In Vivo Imaging Of Human Umbilical Cord Blood Mesenchymal Stem Cells Transplanted Into Myocardium Using Magnetic Resonance Imaging

Part 1 Optimization and identification of in vitro isolation and culture condition of human umbilical cord blood mesenchymal stem cellsOjective: To explore establish an optimized methord to isolate,culture and expend mesenchymal stem cells (MSCs) from human umibilical cord blood (UCB).Method: Under sterile condition, Human UCB sample was harvested from full term delivery by cesarean section. MSCs were isolated from UCB by combination of gradient centrifugation and different adherent time method. And the isolated cells(MSCs) were cultivated in plastic culture flask containing DMEM/F12, 10% fetal calf serum (GM-CSF , IL-3). The cells were observed and their immunophenotypes were determined by flowcytometry(FCM).Results: After 24 hours , there gave rise to a small quantity adherent round cells,after 1 week the adherent cells increased and part of them exhibited single -point -shuttled,after 2 weeks formed cells group, after 3-4 weeks cultured to show parallelly arranged, radiat or whirlpool-shaped fibroblast-like cells and 80%-90% cells were fused. Serial subcultivation cells were found within 12 hours adhered and after about 10 days 80%-90% cells were fused. FCM showed that these cells expressed CD29,CD44,CD105 ,but no hematopoietic lineage markers , such as CD34,CD45,CD14.Conclusion: MSCs can be successfully isolated from human cord blood by using this method,and the cells purification is higher, and the adherent cells have the same immunophenotype with bone marrow derived. Part 2 The Study of the Feasibility of Human Umbilical Cord Blood Mesenchymal Stem CellsObjective:To evaluate the safety and efficiency of a new labeling agent,superparamagnetic iron oxide(SPIO).Methods:MSCs were isolated from UCB by combination of gradient centrifugation and different adherent time method. UCB-MSCs were labeled with different concentration of SPIO agent.The labeling efficiency was tested through Prussian blue staining ,cells viability were tested through Trypan blue rejection method,and the ability of growth was evaluated through MTT.Results:There were intracyto plasmic blue part- icles in nearly every cell in Prussian blue staining.The positive rate was more than 95%.With the concentration of SPIO increasing (14ug/ml~140ug/ml),there was no obvious diffenence in cell viability of UCB-MSCs(p>0.05).When the concentration of SPIO exceeded 280ug/ml,there was a significient difference(p<0.05).Conclusion:The UCB-MSCs could be safety and effi- ciently labledwith suitable concentration of SPIO.Part 3 Development and evaluation of the canine models of acute myocardial infarction using two waysObjective:To explore establish an optimized methord to develop and evaluate the canine model of acute myocardial infarction.Method: The 16 healthy canines were divided into two groups randomly,and each group contained 8 canines . One group was established the myocardial infarction model by using open-chest coronary artery ligation method,and another was received closed-chest coronary artery embolization method of modeling. Then electrocardiogram, CK-MB and histopathologic slide were investigated to confirm AMI. Results: The two methods both can successfully establish canine model of acute myocardial infarction, and be confirmed by the electrocardiogram, CK-MB and pathology.In the group of coronary artery ligation, 4 canines survived and 4 canines died ,while another group of coronary artery embolization,7 canines survived and 1 canines died.Conclusion:The method of coronary artery ligation is reliable,but the operation is difficult and the animal is not easy to survive.While the coronary artery embolization method of modeling is simple, convenient and safe, it is worth to spread.Part 4 Imaging Stem Cells Implanted in Infarcted MyocardiumObjective:To investigate the feasibility of in vivo tracking for umbilical cord blood mesenchymal stem cells delivered to infracted myocardium by MRI.Methods:MSCs were isolated from UCB by combination of gradient centrifugation and different adherent time method and were labeled with different concentration of SPIO agent. The acute myocardial infarction models were established by two methods of coronary artery ligation and coronary artery embolization.Then the labeled cells or SPIO were transplanted into the myocardium in two ways-direct injection and through coronary catheter. The MRI was respectively carried out immediately and 1–4 weeks. After MRI the hearts were excised, the segment in which injections were performed were thin cut and stained with hematoxylin-eosin and Prussian blue staining.Results:The labeled cells transplanted by direct injection could be detected through MRI and were confirmed on pathology. After 4 weeks the injected labeled cells could still be detected through MRI, and the pathology showed the injected cells could survive in the MI area, and parallel in the same direction.While the labeled cells transplanted through coronary catheter could not be detected by MRI and pathology.Conclusion:The labeled cells could be reliably detected by MRI in vivo.