Preliminary Study Of Tumor Stem Cells In Human Brain Glioma

Objective: To isolate and identify tumor stem cells from established human glioma cell lines/strains and glioma tissues.Methods: 1 Human glioma cell line SHG-44, as well as it's subline SHG-44-9 maintained for long term were cultured in DMEM supplemented with 10% FCS, and in serum-free DMEM/F12 containing bFGF, LIF and EGF respectively. Neural stem cell marker CD133 were used to isolated tumor stem cells by magnetic cell sorting. Cells cultured in two different condition described above were also detected with flow cytometry and immunohistochemistrical staining with antibodies against Hoechst33342, Nestin, NSE and GFAP. 2 From 8 glioma samples collected shortly after operation, cell suspensions were made and cultured into tumor spheres. By magnetic sorting, CD133+ cells were acquired and plated at serial dilution Tumor stem cells of passage 5 were induced to differentiate with medium containing 10% fetal bovine serum, and immunocytohis- tologically examined with differentiation related cell surface markers, such as nestin, NSE and GFAP before and after differentiation.Results: 1 Using magnetic cell sorting, CD133 positive cells account for 0.021% of SHG-44 cells and 0.035% of SHG-44-9 cells when cultured in DMEM supplemented with 10% FCS, while in serum-free condition, percentage of cells expressing CD 133 increased to 1.2% and 2.3% respectively. Flow cytometry revealed that 1.5% of SHG-44 cells and 0.37% of SHG-44-9 cells, cultured in medium with serum, were positive for CD133; when serum was removed, CD 133 positive cells were 16.4% for SHG-44 cells, and 29.1% for SHG-44-9 cells respectively. Moreover, CD 133 positive cells have the capacity toproliferate and differentiate to neuronal and glial cells. As far as Nestin, another surface marker was concerned, 51.05% SHG-44 cells and 77.53% of SHG-44-9 cells were positive..2.even cultured in vitro for a long time, CD133+ cells isolated from an anaplastic ependymoma still maintain the capacity to grow into spheres and self-renew, proliferate and differentiate in certain condition, while CD133- could not.Conclusions: Human glioma cell lines cultured for long period still contained tumor stem cells, which could be effectivedly, isolated by both magnetic cell sorting and flow cytometry. Nestin positive cells might represent neural precursors or progenitors, so it was not special for tumor stem cells . Glioma tissues also comprised tumor stem cells that could be kept and passed on for long term, which provids a powerful tool for research of origin cell of glioma.