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。 公共卫生相关性：在这项拨款申请中，建议在培养皿中产生人类内耳细胞。这些细胞将来自人类胚胎干细胞和诱导多能干细胞，这些细胞是从听力损失患者的小皮肤活检中产生的。这种新的策略将建立一种间接形式的“内耳活检”，以允许询问患有各种不同类型听力损失的人类患者的内耳细胞表型。这项拨款提案的重点是感觉毛细胞样细胞和内耳支持细胞样细胞。将详细描述这些细胞类型，并在细胞、细胞生理学和分子水平上测量与特定基因突变相关的病理变化。潜在的治疗方法将直接在患者来源的细胞上进行探索。第二个重点在于识别调节人类遗传性感音神经性听力损失常见形式的严重程度的遗传环境。成功完成拟议的研究将导致一种前所未有的新方法来评估人类内耳功能障碍，诊断，并直接在患者来源的细胞上测试新的治疗策略。

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. PUBLIC HEALTH RELEVANCE: In this grant application it is proposed to generate in the culture dish human inner ear cells. These cells will be derived from human embryonic stem cells and from induced pluripotent stem cells generated from a small skin biopsy taken from hearing loss patients. This novel strategy will establish an indirect form of 'inner ear biopsy' to allow interrogation of the inner ear cellular phenotypes in human patients with a variety of different types of hearing loss. The focus of the grant proposal is on sensory hair cell-like cells and on inner ear supporting cell-like cells. These cell types will be characterized in detail and the pathological changes associated with specific genetic mutations will be measured at the cellular, cell-physiological, and molecular level. Potential ways for therapy will be explored directly on patient-derived cells. A second focus lies on the identification of genetic circumstances that modulate the severity of a common form of human hereditary sensorineural hearing loss. Successful completion of the proposed research will result in an unprecedented novel approach to assess human inner ear dysfunction, diagnosis, and for testing of novel treatment strategies directly on patient-derived cells.