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.

项目总结 我们将验证脉络丛上皮细胞(CPECs)在晚发性阿尔茨海默病中受到影响的假设 与载脂蛋白E(APOE4)4型亚型相关的疾病。至少有一份APOE4是 在56-65%的AD患者中存在，两个拷贝对AD有很高的预测性。ApoE4已被牵连 与AD相关的有毒Aβ肽以及许多其他与AD相关的疾病的错误清除 在各种细胞类型中发挥作用。众所周知，啮齿动物的CPEC可以从脑脊液(CSF)中清除毒素， 表达高水平的载脂蛋白E，并摄取和转运Aβ多肽。然而，人类的CPEC还没有 研究得很好，部分原因是很难获得健康的人类细胞。我们的实验室已经研制出一种 从多能干细胞获得人CPECs的方案，这为研究基础提供了机会 人类CPEC功能及其在AD等疾病中的作用我们在目标1中建议使用以下方法推导CPEC 来自APOE4/4 AD患者及其同基因APOE3/3的现存诱导多能干细胞(IPSCs) 已使用CRISP/CAS9编辑的对应文件。我们将监控中巴经济走廊派生的变化 分化效率，然后检验Aβ摄取在APOE4/4CPEC中受损的特定假设。在……里面 目的2，我们将使用单细胞RNA测序来鉴定单个人CPECs的转录 (ScRNAseq)寻找中巴经济走廊亚群中与Aβ摄取相对应的基因表达变化 AIM的发现1.基于先前的一项研究，该研究检测了来自 APOE等基因IPSCs，我们预计会有广泛的基因表达差异，这些差异是 CPECs和功能改变的预测。然后我们将通过RT-qPCR验证scRNAseq结果和 免疫染色，然后通过死后脉络丛标本的免疫染色在体内交叉验证 APOE基因已知的患者。我们设想这份R21提案将导致后续的R01提交 这将这项工作扩展到来自涉及APOE和其他AD风险的额外同基因对的CPEC 基因，测试关于AD患者CPEC功能改变的其他紧急假说，并探索 纠正中巴经济走廊受损功能的潜在治疗干预。