Differentiation into NPCs and fibroblasts

hESCs were differentiated into NPCs and fibroblasts as previously described (Chiba et al., 2015).

Cells were cultured under feeder free conditions on matrigel-coated plates in mTESR1 medium (Stem Cell Technologies). The dual SMAD inhibition protocol for the differentiation of hESCs into NPCs was adapted from Chambers et al. (2009). In brief, hESCs were cultured for 10 d with increasing amounts of N2 medium (50% DMEM/F12; Lifetech), 50% Neurobasal Medium (Lifetech), 0.75% BSA (wt/vol; Sigma-Aldrich), N2 Supplement (Lifetech), 20 ng/ml insulin (Sigma-Aldrich), 1 mM glutamine (Lifetech), 1,000 U/ml penicillin/streptomycin (25%, 50%, 75%; Lifetech) supplemented with KOSR medium (hESC medium without FGF), 100 ng/µl Noggin (R&D Systems), and 10 µM SB431542 (Tocris). NPCs were maintained in N2 medium supplemented with 25 ng/ml FGF2 (Lifetech) and 40 ng/ml EGF (R&D Systems).

For the formation of embryonic bodies, hESC colonies were grown on ultralow attachment plates (Corning) in fibroblast medium (DMEM/F12; Lifetech) supplemented with 15% fetal bovine serum (Lifetech), 1 mM glutamine (Lifetech), 1% nonessential amino acids (Lifetech), and penicillin/streptomycin (Lifetech). After 9 d, embryonic bodies were transferred to tissue culture dishes coated with 0.2% gelatin and maintained in fibroblast medium (Ahfeldt et al., 2012).