Human pluripotent stem cell-derived inner ear organoids recapitulate otic development in vitro.

Our molecular understanding of the early stages of human inner ear development has been limited by the difficulty in accessing fetal samples at early gestational stages. As an alternative, previous studies have shown that inner ear morphogenesis can be partially recapitulated using induced pluripotent stem cells directed to differentiate into inner ear organoids (IEOs). Once validated and benchmarked, these systems could represent unique tools to complement and refine our understanding of human otic differentiation and model developmental defects. Here, we provide the first direct comparisons of the early human embryonic otocyst and fetal sensory organs with human IEOs. We use multiplexed immunostaining and single-cell RNA-sequencing to characterize IEOs at three key developmental steps, providing a new and unique signature of in vitro-derived otic placode, epithelium, neuroblasts and sensory epithelia. In parallel, we evaluate the expression and localization of crucial markers at these equivalent stages in human embryos. Together, our data indicate that the current state-of-the-art protocol enables the specification of bona fide otic tissue, supporting the further application of IEOs to inform inner ear biology and disease.

Norrie disease protein is essential for cochlear hair cell maturation.

Mutations in the gene for Norrie disease protein (Ndp) cause syndromic deafness and blindness. We show here that cochlear function in an Ndp knockout mouse deteriorated with age: At P3-P4, hair cells (HCs) showed progressive loss of Pou4f3 and Gfi1, key transcription factors for HC maturation, and Myo7a, a specialized myosin required for normal function of HC stereocilia. Loss of expression of these genes correlated to increasing HC loss and profound hearing loss by 2 mo. We show that overexpression of the Ndp gene in neonatal supporting cells or, remarkably, up-regulation of canonical Wnt signaling in HCs rescued HCs and cochlear function. We conclude that Ndp secreted from supporting cells orchestrates a transcriptional network for the maintenance and survival of HCs and that increasing the level of ß-catenin, the intracellular effector of Wnt signaling, is sufficient to replace the functional requirement for Ndp in the cochlea.

Inner ear organoids: new tools to understand neurosensory cell development, degeneration and regeneration.

The development of therapeutic interventions for hearing loss requires fundamental knowledge about the signaling pathways controlling tissue development as well as the establishment of human cell-based assays to validate therapeutic strategies ex vivo Recent advances in the field of stem cell biology and organoid culture systems allow the expansion and differentiation of tissue-specific progenitors and pluripotent stem cells in vitro into functional hair cells and otic-like neurons. We discuss how inner ear organoids have been developed and how they offer for the first time the opportunity to validate drug-based therapies, gene-targeting approaches and cell replacement strategies.

Transcriptional response to Wnt activation regulates the regenerative capacity of the mammalian cochlea.

Lack of sensory hair cell (HC) regeneration in mammalian adults is a major contributor to hearing loss. In contrast, the neonatal mouse cochlea retains a transient capacity for regeneration, and forced Wnt activation in neonatal stages promotes supporting cell (SC) proliferation and induction of ectopic HCs. We currently know little about the temporal pattern and underlying mechanism of this age-dependent regenerative response. Using an in vitro model, we show that Wnt activation promotes SC proliferation following birth, but prior to postnatal day (P) 5. This age-dependent decline in proliferation occurs despite evidence that the Wnt pathway is postnatally active and can be further enhanced by Wnt stimulators. Using an in vivo mouse model and RNA sequencing, we show that proliferation in the early neonatal cochlea is correlated with a unique transcriptional response that diminishes with age. Furthermore, we find that augmenting Wnt signaling through the neonatal stages extends the window for HC induction in response to Notch signaling inhibition. Our results suggest that the downstream transcriptional response to Wnt activation, in part, underlies the regenerative capacity of the mammalian cochlea.

Lin28 reprograms inner ear glia to a neuronal fate.

