Abstract: BACKGROUND: Bioluminescence imaging is an emerging in vivo imaging technology, but there are few studies on the tracing of stem cells after transplantation into living animals in existing reports.
OBJECTIVE: To explore the multiplicity of infection of rat derived bone marrow mesenchymal stem cells transfected with Luciferase lentivirus, and further study the effectiveness and stability of Luciferase + bone marrow mesenchymal stem cells in vitro and the tracing effect in rat calvarial defects. 
METHODS: The logarithmic growth phase of rat bone marrow mesenchymal stem cells was transfected with different multiplicities of infection, and the fluorescence value was quantitatively analyzed in the in vivo imaging system to determine the optimal multiplicity of infection. Luciferase + bone marrow mesenchymal stem cells were transfected under optimal conditions, and fluorescence values corresponding to different numbers of cells and different passages were analyzed to assess the effectiveness and stability of Luciferase + bone marrow mesenchymal stem cells. Finally, Luciferase + bone marrow mesenchymal stem cells were traced in the rat calvarial defect model.   
RESULTS AND CONCLUSION: (1) When the multiplicity of infection increased in the interval of 10-50, the fluorescence quantification increased with the same proportion of multiplicity of infection. The fluorescence quantification did not increase significantly when the multiplicity of infection increased to 100, demonstrating that the optimal multiplicity of infection of Luciferase lentivirus transfected bone marrow mesenchymal stem cells was 50. (2) Luciferase + bone marrow mesenchymal stem cells had a good cell-bioluminescence dose-response relationship and proliferation stability in vitro, demonstrating that fluorescence quantification can indicate the relative number of cells. (3) The transplanted Luciferase + bone marrow mesenchymal stem cells were locally distributed in the skull defect, and the biofluorescence intensity decreased over time and finally disappeared after 35 days. (4) Bioluminescence imaging is an efficient and sensitive technique for tracing stem cells in vivo, which is helpful to characterize the distribution and quantity changes of transplanted stem cells, and provides strong evidence for explaining stem cell function and mechanism research.

Key words: stem cells, bone marrow mesenchymal stem cells, Luciferase lentivirus, bioluminescence imaging, in vivo imaging system, in vivo tracing, rat, calvarial defect, tissue engineering