| Objective: In vivo imaging of the cells transplanted into the beating heart is veryimportant for the study of the cell's retention, migration. This study was designed to find anew cell tracing agent to visualize the transplanted cells in vivo.Method:In vitro study:Bone marrow MSCs were incubated with SPIO for 48 hours. The labeling efficiencywas tested through Prussian blue staining, the growth ability was evaluated through MTT,and cells viability was tested through Trypan blue rejection method, the migratory abilitywas assessed with Costar Transwell plates, the differentiation of labeled cell toadiopogenic and osteogenic cells was induces with induction medium.In vivo study:Intramyocardial injection: After 10 days of coronary ligation of the Chinese miniswine, the cells labeled or unlabeled were transplanted into the myocardium. The MRI wascarried out immediately and 1-4 weeks serially. After MRI the hearts were excised, thesegment in which injection were performed were thin cut and stained withhematoxylin-eosin and Prussian Blue Staining.Intrcoronary artery injection: After the coronary artery was ligated 90 minutes andthen reperfused, the labeled or unlabeled BMSCs were injected into infarction-relatedartery through a catherter-based method. After the BMSCs were injected, tha animals werecarried out MRI image at day 1, and 3 weeks later. The hearts were harvested at the sametime, and other non-targeted organs were also harvested to evaluate the redistribution ofthe transplanted cells.Results:In vitro study:There were intracytoplasmatic blue particles in nearly every cells in Prussian bluestaining. SPIO had no poison effects on the cells growth, proliferation and differentiation.The cells' viability was more than 98%. The migratory ability was not affected also. The labeled cells could differentiate into osteoblasts and adipose cells.In vivo study:Intramyocardial injection: The injected sites containing labeled cells could all bedetected through MRI and were confirmed on pathology. After 4 weeks the injectedlabeled cells could still be detected through MRI. The pathology showed the injected cellscould survive in the MI area, and parallel in the same direction, and the blue stainingparticles all exist in the cytoplasm.Intrcoronary artery injection: The labeled cells injected through coronary arterycould be imaged using routine MRI scan, the hypointense lesions were scattered in the leftventricular anterior area, ventricular septum, and right ventricular anterior area. Thepathology examination confirmed the existence of the transplanted cell in the area. Andpathology also confirmed the redistribution of transplanted cells to non-targeted organs,such as lung, spleen, kidney, but seldom in liver.Conclusion: The cells could be efficiently and safely labeled with the clinicallyapplicable SPIO. The cells loaded with SPIO could be reliably and producibly detected bycardiac MRI, either through intramyocardium injection or coronary artery injection. TheMRI image could reliably reflect the rention of the transplanted cells. Retention, Distribution, Migration of the Bone Marrow Stromal Cells afterTranscoronary Infusion, the Expression of SDF-1 after Myocardial Infarction andthe Study of Migratory Mechanism of transplanted MSCsBackgroud: Transcorocnary infusion of the therapeutic stem cells is the mostpopular delivery approach of cell transplantation to improve cardiac function for theinjured heart, but few data is reported about the retention, distribution, andmigration of the implanted cells. This study is designed to investigate the retainagerate, distribution, emigration of the bone marrow stromal cells (MSCs) aftertranscoronary infusion and further evaluate the time course of SDF-1 expressionafter myocardial infarction, and the primary study of the migratory mechanism oftransplanted MSCs.MethodsBMSCs were isolated, purified, expanded, and labeled with CM-Dil. In theLangendorff model, the infracted heart were harvested, irrigated, after stabilizationfor a10 minutes, and the BMSCs were injected into coronary artery, and thelabeled cells in the coronary effluent were counted with flowcytometry. And thepercentage of rentention were evaluated according to the number of injected andeffluent cells. At the same time the heart function were recorded through a latexballoon passed into the left ventricle through the mitral valve and connected to pressuretransducer.In vivo study, the cells were then infused into the briefly distally clampedascending aorta through a catherter insreted into the left ventricle. The hearts wereharvested at different time points after cell delivery to obtain the direct evidence ofdistribution and emigration of the transplanted cells.SDF-1 expression in the area of myocardial infarction of different time wereevaluated through RT-PCR, and the migratory of BMSCs toward supernantant ofMI were evaluated through Costar Transwell system, and the BMSCs pretreatedwith G-protein inhibitor chemoxtasis were also evaluated. Results:In vitro study, We found that only about 3-5% of transplanted cells returnedinto the right ventricle, more than 90% cells retained in the heart. The heartfunction was not reduced after the cell infusion.In vivo study, we found that the labeled cells were entrapped within thecoronary capillary immediately after cell infusion, mainly in the normal area, after24 hours some cells will migrate through the capillary wall into interstitium, after 1week we could found that most survival cells located at the infarcted area and theborder zone.The time course of SDF-1 expression in the MI area began to increase afterMI, and reach peak level at 48 hours, then remain a relatively high level during thesubsequently 2 weeks, and to normal level after 3 weeks. The migratory ability ofMSCs to rhSDF-1 or supernatant of infracted area was dose dependent or timerelevant, and after the cells pretreated with the G-protein inhibitor, the migratory tothe supernatant was greatly reduced.Conclusion: The majority of BMSCs delivered by transcoronary infusionretained in the heart in the early stage; MSCs could cross the vessel wall andhoming to the interstitium in a few hours, and homing to the injured area. TheSDF-1 was highly expressed in the infracted area for a period. The homingmechanism of MSCs may be G-protein mediated.
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