Sensorineural hearing loss is irreversible and can be caused by loss of auditory neurons. Regeneration of neural cells from endogenous cells may offer a future tool to restore the auditory circuit and to enhance the performance of implantable hearing devices. Neurons and glial cells in the peripheral nervous system are closely related and originate from a common progenitor. Prior work in our lab indicated that in the early postnatal mouse inner ear, proteolipid protein 1 (Plp1) expressing glial cells could act as progenitor cells for neurons in vitro. Here, we used a transgenic mouse model to transiently overexpress Lin28, a neural stem cell regulator, in Plp1-positive glial cells. Lin28 promoted proliferation and conversion of auditory glial cells into neurons in vitro. To study the effects of Lin28 on endogenous glial cells after loss of auditory neurons in vivo, we produced a model of auditory neuropathy by selectively damaging auditory neurons with ouabain. After neural damage was confirmed by the auditory brainstem response, we briefly upregulated the Lin28 in Plp1-expressing inner ear glial cells. One month later, we analyzed the cochlea for neural marker expression by quantitative RT-PCR and immunohistochemistry. We found that transient Lin28 overexpression in Plp1-expressing glial cells induced expression of neural stem cell markers and subsequent conversion into neurons. This suggests the potential for inner ear glia to be converted into neurons as a regeneration therapy for neural replacement in auditory neuropathy.

Transcriptome-Wide Analysis Reveals a Role for Extracellular Matrix and Integrin Receptor Genes in Otic Neurosensory Differentiation from Human iPSCs.

We analyzed transcriptomic data from otic sensory cells differentiated from human induced pluripotent stem cells (hiPSCs) by a previously described method to gain new insights into the early human otic neurosensory lineage. We identified genes and biological networks not previously described to occur in the human otic sensory developmental cell lineage. These analyses identified and ranked genes known to be part of the otic sensory lineage program (SIX1, EYA1, GATA3, etc.), in addition to a number of novel genes encoding extracellular matrix (ECM) (COL3A1, COL5A2, DCN, etc.) and integrin (ITG) receptors (ITGAV, ITGA4, ITGA) for ECM molecules. The results were confirmed by quantitative PCR analysis of a comprehensive panel of genes differentially expressed during the time course of hiPSC differentiation in vitro. Immunocytochemistry validated results for select otic and ECM/ITG gene markers in the in vivo human fetal inner ear. Our screen shows ECM and ITG gene expression changes coincident with hiPSC differentiation towards human otic neurosensory cells. Our findings suggest a critical role of ECM-ITG interactions with otic neurosensory lineage genes in early neurosensory development and cell fate determination in the human fetal inner ear.

Engraftment of Human Stem Cell-Derived Otic Progenitors in the Damaged Cochlea.

Most cases of sensorineural deafness are caused by degeneration of hair cells. Although stem/progenitor cell therapy is becoming a promising treatment strategy in a variety of organ systems, cell engraftment in the adult mammalian cochlea has not yet been demonstrated. In this study, we generated human otic progenitor cells (hOPCs) from induced pluripotent stem cells (iPSCs) in vitro and identified these cells by the expression of known otic markers. We showed successful cell transplantation of iPSC-derived-hOPCs in an in vivo adult guinea pig model of ototoxicity. The delivered hOPCs migrated throughout the cochlea, engrafted in non-sensory regions, and survived up to 4 weeks post-transplantation. Some of the engrafted hOPCs responded to environmental cues within the cochlear sensory epithelium and displayed molecular features of early sensory differentiation. We confirmed these results with hair cell progenitors derived from Atoh1-GFP mice as donor cells. These mouse otic progenitors transplanted using the same in vivo delivery system migrated into damaged cochlear sensory epithelium and adopted a partial sensory cell fate. This is the first report of the survival and differentiation of hOPCs in ototoxic-injured mature cochlear epithelium, and it should stimulate further research into cell-based therapies for treatment of deafness.

Increasing the expression level of ChR2 enhances the optogenetic excitability of cochlear neurons.

