| Part One Effects of HIV-Tat-CLIO on the physiological manner of rat marrow-derivedmesenchymal stem cellsObjectiveTo evaluate the feasibility of HIV transduction domain cross-linked iron oxide (HIV-Tat-CLIO) labeling technique and its labeling efficiency, toxicity and effect on cellular function of rat marrow-derived mesenchymal stem cells (rMSCs).Materials and MethodsOn the basis of the difference of cellular adherent ability, MSCs from primary cultures of rat marrow were isolated and purified by the preplate technique. To verify the nature of cultured rMSCs, the 3rd passage cells were labeled against CD34, CD45 and CD90 and analyzed by flow cytometry. We chose the 3rd passage rMSCs to follow the later study. The cells were labeled by the HIV-Tat-CLIO with 25μg /ml for 24h or by CLIO with 25μg/ml for 24h. The labeling percentage was assessed by the Prussian blue staining to compare the effects of different tag technique. After labeling of rMSCs with HIV-Tat-CLIO complexes, Prussian blue staining and electron microscopy confirmed the presence of iron oxide nanoparticles inside the cells and cellular toxicity, functional capacity were determined by trypan blue exclusion, MTT assay, oil 0 and AKP staining and FITC-and PE-conjugated monoclonal antibodies directed against CD34, CD45 and CD90．We used transwell test to assess the ability of migration.ResultsAfter 3 passages, rMSCs were negative for CD34 and CD45 and positive for CD90．The labeling percentage of HIV-Tat-CLIO was better than the CLIO group. HIV-Tat-CLIO labeled cells demonstrated no short or long-term toxicity, changes in differentiation capacity of the rMSCs, or changes in phenotype when compared with unlabeled cells. There was also no change on the manifestation of cells capability for differentiation and migration.ConclusionThe HIV-Tat-CLIO incorporation did not affect the capability for cell viability, proliferation, differentiation and migration of rMSCs which can be tracked lively by MRI. It was an efficient and effective technique for incorporating the nanoparticles within endosomes, thereby labeling cells that can be detected by MRI. Cell labeling using these methods was likely to enhance the development of cell-based strategies for the repair or replacement of tissues and other novel therapies. Part TwoMagnetic Resonance Tracking of Transplanted Rat Marrow-derived MesenchymalStem Cells Labeled by HIV-Tat-CLIO in Rat Model of Hindlimb IschemiaObjectiveTo investigate the feasibility and effect of using HIV-Tat-CLIO to label rMSCs for monitoring their temporal and spatial migration in vivo by magnetic resonance imaging (MRI) and evaluate the treatment efficiency of the implantation of HIV-Tat-CLIO labeled rMSCs in the model of rat hindlimb ischemia.Materials and MethodsMSCs from primary cultures of rat marrow were isolated and cultured to the 3rd passage. The cells were labeled against CD34, CD45 and CD90 and analyzed by flow cytometry to verify the nature of cultured rMSCs. The cells were labeled by the HIV-Tat-CLIO with 25μg /ml for 24h and another cells were double labeled by BrdU (5-bromodeoxyuridine) with 3μg /ml for 24h, respectively. Twenty eight SD rats were randomly divided into 4 groups, containing 7 ones in each. The femoral arteries of the rats were excised to establish the model of right hindlimb ischemia and the model was check as successful according to the DSA imaging. At the 7th day after the operation, rMSCs implantation was accomplished with the technique of intramuscularly injection. We established 4 group, including A, B, C and D individually injected with HIV-Tat-CLIO labeling rMSCs, double labeled rMSCs with HIV-Tat-CLIO and BrdU, unlabeled rMSCs and same volume PBS liquids. MRI was performed at 4 different time points after the ischemia model procedure (the 3rd day, 1st week, 2nd week and the 4th week, individually) and the group C and D were only checked once on the 3rd day. That is, in vivo MR imaging was used to track their fate. At the 4th week, the skin temperatures of the ischemic hindlimb were measured with infrared thermography and the impaired score of limb function were assessed. Additionally, we carried out DSA to evaluate the hindlimb angiography and used immunohistochemistry technique to record the capillary density of the ischemic muscular tissue to observe the formation of new blood vessels. We united the staining techniques of Prussian blue and BrdU and MRI to assess the labeled effected of transplanted rMSCs. ResultsThe 3rd passage rMSCs were negative for CD34 and CD45 and positive for CD90．During our experiment, there were 3 rats dead. Two rats in group B and C died of incision infection and the other in group B was due to the anethesia incident. The implanted cells were visible on MR images as a hypointense area at the injection site on the 3rd day after the rMSCs implantation. Then gradually separated into small particles and dispersed around the injection site during the follow 3 weeks. The skin temperature of the ischemic hindlimb was higher in A (32．12±0．51℃),B (32．39±0．61℃)and C (31．97±0．56℃) group than in group D (31．16±0．67℃) 4 weeks after the implantation procedure (P<0．05) . There were no group difference between group A and B, B and C or A and C (P>0．05) . Impaired score of limb function was significantly lower in group A (0．68±0．17) , B (0．65±0．24) and C (0．67±0．11) than in group D (1．79±0．22) 4 weeks after the implantation procedure (P < 0．05) . There were no obviously group difference between group A and B, B and C or A and C (P> 0．05) . The hindlimb angiography showed that the average angiographic score was 2．56±0．37 in group A, 2．51±0．29 in group B, 2．58±0．26 in group C and1．33±0．41 in group D. Group A, B and C were significantly higher than group D (P < 0．05) . There were no obviously group difference between group A and B, B and C or A and C (P>0．05) . The capillary density was 570．35±68．17/mm~2 in group A, 563．02±61．26/mm~2 in group B, 576．97±77．87/mm~2 in group C and 513．01 + 70．68/mm~2 in group D. Group A, B and C were significantly higher than group D (P < 0．05) . There were no obviously group difference between group A and B, B and C or A and C (P>0．05) .ConclusionThe technique of MRI in vivo tracking transplanted rMSCs preoperatively labeled by HIV-Tat-CLIO was feasible and effective. The HIV-Tat-CLIO labeled rMSCs implantation could stimulate the formation of the new blood vessels and promote the foundation of the blood flow and rehabilitation of limb function in the model of rat hindlimb ischemia. Using the HIV-Tat-CLIO combination for magnetic cellular labeling should facilitate translation of this approach into clinical trials.
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