Derivation and Differentiation of Oligodendrocyte Progenitor Cells

Derivation of NPCs was performed as described [30 (link), 60 (link)] (also described in Supplementary Methods). Briefly, NPCs were cultured in suspension as neurospheres in Advanced DMEM/F12 containing: 1% N-2 supplement, 1% B27 supplement, 1% Anti-anti, 1% GlutaMAX (all from Life Technologies), FGF2 20 ng/ml (PeproTech), PDGFα 20 ng/ml (PeproTech), purmorphamine 1 μM (Calbiochem), SAG 1 μM (Calbiochem), IGF-1 10 ng/ml (PeproTech), and Tri-iodothyronine (T3) 30 ng/ml (Sigma) for 6–12 weeks. Following papain dissociation of neurospheres (Worthington), cells were plated on 1/100 diluted Matrigel (BD Biosciences), 20 µg/ml Fibronectin (Sigma), and 10 µg/ml Laminin (Sigma) coated coverslips or plates at a density of 20,000–30,000 cells per 0.3 cm². Differentiation of oligodendrocytes was achieved by mitogen withdrawal (except for T3 and IGF-1).