Optogenetics comprise a promising alternative to electrical stimulation for characterization of neural circuits and for the next generation of neural prostheses. Optogenetic stimulation relies on expression of photosensitive microbial proteins in animal cells to initiate a flow of ions into the cells in response to visible light. Here, we generated a novel transgenic mouse model in which we studied the optogenetic activation of spiral ganglion neurons, the primary afferent neurons of the auditory system, and showed a strong optogenetic response, with a similar amplitude as the acoustically evoked response. A twofold increase in the level of channelrhodopsin expression significantly increased the photosensitivity at both the single cell and organismal levels but also partially compromised the native electrophysiological properties of the neurons. The importance of channelrhodopsin expression level to optogenetic stimulation, revealed by these quantitative measurements, will be significant for the characterization of neural circuitry and for the use of optogenetics in neural prostheses.NEW & NOTEWORTHY This study reveals a dose-response relationship between channelrhodopsin expression and optogenetic excitation. Both single cell and organismal responses depend on the expression level of the heterologous protein. Expression level of the opsin is thus an important variable in determining the outcome of an optogenetic experiment. These results are key to the implementation of neural prostheses based on optogenetics, such as next generation cochlear implants, which would use light to elicit a neural response to sound.

Distinct capacity for differentiation to inner ear cell types by progenitor cells of the cochlea and vestibular organs.

Disorders of hearing and balance are most commonly associated with damage to cochlear and vestibular hair cells or neurons. Although these cells are not capable of spontaneous regeneration, progenitor cells in the hearing and balance organs of the neonatal mammalian inner ear have the capacity to generate new hair cells after damage. To investigate whether these cells are restricted in their differentiation capacity, we assessed the phenotypes of differentiated progenitor cells isolated from three compartments of the mouse inner ear - the vestibular and cochlear sensory epithelia and the spiral ganglion - by measuring electrophysiological properties and gene expression. Lgr5+ progenitor cells from the sensory epithelia gave rise to hair cell-like cells, but not neurons or glial cells. Newly created hair cell-like cells had hair bundle proteins, synaptic proteins and membrane proteins characteristic of the compartment of origin. PLP1+ glial cells from the spiral ganglion were identified as neural progenitors, which gave rise to neurons, astrocytes and oligodendrocytes, but not hair cells. Thus, distinct progenitor populations from the neonatal inner ear differentiate to cell types associated with their organ of origin.

ERBB2 signaling drives supporting cell proliferation in vitro and apparent supernumerary hair cell formation in vivo in the neonatal mouse cochlea.

In mammals, cochlear hair cells are not regenerated once they are lost, leading to permanent hearing deficits. In other vertebrates, the adjacent supporting cells act as a stem cell compartment, in that they both proliferate and differentiate into de novo auditory hair cells. Although there is evidence that mammalian cochlear supporting cells can differentiate into new hair cells, the signals that regulate this process are poorly characterized. We hypothesize that signaling from the epidermal growth factor receptor (EGFR) family may play a role in cochlear regeneration. We focus on one such member, ERBB2, and report the effects of expressing a constitutively active ERBB2 receptor in neonatal mouse cochlear supporting cells, using viruses and transgenic expression. Lineage tracing with fluorescent reporter proteins was used to determine the relationships between cells with active ERBB2 signaling and cells that divided or differentiated into hair cells. In vitro, individual supporting cells harbouring a constitutively active ERBB2 receptor appeared to signal to their neighbouring supporting cells, inducing them to down-regulate a supporting cell marker and to proliferate. In vivo, we found supernumerary hair cell-like cells near supporting cells that expressed ERBB2 receptors. Both supporting cell proliferation and hair cell differentiation were largely reproduced in vitro using small molecules that we show also activate ERBB2. Our data suggest that signaling from the receptor tyrosine kinase ERBB2 can drive the activation of secondary signaling pathways to regulate regeneration, suggesting a new model where an interplay of cell signaling regulates regeneration by endogenous stem-like cells.

Bisphosphonate-Linked TrkB Agonist: Cochlea-Targeted Delivery of a Neurotrophic Agent as a Strategy for the Treatment of Hearing Loss.

