Human noggin

NPCs were derived using an optimized protocol previously published^{83 (link)}. iPSCs were dissociated in cell clusters using Accutase solution (Sigma-Aldrich) and seeded onto low-adhesion plates in mTeSR1 supplemented with 0.5X N2 supplement (ThermoFisher Scientific), Pen/Strept (1%, Sigma-Aldrich), human Noggin (0.5 µg/ml, R&D System), SB431542 (5 µM, Sigma-Aldrich) and ROCK inhibitor Y27632 (10 µM). Medium was replaced every 3 days. After 10 days, embryoid bodies were seeded onto matrigel-coated plates (1:100, Matrigel growth factor reduced, Corning) in DMEM/F12 (SigmaAldrich) supplemented with 1X N2 supplement, non-essential amino acids (1%, MEM NEAA, ThermoFisher Scientific) and 1% Pen/Strept. Medium was replaced every other day. After 10 days, rosettes were dissociated with Accutase solution and NPCs (P0) were plated onto matrigel-coated flasks in NPC media containing DMEM/F12, 0.5X N2, 0.5X B27 supplement (ThermoFisher Scientific), Pen/Strept (1%) and bFGF (20 ng/ml, ThermoFisher Scientific). NPCs were passaged twice a week using Accutase solution; experiments were performed with NPCs between P3 and P10.

For OP induction, the EBs were transferred into a plate coated with a poly-l-ornithine and laminin (both were from Sigma-Aldrich Chemical Co.)-coated plate and cultured for 3 days in an advanced Dulbecco’s modified Eagle’s medium (DMEM)/nutrient mixture F12 supplemented with 20% KSR, N2, B27 (ThermoFisher Scientific), human basic fibroblast growth factor (bFGF; 25 ng/mL; R&D Systems), human FGF19 (25 ng/mL; R&D Systems), human noggin (30 ng/mL; R&D Systems), human R-spondin1 (R&D Systems; 50 ng/mL), heparan sulphate (50 ng/mL; Sigma, USA) and ampicillin (50 µg/mL). However, the medium was replaced with the advanced DMEM/F12 supplemented with 15% KSR, N2, B27, bFGF (25 ng/mL), human FGF19 (25 ng/mL), human BMP4 (20 ng/mL; R&D Systems), heparan sulphate (50 ng/mL) and ampicillin (50 µg/mL) and cultured for 3 days^{5 (link),57} .