Induction of cardio-vascular progenitor cells and blood vessel formation by ES cell differentiation.

在脊椎动物胚胎发生过程中，初级神经丛和随后由大小血管组成的维管树的形成机制在很大程度上是未知的。小鼠胚胎干细胞体外分化系统将复杂的血管发育过程分解为可实时监测的细胞事件。我们已经开发了一个培养系统，内皮细胞、心肌细胞和平滑肌细胞从一个来源于胚胎干细胞的中胚层前体细胞分化而来。该培养系统在中胚层细胞群中发现了三能性心血管祖细胞。在基质细胞层上生成的ES细胞衍生的内皮细胞集落已被用作体外模型，以研究单个内皮细胞对各种血管生成刺激（如VEGF）的反应行为。VEGF信号可诱导内皮细胞伸长和分散。我们发现叉头型转录吸收因子Foxo1作为内皮细胞伸长反应的调控分子，这可能是Foxo1缺陷小鼠胚胎中血管生成严重异常的原因。我们制备了在VE-cadherin基因启动子/增强子调控下表达VE-cadherin- venus或claudin 5-VENUS的ES细胞克隆。对ES细胞衍生的内皮细胞集落的延时分析显示内皮细胞的活跃运动。尽管内皮细胞具有高运动性，但VE-cadherin-based粘附连接和claudin - 5-based紧密连接蛋白的完整性得以维持。在移动的内皮细胞的前沿，发现了一个细胞连接的动态重塑过程。内皮细胞运动调节可能是在细胞水平上驱动血管发育的一个重要过程。利用VE-cadherin启动子- cre转基因小鼠进行的命运追踪分析表明，心肌缺血激活了VE-cadherin启动子在原有血管细胞和参与新生血管形成的骨髓细胞中的作用。我们还证明了转录因子c-Myb的过度表达增加了内皮细胞群体中造血前体细胞的频率，这为小鼠胚胎发生过程中造血内皮细胞如何形成最终的造血细胞系提供了线索。少

The mechanism by which primary plexus and subsequent vascular tree which consists of large and small vessels form in the process of vertebrate embryogenesis is largely unknown. In vitro differentiation system of murine ES cells serves as a means to break down the complicated processes of vascular development into cellular events which can be monitored in real time. We have developed a culture system in which endothelial cells, cardiomyocytes and smooth muscle cells differentiate from a single mesodermal precursor cell derived from ES cells. This culture system identified tri-potent cardio-vascular progenitor cells in the mesodermal cell population.ES cell-derived endothelial cell colonies generated on stromal cell layer have been used as an in vitro model to examine the behavior of individual endothelial cells in response to various angiogenic stimuli such as VEGF. The VEGF signal was shown to induce elongation and dispersion of endothelial cells. We identified a forkhead-type transcri … More ption factor Foxo1 as a regulatory molecule in the elongation reaction of endothelial cells, which might account for the severe abnormality of angiogenesis found in foxo1-deficient mouse embryos.We produced ES cell clones which express VE-cadherin-VENUS or claudin 5-VENUS under the regulation of VE-cadherin gene promoter/enhancer. Time-lapse analysis of ES cell-derived endothelial cell colonies revealed an active movement of endothelial cells. Integrity of the VE-cadherin-based adherens junction and the claudin 5-based tight junctin was maintained despite the high motility of endothelial cells. A dynamic remodeling process of cell junctions was identified at the leading edges of moving endothelial cells. Modulation of endothelial cell motility might be an important process which drives the vascular development at the cell level.Fate tracing analyses by using VE-cadherin promoter-Cre transgenic mice showed that cardiac ischemia activate VE-cadherin promoter in both pre-existing vascular cells and bone marrow cells involved in neovascularization. We also demonstrated that over-expression of a transcription factor c-Myb increases the frequency of hemogenic precursors in the endothelial cell population, which provides a clue to how hemogenic endothelial cells commit to definitive hematopoietic cell lineages during the mouse embryogenesis. Less