Study On CD90 Determined Two Subpopulations Of Glioma-associated Mesenchymal Stem Cells With Different Roles In Tumour Progression

Objective:Human glioma-associated mesenchymal stem cells（gbMSCs）are the stromal cell components that contribute to the tumourigenesis of malignant gliomas.Recent studies have shown that gbMSCs consist of two distinct subpopulations（CD90⁺and CD90^-gbMSCs）.However,the different roles in glioma progression have not been expounded.In this study,we found that the different roles of gbMSCs in glioma progression were associated with CD90expression.Methods:In vitro,surface markers of gbMSCs were detected by flow cytometry;the differentiation potential was evaluated by Oil Red O staining,Alizarin Red staining and Alcian blue staining;the proliferation was analyzed by the cck8 test,the migration was by wound-healing and transwell assay;angiogenetic capacity was analyzed by tube formation assay;and levels of cytokines in different supernatant were determined by ELISA.Additionally,RNA was extracted from by CD90^highigh and CD90^lowgbMSCs,and their LncRNA and mRNA were analyzed using the RAffymetrix Clariom D microarray.In vivo,the animals were stereotactically inoculated with a CD90^highigh CM or CD90^lowCM suspension containing glioma cells into the right frontal lobe using a Hamilton syringe.The mice were sacrificed 28 days after injection.The tumour volumes of the nude mice in each group were calculated as V=1/2LW²（L=tumour length,W=tumour width）.Both Ki-67 and CD31 expression were detected by immunohistochemistry.Additionally,we took advantage of the full list of available datasets presented on the GlioVis homepage to analyse the overall survival of patients with gliomas in groups with differing CD90 expression levels from TCGA GBM dataset.Results:gbMSCs expressed MSC markers,including CD73,CD105,CD44,and CD90,but not CD31,CD34,CD14,NG2 and PDGFβ-R;moreover,slightα-SMA and desmin expression was observed,and differentiate into adipocytes,osteoblasts and chondrocytes.The proliferation、migration and tube formation capacity of the CD90^lowow gbMSCs incubated in different conditioned media was significantly stronger than that of CD90^highigh gbMSCs.U87 cells cultured in CD90^highigh CM exhibited a significantly greater proliferation and migration ability than cells cultured in CD90^lowow CM.Angiogenic capacity of HUVECs cultured in CD90^lowow CM was significantly greater than that of the cells cultured in CD90^highigh CM.The tumour size was significantly larger with CD90^highigh CM than with CD90^lowow CM.CD31 expression in the CD90^lowow CM tumour specimens was significantly higher than that of their CD90^highigh CM counterparts,whereas Ki-67 expression was significantly higher in the CD90^highigh CM tumour specimens than CD90^lowow CM counterparts.However,the survival time of the mice implanted with U87 cells co-cultured with CD90^highigh CM was not significantly shorter than that of mice implanted with U87 cells cultured in CD90^lowow CM.Moreover,the overall survival of patients with gliomas from TCGA database was not significantly different when the patients were group based on their CD90 expression levels.The VEGF,FGF-2,and IL-6 levels were higher in the CD90^lowow CM than in the CD90^highigh CM.Conversely,the SDF-1α,CCL5,and MMP9 levels were higher in the CD90^highigh CM than in the CD90^lowow CM.The differentially expressed genes between the CD90^highigh and CD90^lowow gbMSCs,and a total of 4977 genes（2055 up regulated and 2922 down regulated in the CD90 high gb MSCs）were identified.Conclusion: CD90 high gbMSCs significantly drove glioma progression mainly by increasing proliferation,migration,whereas CD90 low gb MSCs contributed to glioma progression chiefly through the transition to pericytes and stimulation of vascular formation via vascular endothelial cells.Furthermore,discrepancies in long non-coding RNAs and m RNAs expression were verified in these two gb MSC subpopulations.