Zeng Gefei, Bautch Victoria L
Department of Biology, Carolina Cardiovascular Biology Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Methods Mol Biol. 2009;482:333-44. doi: 10.1007/978-1-59745-060-7_21.
Embryonic stem (ES) cells, which are derived from developing mouse blastocysts, have the ability to differentiate into various cell types in vitro. When placed in basal medium with added serum, mouse ES cells undergo a programmed differentiation favoring formation of cell types that are found in the embryonic yolk sac, including vascular endothelial cells. These in vitro differentiated endothelial cells form primitive blood vessels, analogous to the first vessels that form in the embryo and the yolk sac. This differentiation model is ideal for both genetic and pharmacological manipulation of early vascular development. We have made mouse ES cell lines that express endothelial-specific GFP or H2B-GFP and used these lines to study the processes of mammalian vessel development by real-time imaging. Here we describe protocols for making transgenic ES cells and imaging the processes of blood vessel development. We also provide methods for ES cell maintenance and differentiation, and methods for analysis of vascular marker expression.
胚胎干细胞（ES细胞）来源于发育中的小鼠囊胚，具有在体外分化为各种细胞类型的能力。当置于添加血清的基础培养基中时，小鼠ES细胞会经历程序性分化，倾向于形成胚胎卵黄囊中发现的细胞类型，包括血管内皮细胞。这些体外分化的内皮细胞形成原始血管，类似于胚胎和卵黄囊中形成的第一批血管。这种分化模型对于早期血管发育的基因和药理学操作来说是理想的。我们已经构建了表达内皮特异性绿色荧光蛋白（GFP）或组蛋白H2B-GFP的小鼠ES细胞系，并利用这些细胞系通过实时成像研究哺乳动物血管发育过程。在此，我们描述了制备转基因ES细胞和对血管发育过程进行成像的方案。我们还提供了ES细胞维持和分化的方法，以及血管标志物表达分析的方法。
