AIMTo amplify mesenchymal stem cells (MSCs) from human bone marrow (HBM) and to induce MSCs differentiated into endothelial cells (ECs) in vitro, which possibility and conditions were to be discussed.

METHODSMSCs were separated by gradient centrifugation on Percoll (density 1.073 g/ml) from HBM, and incubated for purification and amplification in DMEM (low glucose) with 10% fetal bovine serum (FBS). MSCs, which their phenotypic characteristics were analyzed by flow cytometry, were incubated for orientation differentiated into ECs in DMEM (high glucose) with 20% FBS and VEGF (10 ng/ml) for about 14 - 21 days. Afterwards, the cells differentiated were evaluated by immunohistochemistry with Tie-2 monoclonal antibody and by transmission electron microscopy (TEM) for observation Weible-palade corpuscle in the cytoplasm.

RESULTSThe quantity of MSCs was increased from 5.0 - 10(5) in the primary culture to 8.0 x 10(12), or to increase 1.6 x 10(7) times after 15 generations incubated. The purity of MSCs was above 95% and 98% homogeneous at passages 2 and 3, respectively. The typical "cobblestone" of these cells presented from MSCs differentiation culture after 14 - 21 days was observed by light microscopy. More than 90% of the cells were positive stain for Tie-2 related antigen by immunohistochemistry assay. The Weible-palade corpuscle, which form is typical morphology of ECs, was also observed by TEM in the cytoplasm.

CONCLUSIONMSCs from HBM has the capability in differentiation into ECs in vitro, which is possible to provide the seed cells for fabrication of tissue-engineering heart valve.