Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York, USA.
Stem Cells Dev. 2012 Jan 20;21(2):331-42. doi: 10.1089/scd.2010.0587. Epub 2011 Apr 27.
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 β1 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.
血管生成是发育过程中的一个重要事件，它在新血管生成中起着关键作用。建立一种能够对血管生长进行活体成像的体外模型，将有助于研究血管生长的分子和细胞机制。胚胎干细胞（ESCs）被认为是一种新型可再生资源，可用于衍生遗传可操作的内皮细胞（ECs）。为了衍生表达绿色荧光蛋白（GFP）的 ECs，我们使用了一种转基因 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 天胚体衍生的 ESC-EC。将聚集的 ESC-EC 嵌入三维胶原凝胶基质中，导致 ESC-EC 从聚集物中迁移并融合成毛细血管网络。延时显微镜观察到在此过程中 EC 的迁移、增殖、管腔形成和与其他毛细血管的吻合，这使人联想到血管生成过程。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。β1 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，我们的体外 ESC-EC 血管生成模型代表了一个用于观察血管生成级联反应下细胞事件的高分辨率动态视频图像系统。我们还认为该模型是鉴定促血管生成和抗血管生成分子的图像筛选工具。
