Sinerem Labeling Of BMSCs-D-NSCs For MR Imaging And Applying In Ischemical Model Rats

BackgroundIncidence of cerebro-vascular disorders is very high, and cerebral stroke is oneof the three leading causes of mortality and morbidity in China.80% of cerebral strokeis ischemic cerebral infarction. The infarction can be recovered with the tissueplasminogen activator. However, of all patients who suffer from an ischemic stroke,only a small proportion of patients were treated with acute reperfusion therapy to haltprogression of ischemic brain damage because of side effect of reperfusion therapyand shorter time window. Moreover, frequently, these survial patients suffered fromservious morbidity and the effects of therapies are limited, even the best rehabilitationwas applied. Therefore, It is necessary to search for better therapies to cure thecerebral stroke.The recovery progress of structure and function of damaged neural cells wasslow for a long time because an old theory that center nerve can not regrow affectedthe people. Emerge of Stem cells provided a good chance for curing many intractablediseases.SCs have become a research hot point because of its self-renewal andmulti-potential differentiation, which is important for theory research and clinicalapplications. Neural stem cells, as one kind of stem cells, has brought a new way forintractable diseases of center neural system including cerebral stroke. Nowadays, It has been demonstrated that neural stem cells exist in the following parts:(1)Stem cellsfrom the subventricular zone,the hippocampus or dentate gyrus, can be actived andmigrate to the injury site. However, their number and effect are limited.(2)Embryonicstem cells originate as inner mass cells within a blastocyst and can differentiate intoneural stem cells under appropriate conditions.But the embryonic stem cells ispresently only limited to be used in animal trial for ethical issues. (3) Bone marrowstromal cells, brifely, BMSCs, can differentiate into NSCs, neurons and glial cells. Todistinguish them from the stem cells from neural tissue, the neural stem cells inducedfrom bone stromal cells were called bone marrow stromal cells-derived-neural stemcells (BMSCs-D-NSCs). BMSCs are ease of isolation and high expansion potential invitro, and can be induced into neural stem cells.Therefore, BMSCs has been theimportant resource, which overcomes the risk of gaining material, as well as avoid theproblem of ethical issue and immunological rejection. The damaged brain tissuecould be repaired by transplanted NSCs if the exogenous gene was introducedinto them. Consequently, transplantation of BMSCs-D-NSCs is a potential strategyfor treating with the ischemical stroke.Structure change and functional improvement of tissue after NSCstransplantation are our focus, and the outcome is closely related with NSCs status invivo including cell vability, growth, migration,differentiation and integration withsurounding tissues.Hence, It is important to evaluate the process of transplanted cellsin vivo.In the past, the animals must be firstly killed and then the histological andmolecular trials were taken to observe the events and how to promote behaviorimprovement, which restricted the research on relationship between anatomy changeand behavior recovery. The invasive analysis can only provide single snapshot andcan not have a real observation on the tansplanted stem cells and animals serially.Moreover, the migration way and some pathological process were hard to be found and much important information would lost, and the invasive way can not be appliedfor human being. All of the shortcomings above had seriously restricted the clinicalresearch and application.Therefore, it is necessory to explore new method to monitorthe stem cells in vivo and resolve the bottleneck problem.Ultrasmall superparamagnetic iron oxide(USPIO) is a new kind ofsuperparamagnetic iron particulate with nano-scale. USPIO can visualizedistinguishing from surrounding tissue because of its special magnetic property andthe labeled stem cells should be monitored by nontraumatic methods. Up to date,there was no report about rat BMSCs transplantation to cerebral stroke. This issuemainly explored the effect of USPIO sinerem on BMSCs-D-NSCs biologicalcharacteristics and the events about recognition,survial, migration and proliferation ofthe labeled stem cells,and investigated the mechanism of brain ischemic diseasetreated by NSCs transplantation and applicability of nontraumatic imagingtechnology in order to establish a base for clinical