The Effects Of SPIONs-labeled Amniotic Membrane-derived Mesenchymal Stem Cell Transplantation On Tissue Regeneration Of Hippocampal Dentate Gyrus In Rats

Background and ObjectiveTraumatic brain injury, cerebral hemorrhage and cerebral infarction is the main reason for human disability, there is poor prognosis after rescuing the living, No special cure methods have currently been found. In this study, the effects of SPIONs-labeled amniotic membrane-derived mesenchymal stem cell transplantation on tissue regeneration of hippocampal dentate gyrus in rats have been investigated through the following three stages.1?Mesenchymal stem cells among amnion-derived cells have been identified through cell morphology, flow cytometry, MTT and neuron-like differentiation, which then provide stem cells for this study on superparamagnetic iron oxide nanoparticles (SPIONs) labeled cells.2?Use single-and multiple-SPIONs-labeled methods to label amniotic membrane-derived mesenchymal stem cells so as to look for the safe concentration of SPIONs to label amniotic membrane-derived mesenchymal stem cells and do not effect amniotic membrane-derived mesenchymal stem cells differentiation into neuron-like cells; to detect the signal of existence and alteration of SPIONs-labeled stem cells both in vitro and in vivo through MRI, thus providing practical basis for in vivo tracking of the treatment of related diseases by the amniotic membrane-derived mesenchymal stem cell transplantation.3?transplant SPIONs labeled amniotic membrane-derived mesenchymal stem cells into the cortex of the brain with hypoxic-ischemic damage, and track the stem cells by MRI to show their distribution and migration in the host brain after transplantation so as to achieve in vivo non-invasive and continuous dynamic monitoring; to detect hippocampal dentate gyrus capillary regeneration through immunohistochemistry, and explore the roles played in tissue regeneration by stem cells.Materials and methods1. Amniotic membrane-derived mesenchymal stem cells in primary culture and differentiation of neuron-like cellsObtain amniotic cells by Enzyme digestion and adherent tissue culture method and identify amniotic membrane-derived mesenchymal stem cells through cell morphology, flow cytometry, MTT method and neuron-like cells differentiation. P3 of the amniotic membrane-derived mesenchymal stem cells were cultured in Cytokine Induction Medium in three groups. The following were done respectively on the 1st, 2nd,4th, and 7th day.1?MTT method:cell cytotoxicity analysis of induction medium; 2?flow cytometry:analysis of the alteration of specificity surface molecular of mesenchymal stem cells; 3?RT-PCR and immunocytochemistry to understand the potential of amniotic membrane-derived mesenchymal stem cells to differentiate into neuron-like cells.2. SPIONs labeled amniotic membrane-derived mesenchymal stem cells and cells in vitro and in vivo MRICell growth medium (Cytokine Growth Medium, CGM) or cell induction medium (CIM) with concentrations of Schering AG, Germany production of superparamagnetic iron oxide nanoparticles (SPIONs) being 3.5,7,14, and 28 ug/ml respectively was used in this study. Two SPIONs-labeling methods were used in this study:1?single SPIONs labeling method:the cell culture medium replaced for the first time (0 d) contains SPIONs, and that replaced 1d,2d and 4d later without SPIONs; 2?multiple SPIONs labeling:cell culture medium for the first time (0 d) replacement contains SPIONs, and the 1d,2d and 4d later replacement containing SPIONs. According to cell culture medium and different labeling methods, cells were divided into six groups:1?cytokine growth medium group; 2?cytokine growth medium+single SPIONs-labeled group; 3?cytokine growth medium+multiple SPIONs-labeled group; 4?cytokine induction medium group; 5?cytokine induction medium+single SPIONs-labeled group; 6?cytokine induction medium+multiple SPIONs-labeled group. Cells in cytokine growth medium were harvested in 1,2,4, and 7 d. Cells obtained each time were divided into three groups for the performance of MTT, Prussian blue and flow cytometry; cells in cytokine induction medium were harvested in 1,2,4, and 7 d. Cells obtained each time were divided into three groups to perform MTT, Prussian blue, immunocytochemistry, RT-PCR and flow cytometry; cells in cytokine growth medium without SPIONs and cells in cytokine induction medium as control group. Amniotic membrane-derived mesenchymal stem cells were cultured in cytokine growth medium containing SPIONs of different concentrations: 3.5?g/ml,7?g/ml,14?g/ml and 28?g/ml. After 1 day,they were digested, centrifugated, and suspended in 1.5 ml of 1% agarose in Ependoff tube for cooling, solidification, and SIEMEMS 3.0 Trio Tim?