BACKGROUND: Traditional cell-tracking methods fail to meet the needs of preclinical or clinical research. Thus, the aim of the present study was to establish a new method of double labeling bone marrow mesenchymal stem cells (BMSCs) from type 1 diabetic (T1D) minipigs with super-paramagnetic iron oxide (SPIO) and enhanced green fluorescent protein (eGFP) and tracing them using MRI in vitro. METHODS: Isolated BMSCs from T1D minipigs were labeled with eGFP and different concentrations of SPIO. The effects of lentivirus (LV)-eGFP transfection and SPIO on the viability and growth curves of BMSCs were determined by Trypan blue exclusion, the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry. Cellular ultrastructure was evaluated by transmission electron microscopy. Magnetic resonance imaging was used to evaluate BMSCs labeled with SPIO-eGFP complexes 6 weeks after labeling. RESULTS: Expression of eGFP in BMSCs peaked 96 h after transfection with LV-eGFP. Prussian blue staining revealed scattered blue granules in the cytoplasm of SPIO-labeled cells. Transmission electron microscopy revealed that the dense granules aggregated mainly in secondary lysosomes. On MRI, T2* -weighted imaging was far more sensitive for SPIO-labeled BMSCs than other image sequences 3 and 6 weeks after the cells had been labeled with SPIO-eGFP. CONCLUSIONS: We have developed a relatively simple and safe method for double labeling of BMSCs from T1D minipigs using SPIO and LV-eGFP and tracing them in vitro by MRI for 6 weeks.
BACKGROUND: Traditional cell-tracking methods fail to meet the needs of preclinical or clinical research. Thus, the aim of the present study was to establish a new method of double labeling bone marrow mesenchymal stem cells (BMSCs) from type 1 diabetic (T1D) minipigs with super-paramagnetic iron oxide (SPIO) and enhanced green fluorescent protein (eGFP) and tracing them using MRI in vitro. METHODS: Isolated BMSCs from T1D minipigs were labeled with eGFP and different concentrations of SPIO. The effects of lentivirus (LV)-eGFP transfection and SPIO on the viability and growth curves of BMSCs were determined by Trypan blue exclusion, the 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry. Cellular ultrastructure was evaluated by transmission electron microscopy. Magnetic resonance imaging was used to evaluate BMSCs labeled with SPIO-eGFP complexes 6 weeks after labeling. RESULTS: Expression of eGFP in BMSCs peaked 96 h after transfection with LV-eGFP. Prussian blue staining revealed scattered blue granules in the cytoplasm of SPIO-labeled cells. Transmission electron microscopy revealed that the dense granules aggregated mainly in secondary lysosomes. On MRI, T2* -weighted imaging was far more sensitive for SPIO-labeled BMSCs than other image sequences 3 and 6 weeks after the cells had been labeled with SPIO-eGFP. CONCLUSIONS: We have developed a relatively simple and safe method for double labeling of BMSCs from T1D minipigs using SPIO and LV-eGFP and tracing them in vitro by MRI for 6 weeks.
Keywords:
bone marrow mesenchymal stem cells; enhanced green fluorescent protein; magnetic resonance imaging; minipig; type 1 diabetes mellitus.; 骨髓间充质干细胞，增强型绿色荧光蛋白，磁共振成像，小型猪，1型糖尿病