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Aim: To establish an easily-handled method to isolate mesenchymal stem cells (MSCs) from coagulated human bone marrow samples., Methods: Thrombin was added to aliquots of seven heparinized human bone marrow samples to mimic marrow coagulation. The clots were untreated, treated with urokinase or mechanically cut into pieces before culture for MSCs. The un-coagulated samples and the clots were also stored at 4 °C for 8 or 16 h before the treatment. The numbers of colony-forming unit-fibroblast (CFU-F) in the different samples were determined. The adherent cells from different groups were passaged and their surface profile was analyzed with flow cytometry. Their capacities of in vitro osteogenesis and adipogenesis were observed after the cells were exposed to specific inductive agents., Results: The average CFU-F number of urokinase-treated samples (16.85 ± 11.77/10(6)) was comparable to that of un-coagulated control samples (20.22 ± 10.65/10(6), P = 0.293), which was significantly higher than those of mechanically-cut clots (6.5 ± 5.32/10(6), P < 0.01) and untreated clots (1.95 ± 1.86/10(6), P < 0.01). The CFU-F numbers decreased after samples were stored, but those of control and urokinase-treated clots remained higher than the other two groups. Consistently, the numbers of the attached cells at passage 0 were higher in control and urokinase-treated clots than those of mechanically-cut clots and untreated clots. The attached cells were fibroblast-like in morphology and homogenously positive for CD44, CD73 and CD90, and negative for CD31 and CD45. Also, they could be induced to differentiate into osteoblasts and adipocytes in vitro., Conclusion: Urokinase pretreatment is an optimal strategy to isolate MSCs from human bone marrow samples that are poorly aspirated and clotted.