Leukemia inhibitory factor

ESCs were cultured on irradiated mouse embryonic fibroblasts in DMEM (Invitrogen) supplemented with 15% ESC-qualified FBS (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 1 mM sodium-pyruvate (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 100 U/ml penicillin (Invitrogen), 100 µg/ml streptomycin (Invitrogen), and 1,000 U/ml leukemia inhibitory factor (ESGRO). ESCs were replated on gelatin-coated plates without fibroblasts 2 d before differentiation, and differentiation was performed by hanging drops of 1,000 cells in 25 µl differentiation medium (the same medium without leukemia inhibitory factor but containing 0.5 mM ascorbic acid [Sigma-Aldrich]), as previously described (Bondue et al., 2008 (link)). Dox (Sigma-Aldrich) was added to hanging drops at day 2, at the indicated concentration for Mesp1- and Mesp2-inducible ESCs (0.08 and 1 µg/ml, respectively) and at 1 µg/ml for Prickle1-, Prickle1/Mesp2-, RasGRP3-, and RasGRP3/Mesp2-inducible ESCs. After 4 d in hanging drops, EBs were replated on gelatin-coated dishes for further differentiation. For chimeric EB experiments, Mesp1- or GFP-inducible ESC lines were mixed with the control DsRed-inducible ESC line in equal proportions. To inhibit specifically the ERK signaling pathway during ESC differentiation, PD0325901 (Stem Gent) was added to the differentiation medium at a final concentration of 1 µM.

Human fetal brain tissue was obtained from a cadaver at 13 weeks of gestation with full parental consent and the approval of the Research Ethics Committee of Yonsei University College of Medicine, Seoul, Korea (Permit Number: 4-2003-0078)^{28 (link)}. All procedures conformed to the guidelines of the National Institutes of Health and the Korean Government. hNPCs isolated from the telencephalon were grown as neurospheres in serum-free growth medium, which consisted of a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 (DMEM/F12; Gibco, Carlsbad, CA, USA) containing 1% penicillin/streptomycin (P/S; Gibco), 1% N2 formulation (Gibco), 20 ng/ml fibroblast growth factor-2 (R&D Systems, Minneapolis, MN, USA), 10 ng/ml leukemia inhibitory factor (Sigma, St. Louis, MO, USA) and 8 μg/ml heparin (Sigma). All cultures were maintained in a humidified incubator at 37 °C and 5% CO₂ in air, and half of the growth medium was replaced every 3–4 days. Cells were passaged every 7–8 days by dissociation of bulk neurospheres with 0.05% trypsin/EDTA (Gibco) and cryopreserved at each passage in a Good Manufacturing Practice facility. For TNF-α (Peprotech, Rocky Hill, NJ, USA) pretreatment, TNF-α was added to the cell culture medium (final concentration: 0; 5; 10; 20; 50; or 100 ng/ml) for 24 h and then washed out prior to in vitro experiments.