Experimental Study On Cytobiological Characteristics Of Feridex Labeled Rabbit Bone Marrow Stroma Cells

Nowadays, the aim of treatment to the central nervous system (CNS) is mainly torelieve and control the symptom and the development of the diseases. However,Neural stem cells (NSCs) were discovered in the brain of adult mammals and coulddifferentiate into nerve tissue in the past decade, which indicated that CNS has theability of self-recovery. Stem cells (SCs) is defined as cells capable ofmulti-differentiation and self-refreshment, and they are situated in many tissues andembryos. In recent years, bone marrow derived stromal cells (BMSCs) were verifiedto have the potential of differentiating into NSCs, and have the advantages of easyobtaining, abount resource, low immunology, as well as stable genetics. Therefore,SCs have been one of the most ideal candidates for cell transplantation. It has becomeresearch emphasis to apply NSCs, especially BMSCs to repair damaged function ofCNS through their transplantation and regulating their differentiation.So far, whatever CNS or peripheral nervous system (PNS), the major detectionmethod of identification of nerve regeneration and clinical symptom improvementafter NSCs transplantation is immunohistochemical array. This method need obtaintissue and can not evaluate transplantation outcome in vivo, on the contrast, theclinical and scientific research are eager to need a nontraumatic method in vivo,which can monitor the survival, migration and functional differentiation of the NSCs after transplantation.Today, it is accessible to monitor the survival, transplantation, survival conditionof BMSCs in vivo with successful preparing and rapid development of the MRIcontrast agent. Now, MRI can provide the resolution of 25-50μm, access to the levelof single cell, so the cells transplanted in the body may be traced in theory.superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide(USPIO) are the prior tracer agent of MRI and had been successfully employed tolabel various cells in vitro. Feridex is one of the SPIO and was approved by FDA.Feridex had successfully labeled kinds of mammalian SCs by transferring agentpoly-1-lysine (PLL). Presently, scientists had started experimental study on FE allover the world. These system studies included: the culture of BMSCs of rat, rabbit andrhesus monkey and their converting to the NSCs, the transplantation into the model oftraumatic animal with NSCs labelled by FE, and the application of the MRI, theimmunohistochemical technique, the confocal microscopy technique to observe thegrowth, differentiation and synaptic formation of NSCs, as well as the integratingwith brain tissue. But it is not enough for the research of biological viability beforeand after NSCs labeled by feridex.Based on above, this issues proposed to have a study on BMSCs viability,proliferation, differentiating ability and apoptosis, etc, before and after labeling inwith different FE concentration in cell level, and explored the effect of FE on labeledBMSCs and the optimal concentration, in order to provide a reference for furtherresearch.Part One: Experimental study on the isolation, induction and differentiationof the bone marrow stromal cells from New-Zealand rabbitObjective: To observe in vitro culture, proliferation and differentiation intoNSCs of the rabbit BMSCs, and grasp the methods of cell culture to make a base fornext advanced test.Methods:1. Under the sterile condition, BMSCs were acquired from the bone marrow of rabbit thighbone and harvested by means of density gradient centrifugation followingan ilium puncture.2. Cell culture: NSCs culture medium, 1%Fetal bovine serum(FBS, 1%),leukemia inhibitory factor (LIF, 10ng/ml)and basic fibroblast growth factor(bFGF,10ng/ml)were used to cultivate, induce and proliferate BMSCs in vitro after 3 days ofprimary culture. The purified BMSCs were obtained after 1-2 weeks and could bepassaged, during which FBS and Retinoic acid (RA, 0.5μg/ml) were added to induceBMSCs differentiation into NSCs.3. Cell growth was observed and photographed by CK-2 invert phase contrastlight microscope.4. Cell Identification: Special antigens, Nestin of neural stem cell,Neuron-specific endase (NSE) of neuron and Glial fibrillary acidic protein (GFAP) ofastrocyte were detected by immunocytochemical staining respectively with SABCmethod to identify the above cells.Results:1. Observation under inverted phase contrast microscope: The BMSCs started toattach after 10 hours of seeding, and the attached cells began to split and proliferate,then formed island-like clone cell groups, which could differentiate intomultiple-shaped cells with slim, long process.2. Early cultured cells could express Nestin without induction and indicated theywere in characteristics of SCs, which showed that BMSCs-D-NSCs were induced.The NSCs derived from BMSCs could differentiate into neuron-like cells andastrocyte-like cells following the induction. These differentiated cells could expressNSE and GFAP antigen respectively by immunocytochemical staining.Conclusion:1. The results demonstrated the BMSCs from rabbit could be cultured andexposed in vitro with the proliferating abilities, and induced BMSCs could expressNestin antigen with NSCs characteristics.2. The above culture condition could be suitable for rabbit BMSCstrans-differentiating into NSCs, and the NSCs derived from BMSCs have the ability of differentiating and were capable of expressing special