| Background and objectives: Recent years,cell therapy and gene therapy are under investigation as potential treatments for myocardial dysfunction. Most techniques in vivo monitoring the distribution, prolification and migration of transplanted cells. To objectively evaluate curative effect after cells transplantation, most techniques for the study of stem cell transplantation in animal models require histological analysis to determine the fate and migration of cells. Thus, the number and location of marrow stromal cells(MSCs) delivered using intramyocardial delivery techniques can only be estimated postmortem, which is hard to satisfy the status of clinical therapy of cellular transplantation. For this reason, a technique, which can be noninvasively and repeatedly performed in vivo, is being badly need. With the development of molecular biology, mechanism of diseases has be explained from cellular and molecular level. The imaging techniques also improved towards cellular and molecular level. Since the concept of molecular imaging was proposed by Weissleder in 1999, the monitoring of transplanted cells enter a new stage and the mankind can in vivo evaluate cellular biological procedure in molecular level. The greatest advantages of using MR to monitoring transplanted cells is its high tissue and spatial resolution, comparing with isotopic technique. Magnetic resonance imaging (MR) permitting multi-sequences and multi-angles imaging and easily showing anatomic structure of soft tissue. Once delivered to the heart, injected cells are difficult to distinguish from target tissue by MR in vivo.To specifically imaging injected cells in MR, the recent investigation generally using superparamagnetic iron oxid(SPIO) to label MSCs, which should enable serial tracking and quantification of MSC transplantation noninvasively by MR. Since iron is an paramagnetic material, which can generate significantly greater T2 effects, thus enable to obviously lower the signal intensity of T2WI and T2*WI in target area and thereby generate negativity contrast effect. With this characteristic of feridex, SPIO labeled transplanted cells able to be imaging with low signal by MR, which thereby indirectly represent the distribution and localization of transplanted cells.The purpose of this study was to determine whether magnetically labeled MSCs injected intramyocardially could be detected and tracked noninvasively by MRI in a swine model of myocardial infarction. We firstly need to in vitro evaluate labeling efficiency of rabbit MSCs with different labeling concentrations of SPIO nanoparticles as well as detection of characteristics and signal attenuation rules by MRI at 1.5T. We secondly attempt to determine the feasibility of tracing intramyocardia transplanted cells by using a conventional clinical 1.5T MR scanner. The imaging characteristics and rules of intramyocardia injected cells also need dynamic observation.We hope to find a easy and feasible technique and methods for future clinical trials of cellular therapy in MI.Methods: (1)MSCs were derived from bone marrow aspirates of healthy adult rabbits. Mononuclear cells were isolated using density gradient centrifugation , and culture was expanded based on plastic adherence. Cellular growth characteristics were observed and record under light microscopy. To identify most cells were MSCs, detection and analysis of surface antigen of CD34 and CD44 was performed by flow cytometry, as well as the observation of ultramicrostructure of MSCs by transmission electron microscope. (2)After being induced by 5-azacytidine, the capability of MSCs differentiated towards myocytes some were identified by the means of transmission electron microscope technique, immunocytochemistry technique and western blot technique. Immunocytochemicalα-actin positive expression of MSCs was observed and the cellular characteristics were identified by the means of transmission electron microscope technique. ( 3 ) MSCs were isolated from rabbit and incubated with different concentrations of SPIO particles at 37°C in 5% CO2. To measure labeling efficiency of SPIO, samples labeled at a range of SPIO concentrations separately were observed and counted under fluorescent microscop for evidence of distribution and labeling ratio of SPIO particles in cells, optimal incubation time after labeling and morphological evidence of visualization abnormality. To assess the effects of the particles on cell proliferation, MTT growth curves were obtained at a range of SPIO concentrations (18 to 210μg Fe per mL medium). At a propriety concentration, samples of SPIO-labeled and unlabeled MSCs suspension in agar were imaged by MRI with T1 weighted imaging(T1WI), T2 weighted imaging(T2WI) and fast field echo(FFE) sequence, and the signal intensity were measured and statistically analysed.