Study Of Establishment Of A2B5+/CD133-Glioma Stem Cell Line

This study introduces the method of establishing A2B5+/CD133-glioma stem cell line which provides a foundation for the basic research and targeted therapy of glioma stem like cells.The specimens were obtained from a male patient with recurrent glioma,diagnosed as glioblastoma according to the WHO guidelines for CNS,were cultured 10 generations(SHG-140),then A2B5+/CD133-glioma cells were separated and cloned to a cell line(SHG-ZW).After that SHG-ZW were transferred to stem cell line(SHG-ZWs).Protein expression were detected by immunofluorescence,cell cycle analysis by flow cytometry,ability of differentiation and self-renewability by differentiation and cell sphere forming experiment,tumorigenicity by intracranial xenograft model in nude mouse.Pathological characters were assessed,all exons of gene were detected to make a comparison of gene expression in SHG-ZW and SHG-ZWs cell lines.The SHG-ZW and SHG-ZWs cell lines were established successfully.Immunohistochemical detection showed that SHG-ZWs cell line express A2B5,nestin and sox2 while CD133 was negative.Secondary sphere forming and differentiation shows the SHG-ZWs cell line was self-renewalable and could differentiate intooligodendrocytes?astrocytes and neuron cells.The tumorigenicity and metastatic potential of SHG-ZW cells and SHG-ZWs cells were assessed by intracranial transplantation taken in 30 female Balb/SCID mice,and the whole brain tissue was taken to make pathological sections for immunohistochemistry.SHG-W1 s xenograft tumors were particularly much more aggressive than those of SHG-ZW.In summary,the cell line SHG-ZW is a fast growing malignant cell line which express A2B5 and SHG-ZWs is a GSC subgroup which express A2B5 while CD133 is absent.We hope that we could provide a stable and effective way to establish A2B5+/CD133-glioma cell lines from fresh tumor tissues,which may contributes to the research of glioma.Materials and Methods:1.Clinical tissue samples2.Immunohistochemistry was used to detect the expression of glioma related proteins in the clinical tissue samples.3.Primary culture,separation and purification – MAC microbeads were used to establish A2B5+/CD133-stem cell line SHG-ZWs and immunofluorescence were used to detect A2B5 expression.4,MTT experiment,flow cytometry,cell proliferation and cell cycle.5.The secondary cell spheres forming experiments,immunofluorescence experiments and multidirectional differentiation test were used to detect the SHG-ZWs cell stem.6.The xenograft tumors model of nude mice was used to detect the tumorigenicity of SHG-ZW and SHG-ZW s cells.Result:1.The clinical imaging and pathological data of the patients with gliomaThe CT of the patient with brain tumor is diagnosed as recurrent gliom.Histological examination showed obvious nuclear atypia.The diameter of tumor cells was large,nuclear division and nuclear irregula.The highly proliferating tumor cells are small and round with mononuclear or multinucleated giant cells.Immunohistochemistry showed: GFAP(+),S-100(+),vimentin(+),Ki-67(65%),A2B5(-),EGFR(+),MGMT(+),p53(+),Nestin(+).2.Primary culture of human glioma cell SHG-140 and cloning of SHG-ZW cell lineSHG-140 cell line was obtained in the 10 generation after the primary cell culture from the tumor specimens,after passage 20 generations we separated A2B5+ cell by immunomagnetic micro beads,through limited dilution and single cell clone of this subpopulation of cells we get the A2B5+ cell line named SHG-ZW.SHG-ZW cells were adhered to the wall,the nucleus was small and inhomogeneous.Immunofluorescence staining showed some expression of molecules associated of SHG-140 cell line and SHG-ZW cell line,immunofluorescence staining showed that: GFAP(+)vimentin(+)S-100(+)VEGF(+ Ki)-67(52%);and the cell immunofluorescence cell line SHG-ZW results showed that SHG-ZW cell line is obtained from a single cell :GFAP(-)A2B5(+)vimentin(+)S-100(+)VEGF(+)Ki67(48%).3.Proliferation rate and cell cycle of human glioma cell SHG-ZWAfter the digestion of the twentieth generation of SHG-ZW cells,the 2x107 cells were counted in the 10 mm Petri dish,and the SHG-ZW cells increased rapidly within 48 hours.After 96 hours,the SHG-ZW grew slowly and reached the plateau stage after 120 hours.The cell cycle was analyzed by flow cytometry,and the proportion of cells in G1 phase,S phase and G2 phase was 75.08%,8.40%,16.11%,respectively.The twentieth generation of SHG-ZW cells which grew for second days were analyzed by flow cytometry.4,The transformation of SHG-ZW cells into SHG-ZWs cells and their cell cycle analysisThe twentieth generation SHG-ZW cells were cultured in NSCM for 2 weeks,and the stem cell like spheres were suspended.To sixth generations,a large number of SHG-ZWs cells were suspended.The proportion of G1,S and G2 cells in SHG-ZWs cells was 42.65%,12.65% and 45.47%,respectively.The two spheroid formation rate of SHG-ZWs cells was 97.6%,and the average diameter of the spheroid reached 220 mu m after the culture of 21 d.5.Molecular phenotypic characteristics of SHG-ZWs cellsThe tenth generation SHG-ZWs cells were immunofluorescent cytochemical detection.SOX2(+),nestin(+),Nanog(+),CD133 positive ratio were(4.20 + 1.29)%,A2B5 positive ratio was(84.12 + 9.96)%,indicating SHG-ZWs was A2B5 + CD133 GSCs subgroup cells.6.The multidirectional differentiation potential of SHG-ZWs cells: after inducing the differentiation of SHG-ZWs cells 1D,a large number of cells that grow on the wall as the center of stem cell are visible.Cell immunofluorescence results showed that the positive ratios of GFAP,beta-III tubulin and Gal C in adherent cells were respectively,indicating that SHG-ZWs stem cells have the ability of multidirectional differentiation.7.The establishment and molecular pathological characteristics of SHG-ZW and SHG-ZWs cell in xenograftsCell morphology and nuclear atypia and SHG-ZW SHG-ZWs cells of intracranial orthotopic transplantation tumor is similar,but the growth rate of SHG-ZW cells of intracranial orthotopic transplantation tumor,the formation of tumor volume showed expansive growth,has a relatively clear boundary of the tumor,tumor cells and SHG-ZWs cells in diversity,intracranial tumor growth is relatively slow,the volume of tumor the formation of a small invasive tumor growth,no obvious boundary,intratumoral and peritumoral vascular metastasis is more abundant,more obvious.Immunofluorescence,GFAP(+)vimentin(+)A2B5(+)EGFR(+).Conclusions:This study shows that A2B5+/CD133-glioma stem cell line could be established from tumors by microbeads separation and single cell clone,this may provide a stable and effective way to establish A2B5+/CD133-glioma cell lines from fresh tumor tissues,which may contributes to the research of glioma.