Abstract:
Objective:To isolate and identify mouse brain tumor stem cells from Tet-off/Tet-on genetically engineered mouse models with brain tumors, and to explore the origin of brain tumor stem cells. Methods: Brain tumor tissues were obtained from genetically engineered mice with brain tumor symptoms. Cells from the tumor tissue were cultured in DMEM/F 12medium containing fibroblast growth factor-2 (bFGF), epidermal growth factor (EGF) and B27minus retinyl acetate. Differentiated cells of mouse brain tumor spheres were cultured in DMEM/F 12containing 10% FCS. Cell morphology and ultrastructure were observed using phase contrast microscopy and transmission electron microscopy (TEM). To determine whether mouse brain tumor spheres contained candidate cancer stem cells, Hoechst 33342 fluorescent staining was used to sort for the side population (SP) phenotype. Cell cycle stage and chromosome ploidy of mouse brain tumor sphere cells were detected using flow cytometry. Immunofluorescence was used to assay for markers of brain tumor spheres and differentiated cells. Results: Brain tumors were located in the pineal region. The tumors were di-agnosed as medulloblastoma. Cells from genetically engineered mouse brain tumor tissue formed stem-like spheres when cultured in DMEM/F 12medium containing FGF2, EGF and B 27minus retinyl acetate, and dif -ferentiated into neuronal and glial cells when cultured in DMEM/F 12containing 10% FCS. Using phase contrast and transmission electron microscopy, the brain tumor spheres showed stem cell ultrastructural characteristics including undeveloped organelles and a high nucleus:cytoplasm ratio. The percentage of SP cells in brain tumor spheres was 17.27% . Heteroploidy occurred in 26.77% of the mouse brain tumor spheres. The majority of cells in the mouse brain tumor spheres were Nestin positive and a small fraction of cells were NSE and GFAP positive. Most differentiated cells coexpressed Nestin with MAP 2 or GFAP. Conclusion:Tet-off/Tet-on genetically engineered mouse brain tumor contains brain tumor stem cells which have the capacity to self-renew and differentiate into neuronal and glial cells.