-class MR imaging magnetic resonance imaging. SPIONs-labeled amniotic membrane-derived mesenchymal stem cells'in vitro MRI was completed. Wistar rat groups:1?14?g/ml SPIONs-labeled amniotic membrane- derived mesenchymal stem cells transplantation group,2?14?g/ml SPIONs transplantation animal group. Preparation of cells to be transplanted:14?g/ml SPIONs-labeled amniotic membrane-derived mesenchymal stem cells (1 day), 5?l of 1×106/?l cells suspension for transplantation. Cell tansplantation method: Healthy Wistar rats were anesthetized, the anterior fontanel before the 0.5 mm,3.0 mm lateral to the center hole of about 1 mm diameter hole, slowly injected into the left cerebral cortex (2mm away from the dural surface).5?l PBS of 14?g/ml SPIONs was used in the control group for tansplantation.1 day,1,4 and 7 weeks after transplantation MRI scanning was taken respectively.14?g/ml SPIONs transplant animals with the same transplantation method in cells transplantation animals group. Complete SPIONs-labeled amniotic membrane-derived mesenchymal stem cells magnetic resonance imaging in vivo.3. Effects of Amniotic membrane-derived mesenchymal stem cell transplantation on tissue regeneration of hypoxic-ischemic hippocampal dentate gyrus in ratsA total of 487-day newborn rats were randomly divided into 6 groups with 8 in each group:A group (sham group):separate but not ligate the left common carotid artery; B group (model preparation group):succeed in model preparation but not for cell transplantation; C group (model preparation+injured side PBS solution transplantation group):prepare the model successfully and transplant 5 ul PBS solution; D (model preparation+injured side SPIONs liquid transplantation group): prepare the model successfully and transplant 5ul SPIONs; E group (model preparation+injured side stem cell transplantation group):prepare the model successfully and transplant 5ul un-SPIONs-labeled amniotic membrane-derived mesenchymal stem cells; F (model preparation+injured side SPIONs-labeled stem cell transplantation group):prepare the model successfully and transplant 5ul SPIONs-labeled amniotic membrane-derived mesenchymal stem cells; animals in each group went through the MRI examination and GFAP immunohistochemistry in the brain hippocampus at different time points (1 day,1,4 and 7 weeks after transplantation). Amniotic membrane-derived mesenchymal stem cell suspension preparation, transplantation, MRI monitoring and immunohistochemistry are similar to the previous study.Results:1. Amniotic membrane was digested by trypsin and collagenase. The cell suspension obtained was primarily inoculated to the culture flask. After a 24-hour culture a small amount of cell adhesion appeared in PO; a 48-hour culture increased the number of adherent cells; cultured for 72 hours, the number of adherent cells increased significantly; cultured for 7 days, adherent cells covered the bottom of the cell culture flask, close to 50%-60%of fusion, and the adherent cells were fibroblast-like. P3-P10 cells'growth curves are basically of the same. Cell viability of P10 generation later attenuated. Amniotic membrane-derived mesenchymal stem cells by flow cytometry analysis:primary cultures of P3 cells cultured in cytokine growth medium in the expression of surface molecules CD29, CD44, CD90, CD 105; negative expression of CD31, CD34, CD45 and CD 106. Amniotic membrane-derived mesenchymal stem cells were cultured in cytokine induction medium. Cell morphology displayed:cultured for 7 days,50-70%of the cells were similar to neuron-like cell morphology and cells interwove into a network of slender processes. Flow cytometry analysis showed that:on the 7th day, CD29, CD44, CD90 and CD 105 expressed negatively. RT-PCR analysis showed that:on the 1st day Nestin expressed which decreased on the 2nd day; on the 4th day it reduced significantly with no expression at all on the 7th day. There was no expression of NSE on the 1st day, which began to express on the 2nd day; on the 4th day it