Induction Of Umbilical Cord Blood Mesenchymal Stem Cells To Form Urothelium Cells On Fibrin Clot

Objective:To investigate the possibility of umbilical cord blood mesenchymal stem cells to form urothelium cells on fibrin clot.Content:In the experimental group,the UCB-MSCs on umbilical cord blood fibrin clot were co-cultured with fetal urothelium cells. In the control group,the UCB-MSCs on umbilical cord blood fibrin clot were cultured in DMEM/F12culture medium. Morphological changes in UCB-MSCs and fetal urothelium cells were analyzed under a phase contrast microscopy. The urothelium cells were tested under transmission electron microscope. Immunhistochemistry staining for anti-cytokeratins(AE1/AE3) w ere used to verify the cell’s origin.Methods:1. The umbilical cord blood mesenchymal stem cells (UCB-MSCs) were obtained by umbilical cord blood. The higher purity of UCB-MSCs were obtained by keeping the adherent cells and removal of nonadherent cells repeated.2. P3UCB-MSCs populations were collected, detecting expressed the surface marker expression of UCB-MSCs by flow cytometrie analysis was assessed by related method.3. Fetal urothelium cells isolated by enzymatic digestion, fetal urothelium cells were identified by chemical methods dithizone staining and electron microscopy.4. Using the method of calcium chloride solution to join with the umbilical cord blood plasma, preparation fibrin clots.5. Will certain density umbilical cord blood mesenchymal stem cells vaccination on fibrin clot surface, make its growth.6. The UCB-MSCs on umbilical cord blood fibrin clot were co-cultured with fetal urothelium cells.UCB-MSCs subsequently evaluated for antigen expression and ultramicrostructure by immunocytochemistry and electron microscopy.Result:1. The highly purified UCB-MSCs were obtained by density gradient centrifugation and keeping the adherent cells derived from umbilical cord blood and the UCB-MSCs showed long fusiform or spindle and fibroblast-like shaped after repeated passages2. The UCB-MSC surface markers specific CD44.CD90were highly expressed,In contrast,CD35.CD45were barely detectable by flow cytometry. under inductive medium in vitro.3. Fetal urothelium cells were isolated by enzymatic digestion, Immunocytochemical analysis using the primary antibody CKAE1/AE3demonstrated that the resulting cells were urothelial cells. Urothelial cells examined by TEM displayed Urothelial cell features.4. The UCB-MSCs on umbilical cord blood fibrin clot were co-cultured with fetal urothelium cells. The UCB-MSCs gradually became larger and were polygonal.the cells had ultrastructure of urothelium cells under transmission electron microscope and were stained positively for Immunhistochemistry staining of anti-cytokeratins (AE1/AE3).Conclusion:1. Pure UCB-MSCs were obtained from umbilical cord blood. Stem cells are functionally defined by their capacity to self-renew and differentiate into one or more terminal cell types in vitro.2. Pure urothelial cells could be obtained by the method of enzymatic digestion. A new method was provided for culture and purification of urothelial cells.3.The UCB-MSCs on fibrin clot have the potentiality to differentiate into urothelium cells.