Directed Neural Differentiation of hPSCs

The neural differentiation protocol was modified from a previously published method⁶ . hPSCs were detached using TrypLE Select (Thermo Fisher Scientific) and plated at a density of 5 × 10⁵ cells/cm² on 100 µg/ml poly-L-ornithine (PO, Sigma) and 15 µg/ml LN521 or Matrigel matrix (Corning)-coated plates in E8 medium containing 10 µM ROCK inhibitor (Y-27632, Sigma). Neural maintenance medium was used as a basal medium and consisted of 1:1 DMEM/F12 with Glutamax and Neurobasal, 0.5% N2, 1% B27 with Retinoic Acid, 0.5 mM GlutaMAX, 0.5% NEEA, 50 µM 2-mercaptoethanol (all from Thermo Fisher Scientific), 2.5 µg/ml Insulin (Sigma) and 0.1% penicillin/streptomycin (Thermo Fisher Scientific). During the neural induction stage (days 1–12, Fig. 1a), the maintenance medium was supplemented with 100 nM LDN193189 and 10 µM SB431542 (both from Sigma), and the medium was changed daily. At day 12, the cells were detached with StemPro Accutase (Thermo Fisher Scientific) and plated at a density of 2.5 × 10⁵ cells/cm² on PO and either LN521 or mouse laminin (Sigma)-coated well plates in neural induction medium containing 10 µM ROCK inhibitor. For neural proliferation (days 13–25), the maintenance medium was supplemented with 20 ng/ml fibroblast growth factor-2 (FGF2, Thermo Fisher Scientific). At days 17, 21 and 25, the neural progenitor cells were passaged with StemPro Accutase and replated in medium containing 10 µM ROCK inhibitor. At day 21, the NPCs were cryopreserved in the same medium containing 10% DMSO (Sigma). For final maturation (days 26–130), the medium was changed to maintenance medium supplemented with 20 ng/ml brain-derived neurotrophic factor (BDNF, R&D Systems), 10 ng/ml glial-derived neurotrophic factor (GDNF, R&D Systems), 500 µM dibutyryl-cyclicAMP (db-cAMP, Sigma) and 200 µM ascorbic acid (AA, Sigma). At day 32, the cells were plated for experiments at a density of 50,000 cells/cm² on plastic well plates or 1 × 10⁶ cells/cm² on microelectrode arrays (MEAs). Plastic well plates were coated with PO and either LN521 or mouse laminin as before and MEAs with 0.1% poly-ethylene-imide (PEI, Sigma) and either 50 µg/ml LN521 or mouse laminin. Medium changes were performed every two to three days.