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.
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.
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