In Vivo Tracking Of Bone Marrow Mesenchymal Stem Cells Labeled With Superparamagnetic Iron Oxide After Spinal Cord Injury In Rabbits By Magntetic Resonance Imaging

There is an increasingly enthusiastic interest in stem cells, cells with particularcharacteristics, as potential therapeutic reagents for various degenerative diseases and damagedorgans. Stem cells may be defined as cells capable of proliferation, self-maintenance, andproduction of differentiated, functional progeny that are characteristic of the organ which theywere derived from. Many studies have demonstrated the migrational capacity of stem cells inmammalian spinal cord during normal and pathophysiological conditions. The therapeuticpotential of stem cell grafting has recently been studied in various pathological conditions of thespinal cord. The clinical application of stem cell therapy to treat spinal cord injury is dependenton the potential of grafts to promote sustained recovery after transplantation. Stem cells areinfused or transplantated into tissue for therapeutic purposes. It was shown that the bonemesenchymal stem cells (BMSCs) might differentiate into neural cells and develop into neuralcells ultimately. Therefore, the BMSCs derived neural cells can be considered as ideal seed cellsin replacement therapy of the spinal cord injury due to the advantages of plenteous cell-sourceand no the immunorepulsion or ethical limitation during the autotransplantation application.At present, investigating the relationship between anatomical change and behavioralrecovery is hindered by the necessity to use post-mortem tissue to determine how BMSCscontribute to behavioral recovery. The migratory properties and the seamless integration ofBMSCs in vivo are considered to be beneficial to behavior recovery. However, at present, it isstill poorly understood how the migration or integration of stem cells into the damaged hostparenchyma relates to behavioral recovery.Several recent studies have demonstrated that incorporation of contrast agents into cellsdesigned for cellular therapy before transplantation can be identified in vivo by magneticresonance imaging (MRI). Modified dextran coated superparamagnetic iron oxide nanoparticles(SPIO) have been used to label cells ex vivo and monitor the migration of these cells by nanoparticle, approved by the MRI. Feridex is a class of dextran-coated SPIO Food and DrugAdministration (FDA) as a MRI contrast agent for hepatic imaging. Recently, a technique hasbeen developed to efficiently label mammalian cells by compounding, via electrostaticinteractions negatively charged FDA-approved ferumoxides (FE) SPIO nanoparticle topoly-cationic transfection agent poly-L-lysine (PLL), thereby facilitating the detection of thelabeled cells' temporal spatial migration by MRI after transplantation.Based on these, in vivo tracking of BMSCs migration in the rabbit of spinal cord injury model was studied in this paper. This topic took the BMSCs of rabbits as our research object,which were isolated and amplified in vitro. Meanwhile observe the changes of the biologicalcharacteristics of labeled BMSCs and track the labeled BMSCs grafted into the spinal cordinjury models by MRI in vivo.PartⅠIsolation, cultivation and identification of bone marrowmesenchymal stem cell of rabbit in vitroObjective:To explore a new method of isolation, purification,cultivation and identification ofMSC in vitro.Method:1. The bone marrow mesenchymal stem cells(MSCs) were isolated , cultivated andpurified by density gradient centrifugation and plastic adherence. We researched the basiccharacteristics of MSCs when they grew in vitro．And specific surface antigens of MSCs weredetected by flow cytometry to identify whether cultured cells have biological properties ofmesenchymal stem cells．2. MSCs were cultured in the general osteogenic and adipogenicinductive medium respectively, and then detected by ALP staining, mineralization of Von KossaStaining and fat of Oil Red O staining, which could prove the potential of osteogenic cells andadipogenic cells.Result:1. Original generation of MSCs were in form of short spindle ,and could be cultured andexpanded in vitro for a long period. And its shape of growth curve is S．Detection of surfaceantigen of MSCs by flow eytometry demonstrated that the phenotype of MSCs was CD29,CD44positive and CD34, CD45 negative,accorded with biological characteristics of MSCs. 2. MSCshad mineralization deposition in vitro culture under condition of osteogenesis induction, and theresult of Von Kossa staining was positive．Alkaline phosphatase test of cells,ALP activityobviously increased in the cells．The results indicated that MSCs had the potentiality ofosteogenic differentiation．The positive result of Oil Red O indicated that MSCs had thepotentiality of adipogenic differentiation.Conclusion:1 MSCs could be isolated and proliferated by the method of this study, which wascharacteristic of simple operation, high efficiency, economy and practice . 2 Cultured MSCspossessed stable growth, rapid amplification from generation to generation and a high rate ofadherence, which were appropriate for further research． ParPartⅡBiological characteristics and MR imaging ofsuperparamagnetic iron oxide labeled bone marrow mesenchymal stemcellsObjective: To explore the labeling efficiency, and to research the effect of cellular viability andproliferation capacity of rabbit bone marrow mesenchymal stem cells labeled with differentconcentration of superparamagnetic iron oxide (SPIO) particles, and to determine the feasibilityof detection of magnetically labeled stem cells with MR imaging.Methods: The bone marrow MSCs were added to the culture media contained differentconcentration SPIO particles, (the ultimate concentration of SPIO was 25μg/ml,50μg/ml,75μg/ml,100μg/ml,150μg/ml respectively). Prussian blue staining, trypan blue exclusion testand vitro magnetic resonance imaging of labeled cells were performed at 24h, 72h, 1w, 2w, 3w,4w respectively after labeled, to make sure rational labeled concentration and time.Results: MSCs were efficiently labeled with SPIO, and labeled SPIO particles were stained inall cytoplasm of labeled cells groups. With the increase of concentration of Fe2+, blue dyeparticles increased; non-labeled cells group was negative. Trypan