Study of cell-type specific Alzheimer's disease genetic variants using a novel bioengineered model of iPSC-derived neural tissue

使用 iPSC 衍生神经组织的新型生物工程模型研究细胞类型特异性阿尔茨海默病遗传变异

• 批准号: 10399527
• 负责人: PHILIP G HAYDON
• 金额: $ 97.59万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10399527
• 关键词: 3-Dimensional; Abeta clearance; Action Potentials; Affect; Aging; Alleles; Alzheimer like pathology; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease pathology; Alzheimer' s disease patient; Alzheimer' s disease risk; Amyloid beta-42; Amyloid beta-Protein; Animal Disease Models; Animal Model; Apolipoprotein E; Apolipoproteins; Architecture; Astrocytes; Axon; Binding; Biomedical Engineering; Brain; Cell Culture Techniques; Cell Density; Cell Line; Cell Surface Proteins; Cell Transplantation; Cell model; Cells; Cholesterol Homeostasis; Code; Cognitive; Collagen; Complement; Complex; Data; Deterioration; Diffusion; Electrophysiology (science); Family; Functional disorder; Gene Expression; Genes; Genetic Polymorphism; Genotype; Growth; Homeostasis; Human; In Vitro; Induced pluripotent stem cell derived neurons; Late Onset Alzheimer Disease; Learning; Link; Mammals; Memory; Memory impairment; Metabolic; Microglia; Modeling; Mus; Mutation; Myeloid Cells; Necrosis; Nerve Degeneration; Neurodegenerative Disorders; Neuroglia; Neurons; Nutrient; Optics; Oxygen; Pathogenesis; Pathologic; Patients; Phagocytosis; Phenotype; Physiological; Population; Porifera; Proteins; Risk; Risk Factors; Rodent; Senile Plaques; Silk; Sleep; Synapses; Synaptic Transmission; Synaptic plasticity; System; TREM2 gene; Technology; Time; Tissue Model; Tissues; Transplant Recipients; Transplantation; Variant; abeta deposition; apolipoprotein E-4; base; beta secretase; brain behavior; cell type; early onset; exome; extracellular; familial Alzheimer disease; genetic variant; genome editing; genome sequencing; genome wide association study; human subject; in vivo; induced pluripotent stem cell; innovation; lipid metabolism; member; molecular phenotype; mutant; neonatal mice; neuronal excitability; novel; precursor cell; receptor; relating to nervous system; risk variant; scaffold; stem cell technology; synaptic function; synaptogenesis; three dimensional cell culture; trafficking; whole genome

ABSTRACT Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by memory impairments and cognitive deterioration. Aging is the major risk factor for AD. Furthermore, increasing evidence indicates that astrocytes and microglia are implicated in the pathogenesis of AD. The ε4 allele of the apolipoprotein E gene (APOE) has been identified as a major risk factor contributing to the pathogenesis of sporadic AD (SAD) in about 15-20% of the cases. APOE is the major apolipoprotein expressed in the human brain primarily by astrocytes and to a lesser extent by microglia, and is involved in cholesterol homeostasis, and regulates A clearance. Furthermore, genome-wide association studies (GWAS) have identified polymorphisms in genes enriched in microglia (e.g. SORL1, CR1, CD2AP, CD33, TREM2, ABCA7) and astrocytes (e.g. CLU and ABCA7) that increase the risk of developing AD. Recent advances in stem cell technology have allowed the reprogramming of primary cells from human subjects into induced pluripotent stem cells (iPSCs) and their differentiation in neurons, astrocytes and microglia. However, conventional 2D culture systems fail to recapitulate the diversity and maturation of multiple cell types and their interaction under physiological and pathological conditions. To overcome these weaknesses we have developed a novel bioengineered model of iPSC-derived neural tissue. Our silk-collagen protein-based ‘donut’ scaffolds can support compartmentalized, 3D brain-like tissues over a year, without necrosis. This tissue model is highly innovative, supporting the differentiating neurons growth in a donut-shaped porous silk sponge within an optically cleared collagen-filled central region for axon connectivity and synapse formation, that will allow for the first time live in vivo studies (e.g., cell-based electrophysiology, trafficking, synaptic functionality) of an human AD brain-like tissue during ageing (months of cultivation) under controlled experimental conditions. More importantly, the architecture of the scaffolds was optimized to meet the metabolic demand of high-density cell cultures in terms of free diffusion of nutrients and oxygen, a fundamental requisite for long-term cultures and ageing-related studies. Thus, we propose to: 1) Assess genotype-phenotype relationship of AD genetic variants enriched in astrocytes and microglia in patient-derived 3D brain-like cultures; 2) Assess genotype-phenotype relationship of AD genetic variants in vivo after transplantation of patient-derived cells in mice.

摘要 阿尔茨海默病(AD)是一种以记忆障碍为特征的进行性神经退行性疾病 以及认知能力的下降。衰老是AD的主要危险因素。此外，越来越多的证据表明 星形胶质细胞和小胶质细胞参与了AD的发病机制。载脂蛋白E的ε4等位基因 载脂蛋白E基因(APOE)是散发性阿尔茨海默病(SAD)发病的主要危险因素 在大约15%-20%的病例中。载脂蛋白E是在人脑中表达的主要载脂蛋白，主要通过 星形胶质细胞和小胶质细胞，并参与胆固醇稳态，并调节A 通行证。此外，全基因组关联研究已经确定了基因的多态。 富含小胶质细胞(如SORL1、CR1、CD2AP、CD33、TREM2、ABCA7)和星形胶质细胞(如CLU和 ABCA7)，这增加了患AD的风险。干细胞技术的最新进展使 人类原代细胞重编程为诱导多能干细胞及其生物学特性 神经元、星形胶质细胞和小胶质细胞的分化。然而，传统的2D培养系统无法 概述了多种细胞类型的多样性和成熟性及其在生理和社会环境下的相互作用 病理情况。为了克服这些弱点，我们开发了一种新的生物工程模型 IPSC衍生的神经组织。我们的丝胶原蛋白为基础的“甜甜圈”支架可以支持隔间， 一年多的3D脑样组织，没有坏死。这种组织模型具有很高的创新性，支持 在光学透明的胶原蛋白填充的多孔丝海绵中分化神经元生长 轴突连接和突触形成的中心区域，这将首次允许活体研究 (例如，基于细胞的电生理学、运输、突触功能) 在受控实验条件下老化(培养数月)。更重要的是， 对支架进行了优化，以满足高密度细胞培养的代谢需求 营养物质和氧气的扩散，这是长期培养和衰老相关研究的基本要求。 因此，我们建议：1)评估富含星形胶质细胞的AD基因变异体的基因型-表型关系 和小胶质细胞在患者来源的3D脑样培养中；2)评估AD的基因-表型关系 小鼠移植患者来源的细胞后体内的遗传变异。