Investigation Of Oncogenicity Of Bone Marrow Mesenchymal Stem Cell In Vivo And Their Labeling

Objective To investigate the oncogenicity of bone marrow applications on the basis of the study on culture conditions and the biological properties as well as the fluorescence labeling of MSC. To explore the possible mechanisms of tumor cells mutated from MSC and also provide experimental evidence for hypothesis of tumor stem cell.Methods Human MSC was labeled with PKH26, and the growth characteristics and multi-differentiation were analyzed by cell biology and molecular biology. Sprague -Dawley rat MSC were cultured and injected repeatedly into allogeneic rats through vena caudalis. Two tumors were found in the vena caudalis after a long period over 20 months . The tumor tissue was analyzed by HE and immunohistochemistry stain. The tumor cells were cultured using L-DMEM with 10% FBS and the morphological growth features were observed. Limit dilution clone was used to analyze morphological growth features and proliferation of tumor cell strain. The concentration gradient cultural tumor cells were respectively transplanted to BALB/c-nu/nu mice through subcutaneous and abdominal cavity to observe the outgrowth and invasion of formed tumor. Flow cytometry was applied to analyze CD29, CD44, CD45 and CD90 in tumor cells. The characteristics of tumor cells were studied by detection of the Bmi-1, Nucleostemin oncogene expression with RT-PCR and the karyotype analysis.Results The labeled MSC appeared red fluorescence and labeling rate was 100 percent.During serial subcultivation of labeled MSC from passage 1 to passage 7, the fluorescence intensity and labeling rate of MSC gradually decreased. The biological features such as morphology, growth, expression level of nucleostemin and GAPDH gene and capability of differentiation into osteoblast in vitro were not affected by labeling. Two of ten(20%) SD rats formed tumors in tail after injecting allogeneic MSC in vena caudalis at 20 months. The result showed that the tumor tissue had malignant morphology. The features of morphology, growth and the capacity of proliferation for the cultural tumor cells were quite different from normal MSC. The characteristics of each tumor cell line cloned from limit dilution were also quite different . BALB/c-nu/nu mice that had transplanted Only 10~4 tumor cells formed tumors . Both of two BALB/c-nu/nu mice which had been transplanted tumor cells through abdominal cavity got abdominal dropsy. The formed tumors were metastatic and invasion. The tumor cell surface antigens were positive for CD29(+), CD44(+), CD45(+)~W, CD90(+).The karyotype of tumor cells was aneuploid , The expression of Bmi-1, Nucleostemin in these tumor cells was detected.Conclusions The method of labeling human MSC with PKH26 had been successfully established .It was an effective, practical, and an important way for study on homing, plasticity, and transplantation of MSC. The transplanted MSC in host body might spontaneously mutate into tumor cells in certain time. These findings suggested that MSC have promising clinical applications, but it is necessary to carry out studies for their assurance of the long-term safety before MSC clinical application for regenerative medicine and tissue engineering.