Hearing loss affects more than two-thirds of the elderly population, and more than 17% of all adults in the U.S. Sensorineural hearing loss related to noise exposure or aging is associated with loss of inner ear sensory hair cells (HCs), cochlear spiral ganglion neurons (SGNs), and ribbon synapses between HCs and SGNs, stimulating intense interest in therapies to regenerate synaptic function. 7,8-Dihydroxyflavone (DHF) is a selective and potent agonist of tropomyosin receptor kinase B (TrkB) and protects the neuron from apoptosis. Despite evidence that TrkB agonists can promote survival of SGNs, local delivery of drugs such as DHF to the inner ear remains a challenge. We previously demonstrated in an animal model that a fluorescently labeled bisphosphonate, 6-FAM-Zol, administered to the round window membrane penetrated the membrane and diffused throughout the cochlea. Given their affinity for bone mineral, including cochlear bone, bisphosphonates offer an intriguing modality for targeted delivery of neurotrophic agents to the SGNs to promote survival, neurite outgrowth, and, potentially, regeneration of synapses between HCs and SGNs. The design and synthesis of a bisphosphonate conjugate of DHF (Ris-DHF) is presented, with a preliminary evaluation of its neurotrophic activity. Ris-DHF increases neurite outgrowth in vitro, maintains this ability after binding to hydroxyapatite, and regenerates synapses in kainic acid-damaged cochlear organ of Corti explants dissected in vitro with attached SGNs. The results suggest that bisphosphonate-TrkB agonist conjugates have promise as a novel approach to targeted delivery of drugs to treat sensorineural hearing loss.

Diphtheria Toxin-Induced Cell Death Triggers Wnt-Dependent Hair Cell Regeneration in Neonatal Mice.

UNLABELLED: Cochlear hair cells (HCs), the sensory cells that respond to sound, do not regenerate after damage in adult mammals, and their loss is a major cause of deafness. Here we show that HC regeneration in newborn mouse ears occurred spontaneously when the original cells were ablated by treatment with diphtheria toxin (DT) in ears that had been engineered to overexpress the DT receptor, but was not detectable when HCs were ablated in vivo by the aminoglycoside antibiotic neomycin. A variety of Wnts (Wnt1, Wnt2, Wnt2b, Wnt4, Wnt5a, Wnt7b, Wnt9a, Wnt9b, and Wnt11) and Wnt pathway component Krm2 were upregulated after DT damage. Nuclear ß-catenin was upregulated in HCs and supporting cells of the DT-damaged cochlea. Pharmacological inhibition of Wnt decreased spontaneous regeneration, confirming a role of Wnt signaling in HC regeneration. Inhibition of Notch signaling further potentiated supporting cell proliferation and HC differentiation that occurred spontaneously. The absence of new HCs in the neomycin ears was correlated to less robust Wnt pathway activation, but the ears subjected to neomycin treatment nonetheless showed increased cell division and HC differentiation after subsequent forced upregulation of ß-catenin. These studies suggest, first, that Wnt signaling plays a key role in regeneration, and, second, that the outcome of a regenerative response to damage in the newborn cochlea is determined by reaching a threshold level of Wnt signaling rather than its complete absence or presence. SIGNIFICANCE STATEMENT: Sensory HCs of the inner ear do not regenerate in the adult, and their loss is a major cause of deafness. We found that HCs regenerated spontaneously in the newborn mouse after diphtheria toxin (DT)-induced, but not neomycin-induced, HC death. Regeneration depended on activation of Wnt signaling, and regeneration in DT-treated ears correlated to a higher level of Wnt activation than occurred in nonregenerating neomycin-treated ears. This is significant because insufficient regeneration caused by a failure to reach a threshold level of signaling, if true in the adult, has the potential to be exploited for development of clinical approaches for the treatment of deafness caused by HC loss.

Destabilization of Atoh1 by E3 Ubiquitin Ligase Huwe1 and Casein Kinase 1 Is Essential for Normal Sensory Hair Cell Development.

