Materials and Methods:
Glioma Tumor–Derived Neurospheres.
All of the work related to human tissues was performed at The Ohio State University under an institutional review board–approved protocol according to National Institute of Health (NIH) guidelines. Glioma and normal neurospheres were derived from 19 of high-grade glioma (HGG) samples, 3 fetal brain-derived astrocytes (such as 16wf), and neural progenitors (Table S1) as described previously (1–4). Surgeries of brain tumor resections were performed by Drs. I. Nakano and E. A. Chiocca at the Department of Neurological Surgery, The Ohio State University (Columbus, OH). Briefly, freshly resected glioma tumor samples were dissociated into single cells using both mechanical (gently pipet neurospheres with P1000 pipet tips four to five times) and enzymatic methods (TrypLE Express, 1; Invitrogen). The dissociated tumor cells were cultured in DMEM/F12 (Invitrogen) supplemented with B27 (1:50), heparin (5 mg/mL), basic FGF (bFGF) (20 ng/mL), and EGF (20 ng/mL). Growth factors (bFGF and EGF) were added twice a week. To differentiate glioma stem cells (GSCs), neurospheres were cultured in DMEM/F12 supplemented with 10% (vol/vol) FBS for 10 d. Phenotypic characterization of these primary cultures was performed as described previously (1, 5). The human fetal neural stem cell sample (16wf) was established at the University of California at Los Angeles as described previously (6). All of the neurospheres analyzed in this study were cultured <20 passages. In some experiments, various neurospheres were exposed to radiation (5 Gy) after cells were plated at a density of 1 Å~ 106 cells/flask 1 d before radiation treatment. Single cells of various neurospheres were also treated with diethylaminobenzaldehyde (DEAB) (100 μM; Sigma) or DMSO as a vehicle control.
See our recent papers (link to publications) with these patient-derived glioma stem cell samples.
Experimental flow for establishing primary cultures with patient tumor samples
Our first set of patient-derived glioma stem cell cultures (published in PNAS: Mesenchymal glioma stem cells are maintained by activated glycolytic metabolism involving aldehyde dehydrogenase 1A3).
Our second set of patient-derived glioma stem cell cultures (ready to share, unpublished).