Regenerating hair cells in vestibular sensory epithelia from humans.

Human vestibular sensory epithelia in explant culture were incubated in gentamicin to ablate hair cells. Subsequent transduction of supporting cells with ATOH1 using an Ad-2 viral vector resulted in generation of highly significant numbers of cells expressing the hair cell marker protein myosin VIIa. Cells expressing myosin VIIa were also generated after blocking the Notch signalling pathway with TAPI-1 but less efficiently. Transcriptomic analysis following ATOH1 transduction confirmed up-regulation of 335 putative hair cell marker genes, including several downstream targets of ATOH1. Morphological analysis revealed numerous cells bearing dense clusters of microvilli at the apical surfaces which showed some hair cell-like characteristics confirming a degree of conversion of supporting cells. However, no cells bore organised hair bundles and several expected hair cell markers genes were not expressed suggesting incomplete differentiation. Nevertheless, the results show a potential to induce conversion of supporting cells in the vestibular sensory tissues of humans.

Rapid hair cell loss: a mouse model for cochlear lesions.

In comparison to other mammals, mice have proved extremely resistant to aminoglycoside-induced hair cell ablation in vivo. In this paper we examine the pattern and extent of cochlear lesions rapidly induced with a combination of a single dose of aminoglycoside (kanamycin) followed by a loop diuretic (bumetanide). With this protocol, the vestibular system was unaffected, but in the cochlea, there was extensive loss of outer hair cells (OHC) that commenced in the basal coil and progressed apically so that, by 48 h, OHC loss was almost complete. TUNEL-positive nuclei and activated caspase-3 labeling demonstrated that most OHC died via a classical apoptotic pathway. However, scattered debris within the OHC region suggested that many apoptotic cells ruptured prior to completion of apoptosis. Following lesion repair, supporting cells retained characteristics of differentiated cells but positional shift occurred. In comparison to OHC loss, inner hair cell (IHC) death was delayed and only observed in 50% of all cochleae examined even after extensive reorganization of the tissue. The coadmininstration of diuretic with FM1-43, used as a tracer for aminoglycoside uptake, indicated entry into IHC as readily as OHC, suggesting that the differential response to aminoglycoside was not due to differential uptake. Where IHC death was ongoing, there were indications of different modes of cell death: cells with morphological features of autophagy, necrosis, and apoptosis were apparent. In addition to damage to the organ of Corti, there was a significant and progressive decrease in strial thickness beginning as early as 7 days posttreatment. This was due predominantly to degeneration of marginal cells. The strial pathology resembled that reported after noise damage and with aging. This in vivo protocol provides a robust model in which to obtain extensive OHC loss in the mature cochleae of mice and is a means with which to examine different aspects of cochlear pathology in transgenic or mutant strains.