The Isolation, Culture And Differentiation Of Mesenchymal Stem Cells From Wharton's Jelly Of Human Umbilical Cord

Objective:To establish a simple, fast, economic, and efficient procurement method for mesenchymal stem cells from Wharton jelly of human umbilical cord.Methods:remove blood vessels and the adventitia from umbilical cord, shred the remaining jelly organization (Wharton's jelly, WJ) to the size of 1-2mm3, and then place them evenly in 6-well plate with FasGrow culture medium according to a certain percentage. Culture in incubator under 37℃, 5% CO2. After cells swim out, digest with trypsin and then subculture and cryopreservate with liquid nitrogen. Carry on HE and Gimesa staining with the 5th generation of cells, observe morphology.Results: The Wharton jelly organization kept still for seven days or so. After that, some cells swam out from the tissue block. Mostly of them were double-protruding long spindle-shaped, short rod-like or flat-shaped fibroblast-like cells; 14 days or so, the cells presented in a uniform spindle morphology shape, and reached 70%-80% confluence. The cells can maintain undifferentiated state and stability of proliferation after passage, and can expand to at least 20 passages in vitro, with a cell doubling time of about 3d. After P3 generation, cells presented in uniform morphology. The recovered cells grew well, and were basically the same as those before cryopreservation on morphology and growth characteristics.Conclusion:The planting method is simple, fast, economic and efficient, with which, it is able to isolate a large number of fibroblast-like cells from the Wharton jelly of human umbilical cord. Objective:To decide whether the isolated fibroblast-like cells from Wharton jelly of human umbilical cord are mesenchymal stem cells, and to prove their differentiation capability.Methods:carry on immunohistochemistry for the 5th generation cells to detect the immune phenotype. Induced differentiate the fibroblast-like cells into bone cells with FasGrow culture medium containing dexamethasone, vitamin C,β-glycerophosphate, and identify the bone characteristics by alkaline phosphatase staining, Von Kossa staining and tetracycline staining; Induced differentiate the fibroblast-like cells into fat cells with FasGrow culture medium containing dexamethasone, vitamin C, isobutyl-methyl xanthine, and identify the adipogenic characteristics by oil red O staining; Induced differentiate the fibroblast-like cells into neural-like cells with FasGrow culture medium containingβ-mercaptoethanol, and identify the characteristics by check specific antibodies.Results:The surface phenotype analysis showed:CD44, CD 105, CD133, MHC-Ⅰwere positive, CD34, CD45 were negative, and the isolated fibroblast-like cells were MSCs; Culture experiment proved that:the cells were capable of differentiating into fat, bone, and nerve-like cells in vitro.Conclusion: The fibroblast-like cells isolated from Wharton jelly of human umbilical cord expresses mesenchymal stem cell surface phenotype, and has great differentiation capability, which can be used as an important source of seed cells in tissue engineering.