Abstract: Novel inner ear organoid models for studying hair cells in normal development and in the deaf- blindness disease Usher Syndrome Type 1F The fundamental human sense of balance, which comprises both movement through the world and one’s body position in space, begins with specialized biological detectors called “hair cells” responsible for converting mechanical stimuli into electrical signals (also known as mechanotransduction). Hair cells perform this function by converting mechanical stimuli on their apical surface (received by a specialized structure of actin-rich stereocilia) into electrical impulses on their basolateral surface (produced by specialized ribbon synapses). Despite their critical role and decades of study in animal models, many aspects of the hallmarks of hair cell development specifically in developing human tissue have yet to be explored. To circumvent the difficulty in access, collection, and preservation of in vivo or ex vivo human tissue, human induced pluripotent stem cell (hiPSC) derived inner ear organoids (IEOs) have emerged as a new model for studying human development. IEOs are 3D aggregates of cells containing many internal vesicles lined with inner ear sensory epithelia and synapsing neurons. Strong histologic and limited transcriptomic data of IEOs show that the in vitro organoids recapitulate many of the diverse features found in the in vivo inner ear. Unfortunately, the sensory cells of interest are buried by a large population of supporting and mesenchymal cells, preventing their long-term developmental and functional study. My recent work in the lab has shown that perturbation of extracellular matrix proteins reverses the polarity of the sensory epithelia such that the progenitor epithelia remain on the surface and have their apical surfaces pointed outwards into the media. The outwardly facing hair cells with this new method are now both accessible and imageable, overcoming the limitations of previous IEO versions. Based on initial characterization, I hypothesize that inverted apical-out IEOs yield more numerous and more functionally mature hair cells. In Aim 1, I will characterize the genetic and physiologic properties of the human hair cell population that is now accessible through our in vitro model. I will perform single cell RNA sequencing in the mature organoid and bathe the organoids in artificial solutions that mimic in vivo endolymph to attempt to mature the transcriptomic and electrophysiological profiles of the hair cells. In Aim 2, I will use a patient-derived hiPSC line from a patient with a devastating genetic deaf-blindness mutation known as USH1F to investigate the development of critical apical structures and the disrupted effects that damage hair cell function. I will then attempt to use viral-mediated gene therapies before the onset of hair cell dysfunction to restore proper hair cell development. These experiments will reveal the first generation of many applications of this new appr...