In Vitro Imaging of Angiogenesis Using Embryonic Stem Cell-Derived Endothelial Cells
In Vitro Imaging of Angiogenesis Using Embryonic Stem Cell-Derived Endothelial Cells
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DOI：
10.1089/scd.2010.0587
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发表时间：
2012-01-01
影响因子：
4
通讯作者：
Stuhlmann, Heidi
中科院分区：
文献类型：
--
作者：
Li, Jia;Stuhlmann, Heidi
血管生成是发育过程中的重要事件，在新生血管形成中起着关键作用。发展一种可用于血管生长实时成像的体外模型，将有助于研究血管生长的分子和细胞机制。胚胎干细胞（Embryonic stem cells，ESCs）被认为是一种新的可再生来源，可用于遗传操作的内皮细胞（endothelial cells，ECs）的衍生。为了获得表达绿色荧光蛋白（GFP）的EC，我们使用了转基因ESC系，其中GFP报告基因由内皮特异性启动子胎肝激酶1驱动。通过荧光激活细胞分选从11天的胚状体中分离ESC-EC。将聚集的ESC-EC包埋在三维胶原凝胶基质中导致ESC-EC从聚集体中迁移出来并聚结成毛细血管网络。延时显微镜显示EC迁移，增殖，管腔形成，并吻合到其他毛细血管在这个过程中，这是血管生成过程的回忆。血管内皮生长因子在体外诱导ESC-EC血管生成中起主要作用。β 1整合素亚基的阻断严重损害了ESC-EC的存活和迁移。我们证明，我们在体外ESC-EC血管生成模型是一个高分辨率的动态视频图像系统，用于观察血管生成级联反应的细胞事件。我们也认为这个模型作为一个图像筛选工具，用于识别促血管生成和抗血管生成分子。
Angiogenesis is an important event during developmental processes, and it plays a key role in neovascularization. The development of an in vitro model that can be used for live imaging of vessel growth will facilitate the study of molecular and cellular mechanisms for the growth of blood vessels. Embryonic stem cells (ESCs) are considered to be a novel renewable source for the derivation of genetically manipulable endothelial cells (ECs). To derive green fluorescence protein (GFP)-expressing ECs, we used a transgenic ESC line in which a GFP reporter was driven by the endothelial-specific promoter fetal liver kinase 1. ESC-ECs were isolated from 11-day embryoid bodies by fluorescence-activated cell sorting. Embedding the aggregated ESC-ECs in a 3-dimensional collagen gel matrix resulted in ESC-EC migration out of the aggregates and coalescence into a capillary network. Time-lapse microscopy revealed EC migration, proliferation, lumen formation, and anastomosis to other capillary vessels during this process, which were reminiscent of angiogenic processes. Vascular endothelial growth factor plays major roles in the induction of ESC-EC angiogenesis in vitro. Blockage of the beta 1 integrin subunit severely impaired ESC-EC survival and migration. We demonstrate that our in vitro ESC-EC angiogenesis model represents a high-resolution dynamic video-image system for observing the cellular events underlying angiogenic cascades. We also consider this model as an image screening tool for the identification of pro-angiogenic and anti-angiogenic molecules.