Patient derived sensory hair- and supporting cell-like cells

描述（由申请人提供）：该拨款提案的首要目标是将用小鼠胚胎干细胞（ESC）和诱导多能干细胞（iPSC）开发的技术转化为临床环境。更具体地，提出了从ESC和iPSC产生和表征人内耳感觉毛细胞样细胞和支持细胞样细胞。在目标1中，人胚胎干细胞衍生的毛细胞样细胞的特点是免疫细胞化学，形态学和功能。在目的2中，提出了从人iPSC，特别是从携带MYO 15 A基因突变的患者产生和表征毛细胞样细胞。预期突变MYO 15 A基因的结果反映在从听力损失患者产生的毛细胞样细胞的细胞表型中。进一步提出了用野生型MYO 15 A进行拯救实验以恢复患者来源的毛细胞样细胞中的细胞表型。最后，目标3集中于表征来自DFNB 1患者的支持细胞样细胞中的间隙连接，所述DFNB 1患者对于常见的GJB 2 35 delG突变是纯合的。建议将具有重度至极重度听力损失的35 delG纯合子与具有轻度至中度听力损失的35 delG纯合子的细胞特性进行比较。测量包括间隙连接生理学和全转录组分析，这将与DFNB 1患者的大队列中的全基因组关联研究相结合。这项奋进的目标是鉴定临床相关的遗传修饰剂，这是开发策略以改善DFNB 1患者中与35 delG/35 delG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.