Isolation of sphere-forming stem cells from the mouse inner ear

The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies.

Integrity and regeneration of mechanotransduction machinery regulate aminoglycoside entry and sensory cell death

Sound perception requires functional hair cell mechanotransduction (MET) machinery, including the MET channels and tip-link proteins. Prior work showed that uptake of ototoxic aminoglycosides (AG) into hair cells requires functional MET channels. In this study, we examined whether tip-link proteins, including Cadherin 23 (Cdh23), regulate AG entry into hair cells. Using time-lapse microscopy on cochlear explants, we found rapid uptake of gentamicin-conjugated Texas Red (GTTR) into hair cells from three-day-old Cdh23(+/+) and Cdh23(v2J/+) mice, but failed to detect GTTR uptake in Cdh23(v2J/v2J) hair cells. Pre-treatment of wildtype cochleae with the calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) to disrupt tip-links also effectively reduced GTTR uptake into hair cells. Both Cdh23(v2J/v2J) and BAPTA-treated hair cells were protected from degeneration caused by gentamicin. Six hours after BAPTA treatment, GTTR uptake remained reduced in comparison to controls; by 24 hours, drug uptake was comparable between untreated and BAPTA-treated hair cells, which again became susceptible to cell death induced by gentamicin. Together, these results provide genetic and pharmacologic evidence that tip-links are required for AG uptake and toxicity in hair cells. Because tip-links can spontaneously regenerate, their temporary breakage offers a limited time window when hair cells are protected from AG toxicity.

Designer aminoglycosides prevent cochlear hair cell loss and hearing loss.

Bacterial infections represent a rapidly growing challenge to human health. Aminoglycosides are widely used broad-spectrum antibiotics, but they inflict permanent hearing loss in up to ~50% of patients by causing selective sensory hair cell loss. Here, we hypothesized that reducing aminoglycoside entry into hair cells via mechanotransducer channels would reduce ototoxicity, and therefore we synthesized 9 aminoglycosides with modifications based on biophysical properties of the hair cell mechanotransducer channel and interactions between aminoglycosides and the bacterial ribosome. Compared with the parent aminoglycoside sisomicin, all 9 derivatives displayed no or reduced ototoxicity, with the lead compound N1MS 17 times less ototoxic and with reduced penetration of hair cell mechanotransducer channels in rat cochlear cultures. Both N1MS and sisomicin suppressed growth of E. coli and K. pneumoniae, with N1MS exhibiting superior activity against extended spectrum β lactamase producers, despite diminished activity against P. aeruginosa and S. aureus. Moreover, systemic sisomicin treatment of mice resulted in 75% to 85% hair cell loss and profound hearing loss, whereas N1MS treatment preserved both hair cells and hearing. Finally, in mice with E. coli-infected bladders, systemic N1MS treatment eliminated bacteria from urinary tract tissues and serially collected urine samples, without compromising auditory and kidney functions. Together, our findings establish N1MS as a nonototoxic aminoglycoside and support targeted modification as a promising approach to generating nonototoxic antibiotics.

Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection.

Aminoglycosides are commonly prescribed antibiotics with deleterious side effects to the inner ear. Due to their popular application as a result of their potent antimicrobial activities, many efforts have been undertaken to prevent aminoglycoside ototoxicity. Over the years, understanding of the antimicrobial as well as ototoxic mechanisms of aminoglycosides has increased. These mechanisms are reviewed in regard to established and potential future targets of hair cell protection.