antigens of nerve cell lines(neuron and astrocyte).3. BMSCs are of abundant resource, easy isolating and expansion, which can beused as autotransplantation seeds.Part Two: Cytobiology study on intracellular labeling of bonemarrow stromal cells of rabbit with FeridexObjective: The purpose of this study was to explore the feasibility of protocolsusing Feridex and polycationic transfer agent for in vitro magnetic labeling of rabbitBMSCs. To observe the viability/proliferation/differentiation potency and apoptosisof BMSCs before and after labeling, and explore the best FE labeling concentration.Methods:1. Animals, marrow collecting and isolating of BMSCs were omitted (See part one).2. FE labeling concentration and groups: Firstly, the FE were diluted and dividedinto four different concentrations (10μg/ml, 25μg/ml, 50μg/ml and 75μg/ml), whichwere mixed with PLL (1.5μg/ml) in equal volume. Then the complexes were allowed tomix in a rotator for 30 min and were added to the existing media in the cell culture. TheFE (Fe) concentrations would be 5μg/ml, 12.5μg/ml, 25μg/ml and 37.5μg/ml, and PLLconcentration would be 0.75μg/ml. The objective was divided into six groups:a.BMSC-D-NSC without FE; b. 0.75 PLL; c.5FE-0.75PLL; d.12.5 FE-0.75PLL;e.25FE-0.75PLL; f. 37.5FE-0.75PLL(n=10).3. Observation by inverted phase contrast microscope and transmission electronmicroscope: Cellular morphological change observation of FE-PLL labeled and controlcells.4. Identification of iron particles in cell: Prussian blue staining and transmissionelectron microscopy were employed to detect the Fe in cell and the FE-PLL labelingefficiency.5. Leakage detection of labeled BMSCs: FE labeled BMSCs were cocultured withneurons to determine the leak of FE from labeled cells.6. Detection of cell growth curve: Cell viability of labeled and control BMSCs were investigated by CCK-8 method.7. Cellular apoptosis detection: The apoptosis of labeled cells and control cells weredetermined by flow cytometry.8.Celluar MRI: The cells scanned were divided into five groups.The scanningsequence of 4.7T MRI in vitro included T₂ fast spin echo sequence and T₂^*gradient echosequence in pinacoid.9. Statistical analysis: The experimental data of cell absorbency and apoptosis ratiowere analyzed with repeated measurement of variance and SNK analysis comparing twogroups by the software SPSS13.0. The inspection levelαis 0.05.Results:1. Observation by inverted phase contrast microscope and transmission electronmicroscope: Cellular morphology has no significant difference between FE-PLL labeledand control cells under inverted phase contrast microscope. Main difference betweenthem was due to color of labeled BMSCs manifesting light to deep yellow. Transmissionelectron microscopy revealed that cell volume, morphology and cell organs had nosignificant change. The presence of scattered vesicle-like inclusion body structure withmembrane in cytoplasm. There were apoptosis in minor labeled cells, but moreapoptosis occurred in f group.2. Identification of iron particles: Transmission electron microscopy revealed thepresence of numerous vesicles which are filled with the electron-dense magnetic ironparticles in FE-PLL labeled BMSCs. Prussian blue staining showed numerousblue-stained fine particles in the cytoplasm of FE-PLL labeled BMSCs. The colorbecame more and more blue from c to f group.3. Comparison of labeling efficiency between groups: The FE labeled cells showeddifferent labeling degree. Counting under microscopy indicated the labeling efficiencyadded with the increasing of the FE concentration. Electron microscopy observationshowed that vesicle-like structure with membrane involving the FE in cytoplasm becamemore and more with the concentration increasing of various groups, which mean that theiron content also increased. 4. OD value and cell growth curve: OD value has significant difference betweenvarious groups, and there has no significant difference within group a, b, c, d and e.However, there was significant difference between f group and other groups(P＜0.001).The above results showed cellular viability was not influenced by FE-PLLcomplex under concentration of 25μg/ml.5. Leakage detection: Labeled NSCs was blue and unlabeled cerebral cortex neuronsshowed negative after coculture of FE-PLL labeled NSCs with cerebral cortex cells byPrussian blue staining, confirming that there was no leakage and reuptake of Feridex invitro at this time point.6. Cellular apoptosis detection: The flow cytometry assay indicated that there was anincreased tendency in the apoptosis along with culture time elapsed. There was nosignificant difference in the apoptosis rate within a～e groups. However, f group hadsignificant difference (P＜0.001) compared with other groups. Above results illustratedFE-PLL complex under 25μg/ml had no significant influence on cell vigor.7. MRI of labeled BMSCs in vitro: Signal of labeled cells decreased obviously withincreasing concentration of Feridex, and unlabeled cells showed notable high signal byusing T₂ and T₂^*sequence.Conclusion:1. The above results suggested that FE-PLL complex might be used to labelBMSCs of rabbit in vitro.2. The labeling efficiency added with FE concentration increasing and T₂WI/T₂^*WIsignal change also become more obvious. The concentration below 25μ/ml of Magneticlabeling of BMSCs of rabbit with FE-PLL complex is high efficient and safe.3. The appropriate concentration of Magnetic labeling BMSCs with FE-PLLcomplex is 25μg/ml.