Modeling Genetic Inner Ear Disorders with Human Pluripotent Stem Cells

项目摘要 基因突变导致人类先天性或进行性内耳疾病。尽管最近的进展 在人类遗传学中，用于识别综合征和非综合征基因的基因，很少有人知道突变是如何发生的。 这些基因导致耳蜗和/或前庭功能障碍患者的临床特征。为了 为了克服由于可用于实验的人类内耳组织的缺乏而产生的限制，我们 最近建立了一种用于获得具有功能性感觉毛细胞的内耳感觉上皮的方法 从人类多能干细胞的3D培养中分离出来。这些干细胞衍生的组织，被称为"人类内部组织"， 耳类器官"具有一层紧密排列的毛细胞，其结构、生化和功能 这些特性与人内耳中的天然感觉毛细胞无法区分。这个的主要目标 应用是研究两种单基因内耳疾病的病理生理学，CHARGE综合征和 DFNA 36/DFNB 7/11耳聋，以人内耳类器官作为模型系统。CHARGE综合征是一种 以内耳结构的畸形特征为特征的先天性疾病，主要是由先天性耳聋引起的。 CHD7是一种编码ATP依赖性染色质重塑酶的基因。DFNA 36和 DFNB 7/11相关疾病分别由TMC 1的显性和隐性突变引起。 由于TMC 1在感觉毛细胞的机械-电转导中起着关键作用， DFNA 36或DFNB 7/11引起的细胞凋亡被认为起源于感觉转导的缺陷。我们将产生 使用CRISPR/Cas9基因组编辑携带疾病相关突变的人胚胎干细胞系 技术，并检查表型何时以及如何使用组织学， 生物化学和电生理学测定。设计了额外的实验来阐明 病理缺陷的潜在机制，并测试一些缺陷是否可以通过强制补救。 外源基因的表达。据我们所知，这是最早的研究之一，以概括遗传内部 使用人类模型系统的耳部疾病，并将提供有价值的临床信息的病因学 这些紊乱。

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.