Modeling Genetic Inner Ear Disorders with Human Pluripotent Stem Cells

用人类多能干细胞模拟遗传性内耳疾病

基本信息

批准号：
9214594
负责人：
Eri Hashino
金额：
$ 66.41万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-12-04 至 2021-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9214594
关键词：
Achievement Afferent Neurons Binding Biochemical Biological Assay Biological Models Biopsy CHARGE syndrome CHD7 gene CRISPR/Cas technology Cells Clinical Complex Congenital Disorders DNA Sequence Alteration Defect Derivation procedure Disease ES Cell Line Electron Microscopy Electrophysiology (science)Enzymes Epithelium Etiology Exhibits Functional disorder Gene Silencing Gene Targeting Genes Genetic Genetic Models Genetic Predisposition to Disease Goals Hair Cells Histologic Human Human Genetics Impairment In Vitro Labyrinth Mendelian disorder Methods Modeling Mosaicism Mus Mutation Organoids Otic Vesicle Pathogenesis Pathologic Patients Phenotype Play Point Mutation Procedures Property Proteins Role SHH gene Sensory Sensory Hair Stem cells Structure Supporting Cell Syndrome System Technology Testing Tissues Transduction Gene Transgenic Organisms Vestibular Hair Cells Viral Vector WNT Signaling Pathway Zebrafish base chromatin remodeling clinical phenotype deafness design early onset equilibration disorder experimental study genome editing hearing impairment histone methylation histone methyltransferase human embryonic stem cell human embryonic stem cell line human pluripotent stem cell human stem cells in vitro Model inner ear diseases interdisciplinary approach malformation mechanotransduction mutant novel patch clamp progenitor smoothened signaling pathway stem therapeutic target three dimensional cell culture virtual

项目摘要

PROJECT SUMMARY Genetic mutations cause congenital or progressive inner ear disorders in humans. Despite the recent progress in human genetics for identifying syndromic and nonsyndromic genes, little is known about how mutations in these genes contribute to the clinical features of patients with cochlear and/or vestibular dysfunction. In order to overcome limitations stemming from a paucity of human inner ear tissues available for experimentation, we recently established a method for deriving inner ear sensory epithelia harboring functional sensory hair cells from human pluripotent stem cells in 3D culture. These stem cell-derived tissues, designated as “human inner ear organoids,” harbor a layer of tightly packed hair cells whose structural, biochemical and functional properties are indistinguishable from native sensory hair cells in the human inner ear. The primary goal of this application is to investigate pathophysiology of two monogenetic inner ear disorders, CHARGE syndrome and DFNA36/DFNB7/11 deafness, with human inner ear organoids as a model system. CHARGE syndrome is a congenital disorder characterized by dysmorphic features of inner ear structures and caused primary by de novo mutations in CHD7, a gene encoding an ATP-dependent chromatin remodeling enzyme. DFNA36 and DFNB7/11 associated maladies are caused by dominant and recessive mutations in TMC1, respectively. Since TMC1 plays a critical role in mechano-electrical transduction of sensory hair cells, hearing loss caused by DFNA36 or DFNB7/11 is believed to originate from defects in sensory transduction. We will generate human embryonic stem cell lines bearing disease-associated mutations using CRISPR/Cas9 genome editing technology, and examine when and how phenotypes manifest themselves using a combination of histological, biochemical and electrophysiological assays. Additional experiments are designed to elucidate the mechanisms underlying the pathological defects and test if some of the defects can be rescued by forced expression of exogenous genes. To our knowledge, this is one of the first studies to recapitulate genetic inner ear disorders using a human model system and will provide valuable clinical information on the etiology of these disorders.

项目摘要遗传突变会导致人类的先天性或进行性内耳性疾病。尽管最近取得了进展在鉴定综合征和非同步基因的人类遗传学中，对突变如何在这些基因有助于人工耳蜗和/或前庭功能障碍患者的临床特征。为了为了克服由于无法进行实验的人类内耳组织而引起的局限性，我们最近建立了一种衍生内耳感觉上皮的方法，该上皮含有功能性感觉毛细胞来自3D培养的人多能干细胞。这些干细胞衍生的组织，设计为“人体内部耳器官，“藏有一层紧密挤满的毛细胞，其结构，生化和功能性特性与人内耳中的天然感觉毛细胞没有区别。这个主要目标应用是研究两种单基因内耳疾病，电荷综合征和 DFNA36/DFNB7/11耳聋，人体内耳类器官作为模型系统。电荷综合征是先天性疾病的特征是内耳结构的营养不良特征，由DE引起 CHD7中的Novo突变，一种编码ATP依赖性染色质重塑酶的基因。 DFNA36和 DFNB7/11相关的疾病分别是由TMC1中的主要和隐性突变引起的。由于TMC1在感觉毛细胞的机械电气转移中起着至关重要的作用，因此听力损失导致据信，dfna36或dfnb7/11源自感官翻译中的缺陷。我们将生成使用CRISPR/CAS9基因组编辑的人类胚胎干细胞系带有疾病相关的突变技术，并检查表型何时以及如何使用组织学的组合表现出来生化和电生理测定法。其他实验旨在阐明病理缺陷的基础机制并测试是否可以通过强制挽救某些缺陷外源基因的表达。据我们所知，这是概括遗传内部的最早研究之一使用人类模型系统的耳部疾病，将提供有关病因的宝贵临床信息这些疾病。