Ferumoxytol Labeling And In Vivo Tracking Of Human Adipose-Derived Stem Cells In Rats With Ischemic Stroke By MRI

Cerebrovascular diseases are one of the leading causes of mortality and long-term disability, the majority of Cerebrovascular diseases are ischemic.Reperfusion and anti-thrombotic therapies are still of limited benefit.Stem cells, with the capacity to self-renew and differentiate into different cell types, raise the hope for future cell-based regenerative therapies. Multiple stem cells, such as bone marrow derived mesenchymal stem cells (BMSCs), neural stem cells(NSCs) and embryonic stem cells (ESCs), have been tested as potential sources for cell based therapy for ischemic stroke. Several lines of evidence indicate that adipose tissue may represent an ideal source for stem cells:1) the collection of adult adipose tissue is technically easy and safe; 2)the possibility of transplantation with no immune rejections, ethical problems or tumorigenesis. Moreover, adipose-derived stem cells(ADSCs) were demonstrated to exhibit differentiation into neural and glial cells in vivo and in vitro.The successful translation of stem-cell therapies requires a detailed understanding of the fate of transplanted cells. Magnetic resonance imaging (MRI) is a non-invasive tool that has demonstrated a high sensitivity for cell tracking and superparamagnetic iron oxide (SPTO) particles appear to be the preferred material for magnetic labeling of cells. SPIO is a new kind of magnetic resonance contrast agents, which produces negative contrast effects on T2-weighted and T2*-weighted sequences. Ferumoxytol, a colloidal suspension of carbohydratecoated ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) (intravenous iron formulation), is currently approved to treat iron deficiency anemia in patients with chronic kidney disease. We hypothesized that ferumoxytol cell labeling can be effectively used in the experimental ischemic model for clinical 3.0T MRI tracking of hADSCs introduced into rat brain.Part I Culture of human adipose-derived stem cells(hADSCs) in vitroObjective:to explore the method of in vitro culture of human adipose-derived stem cells. Method:fresh human adipose tissue from liposuction were used to isolate hADSCs. hADSCs were proliferated by adherence, growth characteristics and morphology changes of primary as well as different passage cells were observed by phase contrast microscope, phenotypic appraisement of the cells and differentiation potential assessments were also made. Results:passage cells of hADSCs cultured for 24h reached a adherent rate of 90%, purity of the 3rd passage cells were got. hADSCs showed positive of stem cell surface markers CD13, CD44 and CD90. Meanwhile, CD34, CD45 and CD 106 were negative. hADSCs were inducted to lipid bone and cartilage formation. Conclusion:3rd passage cells are ideal cell source for transplantation.Part II Labeling hADSCs with Ferumoxytol in vitro and the effect on the cells’characteristicsObjective:To determine the optimal concentration of new USPIO Ferumoxytol labeling hADSCs in vitro, expolre the feasibility of labeling hADSCs with Ferumoxytol in order to further study for MR imaging. Method:Labeling hADSCs with ferumoxytol-protamine (PF), ferumoxytol-heprin-protamin (HPF) and ferumoxytol-poly-L-lysine (F-PLL) nanocomplexes. We used different concentrations of HPF(1μg/ml,5μg/ml, 10μg/ml,20μg/ml,50μg/ml,100μg/ml, 200μg/ml,500μg/ml)to label hADSCs, then morphology and viability, labeling efficiency, cell proliferation capacity were tested. Results:HPF nanocomplexes showed high labeling efficiency. Compared with unlabeled hADSCs, labeled hADSCs with 50μg/ml HPF were observed with no affected. Conclusion:Labeling hADSCs with HPF had none influence on cell bionomics.Part Ⅲ Labeling hADSCs with Ferumoxytol:a MRI study in vitro and in vivoObjective:To observe hADSCs labeled with Ferumoxytol by MRI in vitro, and transplanting them into normal rat brains to evaluate the feasibility of tracking hADSCs with MRI in vivo. Method:hADSCs labeled with Ferumoxytol were studied in vitro and tracking them in normal rat brains by MRI in vitro an in vivo.Results:The MRI signal in all sequence decreased with the labeling of Ferumoxytol both in vitro an in vivo, the ESWAN sequence is the most obvious. At 28 days post-injecting, the ESWAN images showed a remarkably low signal change still. Conclusion:In vivo tracking hADSCs labeled with Ferumoxytol by MRI is available, and the ESWAN sequence is the most sensitive. After 28 days the hADSCs still present at the transplanted sites.Part IV Making a MCAO rat model with a method of thread embolismObjective:To make middle cerebral artery obstruction (MCAO) model using a method of thread embolism. Method:SD rats were subjected to 1.5 hours of middle cerebral artery occlusion (MCAO). At 1 day after reperfusion, rats with a Zea-Longa score of 2-3 were regarded as successful models. Modified Neurological Severity Scores (NSS) were used to evaluate the behavior changes in rats, MRI was alsoused to detect the changes of infarction focus on different arrays.28 days after reperfusion, rats were sacrificed by decapitation and the brain tissues were taken for HE staining. Results:Success rates in model preparing before skillful operation could be made were 55%. Behavior scores were statistically significant in rats among any time point. T2-weighted images clearly showed the lesion in the right hemisphere as a hyperintense. Conclusion:Thread embolism is able to prepare stable MCAO rat model, which is suitable to be used as the model of cerebral infarction study.Part Ⅴ In vivo tracking of human adipose-derived stem cells labeled with Ferumoxytol in middle cerebral artery occlusion-injured rats by MRIObjective:To track the hADSCs labeled with Ferumoxytol in rats with ischemic stroke by MRI. Method:Rats were randomly divided into5 groups. In sham group, only separated branches of the external carotid artery (ECA), and tip of the suture was not inserted within the right common carotid artery (EEA). In MCAO group, no other treatments were made after MCAO. The other three groups transplanted with PBS, unlabeled hADSCs and hADSCs labeled with HPF after MCAO respectively conducted in PBS, unlabeled-ADSCs, and HPF-hADSCs group at the same time. In all animals behavioral tests were assessed 1,7,14 and 28 days after transplantation and the 3.0T MRI scan were made after assessment. Rats were anesthetized and perfused at 28 days post-transplantation and brains were harvested for histological examination. Prussian blue and HE stain were used. Results:Low signal change was obvious at the transplanted sites at 1 day post-injection, and after 28 days the low signal intensity still present at the transplanted sites, became slightly blurred in T2WI, but still remained stable in ESWAN images. Majority of the Prussian blue (PB) cells were observed in brain tissue sections at or around the injection site. Some blue iron particles located in the ischemic cortex and a few diffused ones in the ipsilateral subarachnoid space. Conclusion:The distribution of hADSCs labeled with Ferumoxytol could be tracked in vivo after transplantion into the MCAO rat brains by clinical 3.0TMRI.