Human choroid plexus epithelial cells derived from APOE isogenic iPSCs

源自 APOE 同基因 iPSC 的人脉络丛上皮细胞

• 批准号: 9810057
• 负责人: EDWIN S MONUKI
• 金额: $ 22.22万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9810057
• 关键词: Abeta clearance; Affect; Aging; Algorithms; Alzheimer' s Disease; Alzheimer' s disease risk; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Apolipoprotein E; Astrocytes; Autolysis; Autopsy; Benign; Biology; Blood; CRISPR/Cas technology; Carrier Proteins; Cell Differentiation process; Cell Line; Cell physiology; Cells; Cerebral Ventricles; Cerebrospinal Fluid; Cleaved cell; Confocal Microscopy; Data Analyses; Databases; Dementia; Derivation procedure; Diagnostic; Disease; Elderly; Embryo; Enzymes; Epithelial Cells; Experimental Designs; Female; Flow Cytometry; Functional disorder; Gene Expression; Gene Expression Profile; Genes; Genotype; Goals; Health; Human; Human Biology; Impairment; Individual; Inherited; Laboratories; Late Onset Alzheimer Disease; Measures; Microglia; Molecular Chaperones; Monitor; Morphology; Mus; Mutation; Neurons; Onset of illness; Ontology; Pathway interactions; Patients; Peptides; Pharmacotherapy; Pluripotent Stem Cells; Population; Procedures; Protein Isoforms; Protocols documentation; Rattus; Research; Rodent; Rodent Model; Role; Services; Specimen; Stem cells; Structure of choroid plexus; Testing; Therapeutic Intervention; Thick; Time; Toxin; United States National Institutes of Health; Validation; Western Blotting; Work; abeta toxicity; apolipoprotein E-4; base; biobank; cell type; data acquisition; early onset; effective therapy; gene correction; human embryonic stem cell; in vivo; induced pluripotent stem cell; male; novel; protein transport; risk variant; single-cell RNA sequencing; transcriptome; uptake

PROJECT SUMMARY We will test the hypothesis that choroid plexus epithelial cells (CPECs) are affected in late onset Alzheimer's Disease (AD) associated with the type 4 isoform of apolipoprotein E (APOE4). At least one copy of APOE4 is present in 56-65% of people with AD, and two copies are highly predictive of AD. APOE4 has been implicated in faulty clearance of the toxic Aβ peptide associated with AD, as well as in numerous other AD-related functions in various cell types. Rodent CPECs are known to remove toxins from the cerebrospinal fluid (CSF), express high levels of APOE, and take up and transport Aβ peptides. However, human CPECs have not been well studied, in part due to the difficulty in acquiring healthy human cells. Our laboratory has developed a protocol to derive human CPECs from pluripotent stem cells, which provides the opportunity to study basic human CPEC functions and their roles in AD and other diseases. We propose in Aim 1 to derive CPECs using existing induced pluripotent stem cells (iPSCs) from APOE4/4 AD patients and from their isogenic APOE3/3 counterparts that have been edited using CRISP/Cas9. We will monitor the CPEC derivations for changes in differentiation efficiency, then test the specific hypothesis that Aβ uptake is impaired in APOE4/4 CPECs. In Aim 2, we will characterize the transcriptomes of individual human CPECs using single cell RNA sequencing (scRNAseq) to look for gene expression changes in CPEC subpopulations that correspond to the Aβ uptake findings from Aim 1. Based on a prior study that examined neurons, astrocytes, and microglia derived from APOE isogenic iPSCs, we anticipate a broad spectrum of gene expression differences that are unique to CPECs and predictive of altered functions. We will then validate scRNAseq findings by RT-qPCR and immunostaining, then cross-validate in vivo by immunostaining postmortem choroid plexus specimens from patients with known APOE genotypes. We envision this R21 proposal leading to subsequent R01 submissions that extend this work to CPECs derived from additional isogenic pairs involving APOE and other AD risk genes, to test additional emergent hypotheses regarding altered CPEC functions in AD, and to explore potential therapeutic interventions to correct impaired CPEC functions.

项目摘要 我们将检验脉络丛上皮细胞（CPEC）在晚发性阿尔茨海默病中受到影响的假设 与载脂蛋白E（APOE 4）4型亚型相关的疾病（AD）。至少有一个APOE 4的拷贝， 存在于56-65%的AD患者中，并且两个拷贝高度预测AD。APOE 4与 在与AD相关的毒性Aβ肽的错误清除中，以及在许多其他与AD相关的 在各种细胞中发挥作用。已知啮齿动物CPEC可从脑脊液（CSF）中清除毒素， 表达高水平的载脂蛋白E，并摄取和转运Aβ肽。然而，人类CPEC尚未被 这是因为很难获得健康的人体细胞。我们的实验室开发了一种 从多能干细胞中获得人类CPEC的协议，这为研究基础 人CPEC功能及其在AD和其他疾病中的作用。我们在目标1中提出，使用 来自APOE 4/4 AD患者及其同基因APOE 3/3的现有诱导多能干细胞（iPSC） 使用CRISP/Cas9编辑的对应物。我们将监测CPEC衍生物的变化， 分化效率，然后检验APOE 4/4 CPEC中Aβ摄取受损的特定假设。在 目的2：利用单细胞RNA测序技术，研究人CPEC的转录组特征 （scRNAseq）寻找CPEC亚群中与Aβ摄取对应的基因表达变化 目标1的发现基于先前的一项研究，该研究检查了来自于神经元，星形胶质细胞和小胶质细胞的神经元， APOE同基因iPSC，我们预期广泛的基因表达差异是独特的， CPEC和预测功能改变。然后，我们将通过RT-qPCR验证scRNAseq发现， 免疫染色，然后通过免疫染色死后脉络丛标本进行体内交叉验证， 已知APOE基因型的患者。我们设想这一R21提案将导致随后的R 01提交 将这项工作扩展到从涉及APOE和其他AD风险的其他同基因对衍生的CPEC 基因，以测试有关AD中CPEC功能改变的其他紧急假设，并探索 潜在的治疗干预，以纠正受损的CPEC功能。