Matrix-mediated endothelial differentiation of induced pluripotent stem cells

描述(申请人提供)：美国有超过800万人患有外周动脉疾病(PAD)。PAD的一个特征是血管内皮细胞功能障碍或损伤，血管内皮细胞(ECs)控制着血管的反应性、重塑和血管生成。以细胞为基础的方法恢复或再生血管内皮细胞，从而增强对缺血的血管生成反应，有望为PAD的治疗带来希望。内皮细胞的一个候选来源是诱导多能干细胞(IPSCs)，它来自重新编程的体细胞。IPSCs保持无限的自我更新和分化为心血管血统的能力，包括内皮细胞。为了将IPSCs用于治疗，必须首先将细胞分化为感兴趣的谱系，然后有效地将其输送到缺血性疾病的部位。干细胞的表型和功能受到微环境信号的影响，包括细胞外基质(ECM)，这是一种生物支架材料，提供结构支持并调节细胞功能和表型。细胞外基质对细胞行为的调控是由整合素跨膜受体介导的，它将细胞外基质连接到细胞内的细胞骨架上，并激活下游的信号通路。ECM已被证明可以提高多能干细胞EC谱系的产量，但这些ECM是否对EC分化是最佳的尚不清楚，因为还没有系统的研究来评估基质介导的分化的作用。本项目的目的是明确在PAD动物模型中，ECMS在IPSCs分化为ECs、维持EC表型和治疗促进血管生成方面的作用。该项目将利用高通量的细胞外基质微阵列平台来优化基质介导的IPSC向内皮细胞分化的效率。ECM-整合素相互作用在EC分化和维持过程中的机制作用也将被研究。最后，IPSC来源的ECs和ECM将在PAD的动物模型中用于血管再生。通过对细胞外基质介导的分化机制和血管生成功能的基本了解，申请人打算为IPSC来源的血管修复内皮细胞的临床开发和应用提供更坚实的知识基础和改进的方法。申请者寻求在使用生物工程和分子细胞生物学技术推进血管疾病治疗方面建立终身教职的学术生涯。申请者是斯坦福大学医学院的博士后研究员，目前正在约翰·库克博士的研究小组接受干细胞和分子细胞技术方面的培训。库克博士是内皮生物学和PAD疗法领域的知名研究员。干细胞开发、基质生物学、组织工程、生物材料和生物统计学领域的知名专家将为她的培训和向独立的过渡提供其他指导。 公共卫生相关性：该项目的目标是确定细胞外基质在诱导多能干细胞分化为内皮细胞、维持内皮细胞表型以及促进血管生成以修复外周动脉疾病方面的作用。该项目可导致开发修复血管疾病的新疗法，这对公众健康具有深远影响。

DESCRIPTION (provided by applicant): Over 8 million people in the US suffer from peripheral arterial disease (PAD). A feature of PAD is dysfunction or damage to the vascular endothelium, a layer of endothelial cells (ECs) that exerts control over vascular reactivity, remodeling and angiogenesis. Cell-based approaches to restore or regenerate the endothelium so as to enhance the angiogenic response to ischemia hold promise for the treatment of PAD. A candidate source of ECs is induced pluripotent stem cells (iPSCs), which are derived from reprogrammed somatic cells. The iPSCs maintain unlimited self renewal and the ability to differentiate into cardiovascular lineages, including ECs. In order to utilize iPSCs therapeutically, the cells must first be differentiated into the lineage of interest and then delivered efficiently to the site of ischemic disease. Stem cell phenotype and function are influenced by microenvironmental cues including the extracellular matrix (ECM), a biological scaffolding material that provides structural support and modulates cellular function and phenotype. ECM regulation of cell behavior is mediated by integrin transmembrane receptors that connect the ECM to the intracellular cytoskeleton and activate downstream signaling pathways. ECMs have been shown to enhance the yields of EC lineages of pluripotent stem cells, but whether these ECMs are optimal for EC differentiation is unknown because there has been no systematic study to assess the role of matrix-mediated differentiation. The goal of this project is to define the role of ECMs in the differentiation of iPSCs into ECs, maintenance of EC phenotype, and therapeutic enhancement of angiogenesis in animal models of PAD. This project will utilize a high-throughput ECM microarray platform to optimize the efficiency of matrix-mediated iPSC differentiation into ECs. The mechanistic role of ECM-integrin interactions during EC differentiation and maintenance will also be examined. Finally, iPSC-derived ECs and ECMs will be assessed in animal models of PAD for vascular regeneration. By gaining fundamental insights into mechanisms of ECM-mediated differentiation and angiogenic function, the applicant intends to provide a stronger foundation of knowledge and improved methods for the clinical development and application of iPSC-derived ECs for vascular repair. The applicant seeks to establish a tenure-track academic career in advancing the treatment of vascular diseases using bioengineering and molecular cell biology techniques. The applicant is a postdoctoral fellow in the Stanford University School of Medicine, where she is being trained in stem cell and molecular cellular techniques in the research group of Dr. John Cooke, a well-established investigator in the field of endothelial biology and PAD therapies. Additional guidance in her training and transition to independence will be provided by renowned experts in the fields of stem cell development, matrix biology, tissue engineering, biomaterials, and biostatistics. PUBLIC HEALTH RELEVANCE: The goal of this project is to define the role of extracellular matrices in the differentiation of induced pluripotent stem cells into endothelial cells, maintenance of endothelial phenotype, and therapeutic enhancement of angiogenesis for repair of peripheral arterial disease. This project can result in the development of new treatments to repair vascular diseases, which has far-reaching impact on public health.