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Human pluripotent stem cells are differentiated into inner ear organoids containing cells similar to hair cells and sensory neurons. The derivation of human inner ear tissue from pluripotent stem cells would enable in vitro screening of drug candidates for the treatment of hearing and balance dysfunction and may provide a source of cells for cell-based therapies of the inner ear. Here we report a method for differentiating human pluripotent stem cells to inner ear organoids that harbor functional hair cells. Using a three-dimensional culture system, we modulate TGF, BMP, FGF, and WNT signaling to generate multiple otic-vesicle-like structures from a single stem-cell aggregate. Over 2 months, the vesicles develop into inner ear organoids with sensory epithelia that are innervated by sensory neurons. Additionally, using CRISPR–Cas9, we generate an ATOH1-2A-eGFP cell line to detect hair cell induction and demonstrate that derived hair cells exhibit electrophysiological properties similar to those of native sensory hair cells. Our culture system should facilitate the study of human inner ear development and research on therapies for diseases of the inner ear.

Efficient gene transfer to the mouse inner ear is achieved with a synthetic adeno-associated viral vector. Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities 1 , 2 . Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector 3 , for transgene delivery to the mouse cochlea. Ex vivo transduction of mouse organotypic explants identified Anc80L65 from a set of other adeno-associated virus (AAV) vectors as a potent vector for the cochlear cell targets. Round window membrane injection resulted in highly efficient transduction of inner and outer hair cells in mice, a substantial improvement over conventional AAV vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.

High-resolution computed tomography (HRCT) has become a widely used tool for studying the inner ear morphology of vertebrates. Amphisbaenians are one of the most specialized groups of fossorial reptiles but are poorly understood relative to other squamate reptile. In this paper we survey the anatomy of the inner and middle ear of these fossorial reptiles using HRCT models and we describe qualitatively and quantitatively (using 3D morphometrics) the anatomy of the inner ear. Amphisbaenians are diverse in skull anatomy, especially in the configuration of the snout, which correlates with digging modes. We demonstrate that the ear also exhibits a diversity of configurations, which are independent of phylogenetic relationships. Results from morphological analyses also allow us to describe 11 new potentially informative phylogenetic characters including some that help to diagnose amphisbaenians, such as: 1) the globular vestibule, ii) semicircular canals arranged in a circular trajectory, and iii) an extensive area of interaction between the columella footplate and the lagenar recess. Among extant amphisbaenians, Rhineura floridana has the most unusual inner ear configuration, including a horizontal semicircular canal that is in the same orientation as the inclined snout. The new morphological information helps us to better understand the morphology of headfirst-burrowing fossorial reptiles and contributes new data for resolution of phylogenetic relationships among amphisbaenians.

Adult-onset hearing loss is very common, but we know little about the underlying molecular pathogenesis impeding the development of therapies. We took a genetic approach to identify new molecules involved in hearing loss by screening a large cohort of newly generated mouse mutants using a sensitive electrophysiological test, the auditory brainstem response (ABR). We review here the findings from this screen. Thirty-eight unexpected genes associated with raised thresholds were detected from our unbiased sample of 1,211 genes tested, suggesting extreme genetic heterogeneity. A wide range of auditory pathophysiologies was found, and some mutant lines showed normal development followed by deterioration of responses, revealing new molecular pathways involved in progressive hearing loss. Several of the genes were associated with the range of hearing thresholds in the human population and one, SPNS2, was involved in childhood deafness. The new pathways required for maintenance of hearing discovered by this screen present new therapeutic opportunities.

Mammalian hearing operates on three basic steps: 1) sound capturing, 2) impedance conversion, and 3) frequency analysis. While these canonical steps are vital for acoustic communication and survival in mammals, they are not unique to them. An equivalent mechanism has been described for katydids (Insecta), and it is unique to this group among invertebrates. The katydid inner ear resembles an uncoiled cochlea, and has a length less than 1 mm. Their inner ears contain the crista acustica , which holds tonotopically arranged sensory cells for frequency mapping via travelling waves. The crista acustica is located on a curved triangular surface formed by the dorsal wall of the ear canal. While empirical recordings show tonotopic vibrations in the katydid inner ear for frequency analysis, the biophysical mechanism leading to tonotopy remains elusive due to the small size and complexity of the hearing organ. In this study, robust numerical simulations are developed for an in silico investigation of this process. Simulations are based on the precise katydid inner ear geometry obtained by synchrotron-based micro-computed tomography, and empirically determined inner ear fluid properties for an accurate representation of the underlying mechanism. We demonstrate that the triangular structure below the hearing organ drives the tonotopy and travelling waves in the inner ear, and thus has an equivalent role to the mammalian basilar membrane. This reveals a stronger analogy between the inner ear basic mechanical networks of two organisms with ancient evolutionary differences and independent phylogenetic histories.

