D0260

OPCs were differentiated to oligodendrocytes with differentiation medium that consisted of DMEM/F12, 1× N2 supplement, 1× B-27 without vitamin A supplement, supplemented with 100 ng/mL noggin (3344-NG, R&D Systems), 10 ng/mL neurotrophin-3 (NT-3) (267-N3, R&D Systems), 50 μM cAMP (D0260, Sigma), 100 ng/mL insulin-like growth factor-1 (291-G1, R&D Systems) NT-3, and 40 ng/mL triiodothyronine (thyroid hormone; T-6397, Sigma).

hESCs were induced into neural progenitor cells (NPCs) according to a previously published protocol [19 (link)]. Briefly, single-cell dissociated hESC at a density of 30,000 cells/cm² was plated in neural induction media (NIM, DMEM/F12:NeuroBasal media 1:1 with 1% N2, 2% B27, 1% PenStrep, 1% GlutaMax, 10 ng/ml hLIF and 5 μg/ml Bovine Serum Albumin) containing 4 μM CHIR99021 (Tocris), 3 μM SB431542 (Sigma) and 0.1 μM Compound E (Millipore) for the first 7 days. The culture was then split at a 1:3 ratio for the next five passages using Accutase in NIM without Compound E on Matrigel-coated plates.
For neuronal differentiation, NPCs were plated at a density of 20,000 cells/cm² on 50 μg/ml poly-L-ornithine/10 μg/ml laminin-coated plates and grown in NeuroDiff media (DMEM/F12/Neurobasal media (1:1) supplemented with 1% N2, 2% B27, 20 ng/ml GDNF (R&D Systems), 20 ng/ml BDNF (R&D Systems), 300 μM dibutyryl-cyclic AMP (D0260, Sigma Aldrich) and 200 nM L-Ascorbic Acid (A4403, Sigma Aldrich)) for at least 3 weeks. Medium was changed every 2–3 days.

Around 40 thousand FACS-enriched ENCCs were seeded as droplets on poly-ornithine/laminin/fibronectin (PO/LM/FN)-coated 24-well plates in N2 medium containing 10 ng ml⁻¹ FGF2, 3 μM CHIR99021 and 10 μM Y-27632. The neuronal differentiation started 48 h later and the attached ENCCs were then cultured with N2 medium containing BDNF (10 ng ml⁻¹ Peprotech, 450-01), GDNF (10 ng ml⁻¹, Peprotech, 450-10) and ascorbic acid (200 μM, Sigma, A4034-100G), NT-3 (10 ng ml⁻¹, Peprotech, 450-03), NGF (10 ng ml⁻¹, PeproTech, 450-01), and cAMP (1 μM, Sigma, D0260). For valproic acid (VPA) treatment, 1 mM VPA (Sigma, P4543) was added during neuronal differentiation. The culture medium was changed every 2 days. ENCC-derived neurons at differentiation day 5 or 9 were fixed for immunocytochemistry analyses.