Institute of Atherosclerosis, Taishan Medical University, Taian, 271000, China.
Cytotechnology. 2011 May;63(3):217-26. doi: 10.1007/s10616-010-9329-2. Epub 2011 Feb 18.
Endothelial progenitor cells (EPCs) derived from bone marrow are known to be heterogeneous. In this study, we tried to find favorable conditions that induce the differentiation of mononuclear cells (MNCs) from bone marrow into EPCs. The differentiation capacity of MNCs from rat bone marrow was investigated in different conditions, such as different media, different induction times and different culture surfaces. The cell morphology and endothelial biomarkers associated with differentiated MNCs were studied. Our results indicated that MNCs cultured in EGM-2MV (Endothelial cell basal medium-2, plus SingleQuots of growth supplements) developed a bursiform shape, a late EPC-like morphology, while MNCs cultured in complete medium (CM, M199 with 10% FBS, 20 ng/mL VEGF and 10 ng/mL bFGF) showed a spindle shape, an early EPC-like morphology. Cells of both morphologies were able to incorporate DiI-ac-LDL and bind lectin in vitro. MNCs cultured in EGM-2MV exhibited a higher proliferation rate and higher eNOS expression than MNCs cultured in CM. MNCs cultured in EGM-2MV had the ability to form tubes on Matrigel. Flow cytometry results indicated that CD133 expression was highest at day 12 and that the greatest number of cells positive for both FLK-1 and CD133 appeared at day 20 from cells cultured in dishes without fibronectin coating. In addition, the expression levels of CD133, CD31 and FLK-1/CD133 were not significantly different between cells of different shapes. Our experiments suggest that MNCs from bone marrow can be differentiated into late EP-like cells in EGM-2MV, which have the ability to rapidly proliferate. These MNCs can also be differentiated into early EP-like cells in CM. Additionally, fibronectin may not be necessary for the differentiation of EPCs to mature ECs after three generations. Differentiated MNCs from bone marrow in EGM-2MV have the characteristics of EPCs, although the expression levels of EPC markers were lower than previously reported.
骨髓来源的内皮祖细胞 (EPC) 是不均一的。在这项研究中，我们试图找到有利的条件，诱导骨髓单个核细胞 (MNC) 分化为 EPC。研究了不同条件下大鼠骨髓 MNC 的分化能力，如不同的培养基、不同的诱导时间和不同的培养表面。研究了与分化的 MNC 相关的细胞形态和内皮生物标志物。我们的结果表明，在 EGM-2MV（内皮细胞基础培养基-2，加生长补充物单剂量）中培养的 MNC 呈泡囊状，具有晚期 EPC 样形态，而在完全培养基 (CM，含 10% FBS、20ng/mL VEGF 和 10ng/mL bFGF 的 M199) 中培养的 MNC 呈梭形，具有早期 EPC 样形态。两种形态的细胞均能在体外摄取 DiI-ac-LDL 并结合凝集素。在 EGM-2MV 中培养的 MNC 的增殖率和 eNOS 表达均高于在 CM 中培养的 MNC。在 EGM-2MV 中培养的 MNC 具有在 Matrigel 上形成管的能力。流式细胞术结果表明，CD133 表达在第 12 天最高，而在无纤连蛋白包被的培养皿中培养的细胞第 20 天出现最多的同时表达 FLK-1 和 CD133 的细胞。此外，不同形态细胞的 CD133、CD31 和 FLK-1/CD133 表达水平无显著差异。我们的实验表明，骨髓中的 MNC 可以在 EGM-2MV 中分化为晚期 EP 样细胞，具有快速增殖的能力。这些 MNC 也可以在 CM 中分化为早期 EP 样细胞。此外，在经过三代后，纤连蛋白可能不是 EPC 向成熟 EC 分化所必需的。在 EGM-2MV 中分化的骨髓 MNC 具有 EPC 的特征，尽管 EPC 标志物的表达水平低于之前的报道。
