Carneiro Giane D, Godoy Juliana A P, Werneck Claudio C, Vicente Cristina P
Department of Structural and Functional Biology, State University of Campinas (UNICAMP), São Paulo, Brazil.
Department of Biochemistry and Tissue Biology, Institute of Biology, State University of Campinas (UNICAMP), São Paulo, Brazil.
Cell Biol Int. 2015 Oct;39(10):1138-50. doi: 10.1002/cbin.10487. Epub 2015 Jun 18.
Endothelial progenitor cells (EPCs) can be isolated from bone marrow and characterized by the expression of cellular markers such as CD34, CD133, VEGFR2, CD31, and VE-Cadherin, by the uptake of acetylated low-density lipoprotein and by in vitro tube formation in tridimensional matrices. These cells are able to differentiate into mature endothelial cells and participate in the re-endothelization of damaged vessels. In this work, we tested different cultured media that can promote the proliferation and differentiation of mononuclear cells (MNCs) into early EPCs, with defined concentrations of growth factors and serum in order to establish a composition that may ensure us the reproducibility of our cultures. MNCs from mice bone marrow were cultivated using selective culture media containing DMEM or M199 supplemented with 10% FBS, VEGF, bFGF, and IGF, for 3, 7, and 14 days. Differentiation into early EPCs was analyzed using immunohistochemistry, FACS and western blotting and by functional parameters as uptake of ac-LDL, and formation of vessel-like structures. The cells cultivated with medium DMEM-M1 (DMEM plus VEGF, bFGF and IGF) expressed CD34, CD133, CD31, VEGFR2, and VE-Cadherin at all culture time-points with increased expression of these markers after 7 days. Only EPCs cultured for 30 days were able to form vessel-like structure. The uptake of ac-LDL was observed after 3, 7, 14, and 30 days, confirming the differentiation of mononuclear cells into early EPCs. DMEM-M1 was able to sustain MNCs proliferation and differentiation, increasing the expression of the characteristic EPC markers, allowing the expansion of early EPCs in culture in a similar way to that observed in commercial available media.
内皮祖细胞（EPCs）可从骨髓中分离出来，其特征在于细胞标志物如CD34、CD133、血管内皮生长因子受体2（VEGFR2）、CD31和血管内皮钙黏蛋白的表达，在于对乙酰化低密度脂蛋白的摄取，以及在于三维基质中的体外管腔形成。这些细胞能够分化为成熟的内皮细胞，并参与受损血管的再内皮化过程。在本研究中，我们测试了不同的培养基，这些培养基可促进单核细胞（MNCs）增殖并分化为早期EPCs，培养基中生长因子和血清浓度明确，目的是确定一种能够确保我们培养物可重复性的成分。使用含有DMEM或M199并补充10%胎牛血清（FBS）、血管内皮生长因子（VEGF）、碱性成纤维细胞生长因子（bFGF）和胰岛素样生长因子（IGF）的选择性培养基培养来自小鼠骨髓的MNCs，培养3天、7天和14天。使用免疫组织化学、荧光激活细胞分选术（FACS）和蛋白质免疫印迹法，并通过功能性参数如乙酰化低密度脂蛋白（ac-LDL）摄取和血管样结构形成，分析向早期EPCs的分化情况。用DMEM-M1培养基（DMEM加VEGF、bFGF和IGF）培养的细胞在所有培养时间点均表达CD34、CD133、CD31、VEGFR2和血管内皮钙黏蛋白，且在7天后这些标志物的表达增加。只有培养30天的EPCs能够形成血管样结构。在3天、7天、14天和30天后均观察到ac-LDL的摄取，证实单核细胞分化为早期EPCs。DMEM-M1能够维持MNCs的增殖和分化，增加特征性EPC标志物的表达，使早期EPCs在培养中得以扩增，其方式与市售培养基中观察到的相似。
