Modeling Genetic Inner Ear Disorders with Human Pluripotent Stem Cells

项目总结 基因突变会导致人类先天性或进行性内耳疾病。尽管最近取得了进展 在人类识别综合征和非综合征基因的遗传学中，人们对基因突变是如何 这些基因有助于耳蜗病患和/或前庭功能障碍患者的临床特征。按顺序 为了克服可用于实验的人类内耳组织稀缺的局限性，我们 最近建立了一种含有功能性感觉毛细胞的内耳感觉上皮细胞的获得方法 来自3D培养中的人类多能干细胞。这些干细胞衍生的组织，被称为“人类内脏” 耳朵类器官含有一层紧密排列的毛细胞，其结构、生化和功能 这些特性与人类内耳的天然感觉毛细胞没有什么区别。这样做的主要目标是 应用于研究两种单基因内耳疾病的病理生理学：Charge综合征和 DFNA36/DFNB7/11耳聋模型，以人内耳器官为模型系统。充电综合征是一种 以内耳结构畸形为特征的先天性疾病，由De引起 CHD7的Novo突变，这是一种编码依赖于ATP的染色质重塑酶的基因。DFNA36和 DFNB7/11相关疾病分别由TMC1的显性突变和隐性突变引起。 由于TMC1在感觉性毛细胞的机电转导中起关键作用，导致听力损失 DFNA36或DFNB7/11的缺陷被认为起源于感觉转导的缺陷。我们将生成 利用CRISPR/Cas9基因组编辑技术获得携带疾病相关突变的人胚胎干细胞系 技术，并研究何时以及如何使用组织学， 生化和电生理检测。我们设计了更多的实验来阐明 病理缺陷的潜在机制，并测试一些缺陷是否可以通过强制修复 外源基因的表达。据我们所知，这是第一批概括遗传内在的研究之一。 使用人体模型系统，将提供有关耳部疾病病因的有价值的临床信息 这些障碍。

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.