Brains of tumor-bearing mice were micro-dissected and glioma tissue was mechanically and enzymatically disaggregated for the generation of ex-vivo cultures. The cultures were plated as single cells in serum-free DMEM/F-12 1:1 media (Life Technologies) supplemented with B27 without Vitamin A (Life Technologies), 10 ng/mL EGF (Peprotech), and 10 ng/mL basic FGF (Peprotech), and grown as tumor neurospheres. Every 3–4 days, the cells were dissociated by triturating with Accutase (Sigma-Aldrich) and sub-cultured, as described previously [39 (link)]. All cell lines were routinely mycoplasma tested using the MycoAlert mycoplasma detection kit (Lonza). For serum-induced differentiation studies, the neurosphere cultures were dissociated into single cells and the growth medium was switched to Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS (Hyclone). The cells were grown under these conditions for 7–10 days and assessed for expression of differentiation markers. For sphere formation assays, cells were dissociated and plated at single-cell density in Nunclon Sphera Surface ultra-low adhesion 96-well plates (Thermo Fisher) in neurosphere medium. Sphere numbers per 96-well plate were quantified after 12 days. Primary astrocytes were isolated from 3-day old pups and cultured in DMEM supplemented with 10% FBS.