Our overarching goal is to use human pluripotent stem cells (PSC) to inform our understanding of and ability to treat hearing loss. Given the challenging anatomy of the inner ear, PSC-based applications hold particular promise in improving our understanding of hearing loss. Specifically, patient-derived induced PSC-based disease modeling could enable improved correlation of genotype mutations with underlying cellular alterations. In addition, recently described sequencing methods using iPSC-based models could enable identification of novel hearing loss causing mutations. Last, stem cells hold promise as regenerative therapies for hearing loss. In order to realize the potential applications of human PSC for hearing loss a number of immediate challenges must be overcome. First, a method for generating an enriched population of inner ear (otic) progenitor-like cells must be outlined. Doing so will lay the foundation for both inner ear developmental studies and future transplantation-based therapies. Next, techniques to differentiate human PSC to functionally mature inner ear hair cell and supporting cell-like cells must be developed. The success of disease modeling and molecular diagnostic approaches will depend on creation of reliable techniques to achieve this goal. The studies in this proposal address two immediate challenges in applying PSC to hearing loss.
In Aim 1 we will systematically evaluate the extrinsic and intrinsic regulators enabling the generation of a purified population of human otic progenitor-like cells from PSC. We will apply innovative gene editing techniques as part of this aim to enable unprecedented control of critical genes in the development of the inner ear. We will perform a sequential, detailed analysis including PCR, immunocytochemistry and flow cytometry, as we refine current methods to direct PSC to an inner ear fate upon differentiation.
In Aim 2 we will generate and functionally characterize human PSC-derived inner ear hair cell-like cells. Building upon recent advances in the field we will sequentially evaluate in detail the process of human PSC long-term differentiation to an inner ear hair cell lineage. As part of this aim we will apply advanced gene editing technique to overexpress the hair cell specific transcription factor Atoh1. In addition, we will characterize the electrophysiological properties of the resultant hair cell-lie cells upon mechanical stimulation. To enable more comprehensive functional analysis, we will use both in vitro and in utero transplantation of PSC-derived otic progenitor-like cells into the mouse otocyst. This novel in vivo model may provide insight into the factors most critical to allow for stem cells to generate functional hair cells upon transplantation. The studies in this proposal address pivotal and immediate challenges in application of human PSC-based techniques for hearing loss. Successful completion of the proposed studies will directly contribute to the pursuit of an improved understanding of and ability to manage hearing loss in the short and long term.
Human pluripotent stem cells offer promise for improving our knowledge of and ability to treat hearing loss. Realizing this potential requires an improved understanding of the factors regulating human stem cell transformation into inner ear cell types. This proposal will systematically study the regulation of this process and will evaluate whether stem cell-derived inner ear hair cells are truly functional on multiple levels.
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| Coughlin, Adam R; Willman, Tyler J; Gubbels, Samuel P (2018) Systematic Review of Hearing Preservation After Radiotherapy for Vestibular Schwannoma. Otol Neurotol 39:273-283 |
| Chow, Cynthia L; Trivedi, Parul; Pyle, Madeline P et al. (2016) Evaluation of Nestin Expression in the Developing and Adult Mouse Inner Ear. Stem Cells Dev 25:1419-32 |
| Chow, Cynthia L; Guo, Weixiang; Trivedi, Parul et al. (2015) Characterization of a unique cell population marked by transgene expression in the adult cochlea of nestin-CreER(T2)/tdTomato-reporter mice. J Comp Neurol 523:1474-87 |
