Isolation, culture and differentiation of mesenchymal stem cells from Wharton's jelly of human umbilical cord

BACKGROUND: Bone marrow is the main source of mesenchymal stem cells (MSCs) at present, but its application has been limited, because of some reasons such as inconvenience of isolation, and the quantity of cells decreases with human increased age. Umbilical cord as a new source of MSCs has been widespread concerned recently. OBJECTIVE: To explore the approach of isolating and culturing MSCs from Wharton's jelly of human umbilical cord, and the methods of identifying the surface antigens and the differentiation potential. METHODS: MSCs were isolated and amplified via tissue-cultivation, and cultured by FasGrow medium. Morphology of MSCs from Wharton's jelly of human umbilical cord was observed under the optical microscope. Its immunophenotypes were detected using immunohistochemistry. The differentiation of MSCs into the osteoblasts was determined utilizing Gomori calcium-cobalt alkaline phosphatase staining, von Kossa calcium node staining, and tetracyclinefluorescence labeling. The differentiation of MSCs into the adipocytes was detected using oil red O staining. RESULTS AND CONCLUSION: MSCs were easily obtained from Wharton's jelly of human umbilical cord via the proposed approach. The primary cells grew up to 70%-80% confluence after 12-16 days of culture, and meanwhile the undifferentiated state was maintained and proliferation was stabilized after passage. The cell cycle of double increase was about 2 days, and proliferation in vitro reached twenty generation above. Surface antigen analysis showed that CD44, CD105, CD133, MHC- I were positively expressed, while CD34, CD45 were negatively expressed. Experiments of differentiation in vitro indicated that the obtained cells were capable of differentiating into fat, osteoblast and nerve-like cells.