In Vivo Tracking Of Mesenchymal Stem Cells Following Intra-coronary Injection Post-myocardial Infarction In Swine Using Magnetic Resonance Imaging

BackgroundCoronary artery occlusion leads to ischemia and cell death in the heart. Cardiomyocyte death results in scar formation and left ventricular dilatation ultimately leads to progressive heart failure.Bone mesenchymal stem cells(MSCs) transplantation provides a potential regenerative therapy for the heart damaged by myocardial infarction. The mechanism of MSCs transplantation therapy remains unclear. Numerous studies have been undertaken in animals and humans to analyze the efficacy of this new approach. The success of cell therapy will depend on the ability to monitor the fate of transplanted cells in vivo. However, rare studies evaluated of survival, migration, and differentiation status of transplantation MSCs. Many methods have been developed to track the destination of the injected stem cells. These tracking methods only confirm the presence of transplantion stem cells in postmortem recipient tissues .To better understand the mechanism of cell therapy and potential beneficial effects observed in clinical trials. There is a need to track the transplanted stem cells in vivo.Recent advances in the field of MR contrast media and cell labeling supports molecular and cellular imaging. One such approach involves the use of superparamagnetic iron oxide (SPIO) particles as a contrast agent for cell tracking. This exciting new area offers the potential for non-invasive tracking of implanted cells, ideal for monitoring in the clinical setting. The present study including: superparamagnetic iron oxide (SPIO) particles and fluorescent CM-DiI dye dual labeled MSCs;a minimal-invasive model of myocardial infarction in swine; intracoronary injection of SPIO and CM-DiI dual labeled MSCs in a porcine infarct model; in vivo magnetic resonance imaging tracking of bone marrow-derived mesenchymal stem cells via intracoronary administration; according to MR findings, sorted the fluorescently labeled transplantation cells from injury myocardium using fluorescence-activated cell sorting (FACS), a potential novel technique for tracking of transplantation cells.Part I Superparamagnetic iron oxide particles and fluorescent CM-DiI dye dual labeled MSCsObjective: To investigate the effects of labeling MSCs with SPIO and CM-DiI.Methods: Porcine MSCs were isolated and cultured by the whole bone marrow method and by Percoll density gradient centrifugation. MSCs were labeled with 50μg/mL SPIO and fluorescent CM-DiI dye. The labeling efficiency was tested through Prussian blue staining and fluorescent imaging.The intracellular iron uptake was also assessed with electron microscopy. Labeled MSCs viability and proliferation were determined using Trypan blue rejection method and MTT. Result: Nearly 100% of the MSCs were labeled with SPIO and CM-DiI . SPIO at 50μg/mL doses and treatment times of 24 h did not statistically affect the viability and proliferation of MSCs.Conclusion: Porcine MSCs could be efficiently and safely labeled with SPIO and CM-DiI.Part II A minimal-invasive model of myocardial infarction in swineObjective: To develop a closed-chest, minimal-invasive swine model of myocardial infarction.Methods: A balloon catheter was advanced into the left descending coronary artery directly beyond the second diagonal branch. The balloon was inflated and occlusion of the vessel, Angiography confirmed while ECG was continuously monitored. The balloon was deflated after 60min.Result: Myocardial infarction was successfully induced in 24 animals. After balloon occlusion, coronary angiography shows blood flow interruption in the distal left anterior descending branch.Post-mortem histological analysis revealed myocardial necrosis.Conclusion:The closed chest,minimally invasive methods represent a useful alternative for studies of myocardial infarction.Part III Intracoronary injection of SPIO and CM-DiI dual labeled MSCs in a porcine infarc modelObjective: To evaluate the effects and safety of intracoronary infusion of mesenchymal stem cells after myocardial infarction.Methods: A total of 27 swine were divided into five groups:healthy animals with dual-labeled MSCs transplantation group(n=3);MI control group(n=6);MI with dual-labeled MSCs transplantation group(n=6); MI with SPIO and PBS transplantation group(n=6); MI with CM-DiI labeled MSCs group(n=6). 1×10~8 MSCs were delivered for one model by intracoronary injection 2 week post myocardial infarction. Heart function were observed 5 weeks after transplantation.The transplantation efficiency was tested through Prussian blue staining and fluorescent imaging.Result: During intracoronary injection of dual labeled MSCs, no adverse events were noted. Prussian blue staining positive cells and fluorescent cells were located in the injury myocardium.Conclusion: This study suggests that intracoronary injection of dual labeled MSCs is probably safe and effective after myocardial infarction.Part IV In vivo magnetic resonance imaging tracking of mesenchymal stem cells in ischemic swine heartObjective: To track in vivo intracoronary injection of MSCs labeled with SPIO by using magnetic resonance imaging (MRI)in swine myocardial infarction model.Methods: Swine MSCs were labeled with SPIO, Cell labeling efficiency was assessed by Prussian blue stain. SPIO labeled cells underwent MRI in vitro. MR examinations were performed at 1,3and 5 week after intracoronary transplantation. Double echo steady state was used to scan four-chamber and cor biloculare at long axis view, which was considered as locating phase to obtain image of left ventricle at short axis view .The swine were euthanized at 5 week after MR examinations. Hearts were excised and sliced from apex to base into 6～8 transverse sections according to MR findings. MR findings were confirmed by histological examinations results. The transplantated labelled MSCs was tested through Prussian blue staining and fluorescent imaging.Result: The SPIO labelled MSCs transplantation intracoronary produced a hypointense signal using T2-weighted MRI and hypointense signal persisted for up to 5 weeks. Histologic analyses confirmed the presence of SPIO labelled MSCs high retention and mainly localized in the injured myocardium.Conclusion: This study demonstrated that labeled MSCs can be reliably detected and tracked in vivo using MR imaging; MR findings consistency to pathohistological results.Part V Sorted the fluorescently labeled transplantated cells from injury myocardium using fluorescence-activated cell sortingObjective:To assess the utility of fluorescence-activated cell sorting (FACS) for separating CM-DiI labeled MSCs from injured myocardium.Methods: Fresh single cell suspensions were generated from injured myocardium .Fresh suspensions were sorted by FACS analysis of CM-DiI labeled fluorescent cell. FACS was performed with a FACS Vantage Flow Cytometer (Beckman Coullter ALTRA). The 488-nm argon laser was used.The purification CM-DiI labeled transplantation cells sorted from injured myocardium single cell suspensions with FACS. Selective transplantation was performed using fluorescence microscope and cells cycle was detected by flow cytometry.Result: Success of MSCs transplantation intracoronary was confirmed by FACS analysis. CM-DiI labeled transplantation cells were disassociated from injured myocardium. The purification CM-DiI labeled transplantation cells sorted from injured myocardium display red-fluorescent under fluorescence microscope. The cell cycle G2+S ( % ) was 41.6%.Conclusion: FACS techniques provide a powerful approach for analyzing and purification of transplantation MSCs. The study might provide some new clues for the design of therapeutic approaches for MSCs transplantation.