increased; on the 7th increased significantly. Immunocytochemistry results showed that:cells in the early stage of induction took on a fibroblast-like change and positive Nestin staining; induced cells were later changed unipolarly or multipolarly. The NSE staining was negative, and cell processes were clear, which were similar to the neuron-like cell morphology; No GFAP staining cells.2. Use different SPIONs-labeling methods to label mesenchymal stem cells, which through Prussian blue staining showed that:the cells SPIONs-labeled were 100%, and the cytoplasms contain blue dye particles in varying amounts. MTT results showed that less than and equal to 14ug/mlSPIONs is safe to label amniotic membrance-derived mesenchymal stem cells, meanwhile cell activity is more than 80%. Single SPIONs-labeling method:use different concentrations of SPIONs to label cells at the same time, and it showed that the amount of Blue dye particles in the cytoplasm was in direct propotion to the concentration of SPIONs, and iron particles were clusters of distribution piles. Use SPIONs of the same concentration to label at different times. When SPIONs concentration is less than and equal to 14ug/ml, blue staining particles in the cytoplasm aggregate with the extension of labeled time; when SPIONs concentration is higher than 14ug/ml, no aggregation of blue staining particles happens in the cytoplasm. Multiple SPIONs labeling method:label cells by different concentrations of SPIONs at the same time, fine blue dye particles scattered in the cytoplasm. With the increase in the concentration of SPIONs, iron particles were aggregated to clusters and distribute in piles. Label cells with the same concentration of SPIONs at different times. When SPIONs concentration is less than and equal to 14ug/ml, blue dye particles aggregate in the cytoplasm, and the aggregation increased with the increase of labeled time and frequency; When the concentration is higher than 14ug/ml, blue dye particles in the cytoplasm did not change. Single SPIONs-labeling method:amniotic membrane-derived mesenchymal stem cells were cultured in the CGM, the concentration of SPIONs being 14 ug/ml.7 days later, there appears positive expression of CD29, CD44, CD90 and CD 105 in the surface molecules of the amniotic membrane-derived mesenchymal stem cells; amniotic membrane-derived mesenchymal stem cells were cultured in the CIM, the concentration of SPIONs being 14 ug/ml.2 d later the expression of CD29, CD44, CD90 and CD 105 in the surface molecules of the amniotic membrane-derived mesenchymal stem cells was significantly lower. Signal intensity of SPIONs-labeled cells in ependoff tube decreased with the increaseing concentration of SPIONs in the SWI sequence images. There existed a high degree of linear correlation.14ug/ml of SPIONs labeled amniotic membrane-derived mesenchymal stem cells transplanted directly into the cerebral cortex of normal rats,after 1 day. transplantation area in the SWI sequence of images shows the limitations of very low signal-after 1 week the signal edge blurred, and 7 weeks later the low signal can still be detected in the SWI. In the control group 1 week after transplantation of PBS with the SPIONs concentration being 14ug/ml, the low signal disappeared.3. Neonatal rat model of cerebral ischemia and hypoxia SWI sequence of images in each group:A group (sham group), B group (model preparation group), C (model preparation+injured side PBS solution transplantation group), and E group (model preparation+injured side stem cell transplantation group) animals underwent MRI examination 1 day,2,4 and 7 weeks after transplantation, the results of which showed no abnormalities in brain signals. D group (model preparation+injured side SPIONs transplantation) animals underwent MRI examination at the same time points after transplantation, the results of which displayed on the SWI the ischemic temporal-parietal cortex ipsilateral injection region was with abnormally low signal, and high-signal areas gradually reduced over time. F (model preparation+injured side SPIONs labeled stem cell transplantation group):animals at different time points went through the MRI examination, and the SWI sequence of images showed the signal of the ischemic temporal-parietal cortex ipsilateral was abnormally low. With the extension of time the low signal in the transplantation site changes