Proneural basic helix-loop-helix transcription factor, Atoh1, plays a key role in the development of sensory hair cells. We show here that the level of Atoh1 must be accurately controlled by degradation of the protein in addition to the regulation of Atoh1 gene expression to achieve normal cellular patterning during development of the cochlear sensory epithelium. The stability of Atoh1 was regulated by the ubiquitin proteasome system through the action of Huwe1, a HECT-domain, E3 ubiquitin ligase. An interaction between Huwe1 and Atoh1 could be visualized by a proximity ligation assay and was confirmed by co-immunoprecipitation and mass spectrometry. Transfer of a lysine 48-linked polyubiquitin chain to Atoh1 by Huwe1 could be demonstrated both in intact cells and in a cell-free system, and proteasome inhibition or Huwe1 silencing increased Atoh1 levels. The interaction with Huwe1 and polyubiquitylation were blocked by disruption of casein kinase 1 (CK1) activity, and mass spectrometry and mutational analysis identified serine 334 as an important phosphorylation site for Atoh1 ubiquitylation and subsequent degradation. Phosphorylation by CK1 thus targeted the protein for degradation. Development of an extra row of inner hair cells in the cochlea and an approximate doubling in the number of afferent synapses was observed after embryonic or early postnatal deletion of Huwe1 in cochlear-supporting cells, and hair cells died in the early postnatal period when Huwe1 was knocked out in the developing cochlea. These data indicate that the regulation of Atoh1 by the ubiquitin proteasome pathway is necessary for hair cell fate determination and survival.

Quantitative imaging analysis of transcanal endoscopic Infracochlear approach to the internal auditory canal.

PURPOSE: A transcanal endoscopic infracochlear surgical approach to the internal auditory canal (IAC) in a human temporal bone model has previously been described. However, the proportion of patients with favorable anatomy for this novel surgical technique remains unknown. Herein, we perform a quantitative analysis of the transcanal endoscopic infracochlear corridor to the IAC based on computed tomography. MATERIALS AND METHODS: High resolution computed tomography scans of adult temporal bones were measured to determine the accessibility of the IAC when using an endoscopic transcanal, cochlear-sparing surgical corridor. RESULTS: This approach to the IAC was feasible in 92% (35 of 38) specimens based on a minimum distance of 3mm between the basilar turn of the cochlear and the great vessels (jugular bulb and carotid artery). CONCLUSIONS: Infracochlear access to the IAC is feasible in the majority of adult temporal bones and has implications for future hearing preservation drug delivery approaches to the IAC.

Generation of hair cells in neonatal mice by ß-catenin overexpression in Lgr5-positive cochlear progenitors.

Mammalian hair cells do not regenerate, and their loss is a major cause of deafness. We recently identified leucine-rich repeat containing, G-protein-coupled receptor 5 (Lgr5)-expressing cochlear supporting cells with the capacity for self-renewal and hair cell differentiation in vitro. We found that these cells, a subset of cochlear supporting cells, were responsive to Wnt signaling. Here we asked whether these Lgr5-positive cells, despite their lack of contribution to hair cell replacement after degenerative loss, could be driven by forced expression of ß-catenin to act as hair cell progenitors in vivo. We showed that forced stabilization of ß-catenin in supporting cells in neonatal animals resulted in proliferation of supporting cells and generation of hair cells. Although ß-catenin expression was increased by genetic means in all supporting cells, entry to the cell cycle and differentiation to hair cells of the normally postmitotic cells was restricted to the Lgr5-positive population. Our finding suggests that Wnt/ß-catenin can drive Lgr5-positive cells to act as hair cell progenitors, even after their exit from the cell cycle and apparent establishment of cell fate.

ß-Catenin is required for hair-cell differentiation in the cochlea.

The development of hair cells in the auditory system can be separated into steps; first, the establishment of progenitors for the sensory epithelium, and second, the differentiation of hair cells. Although the differentiation of hair cells is known to require the expression of basic helix-loop-helix transcription factor, Atoh1, the control of cell proliferation in the region of the developing cochlea that will ultimately become the sensory epithelium and the cues that initiate Atoh1 expression remain obscure. We assessed the role of Wnt/ß-catenin in both steps in gain- and loss-of-function models in mice. The canonical Wnt pathway mediator, ß-catenin, controls the expression of Atoh1. Knock-out of ß-catenin inhibited hair-cell, as well as pillar-cell, differentiation from sensory progenitors but was not required to maintain a hair-cell fate once specified. Constitutive activation of ß-catenin expanded sensory progenitors by inducing additional cell division and resulted in the differentiation of extra hair cells. Our data demonstrate that ß-catenin plays a role in cell division and differentiation in the cochlear sensory epithelium.

A phase I/IIa safety and efficacy trial of intratympanic gamma-secretase inhibitor as a regenerative drug treatment for sensorineural hearing loss.

