A complete list of cell lines and in vivo H3K27M model systems can be found in table S1. Culture methods are briefly described below.

Mouse stem cells and mNEURON differentiation. E14 mESCs were cultured as described previously (17). Differentiation to cervical mNEURONs is detailed in (17), but briefly, ESCs were plated at low density in the absence of mitogenic factors to form embryoid bodies. Two days later, media were supplemented with 1 μM retinoic acid and 0.5 μM smoothened agonist. Cells were harvested 4 days later, with one media replenishment for 2 days.

Human brain tumor models. Patient-derived glioma cell lines, including DIPG cell lines (see table S1 for source), were cultured in tumor stem cell media as described (37, 38). Human neural stem cells were derived and cultured as described previously (39). New York University (NYU) patient–derived tumor cell line generation has been approved by the NYU Institutional Review Board (IRB) (no. S15-01228). Pathological diagnoses of primary tumors, from which cell lines were derived, were performed by a board-certified neuropathologist (M.S.), and molecular characteristics were established at an NYU Clinical Laboratory Improvement Amendment (CLIA)–certified molecular pathology laboratory. Furthermore, relevant tumor sample collection and molecular profiling were approved by the NYU IRB (nos. S12-00865 and S14-00948, respectively). All human tumor samples were reviewed, and diagnosis was performed by a board-certified neuropathologist (M.S.) in a CLIA-certified pathology laboratory.

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