Ascorbic acid

Human recombinant cytokines were supplied from Immunotools (Friesoythe, Germany) and reconstituted in water. JAK inhibitor I (Cayman Chemicals, Ann Arbor, Michigan, USA), EGFR inhibitor PD168393 (MedChem Express, Monmouth Junction, New Jersey, USA) and ML351 (Tocris, Wiesbaden-Nordenstadt, Germany) were dissolved in DMSO. Calcium chloride (Sigma-Aldrich, St. Louis, Missouri, USA) stock solution was prepared at 1.2 M in media. Single-use AA and linoleic acid in ethanol were supplied by Cayman Chemicals. Palmitic acid (Sigma-Aldrich) was dissolved in 0.1% fatty acid-free bovine serum albumin (Sigma-Aldrich) in media. Ascorbic acid (Sigma-Aldrich) stock solution was dissolved in media. Antibody details are available in Table S1.

1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical, 2,4,6-tri (2-pyridyl)-striazine, ferric chloride, ferric chloride 6-hydrate, ferrous sulfate 7-hydrate, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid), potassium persulfate, gallic acid, ascorbic acid, butylated hydroxytoluene, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Folin–Ciocalteu phenol reagent, sodium acetate, anhydrous sodium carbonate, sodium nitrite, aluminum chloride, sodium hydroxide, and ferrozine were obtained from Sigma Aldrich Co. (St. Louis, MO, USA). Methanol, glacial acetic acid, and hydrochloric acid (37% w/w) were used for chemical analysis. All solvents, including 95% ethanol, were purchased from Mallinckrodt-Baker (Phillipsburg, NJ, USA).

For generation of smNPCs from pluripotent stem cells, colonies were detached from the MEFs 3–4 days after splitting, using 2 mg/mL collagenase IV. Pieces of colonies were collected by sedimentation and resuspended in hESC medium (without FGF2) supplemented with 10 µM SB-431542 (Ascent Scientific), 1 µM dorsomorphin (Tocris) for neural induction, as well as 3 µM CHIR 99021 (Axon Medchem) and 0.5 µM PMA (Alexis), and cultured in petri dishes. Medium was replaced on day 2 by N2B27 medium supplemented with the same small molecule supplements. N2B27 medium consisted of DMEM-F12 (Invitrogen)/Neurobasal (Invitrogen) 50∶50 with 1∶200 N2 supplement (Invitrogen), 1∶100 B27 supplement lacking vitamin A (Invitrogen) with 1% penicillin/streptomycin/glutamine (PAA). On day 4, SB-431542 and dorsomorphin were withdrawn and 150 µM Ascorbic Acid (AA; Sigma) was added to the medium. On day 6, the EBs, which showed intensive neuroepithelial outgrowth, were triturated with a 1,000 µL pipette into smaller pieces and plated on Matrigel-coated (Matrigel, growth factor reduced, high concentration; BD Biosciences) 12-well plates at a density of about 10–15 per well in smNPC expansion medium (N2B27 with CHIR, PMA, and AA). For coating, Matrigel was diluted to a final dilution of 1∶100 in Knockout DMEM (Invitrogen) prior to coating 500 µL per well of a 12-ell plate overnight. Coated plates were wrapped with parafilm and kept in the fridge for up to 1 month. The first split was performed at a 1∶5 to 1∶10 ratio on days 2 to 4 after plating. All the remaining splitting ratios were at least 1∶10. The higher splitting ratios selected better for smNPC colonies and led to a high purity with fewer splits. After a maximum of 5 splits, cultures were virtually free of contaminating non-smNPCs.