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），其通常是由于肢体外周动脉的动脉粥样硬化闭塞性疾病，引起诸如缺血性溃疡或坏疽的症状。通过再生内皮来改善肢体血流的生物学方法似乎对PAD的治疗很有希望。内皮再生的潜在细胞来源是胚胎干细胞（ESC），其是可以分化成内皮细胞（EC）的多能干细胞。然而，由于产量低（通常小于5%），直接分化成EC的能力仍然具有挑战性。已知ESC分化受到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。这是一种高通量方法，用于筛选数百种ECM和确定组成的ECM混合物，用于基质介导的ESC分化成EC谱系。因此，本研究的目的是优化有利于ESC向EC分化的基质条件，然后在体外和体内表征ESC衍生的EC的表型和血管生成特性。首先，我将在体外使用高通量ECM微阵列方法表征ESC衍生的EC的分化。微阵列将由含有单一基质蛋白或各种基质蛋白的混合物的微尺度点组成。为了便于纯化和非侵入性跟踪，我将利用ESC转导慢病毒构建体，该构建体由EC特异性启动子VE-钙粘蛋白组成，并含有编码萤火虫荧光素酶的基因用于生物发光成像和红色荧光蛋白用于免疫荧光跟踪。接下来，我将在ECM阵列上分化胚胎干细胞，并选择优化EC分化的基质组成以进行大规模分化实验。然后，我将通过荧光激活细胞分选纯化EC，并表征EC表型形态和功能的细胞。最后，使用PAD（后肢缺血）的体内模型，我将确定是否ESC衍生的EC可以在缺血肢体中存活，纳入血管系统，并通过生物发光跟踪和激光多普勒光谱血流测量来改善功能。总之，该项目将利用创新策略来增强ESCs向EC的定向分化，用于患有PAD的患者的血管再生。这项研究对公共卫生的潜在影响是降低PAD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.