Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of a particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells 1, is responsible for a substantial proportion of patients with hearing impairment 2. While the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss since poor innervation limits the prospective performance of an implant 3. Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here, we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory evoked response (ABR) thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.

Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca(2+) dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca(2+) sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca(2+) dependence of immature spiking cells. However, the nature of the Ca(2+) sensor(s) of vesicle fusion at hair cell synapses is unknown. We found that synaptotagmin IV was essential for establishing the linear exocytotic Ca(2+) dependence in adult rodent IHCs and immature outer hair cells. Moreover, the expression of the hitherto undetected synaptotagmins I and II correlated with a high-order Ca(2+) dependence in IHCs. We propose that the differential expression of synaptotagmins determines the characteristic Ca(2+) sensitivity of vesicle fusion at hair cell synapses.

The transcription factors GATA3 and Pax2 are expressed throughout development of the mouse inner ear. We have used antibodies to study their temporal and spatial expression patterns from embryonic days E8-E16.5. The two factors show reciprocal relationships in the regional patterning of the early otocyst and cellular patterning within the sensory epithelia. GATA3 is expressed in the whole otic placode at E8. In the otocyst at E9.5-10.5, the distribution is lateral and complementary to the medial expression pattern of Pax2. Only Pax2 is expressed in the endolymphatic duct, but both factors are expressed in the cochlea. At E11.5-13.5, GATA3 is expressed strongly in the cochlea, but in the dorsal, vestibular region it is downregulated. In all sensory epithelia, downregulation coincides with sensory innervation. Pax2 is expressed in all sensory and some nonsensory epithelia, but within sensory epithelia at E16.5 it is restricted to hair cells. GATA3 is expressed throughout key periods of cell proliferation, fate determination, and differentiation and is not specifically associated with any of these processes. Expression persists most strongly in the main components of the developing auditory system. These include the auditory sensory epithelium, the afferent and efferent nerves, and the mesenchymal and ectodermal cells in regions that form key parts of the middle and outer ear. GATA3 is thus expressed in functionally distinct groups of cells that integrate to form a complete sensory system. The results suggest that both factors may be involved in tissue compartmentalisation, morphogenesis, and cell signalling.

The function of the zinc finger transcription factor GATA3 was studied in a newly established, conditionally immortal cell line derived to represent auditory sensory neuroblasts migrating from the mouse otic vesicle at embryonic day E10.5. The cell line, US/VOT-33, expressed GATA3, the bHLH transcription factor NeuroD and the POU-domain transcription factor Brn3a, as do auditory neuroblasts in vivo. When GATA3 was knocked down reversibly with antisense oligonucleotides, NeuroD was reversibly down-regulated. Auditory and vestibular neurons form from neuroblasts that express NeuroD and that migrate from the antero-ventral, otic epithelium at E9.5-10.5. On the medial side, neuroblasts and epithelial cells express GATA3 but on the lateral side they do not. At E13.5 most auditory neurons express GATA3 but no longer express NeuroD, whereas vestibular neurons express NeuroD but not GATA3. Neuroblasts expressing NeuroD and GATA3 were located in the ventral, otic epithelium, the adjacent mesenchyme and the developing auditory ganglion. The results suggest that auditory and vestibular neurons arise from different, otic epithelial domains and that they gain their identity prior to migration. In auditory neuroblasts, NeuroD appears to be dependent on the expression of GATA3.

Mammalian auditory hair cells are few in number, experimentally inaccessible, and do not proliferate postnatally or in vitro. Immortal cell lines with the potential to di¡erentiate into auditory hair cells would substantially facilitate auditory research, drug development, and the isolation of critical molecules involved in hair cell biology. We have established two conditionally immortal cell lines that express at least ¢ve characteristic hair cell markers. These markers are the transcription factor Brn3.1, the a9 subunit of the acetylcholine receptor, the stereociliary protein ¢mbrin and the myosins VI and VIIA. These hair cell precursors permit functional studies of cochlear genes and in the longer term they will provide the means to explore therapeutic methods of stimulating auditory hair cell regeneration.