Human pdgf ab

SU-DIPG-XIII, SU-DIPG-XXXV, and SU-DIPG-L patient cells heterozygous for the H3-3A mutation (A>T) and derived from autopsy tissue, were a generous gift from Dr. M. Monje at Stanford University, in accordance with informed-consent protocols and in compliance with Stanford University and Cold Spring Harbor Laboratory Institutional Review Board human-subject protocols. The cells were grown as neurospheres in tumor stem media (TSM) consisting of DMEM/F12 (Invitrogen), Neurobasal (-A) (Invitrogen), B27 (-A) (Invitrogen), human-bFGF (20 ng/mL; Protech), human- EGF (20 ng/mL; Peprotech), human PDGF-AB (20 ng/mL; Peprotech), and heparin (10 ng/mL; Stemcell). The point mutation in H3-3A^K27M was confirmed by Sanger sequencing using primers listed in Supplementary Table S2.

iPSCs were maintained feeder-free on Geltrex-coated tissue culture plastic in TesR1 (STEMCELL Technologies). Differentiation to MSCs was initiated by transferring mid-passage iPSCs at 50% confluency to MSC medium, which consisted of Minimal Essential Medium Alpha supplemented with 5% fetal bovine serum, 5% horse serum and 10 ng/ml each of human PDGF-AB, EGF, and bFGF (all from PeproTech, Rocky Hill, NJ, USA). Media was changed every 48 h until cells with fibroblastic morphology were apparent and cultures neared confluency. At this point, MSC cultures were pre-treated with 10 μM ROCK inhibitor for at least 1 h and dissociated using a 50:50 mixture of Accutase and 0.25% Trypsin-EDTA incubated at 37 ° C. When cells had detached, the Accutase/trypsin mixture was diluted with 2 × volume of PBS +1% FBS +3 mM EDTA to prevent clumping. Cells were centrifuged at 400g for 5 min and re-plated onto gelatin-coated plates in growth media plus cytokines containing 5 μM ROCK inhibitor to enhance plating efficiency. Between passages 3–5, cultures can be weaned off of ROCK inhibitor during passage. Flow cytometry analysis was performed using the following antibodies, all from eBioscience (San Diego, CA, USA): Anti-Human CD73 eFluor 450, 48-0739; anti-Human CD105 (Endoglin) PE, 12-1057; and anti-Human CD90 (Thy-1) APC, 17-0909.