Amplification of mesenchymal stem cells from human bone marrow and orientation to induce MSCs differentiating into endothelial cells in vitro

AIM: Our purpose was to induce MSCs differentiating into endothelial cells (EC) in vitro and to provide the seed cells for study of cardiovascular tissue-engineering. METHODS: MSCs were separated by gradient centrifugation on Percoll (density 1 073 g/L) from human bone marrow (HBM), and incubated for purification and amplification in DMEM (low glucose) with 10% fetal bovine serum (FBS). Then, the MSCs were incubated for orientation differentiated into EC in DMEM (high glucose) with 20% FBS, VEGF (10 μg/L), bFGF (5 μg/L), L-glutamine (2 mmol/L), penicillin (1×105U/L) and streptomycin (100 mg/L) for about 14-21 days and their phenotypic characteristics were analyzed by flow cytometry. Afterwards, the differentiating cells were evaluated by histology and immunohistochemistry. RESULTS: The quantity of MSCs was increased from 5.0×105 in the primary culture to 8.0×1012, or to increase to 1.6×107 times after 15 generations of incubation. The purity of MSCs was above 95% and 98% homogeneous at passages 2 and 3, respectively. About 80%-90% of the differentiating cells from MSCs after 14-21 days were positively stained for Ⅷ factor (vWF) related antigen by immunohistochemistry assay, and Weible-palade corpuscle was also observed by transmission electron microscopy in the cytoplasm. CONCLUSION: MSCs from HBM have the capability of differentiation into ECs in vitro, which may be a potential source of seed cells for fabrication of tissue-engineering heart valve, particularly in children with congenital heart disease.