhibitor SB (10 μM; STEMCELL Technologies) and the BMP inhibitor LDN (0.1 μM; STEMCELL Technologies).

Company name and catalog number of some reagents: