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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.

The adult mammalian inner ear lacks the capacity to divide or regenerate. Damage to inner ear generally leads to permanent hearing loss in humans. Here, we present that reprogramming of the adult inner ear induces renewed proliferation and regeneration of inner ear cell types. Co-activation of cell cycle activator Myc and inner ear progenitor gene Notch1 induces robust proliferation of diverse adult cochlear sensory epithelial cell types. Transient MYC and NOTCH activities enable adult supporting cells to respond to transcription factor Atoh1 and efficiently transdifferentiate into hair cell-like cells. Furthermore, we uncover that mTOR pathway participates in MYC/NOTCH-mediated proliferation and regeneration. These regenerated hair cell-like cells take up the styryl dye FM1-43 and are likely to form connections with adult spiral ganglion neurons, supporting that Myc and Notch1 co-activation is sufficient to reprogram fully mature supporting cells to proliferate and regenerate hair cell-like cells in adult mammalian auditory organs. The adult mammalian inner ear cells cannot regenerate nor proliferate. Here, the authors show that co-activation of Myc and NOTCH pathways can stimulate proliferation of inner ear sensory epithelial cells, which can be induced to become hair cell-like cells in vitro and in vivo.

Phylogenomics of bats suggests that their echolocation either evolved separately in the bat suborders Yinpterochiroptera and Yangochiroptera, or had a single origin in bat ancestors and was later lost in some yinpterochiropterans 1 – 6 . Hearing for echolocation behaviour depends on the inner ear, of which the spiral ganglion is an essential structure. Here we report the observation of highly derived structures of the spiral ganglion in yangochiropteran bats: a trans -otic ganglion with a wall-less Rosenthal’s canal. This neuroanatomical arrangement permits a larger ganglion with more neurons, higher innervation density of neurons and denser clustering of cochlear nerve fascicles 7 – 13 . This differs from the plesiomorphic neuroanatomy of Yinpterochiroptera and non-chiropteran mammals. The osteological correlates of these derived ganglion features can now be traced into bat phylogeny, providing direct evidence of how Yangochiroptera differentiated from Yinpterochiroptera in spiral ganglion neuroanatomy. These features are highly variable across major clades and between species of Yangochiroptera, and in morphospace, exhibit much greater disparity in Yangochiroptera than Yinpterochiroptera. These highly variable ganglion features may be a neuroanatomical evolutionary driver for their diverse echolocating strategies 4 , 14 – 17 and are associated with the explosive diversification of yangochiropterans, which include most bat families, genera and species. The presence of a variety of highly derived spiral ganglion structures of the inner ear is associated with diverse echolocation strategies in yangochiropteran bats and distinguishes them from Yinpterochiroptera.

Estimation of cochlear length is gaining attention in the field of cochlear implants (CIs), mainly for selecting of CI electrode lengths. The currently available tools to estimate the cochlear duct length (CDL) are only valid for normal inner anatomy. However, inner ear malformation (IEM) types are associated with different degrees of cystic apices, limiting the application of CDL equations of normal anatomy inner ear. Therefore, this study aimed to understand the degree to which the outer wall (OW) is observed in different malformation types and to formulate mathematical equations to estimate the OW length (OWL) from cochlear parameters, namely the basal turn diameter (A-value) and width (B-value). Three-dimensional (3D) segmentation of promontory and fluid parts of the inner ear was performed to understand the extent to which the OW is visible to measure the OWL manually. Enlarged vestibular aqueduct syndrome (EVAS) was diagnosed in 37 ears, which consistently showed the extent of the OW to an angular depth of 540°, beyond which the cystic apex starts. Incomplete partition (IP) type I was observed in 30 ears, with the OW extending to only 360° of angular depth. IP type II was observed in 35 ears, with the OW extending to 450° of angular depth. IP type III was identified in 24 ears, with the OW observed for 540° of angular depth. Cavity-type malformations were observed in 36 ears, and circumference was measured in the axial view. A strong positive linear correlation was observed between the manually measured OWL and cochlear parameters for all malformation types analyzed. A multiple linear regression model was applied to formulate mathematical equations, which was further used to create a software application for estimating OWLs in IEM types, using cochlear parameters as inputs.

Endothermy underpins the ecological dominance of mammals and birds in diverse environmental settings 1 , 2 . However, it is unclear when this crucial feature emerged during mammalian evolutionary history, as most of the fossil evidence is ambiguous 3 – 17 . Here we show that this key evolutionary transition can be investigated using the morphology of the endolymph-filled semicircular ducts of the inner ear, which monitor head rotations and are essential for motor coordination, navigation and spatial awareness 18 – 22 . Increased body temperatures during the ectotherm–endotherm transition of mammal ancestors would decrease endolymph viscosity, negatively affecting semicircular duct biomechanics 23 , 24 , while simultaneously increasing behavioural activity 25 , 26 probably required improved performance 27 . Morphological changes to the membranous ducts and enclosing bony canals would have been necessary to maintain optimal functionality during this transition. To track these morphofunctional changes in 56 extinct synapsid species, we developed the thermo-motility index, a proxy based on bony canal morphology. The results suggest that endothermy evolved abruptly during the Late Triassic period in Mammaliamorpha, correlated with a sharp increase in body temperature (5–9 °C) and an expansion of aerobic and anaerobic capacities. Contrary to previous suggestions 3 – 14 , all stem mammaliamorphs were most probably ectotherms. Endothermy, as a crucial physiological characteristic, joins other distinctive mammalian features that arose during this period of climatic instability 28 . The functional morphology of the fluid-filled semicircular ducts of the inner ear is adapted to body temperature and behavioural activity and can be used to investigate the evolution of endothermy.

This study investigated the potential of mucoadhesive polymeric nanoparticles coated with polydopamine (Dopa-NPs) for inner ear drug delivery. Dopa, inspired by mussel adhesion proteins, leveraged the mucoadhesive properties of NPs and enhanced their retention within the cochlea. Dopa-NPs were compared with non-adhesive uncoated control NPs to reveal their safety and drug delivery efficiency. The safety evaluation demonstrated no toxicity during in vitro test using HEI-OC1 cells and in vivo test using mouse with auditory function assessment. When coumarin-encapsulated NPs were administered through intratympanic injection to mice, Dopa-NPs provided intense fluorescence in the inner ear compared to non-adhesive control NPs. Furthermore, dexamethasone (Dex)-encapsulated Dopa-NPs exhibited significantly higher drug concentrations in the cochlea than dexamethasone sodium phosphate and uncoated NPs. Finally, in vivo test using an ototoxicity-induced animal model showed that Dopa-NPs improved hearing protection, as indicated by the auditory brainstem response (ABR) test. In conclusion, mucoadhesive Dopa-NPs exhibit enhanced drug delivery efficiency and safety, offering a promising strategy for inner ear drug delivery and chemotherapy. Graphical abstract

In vivo gene delivery to tissues using adeno-associated vector (AAVs) has revolutionized the field of gene therapy. Yet, while sensorineural hearing loss is one of the most common sensory disorders worldwide, gene therapy applied to the human inner ear is still in its infancy. Recent advances in the development recombinant AAVs have significantly improved their cell tropism and transduction efficiency across diverse inner ear cell types to a level that renders this tool valuable for conditionally manipulating gene expression in the context of developmental biology studies of the mouse inner ear. Here, we describe a protocol for in utero micro-injection of AAVs into the embryonic inner ear, using the AAV-PHP.eB and AAV-DJ serotypes that respectively target the sensory hair cells and the supporting cells of the auditory sensory epithelium. We also aimed to standardize procedures for imaging acquisition and image analysis to foster research reproducibility and allow accurate comparisons between studies. We find that AAV-PHP.eB and AAV-DJ provide efficient and reliable tools for conditional gene expression targeting cochlear sensory and supporting cells in the mouse inner ear, from late embryonic stages on.

Major evolutionary transitions, in which animals develop new body plans and adapt to dramatically new habitats and lifestyles, have punctuated the history of life. The origin of cetaceans from land-living mammals is among the most famous of these events. Much earlier, during the Mesozoic Era, many reptile groups also moved from land to water, but these transitions are more poorly understood. We use computed tomography to study changes in the inner ear vestibular system, involved in sensing balance and equilibrium, as one of these groups, extinct crocodile relatives called thalattosuchians, transitioned from terrestrial ancestors into pelagic (open ocean) swimmers. We find that the morphology of the vestibular system corresponds to habitat, with pelagic thalattosuchians exhibiting a more compact labyrinth with wider semicircular canal diameters and an enlarged vestibule, reminiscent of modified and miniaturized labyrinths of other marine reptiles and cetaceans. Pelagic thalattosuchians with modified inner ears were the culmination of an evolutionary trend with a long semiaquatic phase, and their pelagic vestibular systems appeared after the first changes to the postcranial skeleton that enhanced their ability to swim. This is strikingly different from cetaceans, which miniaturized their labyrinths soon after entering the water, without a prolonged semiaquatic stage. Thus, thalattosuchians and cetaceans became secondarily aquatic in different ways and at different paces, showing that there are different routes for the same type of transition.

The in vivo mechanisms underlying dominant syndromes caused by mutations in SRY-Box Transcription Factor 9 (SOX9) and SOX10 (SOXE) transcription factors, when they either are expressed alone or are coexpressed, are ill-defined. We created a mouse model for the campomelic dysplasia SOX9Y440X mutation, which truncates the transactivation domain but leaves DNA binding and dimerization intact. Here, we find that SOX9Y440X causes deafness via distinct mechanisms in the endolymphatic sac (ES)/duct and cochlea. By contrast, conditional heterozygous Sox9-null mice are normal. During the ES development of Sox9 Y440X/+ heterozygotes, Sox10 and genes important for ionic homeostasis are down-regulated, and there is developmental persistence of progenitors, resulting in fewer mature cells. Sox10 heterozygous null mutants also display persistence of ES/duct progenitors. By contrast, SOX10 retains its expression in the early Sox9 Y440X/+ mutant cochlea. Later, in the postnatal stria vascularis, dominant interference by SOX9Y440X is implicated in impairing the normal cooperation of SOX9 and SOX10 in repressing the expression of the water channel Aquaporin 3, thereby contributing to endolymphatic hydrops. Our study shows that for a functioning endolymphatic system in the inner ear, SOX9 regulates Sox10, and depending on the cell type and target gene, it works either independently of or cooperatively with SOX10. SOX9Y440X can interfere with the activity of both SOXE factors, exerting effects that can be classified as haploinsufficient/hypomorphic or dominant negative depending on the cell/gene context. This model of disruption of transcription factor partnerships may be applicable to congenital deafness, which affects ∼0.3% of newborns, and other syndromic disorders.

While inner ear disorders are common, our ability to intervene and recover their sensory function is limited. In vitro models of the inner ear, like the organoid system, could aid in identifying new regenerative drugs and gene therapies. Here, we provide a perspective on the status of in vitro inner ear models and guidance on how to improve their applicability in translational research. We highlight the generation of inner ear cell types from pluripotent stem cells as a particularly promising focus of research. Several exciting recent studies have shown how the developmental signaling cues of embryonic and fetal development can be mimicked to differentiate stem cells into “inner ear organoids” containing otic progenitor cells, hair cells, and neurons. However, current differentiation protocols and our knowledge of embryonic and fetal inner ear development in general, have a bias toward the sensory epithelia of the inner ear. We propose that a more holistic view is needed to better model the inner ear in vitro. Moving forward, attention should be made to the broader diversity of neuroglial and mesenchymal cell types of the inner ear, and how they interact in space or time during development. With improved control of epithelial, neuroglial, and mesenchymal cell fate specification, inner ear organoids would have the ability to truly recapitulate neurosensory function and dysfunction. We conclude by discussing how single-cell atlases of the developing inner ear and technical innovations will be critical tools to advance inner ear organoid platforms for future pre-clinical applications.