Cellular And Molecular Biological Characteristics Of Glioma Stem Cells/Progenitors

Part I Long-term Culture and Characteristics of Glioma Stem Cells/ProgenitorsObjective: Most current research on human brain tumors is focused on the short term cultured human Glioma Stem Cells/ Progenitors (hGSCPs). The biological characteristics of these hGSCPs in most reports were different. Therefore, Establishment of a permanent GSCPs line will provide a powerful tool to investigate the biological characteristics of GSCPs.Methods: GSCPs derived from the primary and recrudescent brain tumor tissues of the same patient respectively. CD133-positive cells were isolated by the Miltenyi Biotec CD133 cell isolation kit .The cells were culured in medium of neural stem cell, passaged , frozen in liquid nitrogen, resuscitate and re-cultued. The capability of indefinite proliferation and multi-directional differentiation were identified. The cell amplification, cell differentiation, molecular genetics and cytogenetics were investigated.Results: CD133-positive cells derived from the primary and recrudescent human brain tumor tissue were maintained for 58 months and 64 months. In serm-free medium, the cells can form tumor spheres and possess the feature of long-term proliferation and seldom differentiation; in medium contained serm they attached to the culture dishes and can differentiate polymorphic tumour cell. In tumor spheres, minority are CD133-positive and majority are Nestin-positive. In adherent differentiated cells, majority are Nestin-positive and some coexpress GFAP orβ-TubulinIII and Nestin. The chromatosomes of cell speres and adherent cells both are heteroploid. Nude mice intracranial xenograft tumors of tumor spheres derived from the primary brain tumor tissues showed invasive growth in injected location and derived from recrudescent brain tumor tissues were disseminated foci and generally infiltrated meningina.Conclusion: Establishment of two GSCPs lines that are able to maintain the properties of indefinite proliferation, multi-directional differentiation and disseminated will lay the foundation to deeply investigate the mechanisms of glioma tumorigenesis ,growth, invasion , disseminateion .Part II Characteristics of Primary and Recurrent Human Glioma Stem Cells/Progenitors Transplantation Tumors in Nude Mice IntracalvariumObjective: Human Glioma Stem Cells/ Progenitors (hGSCPs) can differentiate into all subtypes of parent tumor cells and have high tumorigenicity in nude mice, but the relationship transplantation tumor invasiveness and parent tumor and the relationship of GSCPs and vascular endothelial cells in tumor tissues is still unknown. To elucidate the high invasiveness of hGSCPs and the role of hGSCPs in brain tumor tissue remodeling, we investigated characteristics of primary and recurrent hGSCPs transplantation tumors in nude mice intracalvarium and the origin of vascular endothelial cells in GSCPs intracranial xenografts.Methods: Primary and recurrent hGSCPS spheres were injected into the right caudate nucleus of nude mice. The mice were killed at the time of cachexia. Xenograft tissues were obtained, performed routine paraffin embedding, sliced and conducted HE staining, human leucocyte antigen(HLA)staining and matrix metalloproteinase-1(MMP1) staining. The tumorigenesis ratio, invision and the origin of vascular endothelial cells were observed under light microscope. Results: All forty injected nude mice formed tumor in 30 days. Xenografts show invasive growth in injected location and spread other area. The invision of xenografts of primary and recurrent GSCPs is different. Primary GSCPs xenografts were concentrated in injected hemicerebrum. Recurrent GSCPs xenografts displayed dissemination growth, even to reach hemicerebrum and cerebral pia mater of opposite side. Minority of primary GSCPs xenograft cells are MMP1-positive and majority of recurrent GSCPs xenograft cells are MMP1-positive.In HLA staining, tumor cell-derived vascellums were observed.Conclusion: HGSCPs have strong oncogenicity and invasiveness. The more invasiveness of recurrent hGSCPs could relate to high expression of MMP1. In the intracranial xenografts of GSCPs, tumor cells widely distributed and constructed vascellums. The GSCPs could play an important role in glioma tissue remodeling by differentiating into tumor vascular endothelial cells.Part III Isolation and Culture of Glioma Stem Cells/ Progenitors and Neural Stem Cells/Progenitors in Genetically Engineered Mouse Brain TumorObjective: Glioma Stem Cells/Progenitors(GSCPs) could derive from Neural Stem Cells/ Progenitors (NSCPs). To deeply investigate the relationship of GSCPs and NSCPs, we isolated and cultured GSCPs and NSCPs from genetically engineered mouse and NSCPs from embryo mouse.Methods: Cells from brain tumor tissue and normal brain tissue under ependyma of lateral ventricle away from tumor of genetically engineered mouse and brain tissue of embryo mouse were cultured in serum-free medium and serum medium, respectively. The cells were obeserved under phase-contrast microscope. The ultrastructures of GSCPs spheres were obeserved under transmission electron microscope (TEM). Hoechst 33342 was used to sort for the side population (SP) phenotype. Cell cycle and chromosome ploidy were performed on Flow cytometry. Multiple immunofluorescence was used to assays the cell markers of GSCPs spheres and differentiated cells.Results: All cells from genetically engineered mouse brain tumor tissue and nomal brain tissue and embryo mouse brain tissue formed stem-like spheres when cultured in serum-free medium and express Nestin, and differentiated into neuronal and glial cells when cultured in medium containing 10% FCS. The spheres were passaged in vitro.The ultrastructures of GSCPs sphere indicated stem cells characteristics of undevelopped organelles, high nuclear-cytoplasmic ratio. The average first sort percentage of SP cells was 17.27% in GSCPs spheres and 2.48% in NSCPs spheres. 26.77% cells in GSCPs spheres cells and 0% cells in NSCPs spheres were heteroploid.Conclusion: Isolatin and culture of cells from brain tumor tissue and normal brain tissue under ependyma of lateral ventricle away from tumor of genetically engineered mouse and brain tissue of embryo mouse will lay the foundation to deeply investigate molecular biology relationship of GSCPs and NSCPs.Part IV Molecular information assays of Glioma Stem Cells/ ProgenitorsObjective: The difference in molecular signal pathway of Glioma Stem Cells/ Progenitors (GSCPs) and Neural Stem Cells/ Progenitors (NSCPs )could be the reason the transformation from NSCPs into GSCPs. The differential genes of gene expression profile will lay the foundation to deeply investigate molecular etiopathogenisis of gliomas formation and development.Methods: Spheres of GSCPs and NSCPs from genetically engineered mouse brain tumor tissue and nomal brain tissue and embryo mouse brain tissue were collected. RNA was extracted and purified. The Affymetrix Mouse Expression Array 430 2.0 gene chips were used to detected the significant gene expression changes. After selected the genes of up-regulation and down regulation greater than or equal to 2 folds, cell-cycle, Wnt, Notch and TGF-βpathway analysis were perform.Results: Up-regulation of genes greater than or equal to 2 folds were 3423 genes,1014 genes and 2441 genes, down regulation genes greater than or equal to 2 folds were 2524 genes,2546 gene and 4266 genes, respectively. Cell-cycle, Wnt, Notch and TGF-βpathway analysis display the changed genes in cell-cycle pathway were more than others and the changed genes and in Notch pathway were less than others.Conclusion: Bioinformatics is powerful tool to analyze gene expression profile. Cell-cycle, Wnt, Notch and TGF-βpathway analysis of three gene chips will provide powful tool to deep investigation.