(4)After creating a rabbit myocardial infarction (MI), allogeneic MSCs were given by intramyocardial injection using a fine needle. MR imaging were obtained on a 1.5T MR scanner to demonstrate the location, distribution and migration of the MSCs at immediately after injection and 3, 7, 14, 21and 28 days after injection. MRI contrast characteristics of SPIO-labeled MSCs were studied by measuring spin-lattice (T1) and spin-spin (T2 and T2*) relaxation time constants in cell suspensions and in vivo after endomyocardial injection. Left heart function were determined before and after intramyocardia injection by using ultrasonic cardiogram technique. Histology eventually was performed to demonstrated the survival and migration of injected MSCs in infarcted myocardium.Results: (1)Cells start to adherence 24 hours after postinoculation of mononuclearcell suspension, and the adherenced cells not easily to defluvium while jolting bottle. Initial adherenced cells mostly are round or short fusiform. Division growth of adherenced cells was shown 72 hours after fully changing suspension(.2)Flow cytometry analysis of the adherenced MSCs indicated good homogenicity of passage cells. More than 95% CD34-(Hematopoetic) and CD44+(Mesenchymal) demonstrated the cells we get were purified MSCs.(3)MSCs were efficiently labeled (>99% by confocal microscopy)by SPIO in culture at a range of exposure times and particle concentrations for 24 hours. Endocytosis of iron particles strongly depended on the cell quantity of MSCs in media, cellular labeling concentrztion, and incubation time. 20～50μg Fe per mL medium was an appropriate concentration liminal value. MSCs loaded with SPIO compared to unlabeled cells had similar viability and proliferation profiles at this proper range of labeling concentration. No alterations to proliferation, or increased toxicity for MSCs at this range of labeled concentration. Proliferation of MSCs was restrained when labelling concentration more than 50μg Fe per mL medium. (4)SPIO labeled MSCs represent clear contrast characteristics with lower signal intensity by using T2WI and T2WI/FFE sequence in MR. SPIO labeling caused a stronger lower signal attenuation effect in FFE and T2WI than in T1WI. At a labeling concentration of 35μg Fe per mL medium, MRI demonstrated that 105 SPIO-labeled cells/ml medium was the lowest observable cells quantity. 5×104 to 1×105 SPIO-labeled cells/ml medium were able to lower the signal without magnetic sensitivity artificials in both T2WI and FFE(.5)(6)SPIO labeled cells could be identified on T1WI , T2WI and T2WI/FFE sequence after injection into both normal and infarcted myocardium by using 1.5T MR. Hypointense lesions attributable to labeled MSCs were better visualized on T2WI/FFE images. Locolization, survival and migration of labeling cells were observable in MR.MSCs were observable and could be seen for at least 3 weeks on T2WI/FFE , 2 weeks on T2WI and 1 week on T1WI imaging after injection. The measurement of signal decay in injection points consistent with MR findings and were verified by statistics. histology. Expanded cellular morphous 2 weeks after injection may be explained as the migration of MSCs. (7)Cardiac function analysis by echocardiography before and after intramyocardia injection indicated that there were partly improved of global cardiac function 4 weeks after MSCs embedding into marginal area of MI. Systolic wall thickening of left ventric(LV) greatly improved 4 weeks after infarction compared with immediately after M(IP<0.05). Echocardiography also revealed average regional thickening of wall, ejection fraction improving and end-diastolic dimension minificating of LV. (8)SPIO labeled MSCs were demonstrated on histological sections adjacent to lesions seen on MRI and the migration of MSCs 2 weeks after injection were also verified by histology. When injected into the scar area, MSCs are more likely to differentiate into fibroblast. When transplanted into the marginal area however, MSCs are more likely to differentiate into cardiomyocytes with direct cell-cell interaction of survival cardiomyocytes. Conclusion: MSCs is easy to be isolated and purified by combined using density gradient centrifugation and plastic adherence. The differentiate towards myocyte of MSCs after induced by 5-azacytidine in vitro indicates that MSCs are multipotential stem cell and be of specific function of expressing myocyte. MSCs can be easily and efficiently labeled by SPIO without restriction of cells viability and proliferation. Visualization of SPIO-labeled MSCs in rabbit infracted myocardium by using a conventional 1.5T MR is feasible and the ability may be determinant for future clinical studies. The location of MSCs injection plays a key role in LV function improving. The border between normal and infarcted tissue may represent a suitable environment for engraftment and cardiomyogenic differentiation. It may be useful, therefore, to deliver MSCs directly to this border region.
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