The long-term aim of this project is to develop a stem cell-based approach to regenerate the function of damaged auditory cells and synaptic connections. Our previous application focused on hair cell regeneration using stem cell-based epigenetic approaches. The major objectives of this renewal proposal are to determine the molecular mechanism critical for mouse hair cell synapse formation during normal development and develop a novel stem cell-based approach to reinnervate mouse auditory hair cells. Hair cell regeneration has obtained significant progress. Compared to hair cell regeneration, spiral ganglion neuron (SGN) regeneration has been reported but with relatively limited results. We have identified a stepwise method to guide mouse embryonic stem cells (ESCs) to differentiate into ESC-derived SGN-like neurons (ESNs) that showed SGN features. Up to date, only a few reports show that stem cell-derived neurons can form neural contacts with mouse hair cells. However, whether these neural contacts are bona fide SGN-hair cell synapses remains unclear. The function of these neural contacts has not been determined at the synaptic level. The molecular mechanism critical for regenerated neurons to functionally reinnervate hair cells remains obscure. Therefore, there is a critical need to use ESNs to reinnervate hair cells and determine the molecular mechanism critical for reinnervation. EphrinB family proteins bind to the EphB receptors, a family of transmembrane receptor tyrosine kinases. EphrinB signaling is involved in a variety of developmental programs, including synaptogenesis. Previous studies show that EphrinB1 signaling is critical for excitatory synaptic induction. Our preliminary data suggest the role of EphrinB signaling in ESN-hair cell reinnervation. Based on previous and our preliminary data, we hypothesize that EphrinB signaling may play a critical role in regulating neurons to form functional synapses with sensory hair cells. To test this hypothesis, three complementary specific aims are proposed. Aim 1 will determine the role of neuronal Efnb1 in SGN-hair cell synapse formation during development. Aim 2 will determine the molecular mechanism of ESN-hair cell reinnervation. Results of Aims 1 and 2 will guide the design of hair cell synapse regeneration research. In Aim 3, we will determine the extent to which EphrinB1 regulates ESN-hair cell reinnervation. Completion of this proposal will determine the molecular mechanisms critical for stem cell-derived neurons to reinnervate sensory hair cells. Identifying the role of Efnb1 in hair cell reinnervation will guide the synapse regeneration research, which will be translated into clinical trials to treat hearing loss patients. Therefore, the outcomes of this work will open new avenues to explore a stem cell-based multidisciplinary approach to regenerate hair cell synapses and restore the hearing function of Veteran and civilian patients.