Malignant Transformation Of Bone Marrow Mesenchymal Stem Cells Under Tumor Microenvironment

ObjectiveTo investment whether rat bone marrow mesenchymal stem cells have transformed into malignant cells because of cytokines, signaling molecules and cell interactions under tumor microenvironment as gene delivery vehicles against tumor, and to certify to be able to form tumors when we injected transformed cells into immunodeficient mice,and further,MSCs were then modified in order to avoid the risk of malignant transformation and provide an initial test basis to make it more secure for clinical application.Methods1. Cell culture: Took Wistar rat to dissociate femur and tibia , and then isolated and cultured and amplified MSCs in vitro by adhesion methods; C6 glioma cells lines were cultured using the same medium as MSCs.2. Establishment of co-cultural system and grouping: the experiment is divided into two groups , the control group (alone cultural MSCs); experimental group (including indirect co-culture groups and direct co-culture groups with C6, and indirect co-culture groups were by using six plate and transwell chamber, direct co-culture groups were that MSCs marked with GFP adenovirus were co-cultured with C6 cells).3. Experimental methods: (1)observed morphological changes of MSCs by phase-contrast microscope; (2) detected changes of cell proliferating capacity by FCM and the expression change of NS protein ; (3) p53 and mdm2 mRNA were examined by real-time quantitative polymerase chain reaction (PCR)method based SYBR.GREENI;(4) analysised mutant p53 protein and mdm2 protein expression changes by immunofluorescence and westernblot; (5) Detected the expression and localization of glial cell-specific glial fibrillary acidic protein(GFAP) by immunofluorescence. (6)Regular chromosome assays and G band were used to analyze the karyotypes of the two group cells; (7) transformed MSCS in vitro tumor microenvironment were transplanted into immunodeficient mice subcutaneously by means of injection, 4-8 weeks later, observed cell survival and analyzed tissue sections.Results1. MSCs had a high purity by adherence methods; the morphology of the 4th generated MSCs were uniform and flat and fibro-shape, MSCs evenly distributed and arranged orderly. After 7d co-cultured with C6 glioma, the cell morphology of experimental groups were significant changed , cytoplasm contracted toward perinuclear place and cells became longer and thinner, cell nuclei became smaller, cell morphology were similar to the glioma cell.2. compared with the control groups,the proportion of cells in G1 phase in experimental cells was decreased,while the proportion of cells in S phase was increased,the difference was no significant (p>0.05) , the level of nucleostemin(NS) protein was not changed.3. p53 mRNA decreased about 0.24 times compared with the control groups((p<0.01),some cells of experimental groups expressed mutant p53 protein,but there were no mutant p53 protein in control groups,the difference was significant (p<0.05),mdm2 mRNA increased about 3.18 times and mdm2 protein expressions of experimental groups significantly increased as compared to control groups (p<0.05)after 7 days co-cultured with C6 .4. After 7 days MSCs were co-cultured with C6,the karyotypes assays showed their model number were 42 in the control groups ,while were 38～42 in experimental groups and displayed predominantly non-clonal numerical aberrations, and the proportion of 41 chromosomes increased to 33% of the split phase, the chromosomal structural had no distinguished aberrations by G-banding in two groups.5. Almost all experimental cells expressed GFAP protein, but the ratio of the expression of GFAP was only 13%.6. Transformed MSCs were transplanted into immunodeficient mice subcutaneously , 4 and 8 weeks later, neoplasm could be seen by gross observation in the imbedded locus, took the neoplasm and stained by HE , the neoplasm were composed of the undefferentiated cells and infiltrated to peripheral musculus, and the neoplasm expressed GAFP.Conclusion1. The microenvironment of glioma can cause rat bone marrow-derived mesenchymal stem cells to obtain tumor characteristics and MSCs have been transformed into glioma-like cells.2. Malignant transformated MSCs were transplanted into immunodeficient mice subcutaneously and could form tumors.