B27 gibco

MenSCs were seeded at a density of 1.5 × 10⁴ cells/cm² in a multi-well plate (Greinner-Bio-one, cat# 662102) using Fast-N-spheres medium (DMEN F-12 GIBCO®, cat#11,330–032; supplemented with 2% B27® GIBCO® (cat #17504-044), 20 ng/ml basic fibroblast growth factor (bFGF, R&D Systems, Inc., MN), 20 ng/ml epidermal growth factor (EGF, Sigma cat#E9644), 1 µg/ml heparin sodium salt®, and 100 U/ml penicillin/streptomycin for 11 days according to^{49 (link)}.

Human i³ cortical neurons were generated as previously described (Wang et al., 2017 (link); Fernandopulle et al., 2018 ). In brief, human iPSCs reprogrammed from a wild type male containing a doxycycline-inducible NGN2 integrated into the AAVS1 safe harbor locus were cultured on matrigel-coated (Corning) plates in supplemented Essential 8 medium (Gibco). Once iPSC cultures reached confluence, they were split and induced toward neuronal differentiation with doxycycline-containing induction media (DMEM/F12-Gibco, N2, and NEAA-Invitrogen) for 3 days. On DIV 3, induced neurons were lifted and plated on the final experimental plates for maturation (PLO/Laminin-coated Corning Cell-Bind plates or PEI/Laminin-coated Axion CytoView MEA plates). Neurons were plated at a density of 1.2 × 10⁵ cells per ml for imaging experiment and 2 × 10⁵ cells per ml for MEA experiments. Cells were matured in cortical maturation media (BrainPhys-Stem Cell Technologies, B-27-Gibco, BDNF, NT3, and laminin) for an additional 14–18 days prior to experiment initiation.

The PC-9 cell line used in this study was provided by the Cell Bank of the Chinese Academy of Science (Shanghai, China). SPF female BALB/c-Nude mice (4–6 week old, 18–20 g) were purchased from Vital River Laboratory Animal Technology (Beijing, China). Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from PeproTech China (Suzhou, China). The human antibodies, CD44, ABCG2, CD133, ALCAM, and EpCAM, as well as Annexin-V apoptosis kits, were purchased from American BD (Pasadena, CA, USA). The DMEM/F12 medium, RPMI-1640 medium, TRIzol reagent, GIBCO^TM B27, and fetal bovine serum were purchased from Thermo Fisher Scientific China (Shanghai, China). Gefitinib was purchased from Sigma-Aldrich China (Shanghai, China). The CCK8 kits were purchased from Dojindo Laboratories China (Shanghai, China). Chromium Single Cell 3′ v3 Reagent Kit was purchased from 10× Genomics China (Shanghai, China). This study was approved by the Ethical Committee of Tianjin University General Hospital.