Research Of Telomerase-Immortalized Rat Bone Marrow Mesenchymal Stem Cells In Vitro And Vivo

As a hot new topic , the use of stem cell in therapy of central nervous system impairment has been paid close attention generally,which might be bring a breakthrough in nervous renovation treatment after traumatic brain injury , Howevre , the safety and effectivity of stem cell intervention is an bottleneck which has been limiting itself s development. It is a problem need to be solved urgently how to avoide the adverse effects such as aging and malignant transformation., when we use stem cell to repair nervous impairment,Because of the chemotaxic characteristics of stem cells, there are some reports on that NSCs(Neural stem cells) and BMSCs(bone marrow-derived mesenchymal stem cells) have been used for the therapy of TBI in recent years. However, BMSCs are more readily accessible than NSCs for their unlimited utilization and from logistic and ethical considerations. Some of researches show Stem cells are self-renewable populations that can differentiate into single or multiple cell types. Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into neural cells in vivo and in vitro and secrete some types of neurotrophins. BMSCs can be used as seed cells for central nervous system transplantation. However ,it has been reported that BMSCs lose proliferating and differentiating capacities in long term culture. This will greatly restrict transplantation treatment because of decrease in qualities and quantities of BMSCs. Overcoming this limitation, this study established the immortalized rat bone marrow-derived mesenchymal stem cell line by transfection of expression plasmids of human telomerase reverse transcriptase (hTERT) mediated with cationic liposome. However, enhanced telomerase expression is often a feature of many tumors. To investigate morphology, tumorigenicity, neural differentiation potential and other biological features of immortalized rat BMSCs in vitro and vivo, we upregulated telomerase activity of rBMSCs by using mifepristone regulatory recombined expression plasmid (pGene/V5-His-hTERT) and regulatory expression plasmid (pSwitch).Taking the above considerations as a starting point, this study was divided into 3 parts. In the first part, Rat BMSCs was obtained by complete marrow cells culture and identified by their surface antigens CD29(99.83%), CD44(99.77%), CD90(99.86%), CD31(0.83%), CD34(1.78%), CD45(2.90%). Then we studyed the neural, osteogenic and adipocytes differentiation potential of rat bone marrow mesenchymal stem cells(rBMSCs) in vitro. The results show that rBMSCs could be isolated , pured and expanded easily, which can be induced to differentiate into adipocytes, osteoblasts and neural-like cells, and express neural stem cells protein spontaneously in vitro. These results indicate that rBMSCs have mutipotency and may be primed toward a neural fate.In the second part, To establish immortalized bone marrow-derived mesenchymal stem cell line and investigate tumorigenicity of immortalized rat BMSCs in vitro. pLXSN-S-hTERT were transfected, and mifepristone regulatory recombined expression plasmid (pGene/V5-His-hTERT) and regulatory expression plasmid (pSwitch) were cotransfected to passage 5 rBMSCs. RT-PCR, Western Blot were used to evaluate the effect of transfection and the activity of telomerase. Flow cytometry and MTT assary were performed to identify the immune phenotype and proliferation condition of cells in each experimental group. The tumorigenetic propensity of cells in experimental group were assessed in vitro by serum dependence experiment, soft agar colony assary, cell cycle analysis by flow cytometry and Western Blot of proto-oncogene c-Myc. Results show The activity of telomerase in pLXSN-S-hTERT-rBMSCs group and pGeneSwtch-V5-His-hTERT-rBMSCs mifepristone successive induction group were increased significantly . The immortalized rat bone marrow-derived mesenchymal stem cell line were establish in group of transfected rBMSCs. Flow cytometry analysis demonstrated that CD29, CD44 and CD90 were positive and CD31, CD34 and CD45 negative; MTT analysis illustrated that the cells maintained good growth ability and vitality in experimental group; Serum dependence experiment and soft agar colony assary both indicated that the cells did not have the same ability of resistance to nutrient starvation and clones formed in resistance medium as tumor cells ; Western Blot proved that c-Myc had no significant expression in each group; The results from in vitro study displayed the BMSCs Telomerase-Immortalized rBMSCs have no tumorigenicity in vitro. Then, pLXSN-S-hTERT transfecting BMSCs may provide a safe method to establish BMSCs immortalized cell line.In the third part, The present study was designed to determine the tumorigenicity and he neural differentiation potential of Telomerase-immortalized Rat bone marrow Mesenchymal Stem Cells in vivo. Firstly ,we complete successfully moderate fluid percussion brain injury model of Sprague-Dawley (SD) rat BMSCs. Immortalized pLXSN-hTERT-rBMSCs and pGeneSwtch-V5-His-hTERT-rBMSCs mifepristone successive induction group were transplanted stereotaxically to the parietal cortex of normal and brain injury SD rat, and to left subcutaneous tissue of groin of nude rats at the same time respectively. Hematoxylin-Eosin (HE) staining were executed to estimate the tumorigenicity of transplanted cells in SD rats. Simultaneously, the skin of cell transplanted region was observed in nude rats. Labled by Hoechst33258, rBMSCs and Immortalized pLXSN-hTERT-rBMSCs were transplanted stereotaxically to the parietal cortex of normal and brain injury SD rat. The expression of The markers of neural cells were detected by immnofluorecsence, and transplanted viable cells were count and analyzed . The Results showed Tumor cells had not been observed in the transplanted region of SD rats by H.E.None of tumor had been observed in left subcutaneous tissue of groin of nude rats. Hoechst 33258 partially in the transplanted region of parietal cortex of rats were Nestin, MAP2, GFAP and NSE positive, and the number of Hoechst 33258 positive cells in the region where immortalized pLXSN-hTERT-rBMSCs were transplanted were significantly more than that in the region where rBMSCs were transplanted in normal and brain injury SD rat(F =57.888 , P < 0.05) .these results suggest Telomerase-Immortalized rBMSCs which are transplanted into the brain of normal and brain injury SD rat grow well and express neurocyte identification protein. Furthermore, the number of survival cells in transplanted region is significantly more than that in rBMSCs transplanted region. Their differentiation was circumstance influenced and exogenous hTERT expression can improve survival of rat BMSCs after transplantation. Telomerase-Immortalized rBMSCs have no tumorigenicity in vivo.The new findings based on our preliminary study indicateTelomerase-Immortalized rBMSCs have no tumorigenicity in vitro and vivo. Elevation of activity of telomerase accelerates proliferation of BMSCs. Then, pLXSN-S-hTERT transfecting BMSCs may provide a safe method to establish BMSCs immortalized cell line. Consequently, Telomerase-Immortalized BMSCs might be one of promising and safety means of traumatic brain injury stem cell therapy.