High throughput screening of embryonic stem cell differentiation

描述（由申请人提供）：超过800万美国人患有外周动脉疾病（PAD），这通常是由于四肢外周动脉的动脉粥样硬化性闭塞性疾病，引起缺血性溃疡或坏疽等症状。通过再生内皮细胞来改善肢体血流的生物学方法似乎有望治疗PAD。胚胎干细胞（ESCs）是内皮细胞再生的潜在细胞来源，胚胎干细胞是一种多能干细胞，可以分化为内皮细胞（ECs）。然而，由于产量低（通常低于5%），直接分化为ec的能力仍然具有挑战性。已知ESC分化受条件培养基中的生长因子和它们生长的基质的影响。关于确定ESC分化的最佳基质组成，我建议利用细胞外基质（ECM）微阵列。这是一种高通量的方法，用于筛选数百种ECM和定义成分的ECM混合物，用于基质介导的ESC分化为EC谱系。因此，本项目的目标是优化有利于ESC分化为内皮细胞的基质条件，然后在体外和体内表征ESC衍生的内皮细胞的表型和血管生成特性。首先，我将使用高通量ECM微阵列方法表征体外esc来源的ECs的分化。微阵列将由含有单一或混合各种基质蛋白质的微尺度斑点组成。为了便于纯化和非侵入性跟踪，我将利用由慢病毒结构转导的ESCs，该结构由ec特异性启动子ve -钙粘蛋白组成，并包含编码萤火虫荧光素酶的基因，用于生物发光成像和用于免疫荧光跟踪的红色荧光蛋白。接下来，我将在ECM阵列上分化esc，并选择优化EC分化的基质组成进行大规模分化实验。然后，我将通过荧光活化细胞分选纯化EC，并对EC的表型形态和功能进行细胞表征。最后，利用PAD（后肢缺血）的体内模型，我将通过生物发光跟踪和激光多普勒光谱血流测量来确定esc来源的内皮细胞是否能在缺血肢体中存活，融入血管系统，并改善功能。总之，该项目将利用创新的策略来增强内皮细胞向内皮细胞的定向分化，用于PAD患者的血管再生。这项研究对公共卫生的潜在影响是降低患PAD的患者的死亡率和发病率。

DESCRIPTION (provided by applicant): Over 8 million Americans suffer from peripheral arterial disease (PAD), which is typically due to atherosclerotic occlusive disease of the peripheral arteries of the limbs, causing symptoms such as ischemic ulcerations or gangrene. Biological approaches to improve limb blood flow by regenerating the endothelium seem promising for treatment of PAD. A potential cell source for endothelial regeneration is embryonic stem cells (ESCs), which are pluripotent stem cells that can differentiate into endothelial cells (ECs). However, the ability to direct differentiation into ECs remains challenging due to the low yields (typically less than 5%). ESC differentiation is known to be affected by growth factors in the conditioned medium and by the matrix upon which they are grown. With respect to determining the optimal matrix composition on ESC differentiation, I propose to utilize extracellular matrix (ECM) microarrays. This is a high-throughput approach for screening several hundreds of ECMs and ECM mixtures of defined compositions for matrix- mediated ESC differentiation into EC lineage. Therefore, the goal of this project is optimize matrix conditions that favor ESC differentiation into ECs, and then characterize the phenotype and angiogenic properties of ESC-derived ECs in vitro and in vivo. First, I will characterize the differentiation of ESC-derived ECs in vitro using a high throughput ECM microarray approach. The microarray will be comprised of micro-scale spots containing single or mixtures of various matrix proteins. For easy purification and non-invasive tracking, I will utilize ESCs transduced with a lentiviral construct that consists of an EC-specific promoter, VE-cadherin, and contains genes encoding firefly luciferase for bioluminescence imaging and red fluorescent protein for immunofluorescent tracking. Next, I will differentiate ESCs on the ECM array and select the matrix composition that optimizes EC differentiation for large-scale differentiation experiments. I will then purify ECs by fluorescently activated cell sorting and characterize the cells for EC phenotypic morphology and function. Finally, using an in vivo model of PAD (hindlimb ischemia), I will determine whether ESC-derived ECs can survive in the ischemic limb, incorporate into the vasculature, and improve function by bioluminescence tracking and laser Doppler spectroscopy flow measurement. In summary, this project will utilize innovative strategies to enhance directed differentiation of ESCs into ECs for vascular regeneration for patients suffering from PAD. The potential impact of this research to public health is a reduction in mortality and morbidity among patients suffering from PAD.