application and safety and tosupply benefical revelation for other neural system diseases.Partâ… : Research on inducement of SD rat BMSCs into neural stem cells invitroObjective: To induce the isolated rat BMSCs into neural stem cells by cellculture.Methods: Bone marrow stem cells isolated from thigh bone marrows of SD ratsby density gradient centrifugation with lymphocyte separating medium. Then theBMSCs were cultured and induced into BMSCs-D-NSCs by NSCs culturemedium/basic fibroblast growth factor/leukemia inhibitory factor/fetal bovine serum,and the control BMSCs were cultured with DMEM/F12 medium/fetal bovine serumat the same time. The cultured cells were observed by invert phase contrast lightmicroscope to investigate their morphology and quantity.Cell growth curves were destribed by counting progress. Identification of neural stem cell was performed byspecial nestin marker immunocytochemical staining.Results: The separated BMSCs suspeneded and their shape was round,size-equal, outline-clean and intact under inverted phase contrast microscope at thebeginning of culture.The BMSCs started attaching and more cells attached with timeextending.The cell attachment would stop after 3 days and the unattached cells stillsuspended. The purified cells were left after discarding the unattached cells and theattached cells were more dioptric, bigger and emerged nuclear splitting.There weremore splitting cells during 5days～7days and proliferation was obvious and fewcloning mass appeared. Cell growth became more strong and more cloning massappeared in the day of 9～12, which surrounded one center point with radiating orswirling shape, most of them were round or elliptical.Up to 18days～20days,morphology of most cells started changing, appearing polygon or fusiform, etc.Processus from cell body extended to far place and connected with each other, similarto contact between neural cells.However, only very few cloning mass appeared ineighth day and did not increase with time extending.The growth curving indicated that the experimental group started logarithmicgrowth during 7～9days and merged to 80% during 14～16days.In contrast, controlgroup began logarithmic growth until 9～11days.The experimental group could express special NSCs antigen nestin proteinwhen they were detected by eighth day.Conclusion: The strategy of isolating BMSCs was easy and accessible, and therat BMSCs could be induced into neural stem cells with strong self-renewal andmulti- potential differentiation ability, which could be used as donor cells.Partâ…¡: Sinerem labeling rat BMSCs-D-NSCs and its effect on the cells'characteristics Objective: To determine the optimal concentration of new type USPIO Sineremlabeling BMSCs-D-NSCs in vitro and investigate the metabolism and retaining of Feparticle, and expolre the feasibility of labeling rat BMSCs-D-NSCs by Sinerem inorder to establish base for MR imaging.Methods: Bone marrow stem cells isolated from thigh bone marrows of SD ratsby density gradient centrifugation with lymphocyte separating medium. Then theBMSCs were cultured and induced into BMSCs-D-NSCs in vitro. Firstly, Sineremand poly-l-lysine(PLL) formed complex by static electricity.Then, theBMSCs-D-NSCswere incubated with the comples with different Sineren concentration as 0, 25,50, 100,200and500μg/ml for one night. The proliferation of labeled cells wereobserved by MTT test at the timepoint of 1,3,5,7,9,11～17day. The growth cycle andapoptosis of labeled cells were tested by flow cytometry. The effect on related keygene expression was investigated by RT-PCR. Moreover, the effect of Fe labeling ondifferentiation ability was analized by immunocytochemistry. Prussian blue stainingand transmission electron microscope were conducted for demonstrating theuptake,location and labeling ratio of intracytoplastic nanoparticles.Results: Morphologic difference of labeled and unlabeled BMSCs-D-NSCs wasnot significant.The density of Prussian blue staining increased with the lifting ofSinerem concentration,and the control group has no blue staining. In contrast tounlabeled group, the vitality and apoptosis ratio of labeled cells had no obviouschange when the Sinerem concentration was 200μg/ml or less than 200μg/ml(P＞0.05),but this concentration has very closely near the threshold value.It was significantwhen the Sinerem concentration was 500μg/ml or more than 500μg/ml(P＜0.05). Thegene expression level has no significant change after labeled by the appropriateconcentration(200μg/ml) contrast to the control group. The cell cycle had no