Human Umbilical Cord Mesenchymal Stem Cells Cooperate With Cord Blood Cells In Therapy Of Type Ⅰ Diabetes

Chapter 1 Optimization of Isolation Method for Mesenchymal Stem Cells Derived from Human Umbilical CordObjective:To establish a simple and efficient method for isolating mesenchymal stem cells from human umbilical cord.Methods:After removing the vessels in the umbilical cord,the remaining tissues were digested by different enzyme solutions,then the digested cell suspensions were plated and cultured in DMEM/F12 medium containing 10ng/ml bFGF and 10%FBS.The adherent cells were digested with trypsin/EDTA for passage culture.The immunophenotypic characterization of adherent cells were analyzed using flow cytometry and immunofluorescence staining,the differentiation potential of cells were tested using standard differentiation conditions for osteoblasts and adipocytes.Results:1×10~6 mononuclear cells could be harvested every 5 cm of umbilical cord after digestion using a mixture solution of dispase, collagenaseⅠand hyaluronidase,and the number of adherent cells could reach up to 2.2×10~6 after 10 day culture.A homogenous culture of fibroblast-like was generated after passages.This isolation method which was simple and efficient was significant excelled from other that used in our experiment.The population doubling time of these fibroblast-like cells was(34±4) hours,they revealed a surface antigen profile that is similar to that defined for BM-MSC,that is,CD29~+,CD90~+,Vimentin~+, SMA~+,desmin~+,and do not express surface markers of hematopoetic cells and endothelial cells,namely CD34~-,CD45~-,CD31~-,KDR~-.They also expressed multipotent cell marker such as SSEA4 and OCT4.Moreover, these cells could differentiate into osteoblasts and adipocytes under specified culture condition.Conclusions:We have developed a simple and efficient isolation method to reproducibly generate a large number of high qualified MSC from umbilical cord.Chapter 2 Differentiation of Human Umbilical Cord Mesenchymal Stem Cells to Insulin-Secreting CellsObjective:To explore whether human umbilical cord mesenchymal stem cells are capable of differentiating into insulin-secreting cells.Methods:Human umbilical cord mesenchymal stem cells were induced to differentiate into insulin-secrecting cells with high glucose DMEM containingβ-mercaptoethanol,epidermal growth factor,basic fibroblast growth factor,B27 and niacinamide.Morphological changes of hUCMSCs were observed under a phase contrast microscopy;the expressions of PDX-1 were detected during the differentiation procedure by flow cytometry;the expressions of insulin and C-peptide were detected by immunocytochemistry;The insulin levels under the stimulation of high or low glucose were analyzed by chemical-luminescent assay;reverse transcription-polymerase chain reaction(RT-PCR) was performed to detect multiple genes which related to pancreatic cell development and function,such as ngn3,insulin,glucagon and somatostatin.Results:The morphology of hUCMSCs gradually transformed from spindle-shape to round during the differentiation period and also displayed the tendency of forming clusters.The expression of PDX-1 in cells cluster after stage 2 could be detected.After the stage 3 induction culture,hUCMSCs could express insulin and c- peptide and secrete insulin upon glucose stimulation,and also express ngn3,insulin,glucagon and somatostatin genes.Conclusions:Human umbilical cord mesenchymal stem cells are capable of differentiating into islet -like cells with insulin secreting function under specific culture condition. Chapter 3 Human Umbilical Cord Mesenchymal Stem Cells Cooperate with Cord Blood Cells in Therapy of TpyeⅠDiabetesObjective:To observe the effects of infusion of hUCMSCs and cord blood cells in therapy of tpyeⅠdiabetes.Methods:The models of type 1 diabetes were produced in C57BL/6 male mice by intraperitoneally injecting with daily low doses of streptozotocin on day 1-5.2×10~6 of Umbilical cord mesenchymal stem cells and cord blood cells in different ratio were concomitantly infused into sublethal irradiated diabetic mice through tail vein on day 12 and 17. The blood glucose levels were detected after transplantation;HE staining and immunohistochemical staining was performed to detect the damaged pancreatic islet structure and function;the pancreatic and kindey tissues from cell-treated diabetic mice were assayed for engraftment by PCR for human specific Alu sequences.Results:After adminstrating STZ for 7 days,detecting blood glucose level of mice.If blood glucose concentration rose over 16 mmol/L twice in random,the mice are treated as typeⅠdiabetes mice. The administration of umbilical cord mesenchymal stem cells cooperated with cord blood cells in the ratio of 1:4 significantly improved pancreatic function and structure.The blood glucose levels in the diabetes mice decreased significantly and the cells producing mouse insulin increased related to untreated controls.However,human insulin and human nuclei antigen were not detected in transplanted mice pancreas,but PCR assays detected human- specific Alu sequences in DNA in pancreas and kidney,which suggested that adonor cells were engrafted into the damaged pancreas and kidney.Conclusions:The transplanted hUCMSCs and cord blood cells could reside in the pancreas and kidney of typeⅠdiabetic mice,repairing impaired pancreas tissues,decreasing blood glucose concentraton.