Research on derivation of stem cells and their differentiation into desirable inner ear phenotypes is rapidly progressing. However, practical use of cell replacement therapy for hearing loss will depend on the ability to insert these cells into the inner ear and incorporate them into the native tissues. Inserting cells into the layer of cells making the auditory epithelium is complex because the epithelium forms a confluent and extremely tight barrier. Here we propose novel strategies for starting to design methods for integrating exogenous cells in the auditory epithelium of the deaf ear, in vivo. The challenges include keeping the therapeutic cells alive until they are inserted, opening gaps in the tissue to allow insertion, and maintaining essential native cells intact. Our overall hypothesis is that the deaf ear can be manipulated to receive and incorporate stem cells. To test this hypothesis, we will investigate and optimize means for transient decoupling of inter-cellular junctions in order to facilitate insertion of exogenous cells (Aim 1), test means to transiently reduce potassium concentration in the scala media in order to promote survival of implanted cells and to protect sensitive and indispensable components of the cochlea while junctions are open (Aim 2), and test extent of integration of cells inserted into the endolymph while potassium levels are low and intercellular junctions are open (Aim 3). The work we propose will improve our understanding of the biology of the severely deaf cochleae, and facilitate development of future therapies for deaf ears needing cell replacement therapy. While the ideas and goals of the proposed work are innovative and never before attempted, the expertise and reagents are available, promising high feasibility for successful experiments. It is therefore expected that the results of this study will ultimately benefit a large proportion of patients with severe sensorineural hearing loss from environmental and genetic etiologies. PUBLIC HEALTH RELEVANCE: Profound hearing loss is common and often results from loss or dysfunction of specific cells in the inner ear. Stem cell technology is rapidly advancing with the promise of availing new cells to replace lost or defective cells. Inserting and integrating exogenous cells into the epithelium of the inner ear is a complex and non-trivial task which has yet to be accomplished. Here we propose novel experimental approaches to facilitate insertion of exogenous cells into the epithelium of deaf inner ears.