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Objective: To establish a reliable method of isolation, culture and characterization of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) and study its multiple differentiation potency., Methods: HUCMSCs were isolated and cultured using Trypsin-type II collagen and hyaluronidase digestion method and tissue explant culture method, respectively. The cell growth of hUCMSCs was observed under an inverted microscope. Cell viability rate of the different passages was evaluated by trypan blue staining. The proliferation profile of hUCMSCs was analyzed by growth curve and MTT assay. Flow cytometry was used to study the cell cycle and immunophenotypage change. The differentiation potency of hUCMSCs towards the osteoblasts, adipocytes was assayed using the differentiation kits. The differentiation towards the cardiomyocytes and endothelial cells was tested by immunofluoresence staining with the specific markers., Results: After 1-day culture of the enzyme digested cells, under the inverted microscope, the adherent cells were round, and 4 days later, they grew quickly and presented fusiform. Seven days later, the cells proliferated from the center to the peripheral and fused by 80% on day 10. With the tissue explant culture method, the cells started to proliferate gradually from the periphery of the tissue and grew quickly and arrayed closely in monolayer after 10 days. The cell viability in both isolation methods were more than 96% as tested by trypan blue staining. The growth curve of the third passage presented an "S" shape. MTT assay showed that the optimal cell proliferation occured on day 3 to 5. The ratios of G0/G1 phase and S+G2/M phase was 88.78% and 10.21% respectively by enzyme digestion, and 84.82% and 13.87% respectively by explant culture method. There was no significant difference in cell cycle. The positive rates of CD90, CD105, CD73 were more than 99% and the expressions of CD45, CD34, CD14, CD11b, CD79a, CD19, HLA-DR were lower than 1%. The hUCMSCs isolated by the two methods could efficiently differentiate towards the osteoblasts, lipocytes, cardiac myocytes and endothelial cells, and the positive rates were all above 90%., Conclusion: The hUCMSCs can be effectively isolated by both enzyme digestion and explant culture methods. The enzyme isolation method presents a better method regarding the cell number obtained. This study showed the enzyme isolation method may be an optimal method to isolate the hUCMSCs for the cellular therapy and stem cell bioengineering.