In Vitro And In Vivo Studies:Dual-modality Labeled Bone Mesenchymal Stem Cells Of F344Rats

Objective:Utility of red fluorescent protein (RFP) and fluorescent dyes named Cyl (Cy5derivatives) in labeling bone marrow mesenchymal stem cells (BMSCs) of F344rats, to investigate the efficacy and safety of fluorescent labeling BMSCs, and the feasibility of imaging labeled cells injected in the subcutaneous and intramuscular of rats, and combined with ultrasmall superparamagnetic iron oxide (USPIO) to explore the possibility of in vivo stem cell tracking with both fluorescent imaging and MRI.Methods and materials:We use bone marrow mesenchymal stem cells (BMSCs) of F344rats successfully transfected with RFP (BMSCs/RFP), meanwhile, transfected BMSCs were incubated with culture medium containing different concentrations of CyI for24h. The fluorescence detector was used to detect the fluorescence intensity of these two fluorochromes, and optical imaging system to image the F344rats injected with labeled BMSCs in subcutaneous and intramuscular. In addition, RFP transfected BMSCs (BMSCs/RFP) were incubated with culture medium containing40μg/ml USPIO and1.5μg/ml poly-1-lysine (PLL) for24h. The distribution of iron particles in cells was studied by Prussian blue staining. In vivo, F344rat model of acute myocardial infarction was established by ligating the left anterior descending coronary artery (LAD). Those labeled BMSCs (BMSCs/RFP, CyI or USPIO labeld BMSCs/RFP) were transplanted through myocardial direct injection, then optical imaging and MR was performed to trace the cells. Postmortal study was carried out to observe the distribution of USPIO particles and RFP in heart with Prussian blue stain and fluorescent imaging.Results:In Vitro, fluorescence intensity of RFP or CyI (concentration in culture medium is5.0×10-5mol/L) labeled BMSCs, which in a cell concentration of5.0×105/ml, are strongly detectable. Morphology and death number of BMSCs hadn’t been changed significantly after incubated with different concentration of CyI (5.0×10-6\1.0×10-5\5.0×10-5mol/L). Optical imaging of F344rats injected with a total number of1.0×106fluorescent labeled cells in their body surface are successful, and the images obtained by the CyI labeled cells are better than the RFP labeled ones. Under mechanical ventilation, the F344rat model of acute myocardial infarction was successfully established by ligating left anterior descending coronary artery (LAD). In vivo cell tracking with optical imaging are unsuccessful while in vitro optical imaging of isolated heart from F344rats are successful. The signal intensity of MRI in myocardium decreased significantly when labeled cells were injected directly into myocardium in two weeks. Prussian blue staining and fluorescent imaging displayed the USPIO and RFP distribution as same as the BMSCs in the infracted myocardium.Conclusions:1. RFP or CyI (concentration in culture medium is5.0×10-5mol/L) could safely and effectively label BMSCs;2. Optical imaging of F344rats injected with RFP or CyI labeled BMSCs in the body surface is possible;3. Optical imaging could detect the fluorescent intensity of isolated heart injected with BMSCs/RFP;4. MRI could sensitively detect USPIO labeled cells in vivo in two weeks.