The isolation, culture, identification, and purification of stem cells from primary human brain tumors of different phenotypes have marked capacity for proliferation, self-renewal, and differentiation. These cells represents a minority of the tumor cell population and are identified by expression of the cell surface marker the positive CD133. These positive CD133 cells, termed the brain tumor stem cells (BTSCs), which are the expression of neural differentiation markers, and are necessary for the proliferation and self-renewal of the tumor in culture.

1. Tumor samples were obtained from the informed consenting patients.

2. Tumors were washed, acutely dissociated in oxygenated artificial cerebrospinal fluid and subject to enzymatic dissociation.

3. Tumor cells were then resuspended in tumor sphere medium (TSM)
    • Serum-free neural stem cell medium
    • Human recombinant EGF (20 ng/ml)
    • bFGF (20 ng/ml)
    • Leukemia inhibitory factor (10 ng/ml)
    • Neuronal Survival Factor (NSF) (10 ng/ml)
    • N-acetylcysteine (60 μg/ml)

4. Plated at a density of 3 × 10⁶ live cells/60-mm plate.

5. RBCs were removed using lympholyte-M.

6. After primary sphere formation was noted, sphere cells were dissociated and plated in 96-well microwell plates in 0.2 ml volumes of TSM.

7. Final cell dilutions ranged from 200 cells/well to 1 cell/well in 0.2-ml volumes.

8. Cultures were fed 0.025 ml of TSM every 2 days until day 7, when the percentage of wells not containing spheres for each cell plating density was calculated and plotted against the number of cells per well.

9. Regression lines were plotted and x-intercept values calculated, which represent the number of cells required to form at least 1 tumor sphere in every well.

10. CD133-adherent tumor cells were trypsinized before collection for assays.

11. For primary sphere formation assays, this analysis was performed on the entire acutely dissociated tumor cell population on day 0 to quantify stem cell frequency within the tumor.

12. Cells were plated in 96-well microwell plates in 0.1-ml volumes of SFM supplemented with growth factors, at a density of 1000 cells/well.

13. Cell proliferation assays were performed on days 0, 3 5, and 7 postplating using the Roche 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based Colorimetric Assay Cell Proliferation kit 1.

14. Quantification of viable cells through reading of UV absorption spectrums at 575 nm was performed on a Versamax microplate reader.

15. Two days after primary culture, cells were plated onto glass coverslips coated in poly-L-ornithine in medium with 10% FBS in individual wells of a 24-well culture plate.

16. Cells were fed with FBS-supplemented medium every 2 days, and coverslips were processed 7 days after plating using immunocytochemistry.