To obtain hNES cells, few modifications were made to the protocol described by Shi et al., 2012 [25]. In short, high-density hiPSC cultures were passaged onto Geltrex (GIBCO)-coated 12 well plates. When hiPSC cultures reached confluence, they were neural induced with Noggin (500 ng/ ml; Peprotech) or its small molecule agonist dorsomorphine (1 μM; R&D), and SB431542 (10 μM; Stegment and Selleck chemicals). Neural rosettes were picked manually and cultured in neural maintenance medium (NMM) with FGF2 (20 ng/ ml) and EGF (20 ng/ ml; Peprotech) on PLO (20 μg/ ml)/mouse laminin (20 μg/ ml; both from Sigma) pre-coated plates.
