Mek inhibitor

ES cells were maintained using mouse primary embryonic fibroblasts (19 (link)). We used ES cell culture medium containing Knockout DMEM/F12 (Gibco) supplemented with 20% knockout serum replacement (Gibco), penicillin (100 μg/ml), streptomycin (100 μg/ml), 0.1 mM 2-mercaptoethanol, 1× L-glutamine, 1× nonessential amino acids (Sigma), 10 μM MEK inhibitor (PD 0325901, Cayman Chemical Company), 10 μM GSK3 inhibitor (CHIR99021, Cayman Chemical Company), and 10 ng/ml leukemia inhibitory factor. The procedure of differentiation of ES cells into neurons was described previously (19 (link), 20 (link)). In brief, ES cells were suspended in CDM medium containing Iscove's modified Dulbecco's medium/Hams F12 1:1 (Gibco), 1× lipid concentrate (Gibco), penicillin (100 μg/ml), streptomycin (100 μg/ml), transferrin (150 μg/ml final, Sigma), insulin (7 μg/ml, Sigma), 450 μM monothioglycerol (Sigma), and plated onto a 10-cm Lipidure-coated dish (Sumitomo Bakelite). After 8 days, aggregated cells were dissociated using 25% Accumax (Innovate Cell Technologies) in PBS, plated onto a 10-cm dish coated with laminin and poly-L-lysine using N2/B27 medium containing 0.5% N-2 Supplement (Gibco) and 1% B-27 Supplement (Gibco) in DMEM/F12, and cultured for 12 to 13 days.