blue exclusion test (cellularviability test) showed the viability of the labeled cells with SPIO of 0μg/ml and 25μg/ml,0μg/mland 50μg/ml, 25μg/ml and 50μg/ml was not significantly statistical different(P>0.05). MTTdetection displayed that there was no effect on cellular proliferation when SPIO concentrationwas 25ug/ml and 50ug/ml. MRI test in vitro showed: there was no difference among all groupslabeled with different SPIO concentration in T1WI. However, in T2WI all labeled groups causedsignal to reduce, and with the labeled concentration increasing, the signal reduction became moreapparent. When the concentration was 50, 75, 100 and 150ug/ml, the degree of reduction was notstatistically different.Conclusion: MSCs can be easily and efficiently labeled by SPIO without interference on thecell viability at the labeled concentration of 20 to 50μg Fe per ml and the labeled time of 24 to72h. MRI visualization of MSCs labeled SPIO is feasible, which may be critical for futureexperimental studies. ParPartⅢThe research of inducing bone marrow mesenchymalstem cells labeled by Superparamagnetic iron oxide to differentiate intoneuron-like cells in vitroObjective: To explore the feasibility of inducing the bone marrow mesenchyrnal stemcells(BMSCs) to differentiate into neuron-like cells in vitro, after BMSCs labeled withsuperparamagnetic iron oxide particles．To research whether there is any effect on the survivalrate and cellular activity of neuron-like cells differentiated from the BMSCs.Methods: With super-paramagnetic iron oxide labeling BMSCs, the labeled concentration ofSPIO was 25μg/ml. MSCs were induced byβ-Mercaptoethanol (BME) and basic fibroblastgrowth factor (bFGF),so that the change of morphology and the number of cells could beobserved during the differentiation．Immunocytochemistry was used to detect the expression ofneuron specific enolase (NSE) and microtubule associated protein-2 (MAP-2). Prussian bluestaining was used to identify whether the iron particles exist in neuron like cells again. Thesurvival rate of neuron like cells was detected by Calceein AM/PI staining. Observe the changesof intracellular calcium concentration ([Ca2+]i) in the neuron like cells which subjected to thedrug administration by Flu0-3/AM staining, to observe the cellular functional state. Take theunlabeled as a control group.Results: When induced at 6h,the cells'bodies began to retract like a round．When at 24h,bodies were round and part of extended 2-3 grade processes and overlapped like a network.Cellular shape accorded with the biological characteristics of neuron-like cells. NSE and MAP-2of Neuron-like cells were positive. By Prussian blue staining, we can observe the iron particles inneuron-like cells again. Calcein-AM/PI staining indicated that the survival rate of labeled andunlabeled neuron-like cells was 93.5% and 94.1%. There was no significantly statisticaldifference (P>0.05). By using Flu0-3/AM staining, the changes of intracellular calciumconcentration between labeled and unlabeled neuron-like cells were not significantly statisticaldifferent (P>0.05),which indicated both the labeled and the unlabeled cells kept in good state.Conclusion: At the labeled concentration of 25μg Fe per ml, the BMSCs could differentiate intoneuron-like cells in vitro. There still existed iron particles in the cytoplasm of cells. The survivaland functional condition of neuron-like cells kept in good state. ParPartⅣPreparation of spinal cord injuryObjective: Build and study the pathological, ethology and imaging changes of spinal cordinjury(SCI) models to provide important data for anaphase stem cell therapy．Methods: L2 spinal process and corresponding lamina of vertebra were removed from rabbits;Spinal cord injury (SCI) models were made by ourselves-designed experimental devices. AfterSCI models were established, the separated muscles were sutured to close spinal canal deletion,then skin sutured. Other rabbits were dealt in the same way but without SCI, served as controls.Tarlov scores and pathologic and MRI examinations were performed after SCI establishment．Results: After SCI model establishment, Tarlov scores was 2．Obvious pathological and imagingchanges were noted in SCI area of experimental group．Conclusion: There were obviously histological and imaging changes, significant difference fromSCI rabbits in ethology. The SCI models were successfully established, which could be appliedto spinal cord degeneration research．PartⅤIn vivo MR tracking of labeled bone marrow stem cellsgrafted via subarachnoid space in rabbit spinal cord injury modelsobjective: Graft bone marrow mesenchymal stem cells labeled superparamagnetic iron oxide(SPIO) into the spinal cord injury models via subarachnoid space, to observe the restore oflocomotive function and the feasibility of MRI tracking of grafted cells.Methods: BMSCs were labeled with SPIO．Rabbit spinal Cord injury models were made and themicro tubes were inserted into subarachnoid space. Rabbits were randomly divided into 3 group．Group A received SPIO labeled cells via subarachnoid spaces; Group B received unlabeled cells;Group C was injected by PBS liquid as the control group．When cells were grafted at 3, 7, 14, 21,28and 35 days, the BBB scores were performed to observe the locomotive function of all spinalcord injury groups. Grafted BMSCs were tracked by Serial 1.5T MRl in 3,7 and 14 days aftertransplantation and compared with tissue slices of injured spinal cord．Results: The BBB scores of group A and B were higher than group C, whose difference wasstatistically significant; But there was no statistical difference between group A and B. AfterBMSCs labeled with SPIO were grafted into the spinal cord injury, MRI test was performed at 7,14, 21 days respectively. At 7 days, there appeared point-like low signal shadow in the injuredspinal cord segment in T2WI; At 14 days, this low signal shadow increased; At 21 days, thisshadow decreased. Cells containing blue iron particles in the area of spinal cord injury could be seen in tissue sections with Prussian blue staining.Conclusion: Labeled BMSCs could migrate to the injured spinal cord segment after grafting viasubarachnoid space, which was beneficial to the restore of the spinal cord injury groups'locomotive function; and could be tracked with MRI in vivo.