Isolation Of Human Glioma Stem Cells (GSCs) And The Methylation Pattern Of Genomic DNA

With the rapid development in the research field of stem cells and the progress in related methodology, Tumor stem cells (TSCs) hypothesis has been confirmed by the isolation of TSCs from acute myeloid leukemia (AML) and various malignancies of the breast, brain, colon, liver and prostate. TSCs has been found crucial for tumorigenesis, progression, metastasis and the population of recurrence of cancer. Because of the low percentage of TSCs and demanding conditions of culture, the establishment of ideal methodology for the isolation of TSCs is essential to the research on TSCs.A number of reports have confirmed the existence of glioma stem cells (GSCs), oligoastrocytoma is a entity of CNS tumors composed of a mixture of neoplastic astrocytes and oligodendrocytes. The existence of TSCs needs to be investigated for the tumorigenesis, progression and the histogenesis of the mixed components.The differentiation of embryonic stem cells and adult stem cells is controlled by epigenetic mechanism. DNA menthylation pattern is an important constituent of epigenetics. However, the epigenetic mechanism in TSCs remains unknown.In this study, we isolated tumor spheres from human primary oligoastrocytoma tissues with conditioned medium and monoclonal culture, identified their potential for self-renewal and multipotentiality with limiting dilution and immunocytochemistry. In order to confirm tumorigenicity of GSCs isolated from oligoastrocytoma, we implanted tumor spheres formed by GSCs into nude mice. Moreover, we tested the mRNA level of DNMT1 in GSCs and adhesive tumor cells with RT-PCR, measured the protein level of DNMT1 in GSCs and adhesive tumor cells with Western blot and immunocytochemistry. We also assessed the DNA methylation pattern of GSCs and adhesive tumor cells with HPLC. The main results and conclusions are as follows:1. The tumor cells expressing stem cell markers in primary human anaplastic oligoastrocytoma and the culture of GSCs (1) We found a few tumor cells positive for nestin in the cytoplasm or CD133 on the cell membrane.(2) We acquired primary tumor cells from human oligoastrocytoma tissues with tissue dry-up culture, which was simple and easy to manipulate. The success of the culture depended on pretreatment to the bottom of culture flasks with fetal calf serum, the proper size of tissue pieces and the suitable space between two pieces.(3) Resorting to conditioned medium and monoclonal culture, we acquired GSCs from monolayer primary tumor cells. In conditioned medium which if free of fetal calf serum, proliferation of adhesive tumor cells was inhibited and GSCs could form spheres. After passage, the single cell from tumor spheres could form sphere which was made up of hundreds of GSCs.2. The identification of GSCs from primary human oligoastrocytoma tissuesWe stained tumor cells in the spheres with immunofluorescence of glioma stem cell marker CD133 and neural stem cell marker Nestin. Tumor cells in the spheres expressed both Nestin and CD133, implying stem cell phenotype. For analysis of cell differentiation, tumor spheres were cultured in DMEM containing 10% FCS. The proliferating tumor cells which differentiated from GSCs expressed a moderate level of GFAP and MBP as compared to primary cells. These results indicated that sphere forming primary tumor cells were progeny cells capable of proliferation and differentiation.The results of limiting dilution showed that the cells which had the property of self-renewal in primary tumor cells accounted for 2.49%±0.13% and the cells which had the property of self-renewal in tumor spheres accounted for 36.54%±1.41%, so GSCs could be enriched by conditioned medium.3. The in vivo tumorgenic model constructed by GSCs from primary human oligoastrocytoma tissuesTumor spheres were transplanted to SCID mices and tumor nodules were palpable after 9 weeks in mice implanted with 5x104 GSCs. The average size of the tumors reached 0.64±0.02 cm3 at the 10th week after implantation, significantly larger compared with 0.38±0.08 cm3 in mice implanted with 5x103 GSCs cells (P<0.05). No tumors were formed in mice implanted with adhesive cells. These results suggest that GSCs derived from anaplastic oligoastrocytomas are tumorigenic at low cell numbers. Moreover, immunostaining showed various numbers of GFAP positive tumor cells mixed with MBP positive tumor cells. Tumor tissue sections were found to contain dispersed CD133 and Nestin positive tumor cells, with the positive rate significantly higher than in the primary tumors.4. Resorting to RT-PCR Western blot and immunocytochemistry, we tested the mRNA level of DNMT1 in GSCs and adhesive tumor cells, measured ed the protein level of DNMT1 in GSCs and adhesive tumor cells. We also assessed the DNA methylation pattern of GSCs and adhesive tumor cells with HPLC.The results showed there was no difference in mRNA level of DNMT1 between adhesive tumor cells and GSCs, but GSCs expressed a higher protein level of DNMT1 than adhesive tumor cells. Additionally, there was also no difference in DNA methylation pattern between adhesive tumor cells and GSCs.In summary, Anaplastic human oligoastrocytomas contained GSCs which exhibit neural stem cell properties with tumorigenicity. These stem cell-like precursors might be responsible for the oligodendroglial and astrocytic components of human oligoastrocytoma. There was no difference in DNA methylation pattern between adhesive tumor cells and GSCs, but GSCs expressed a higher protein level of DNMT1 than adhesive tumor cells. The results suggested that inhibiting expression or activity of DNMT1 might promote the differentiation of GSCs and DNMT1 might be as the target in differentiation therapy of GSCs.