Isolation and Characterization of CD133+ In Glioblastoma Multiforme

Glioblastoma multiforme, the most common lethal primary brain tumor, is comprised of a phenotypically diverse population of cells.  The cancer stem cell hypothesis suggests that the bulk tumor mass may contain a subpopulation of cells with stem-like characteristics.  Recently, a CD133+ stem cell-like subpopulation was identified in glioblastoma multiforme.  This subpopulation of cells can recreate the heterogeneity of the original tumor when injected intracranially into immunodeficient mice.  It is expected that 1 X 10³ CD133+ cells injected intracranially into C57BL/6 mice will be more tumorigenic than 1 X 10⁴ heterogeneous GL261 murine glioma cells injected intracranially into C57BL/6 mice.  In order to test this hypothesis, GL261 cells were grown in vitro in tumporsphere media, collected and labeled with PE Anti-mouse CD133 antibody.  Using flow cytometry, it was determined that CD133+ cells comprised 0.3% of the GL261 population.  Sorting CD133+ using Fluorescence-activated Cell Sorting (FACS) achieved a 95% pure population of CD133+.  Post sorting, 1 X 10³ CD133+ cells and 1 X 10⁴ unsorted GL261 cells were injected intracranially into C57BL/6 mice.  When possible, the brains of the mice were collected and stained to confirm tumor. All tumors were visible upon harvest.  Survival data collected for Kaplan-Meier curves indicated that the sorted  CD133+ and unsorted GL261 groups had nearly identical survival curves.  Thus, CD133+ cells can be successfully separated and purified through FACS and the similar survival curves of mice with either unsorted glioma cells or purified CD133+ demonstrates that CD133+ cells have superior tumorigenicity in this mouse implantation model.