Surgical specimens of GBM were collected at Massachusetts General Hospital (GBM series) with approval by the Institutional Review Board. BT74, originally obtained from Dr. C. David James (University of California, San Francisco, San Francisco, CA; as GBM6; ref. 19 (link)), was maintained as s.c. xenografts in mice at Brigham and Women's Hospital. Mechanically minced tissues were digested with 0.1% Trypsin and 10 U/mL of DNaseI at 37°C for 45 min. After washes, tissues were triturated and passed through a 100-μm cell strainer. Cells were plated in EF medium composed of Neurobasal medium (Invitrogen) supplemented with 3 mmol/L L-Glutamine (Mediatech), 1× B27 supplement (Life Technologies), 0.5 × N2 supplement (Life Technologies), 2 μg/mL heparin (Sigma), 20 ng/mL recombinant human EGF (R & D systems), 20 ng/mL recombinant human FGF2 (Peprotech), and 0.5 × penicillin G/streptomycin sulfate/amphotericin B complex (Mediatech). Part of the digested tissue was also grown in DMEM supplemented with 10% FCS to generate standard primary adherent cultures. The cultures for CSCs were fed every third day with 1/3 volume of fresh medium. Passaging of the cultures was performed by dissociating neurospheres using NeuroCult Chemical Dissociation kit (StemCell Technologies).