Patient derived sensory hair- and supporting cell-like cells

描述（由申请人提供）：本拨款提案的主要目的是将小鼠胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）开发的技术转化为临床环境。更具体地说，建议从ESCs和iPSCs中生成和表征人内耳感觉毛细胞样细胞和支持细胞样细胞。在Aim 1中，人类esc来源的毛细胞样细胞在免疫细胞化学、形态学和功能上被表征。在Aim 2中，研究人员提出从人类iPSCs中，特别是从携带MYO15A基因突变的患者中，产生和表征毛细胞样细胞。预计MYO15A基因突变的后果会反映在听力损失患者产生的毛细胞样细胞的细胞表型上。进一步提出用野生型MYO15A进行修复实验，恢复患者来源的毛细胞样细胞的细胞表型。最后，Aim 3重点研究来自于常见GJB2 35delG突变纯合的DFNB1患者的支持细胞样细胞的间隙连接特性。我们建议比较重度至重度听力损失的35delG纯合子与轻度至中度听力损失的35delG纯合子的细胞特性。措施包括间隙连接生理学和全转录组分析，这将与DFNB1患者大队列的全基因组关联研究相结合。这项工作的目标是鉴定临床相关的遗传修饰因子，这是制定策略改善DFNB1患者中与35delG/35delG基因型相关的典型严重表型的第一步。

DESCRIPTION (provided by applicant): The overriding aim of this grant proposal is to translate technology developed with murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to the clinical setting. More specifically, it is proposed to generate and characterize human inner ear sensory hair cell-like cells and supporting cell-like cells from ESCs and iPSCs. In Aim 1, human ESC-derived hair cell-like cells are being characterized immunocytochemically, morphologically, and functionally. In Aim 2, it is proposed to generate and characterize hair cell-like cells from human iPSCs, specifically from patients carrying mutations in the MYO15A gene. It is expected that the consequences of the mutant MYO15A gene are reflected in the cellular phenotype of hair cell-like cells generated from hearing loss patients. Further proposed is a rescue experiment with wild type MYO15A to restore the cellular phenotype in patient-derived hair cell-like cells. Finally, Aim 3 focuses on characterization of gap junctions in supporting cell-like cells derived from DFNB1 patients homozygous for the common GJB2 35delG mutation. It is proposed to compare cellular properties of 35delG homozygotes with severe-to-profound hearing loss to 35delG homozygotes with mild-to-moderate hearing loss. Measures include gap junction physiology and whole transcriptome analysis, which will be combined with genome wide association studies in a large cohort of DFNB1 patients. The goal of this endeavor is the identification of clinically relevant genetic modifiers, which is a first step toward developing strategies to ameliorate the typical severe phenotype associated with the 35delG/35delG genotype in DFNB1 patients.