Feng Bin, Liu Ying-Long, Feng Kai, Gong Ru, Chen Hu
Department of Cardiac Surgery, Cardiovascular Institute and Fuwai Hospital, CAMC & PUMC, Beijing 100037, China.
Zhongguo Ying Yong Sheng Li Xue Za Zhi. 2005 Aug;21(3):340-3.
To 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.
MSCs 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.
The 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.
MSCs 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.
从人骨髓（HBM）中扩增间充质干细胞（MSCs），并在体外诱导MSCs分化为内皮细胞（ECs），探讨其可能性及条件。
采用Percoll（密度1.073 g/ml）梯度离心法从HBM中分离MSCs，接种于含10%胎牛血清（FBS）的低糖DMEM培养基中进行纯化和扩增培养。采用流式细胞术分析其表型特征，将MSCs接种于含20% FBS和血管内皮生长因子（VEGF，10 ng/ml）的高糖DMEM培养基中进行定向诱导分化为ECs，诱导培养14 - 21天。之后，采用抗Tie-2单克隆抗体免疫组织化学法及透射电子显微镜（TEM）观察细胞质内的W-P小体对分化细胞进行鉴定。
原代培养时MSCs数量为5.0 - 10⁵，经15代培养后增加至8.0×10¹²，扩增了1.6×10⁷倍。第2代和第3代MSCs纯度分别高于95%和98%。光学显微镜下观察到MSCs经14 - 21天诱导分化培养后呈现典型的“鹅卵石”样外观。免疫组织化学检测显示，超过90%的细胞Tie-2相关抗原呈阳性染色。TEM观察到细胞质内有典型ECs形态的W-P小体。
HBM来源的MSCs在体外具有分化为ECs的能力，有望为组织工程心脏瓣膜构建提供种子细胞。