are as follows:1 day after transplantation, the transplantation site had a circle-like signal with clear boundary; 1 week after transplantation, the circle-like signal in the transplantation site became larger and boundary somewhat blurred than that 1 day after transplantation; weeks after transplantation, the circle-like low signal in the transplantation site was with linear opacities Traveling along the lateral corpus callosum; 7 weeks after transplantation, the boundary of the circle-like and linear opacities signal became obviously blurred than before in the transplantation site. Experimental animals in each group GFAP-positive cell density of hippocampal is dentate gyrus> CA3 area> CA2 area> CA1 region. Vascular density of CA1 area in order was:lacunar molecular layer, the radiation layer, polymorphic layer and the pyramidal layer. CA1 and CA2 areas have the most obvious hierarchy, with the most abundant capillaries in the polymorphic layer and the least in the pyramidal layer:hierarchy in CA3 area is slightly less clear, where the dentate gyrus molecular layer has the capillary to the most intensive and the granular layer the least. A group (sham group) animals'hippocampus GFAP-positive cells at different time points were highly expressed with intensive; capillary density and diameter at each time point was of no significant difference in statistics, P> 0.05. Compare B (model preparation group), C (model preparation+injured side PBS solution transplantation group) and D (model preparation+injured side SPIONs transplantation group), groups at each time point the same capillary density and diameter in each group was statistically the same at the same time point, P> 0.05; on the 1st day the expression of hippocampus GFAP-positive cells of animals in each group was low and the cells arranged sparsely. Compared with that on the 1 st day, the expression increased on the 1 st week but continued to be low levels of expression with a significant statistic difference, P<0.05; compared with the 1 st week, there was no statistic difference on the 4th and 7th week, P> 0.05. There was no statistic difference in capillary density and diameter between E (model preparation+injured side stem cell transplantation group) and F (model preparation+injured side SPIONs labeled stem cell transplantation group) group at the same time point, P> 0.05. Animals in each group at 1 day hippocampus the expression of GFAP-positive cells were low and sparse arrangement,1 week,1 day compared with higher expression levels continued to show a moderate expression, a significant difference, P<0.05; compared with the 1st week, the expression of the cells on the 4th week increased but continued to be in the moderate level of expression with statistic difference, P<0.05.Conclusion1. Enzymatic digestion and cell adherent culture method was used to isolate cells from amniotic membrane, and then amniotic membrane-derived mesenchymal stem cells were identified. It was confirmed that the amniotic membrane-derived mesenchymal stem cells possess the ability to differentiate into neuron-like cells.2. Through the application of single and multiple SPIONs-labeling methods, it became clear that the safe concentration of SPIONs to label amniotic membrane-derived mesenchymal stem cells is less than and equal to 14ug/ml, which did not affect the biological characteristic of SPIONs-labeled mesenchymal stem cells differentiating into neuron-like cells.14ug/ml SPIONs-labeled amniotic membrane-derived mesenchymal stem cells in vitro and in vivo magnetic resonance detection of SWI image sequences and the presence and changes of the low signal can provide practical basis for in vivo tracking of the treatment of related diseases by amniotic membrane-derived mesenchymal stem cell transplantation.3. Safe concentrations of SPIONs-labeled amniotic membrane-derived mesenchymal stem cell transplantation for treatment of hypoxic-ischemic brain damage animal model, magnetic resonance imaging applications SWI sequences, magnetic susceptibility difference can show the differences between organizations and achieve in vivo tracking purposes.4. Amniotic membrane-derived mesenchymal stem cell transplantation provides treatment for hypoxic-ischemic brain injury. Enhancement of the activation of the hippocampus dentate tissue regeneration provides a favorable environment for nerve regeneration.