Investigating the pathogenesis of Moyamoya Disease using patient derived induced pluripotent stem cells

使用患者来源的诱导多能干细胞研究烟雾病的发病机制

基本信息

批准号：
10487543
负责人：
GARY K STEINBERG
金额：
$ 19.68万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-15 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10487543
关键词：
3-Dimensional Adult Affect Angiogenic Factor Animal Model Arteries Biological Assay Blood Vessels Brain hemorrhage Bypass Caliber Cell Communication Cell Culture Techniques Cell Differentiation process Cell Proliferation Cell Survival Cell model Cells Cerebral Infarction Cerebrospinal Fluid Cerebrovascular Disorders Cerebrovascular system Characteristics Child Chronic Clinical Coculture Techniques Complex Cues Data Development Disease Disease Progression Disease model Endothelial Cells Environmental Risk Factor Etiology Extracellular Matrix Future Gene Expression Genetic Grant Growth High-Throughput Nucleotide Sequencing Human Hypoxia In Vitro Inflammation Ischemic Stroke Knowledge Lead Length Measures Mediating Mediator of activation protein Methods Modeling Molecular Molecular Target Moyamoya Disease Nervous System Physiology Operative Surgical Procedures Organoids Paralysed Pathogenesis Pathogenicity Pathologic Pathology Pathway Analysis Pathway interactions Patients Pattern Pharmaceutical Preparations Phenotype Platelet-Derived Growth Factor Play Process Proliferating Property Reporting Research Role Sampling Serum Signal Transduction Smooth Muscle Myocytes Stroke Techniques Technology Thinness Transient Ischemic Attack Translating Tube Up-Regulation Vascular Proliferation Vascular Smooth Muscle angiogenesis base cell type cerebral artery clinically relevant functional disability induced pluripotent stem cell insight migration novel therapeutics phenotypic biomarker revascularization surgery transcriptome transcriptome sequencing vascular smooth muscle cell proliferation

项目摘要

PROJECT SUMMARY Moyamoya Disease (MMD) is a rare, chronic cerebrovascular disease that affects the blood vessels of the brain, causing occlusion of major cerebral arteries and formation of fragile vessels in the vicinity. Clinical manifestations of MMD are transient ischemic attacks and cerebral infarctions, often leading to ischemic or hemorrhagic stroke. Invasive revascularization surgery is the only current treatment available. There may be a combination of genetic, circulating and environmental factors involved in the pathogenesis of MMD, however, the molecular mechanisms underlying MMD is largely unknown, mainly due to the lack of established MMD-specific cellular or animal models. In this proposal we aim to understand the pathogenesis of MMD by using MMD patient- derived iPSCs cellular models in combination with functional assays and high throughput sequencing approaches. The main histopathological finding in MMD is the fibro-cellular thickening of the innermost layer of the vessel (intima) which causes narrowing and occlusion of the vessel. This is likely due to an increase in proliferating vascular smooth muscle cells (VSMCs) or endothelial cells (ECs) and extracellular matrix components. Cues from ECs could cause VSMCs to switch to a phenotype that is proliferative and migrates from media to the intima, thus contributing to the thickening of the intima. Thus, we hypothesize that dysregulated signaling between VSMCs and ECs drive MMD pathology. Using MMD patient iPSC-derived ECs and VSMCs, we have established co-culture model and 3D cellular model by generating vascular organoids. Preliminary co-culture data show that both MMD ECs and VSMCs are functionally impaired when compared to healthy controls, with respect to cell proliferation and in vitro angiogenic tube stabilization. In Aim 1, we aim to characterize the functional properties of MMD iPSC-derived ECs and VSMCs in co-cultures by assessing their ability in cell proliferation, migration and tube formation in normal and hypoxic conditions. VSMC phenotype switching will be examined using specific phenotypic markers and contractility assay. We will also characterize vessel structural characteristics using our established vascular organoids generated from MMD iPSCs. In Aim 2, we will use RNA sequencing technology to investigate the transcriptome of VSMCs and ECs and identify potential molecular mediators involved in MMD. Top targets will be validated using quantitative PCR and their expression pattern will be investigated in our cellular models using immunostaining. Our study will elucidate cell-type specific factors that may drive MMD pathology. Vascular organoids from MMD may be an efficient human in vitro MMD model and provide invaluable information on MMD mechanisms. Data from our studies will advance the knowledge in MMD pathogenesis and open up new avenues of research to yield clinically relevant drug-based methods to treat MMD.

项目摘要 Moyamoya病（MMD）是一种罕见的慢性脑血管疾病，会影响大脑的血管，引起大脑主要动脉的阻塞和附近脆弱血管的形成。临床表现 MMD的是短暂的缺血发作和脑梗塞，通常导致缺血性或出血性中风。侵入性血运重建手术是唯一可用的治疗方法。可能有遗传的组合，但是，MMD发病机理涉及的循环和环境因素，但是分子机制潜在的MMD在很大程度上是未知的，这主要是由于缺乏已建立的MMD特异性细胞或动物型号。在此提案中，我们旨在通过使用MMD患者衍生的IPSC来了解MMD的发病机理蜂窝模型与功能测定和高通量测序方法结合使用。 MMD中的主要组织病理学发现是血管最内层的纤维细胞增厚（intima）会导致船只的狭窄和阻塞。这可能是由于增殖的增加血管平滑肌细胞（VSMC）或内皮细胞（ECS）和细胞外基质成分。提示从ECS可能会导致VSMC切换到增殖的表型，并从媒体迁移到内膜，因此有助于内膜的增厚。因此，我们假设信号失调在VSMC和ECS之间驱动MMD病理学。使用MMD患者IPSC衍生的ECS和VSMC，我们有通过产生血管类器官建立的共培养模型和3D细胞模型。初步共培养数据表明，与健康对照组相比，MMD ECS和VSMC在功能上都受损涉及细胞增殖和体外血管生成管稳定。在AIM 1中，我们旨在表征 MMD IPSC衍生的EC和VSMC在共培养中的功能特性通过评估其在细胞中的能力在正常和低氧条件下的增殖，迁移和管形成。 VSMC表型切换将是使用特定的表型标记和收缩性测定法检查。我们还将表征船只结构使用MMD IPSC产生的我们已建立的血管类动物的特征。在AIM 2中，我们将使用RNA 测序技术研究VSMC和EC的转录组并识别潜在的分子参与MMD的调解人。最高目标将使用定量PCR及其表达模式进行验证将在我们的细胞模型中使用免疫染色进行研究。我们的研究将阐明细胞类型的特定因素这可能会驱动MMD病理。来自MMD的血管器官可能是有效的人体体外MMD模型并提供有关MMD机制的宝贵信息。我们研究的数据将提高知识 MMD发病机理并开放了新的研究途径，以产生临床上相关的药物方法治疗MMD。