Engineering of functional auditory neurons from human induced pluripotent stem cells

Background Spiral ganglion neurons (SGNs) relay auditory sensory information from the cochlea to the brain. Their loss results in permanent hearing impairment in humans due to their limited regenerative capacity. Progress in hearing restoration has been constrained by the inaccessibility of human inner ear tissue and challenges in generating functionally mature human SGN-like neurons from stem cells in vitro . Methods To generate human SGN-like neurons from human induced pluripotent stem cells (hiPSCs), we recapitulated key signaling pathways involved in human inner ear development. On day (D) 11 of differentiation, nerve growth factor receptor-positive cells (precursors of pre-placodal ectoderm and neural crest) were isolated using magnetic sorting. From D18 to D25, cultures were treated with sonic hedgehogs to induce otic neural progenitors. Neuronal maturation was subsequently promoted by a cocktail of brain-derived neurotrophic factor, neurotrophin-3, and insulin-like growth factor-1, which supports SGN development. Cellular identity and functionality were assessed using single-cell RNA sequencing, immunocytochemistry, whole-cell patch-clamp electrophysiology, co-culture assays, and calcium ion (Ca²⁺) imaging. Results hiPSC-derived SGN-like neurons exhibited morphological, molecular, electrophysiological, and functional characteristics of SGNs in vivo . Neurons acquired bipolar morphology and were wrapped by glial cells. Transcriptomic analysis revealed that SGN-like neurons were distinct from other neuronal lineages and showed similarity to type I and type II SGNs based on expression of synaptic and intrinsic excitability-related genes. Electrophysiological recordings revealed progressive hyperpolarization of resting membrane potential and emergence of overshooting action potentials, consistent with neuronal maturation. In co-culture systems, human SGN-like neurons formed functional synaptic connections with mouse cochlear hair cells and cochlear nucleus neurons, evidenced by Ca 2+ transients and induction of the immediate early gene c-Fos . Conclusions This study reports a robust and reproducible protocol for generating human SGN-like neurons from hiPSCs, providing a versatile platform for studying human auditory development, disease modeling, drug screening, and cell-based therapies for hearing restoration.