Neural Progenitor Cell Derivation and Characterization

NPCs were derived from embryonic stem cell line BG02, provided by Yunnan Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine [41 (link)]. NPCs culture medium contained Neurobasal medium (Gibco, Thermo Fisher Scientific), 1% N2 (Gibco), 2% B27 (Gibco), 1% NEAA (Sigma), 1% Glutmax (Sigma), 10 ng/mL bFGF (1:500; Millipore Ltd., Burlington, MA, USA), and 1000 U/mL LIF (Millipore), 3 μM CHIR99021 (Selleck, Houston, TX, USA), and 5 μM SB431542 (Cellagentec, San Diego, CA, USA). NPCs were cultured with 5 μg/mL laminin (Gibco) in poly-ornithine-coated 12-well plates and were passaged for every three days during the experiment [41 (link)]. Staining was carried out with PAX6 (1:500, Biolegend Covance, Dedham, MA, USA), SOX2 (1:500, Millipore), and Nestin (1:200, Millipore) [42 (link)], which are specific NSC markers. Primary cortical neuron culture was prepared from cerebral cortical tissue from postnatal day one (P1) wild-type C57BL/6 mice. The tissue was digested with 0.25% trypsin for 30 min followed by centrifugation at 1000 rpm for 5 min and filtering cells with 40 μm filter. Astrocytes culture medium contained DMEM (Gibco), 10% fetal bovine serum (FBS, Northvale, NJ, USA), and 1% NEAA (Sigma). Neuron culture medium contained Neurobasal (Gibco), 2% B27 (Gibco), 1% N2 (Gibco), and 1% NEAA (Sigma).