Pen strep 1

To generate murine DIPG neurospheres, whole tumors were extracted and incubated with 5mL papain solution (Papain 4.6 mg; EDTA 0.9 mg; Cysteine 0.9 mg; 5 mL Earls Balanced Salt Solution) plus 30μL DNase (10mg/mL, Sigma Aldrich) with trituration, followed by 2 mL of Ovomucoid solution (Ovomucoid stock solution: Ovomucoid protein 10 mg; 20 μL DNase) and centrifuged at 1100rpm. The pellet was then triturated in 0.5mL of ovomucoid, brought up with 5mL of Neurocult media (Stem Cell Technologies, #05700), centrifuged at 600rpm, and repeated 3 times. The cell pellet and aggregate supernatant were filtered and plated at a density of 500,000 cells per 25 cm² flasks in serum free neurosphere media (per 50 mL total volume: 44.5 mL Neurocult basal media (Stem Cell Technologies); 10% proliferation supplement (Stem Cell Technologies #05701); Pen-strep 1% (Invitrogen #15140–122); Human basic FGF, 20ng/mL (Invitrogen #13256–029); Human EGF, 10ng/mL (Invitrogen #PHG0314); Heparin, 2μg/mL (Stem Cell Technologies #07980). Cells were incubated at 37° C and split as required approximately once a week with Accutase (Stem Cell Technologies).

Human iPSCs (BJ-SiPs and 1016A) were maintained feeder-free on Matrigel hESC-Qualified Matrix (catalog #354277; Corning) coated plates in StemFlex (catalog #A3349401; ThermoFisher) or mTeSR1 (catalog #85857; STEMCELL Technologies) medium to maintain pluripotency and for expansion. Both media were supplemented with pen/strep (1×; catalog #15140122; Gibco). Media were changed every other day for StemFlex and every day for mTeSR1, with cells passaged when they reached >80% confluence.