Reverse engineering zonation-specific and age-specific iPSC-derived cerebrovascular models based on transcriptomic profiling of the human brain

基于人脑转录组分析的逆向工程分区特异性和年龄特异性 iPSC 衍生脑血管模型

• 批准号: 10321473
• 负责人: Myriam Heiman
• 金额: $ 83.31万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 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/10321473
• 关键词: 3-Dimensional; Age; Aging; Automobile Driving; Biochemical Process; Blood; Blood Vessels; Blood capillaries; Blood flow; Brain; Brain Diseases; Cell Line; Cell Nucleus; Cells; Cerebrovascular Disorders; Cerebrovascular system; Collaborations; Cues; Development; Disease; Disease Progression; Engineering; Functional disorder; Gene Expression; Gene Expression Profiling; Genetic Screening; Genomics; Geometry; Goals; Health; Human; Human Engineering; In Vitro; Individual; Intrinsic factor; Libraries; Maintenance; Modeling; Molecular; Neurodegenerative Disorders; Nutrient; Pathology; Perfusion; Phase; Phenotype; Play; Process; Resected; Role; Serum; Small Nuclear RNA; Source; Supporting Cell; Tissue Engineering; Tissues; Translations; Western Blotting; age related; aged; arteriole; base; brain endothelial cell; brain health; cellular transduction; cerebrovascular; induced pluripotent stem cell; next generation; physical process; protein expression; response; shear stress; stem cell technology; trafficking; transcription factor; transcriptome sequencing; transcriptomics; venule; wasting

Project Summary Cerebrovascular dysfunction is emerging as a common pathology in many diseases of the brain, including neurodegenerative diseases, cerebrovascular diseases, as well as in aging. Therefore, understanding the role of cerebrovascular dysfunction in disease progression and aging will be key to long-term maintenance of brain health. With developments in tissue engineering and stem cell technology, in vitro cerebrovascular models can play an important role in understanding the role of cerebrovascular dysfunction in disease progression and aging. Next-generation cerebrovascular models should take into account three key factors: (1) differences in phenotype of brain microvascular endothelial cells along the arterio-venous axis, (intrinsic factors), (2) differences in microenvironmental cues along the arterio-venous axis (extrinsic factors), and (3) changes in zonation-specific cerebrovascular phenotype during aging and in response to aged serum. Therefore, the overall goal of this project is to use zonation- and age-specific intrinsic and extrinsic cues to reverse engineer human cerebrovascular models, and to use these models to understand cerebrovascular phenotype during aging. We will first perform a pooled genetic screen to identify transcription factor combinations that are capable of driving source cells towards gene expression profiles of human brain microvascular endothelial cells along the arterio-venous axis (Aim 1). Three candidate induced brain microvascular endothelial cells (iBMECs) for each zone will be generated using lentiviral transduction. The top candidate for each zone will then be selected from analysis of gene and protein expression profiles (Aim 2). We will then use the three iBMECs to demonstrate zonation-specific cerebrovascular phenotype in zonation-specific models (arteriole, capillary, venule) (Aim 3). Next, we will assess the influence of young and old serum on cerebrovascular phenotype in the zonation-specific models (Aim 4). Finally, we will use the same approach to create an aged cerebrovascular model in one zone. We will create iBMECs that match the transcription factor profile of human brain microvascular endothelial cells in the aged cerebrovasculature, and then assess the role of microenvironmental cues and young/old serum on cerebrovascular phenotype. This project is a collaboration between the Searson group (JHU) with expertise in tissue-engineered microvascular models, and the Heiman group (MIT) with expertise in genomics and molecular mechanisms of neurodegenerative disease. This project builds upon key recent accomplishments from the two labs: (1) the creation of a library of zonation-specific transcription factor profiles for brain microvascular endothelial cells from the human brain, (2) identification of key transcription factors to enable reverse engineering of zonation- specific human brain microvascular endothelial cells, and (3) tissue-engineered platforms for zonation-specific cerebrovascular models.

项目摘要 脑血管功能障碍正在成为许多大脑疾病的常见病理，包括 神经退行性疾病，脑血管疾病以及衰老。因此，了解角色 疾病进展和衰老的脑血管功能障碍将是长期维持大脑的关键 健康。随着组织工程和干细胞技术的发展，体外脑血管模型可以 在理解脑血管功能障碍在疾病进展中的作用和 老化。 下一代脑血管模型应考虑三个关键因素：（1） 沿着动脉轴的脑微血管内皮细胞的表型，（内在因素），（2） 沿动脉轴（外部因素）的微环境线索的差异，以及（3）变化 衰老期间和对老化血清的反应，分区特异性的脑血管表型。因此， 该项目的总体目标是使用分区和特定年龄的内在和外在提示来反向工程师 人脑脑血管模型，并使用这些模型来理解脑血管表型 老化。我们将首先执行一个合并的遗传筛选，以识别转录因子组合 能够将源细胞驱动到人脑微血管内皮细胞的基因表达谱 沿着动脉轴轴（AIM 1）。三个候选者诱发脑微血管内皮细胞（IBMEC） 对于每个区域，将使用慢病毒转导生成。每个区域的最高候选人将是 从基因和蛋白质表达谱分析中选择（AIM 2）。然后，我们将使用三个IBMEC进行 在分区特异性模型中展示了分区特异性脑血管表型（动脉，毛细管，， 静脉）（目标3）。接下来，我们将评估年轻和老血清对脑血管表型的影响 分区特异性模型（AIM 4）。最后，我们将使用相同的方法来创建老年人 一个区域中的脑血管模型。我们将创建与人类的转录因子概况相匹配的IBMEC 老年脑血管造期的脑微血管内皮细胞，然后评估 微环境提示和脑血管表型上的年轻/老血清。 该项目是Searson Group（JHU）与组织工程专业知识之间的合作 微血管模型，以及具有基因组学和分子机制专业知识的Heiman组（MIT） 神经退行性疾病。该项目以两个实验室的最新成就为基础：（1） 创建用于脑微血管内皮细胞的分区特异性转录因子曲线库的创建 （2）识别关键转录因子以实现分区的逆向工程 - 特定的人脑微血管内皮细胞和（3）针对分区特异性的组织工程平台 脑血管模型。