The energy of the electrochemical potential in the guinea pig cochlea is harvested and used to power a wireless transmitter. Endocochlear potential (EP) is a battery-like electrochemical gradient found in and actively maintained by the inner ear 1 , 2 . Here we demonstrate that the mammalian EP can be used as a power source for electronic devices. We achieved this by designing an anatomically sized, ultra-low quiescent-power energy harvester chip integrated with a wireless sensor capable of monitoring the EP itself. Although other forms of in vivo energy harvesting have been described in lower organisms 3 , 4 , 5 , and thermoelectric 6 , piezoelectric 7 and biofuel 8 , 9 devices are promising for mammalian applications, there have been few, if any, in vivo demonstrations in the vicinity of the ear, eye and brain. In this work, the chip extracted a minimum of 1.12 nW from the EP of a guinea pig for up to 5 h, enabling a 2.4 GHz radio to transmit measurement of the EP every 40–360 s. With future optimization of electrode design, we envision using the biologic battery in the inner ear to power chemical and molecular sensors, or drug-delivery actuators for diagnosis and therapy of hearing loss and other disorders.

The position and orientation of the head is maintained to be relatively similar during the CT / MR imaging process. However, the position / orientation dissimilarities present in the resulting images between patients, or between different scans of the same patient, do not allow for direct comparison of the images themselves or features / metrics extracted from them. This paper introduces a method of defining a coordinate system which is consistent between patients and modalities (CT and MR) for images of the temporal bone, using easily identifiable landmarks within the semicircular canals. Cone Beam CT and high resolution MRI (T2) images of the temporal bone from 20 patients with no cochlear or temporal bone pathology in either modality were obtained. Four landmarks within the semicircular canals were defined that can be identified in both modalities. A coordinate system was defined using these landmarks. Reproducibility of landmark selection was assessed using intra- and inter-rater reliability (for three expert raters and two repeats of the landmark selection). Accuracy of the coordinate system was determined by comparing the coordinates of two additional landmarks in CT and MR images after their conversion to the proposed coordinate system. Intraclass Correlation Coefficients at a 95% level of confidence showed significant agreement within and between raters as well as between modalities. The differences between selections, raters, and modalities (as measured using mean, standard deviation, and maximum) were low and acceptable for clinical applications. The proposed coordinate system is suited for use in images of the temporal bone and inner ear. Its multi-modal nature enables the coordinate system to be used in tasks such as image co-registration.

Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness.

Exosomes are nanovesicles involved in intercellular communications. They are released by a variety of cell types; however, their presence in the inner ear has not been described in the literature. The aims of this study were to determine if exosomes are present in the inner ear and, if present, characterize the changes in their protein content in response to ototoxic stress. In this laboratory investigation, inner ear explants of 5-day-old Wistar rats were cultured and treated with either cisplatin or gentamicin. Hair cell damage was assessed by confocal microscopy. Exosomes were isolated using ExoQuick, serial centrifugation, and mini-column methods. Confirmation and characterization of exosomes was carried out using transmission electron microscopy (TEM), ZetaView, BCA protein analysis, and proteomics. Vesicles with a typical size distribution for exosomes were observed using TEM and ZetaView. Proteomic analysis detected typical exosome markers and markers for the organ of Corti. There was a statistically significant reduction in the exosome protein level and number of particles per cubic centimeter when the samples were exposed to ototoxic stress. Proteomic analysis also detected clear differences in protein expression when ototoxic medications were introduced. Significant changes in the proteomes of the exosomes were previously described in the context of hearing loss and ototoxic treatment. This is the first report describing exosomes derived from the inner ear. These findings may present an opportunity to conduct further studies with the hope of using exosomes as a biomarker to monitor inner ear function in the future.