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

基本信息

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
项目类别：
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&apos s Disease Alzheimer&apos 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

项目摘要

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.

项目概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 (APOE4) 4 型亚型相关的疾病 (AD) 至少有一个 APOE4 拷贝。 APOE4 存在于 56-65% 的 AD 患者中，其中两个拷贝与 AD 高度相关。与 AD 相关的有毒 Aβ 肽的错误清除，以及许多其他与 AD 相关的疾病已知啮齿类动物 CPEC 可以清除脑脊液 (CSF) 中的毒素，表达高水平的 APOE，并吸收和转运 Aβ 肽。然而，人类 CPEC 尚未被研究。经过充分研究，部分原因是由于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衍生人类 CPEC 的协议，提供了研究基础的机会我们在目标 1 中建议使用人类 CPEC 功能及其在 AD 和其他疾病中的作用来推导 CPEC。来自 APOE4/4 AD 患者及其同基因 APOE3/3 的现有诱导多能干细胞 (iPSC) 我们将监控 CPEC 推导中的变化。分化效率，然后测试 APOE4/4 CPEC 中 Aβ 摄取受损的具体假设。目标 2，我们将使用单细胞 RNA 测序来表征个体人类 CPEC 的转录组 (scRNAseq) 寻找 CPEC 亚群中与 Aβ 摄取相对应的基因表达变化目标 1 的发现。基于之前一项检查神经元、星形胶质细胞和小胶质细胞的研究 APOE 同基因 iPSC，我们预计存在广泛的基因表达差异，这些差异是然后，我们将通过 RT-qPCR 和功能验证 scRNAseq 结果。免疫染色，然后通过对死后脉络丛标本进行免疫染色进行体内交叉验证我们预计 R21 提案将导致后续 R01 提交。将这项工作扩展到源自涉及 APOE 和其他 AD 风险的其他同基因对的 CPEC 基因，测试有关 AD 中 CPEC 功能改变的其他新假设，并探索纠正受损的 CPEC 功能的潜在治疗干预措施。