significant difference between the labeled and unlabeled cells, which showed Sineremlabeling has no hurt on cell cycle. The differentiation ability of BMSCs-D-NSCs intoadipose cell has no obvious difference between two groups under the concentration of200μg/ml.The ratio of Sinerem labeling was about 98%～100% whenBMSCs-D-NSCs was labeled with 200μg/ml. Prussian blue staining showed that Feparticle lied in cytoplasm and transmission electron microscope indicated that thesenano-particles concentrated in endosomes/lysosomes.Conclusion: BMSCs-D-NSCs labeling by Sinerem was safe under appropriateconcentration of 200μg/ml and has no poisonous for short or long time, whichestablished base for tracking NSCs in vivo.Partâ…¢: Experimental study on Sinerem labeling of rat BMSCs-D-NSCswith MRI in vitroObjective: To observe the MRI of rat BMSCs-D-NSCs labeled by Sinerem invitro and evaluate the feasibility of MRI tracking of labeled NSCs.Methods: The SD rat BMSCs were isolated and induced into BMSCs-D-NSCsin vitro. 10⁵cells labeled with 200μg/ml Sinerem were immerged in 2% gel andscanned by different SE sequence T₁WI,T₂WI与T₂^*WI respectively to determine thebest scanning sequence with 4.7T MR system.10⁶ cells labeled with differentconcentration(0,25,50,100,200,500μg/ml) were scanned in 2% gel by T₂^*WI sequence.Different number(10¹,10²,10³,10⁴,10⁵)of BMSs-D-NSCs labeled with 200μg/mlSinerem were scanned to determine the threshold of cell quantity. In the end, thesignal change of 200μg/ml Sinerem labeled cells was observed at differenttimepoints.Results: MR result indicated that T₂^*WI was the most sensitive sequence. TheMR signal intensity of Sinerem labeled cells with different concentration was various, which reduced progressively with the increasing of Sinerem concentration. The signalintensity was lowest when the concentration was 500μg/ml. The MR signal intensityof more than 100μg/ml Sinerem labeling cells was much lower than control sampleand could be distinguished.The MR signal change could be observed when the cell number was and 10³ andmore. The signal intensity of 10⁶ labeled cells was increaseing with the timeextending, which could still be displayed as different low signal for 4 weeks.Conclnsion:Sinerem labeled BMSCs-D-NSCs in vitro appeared visually lowsignal area. The best sensitive sequence was T₂^*WI. The level of signal could beapplied for evaluating cell numbers. Sinerem labeled cells could be tracked for a longtime.Partâ…£: MR tracking of Sinerem labeled rat BMSCs-D-NSCs in brain andhistological detectingObjective: To observe the survial, migration and integration with surroundingtissue of USPIO labeled BMSCs-D-NSCs in normal or ischemic brain, and todetermine the feasibility of MRI tracking of the labeled stem cells.Methods: The SD rat BMSCs were isolated and induced into BMSCs-D-NSCsin vitro firstly. The BMSCs-D-NSCs were transplanted into normal cortex andstriatum of ischemic (middle cerebral artery occlusion, MCAO)rat bymicrotransplantation method after 10⁶ cells were labeled with Sinerem. The unlabeledcells were transplanted into other side brain as the control group in normal rat. Thelabeled cells were scanned at different timepoints by T₂^*WI with 4.7 T MR system,and then the animals were killed to have a histological detection for the Sineremlabeled cells in vivo.Results: Normal animal: The MR imaging of labeled BMSCs-D-NSCs in ratcortex appeared low signal area and the unlabeled cells had no significent signal change, which could not be distinguished from the surrounding tissue. The low signalarea did not migrate obviously at one week, but they had a slight migration along thecarpus callosum by 4 weeks.Ischemical model animal: The MR imaging of transplanted BMSCs-D-NSCs instriatum appeard low signal area. The low sinai area started to migrate to otherhemisphere by one week and reached carpus callosum fiber by 2 weeks, migratingmuch far along carpus callosum fiber by 4 weeks.The transplanted labled cells were blue with Prussian blue staining and thestaining cells distributed in the same place of low signal area. Fe particles could beobserved in endosomes/lysosomes and the labeled cells contacted closely with thesurrounding tissue by transmission electron microscope. Although we could not findthe synaptic-like structure, some cell bodies had the trendency to extend toward twopoles.Conclusion: Sinerem labeled BMSCs-D-NSCs could display in vivo with MR,whose status of survival and migratin could be tracked specially. This technologycould monitor the labeled stem cells in vivo.