Inhibition of Notch signalling with a gamma-secretase inhibitor (GSI) induces mammalian hair cell regeneration and partial hearing restoration. In this proof-of-concept Phase I/IIa multiple-ascending dose open-label trial (ISRCTN59733689), adults with mild-moderate sensorineural hearing loss received 3 intratympanic injections of GSI LY3056480, in 1 ear over 2 weeks. Phase I primary outcome was safety and tolerability. Phase lla primary outcome was change from baseline to 12 weeks in average pure-tone air conduction threshold across 2,4,8 kHz. Secondary outcomes included this outcome at 6 weeks and change from baseline to 6 and 12 weeks in pure-tone thresholds at individual frequencies, speech reception thresholds (SRTs), Distortion Product Otoacoustic Emissions (DPOAE) amplitudes, Signal to Noise Ratios (SNRs) and distribution of categories normal, present-abnormal, absent and Hearing Handicap Inventory for Adults/Elderly (HHIA/E). In Phase I (N = 15, 1 site) there were no severe nor serious adverse events. In Phase IIa (N = 44, 3 sites) the average pure-tone threshold across 2,4,8 kHz did not change from baseline to 6 and 12 weeks (estimated change -0.87 dB; 95% CI -2.37 to 0.63; P = 0.252 and -0.46 dB; 95% CI -1.94 to 1.03; P = 0.545, respectively), nor did the means of secondary measures. DPOAE amplitudes, SNRs and distribution of categories did not change from baseline to 6 and 12 weeks, nor did SRTs and HHIA/E scores. Intratympanic delivery of LY3056480 is safe and well-tolerated; the trial's primary endpoint was not met.

Large-scale annotated dataset for cochlear hair cell detection and classification.

Our sense of hearing is mediated by cochlear hair cells, of which there are two types organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains 5-15 thousand terminally differentiated hair cells, and their survival is essential for hearing as they do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. Machine learning can be used to automate the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, rat, guinea pig, pig, primate, and human cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 107,000 hair cells which have been identified and annotated as either inner or outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair-cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to give other hearing research groups the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

Wnt-responsive Lgr5-expressing stem cells are hair cell progenitors in the cochlea.

Auditory hair cells are surrounded on their basolateral aspects by supporting cells, and these two cell types together constitute the sensory epithelium of the organ of Corti, which is the hearing apparatus of the ear. We show here that Lgr5, a marker for adult stem cells, was expressed in a subset of supporting cells in the newborn and adult murine cochlea. Lgr5-expressing supporting cells, sorted by flow cytometry and cultured in a single-cell suspension, compared with unsorted cells, displayed an enhanced capacity for self-renewing neurosphere formation in response to Wnt and were converted to hair cells at a higher (>10-fold) rate. The greater differentiation of hair cells in the neurosphere assay showed that Lgr5-positive cells had the capacity to act as cochlear progenitor cells, and lineage tracing confirmed that Lgr5-expressing cells accounted for the cells that formed neurospheres and differentiated to hair cells. The responsiveness to Wnt of cells with a capacity for division and sensory cell formation suggests a potential route to new hair cell generation in the adult cochlea.

Loss of osteoprotegerin expression in the inner ear causes degeneration of the cochlear nerve and sensorineural hearing loss.

Osteoprotegerin (OPG) is a key regulator of bone remodeling. Mutations and variations in the OPG gene cause many human diseases that are characterized by not only skeletal abnormalities but also poorly understood hearing loss: Paget's disease, osteoporosis, and celiac disease. To gain insight into the mechanisms of hearing loss in OPG deficiency, we studied OPG knockout (Opg(-/-)) mice. We show that they develop sensorineural hearing loss, in addition to conductive hearing loss due to abnormal middle-ear bones. OPG deficiency caused demyelination and degeneration of the cochlear nerve in vivo. It also activated ERK, sensitized spiral ganglion cells (SGC) to apoptosis, and inhibited proliferation and survival of cochlear stem cells in vitro, which could be rescued by treatment with exogenous OPG, an ERK inhibitor, or bisphosphonate. Our results demonstrate a novel role for OPG in the regulation of SGC survival, and suggest a mechanism for sensorineural hearing loss in OPG deficiency.