The mechanoreceptive sensory hair cells in the inner ear are selectively vulnerable to numerous genetic and environmental insults. In mammals, hair cells lack regenerative capacity, and their death leads to permanent hearing loss and vestibular dysfunction. Their paucity and inaccessibility has limited the search for otoprotective and regenerative strategies. Growing hair cells in vitro would provide a route to overcome this experimental bottleneck. We report a combination of four transcription factors (Six1, Atoh1, Pou4f3, and Gfi1) that can convert mouse embryonic fibroblasts, adult tail-tip fibroblasts and postnatal supporting cells into induced hair cell-like cells (iHCs). iHCs exhibit hair cell-like morphology, transcriptomic and epigenetic profiles, electrophysiological properties, mechanosensory channel expression, and vulnerability to ototoxin in a high-content phenotypic screening system. Thus, direct reprogramming provides a platform to identify causes and treatments for hair cell loss, and may help identify future gene therapy approaches for restoring hearing.

During embryonic development, hierarchical cascades of transcription factors interact with lineage-specific chromatin structures to control the sequential steps in the differentiation of specialized cell types. While examples of transcription factor cascades have been well documented, the mechanisms underlying developmental changes in accessibility of cell type–specific enhancers remain poorly understood. Here, we show that the transcriptional “master regulator” ATOH1—which is necessary for the differentiation of two distinct mechanoreceptor cell types, hair cells in the inner ear and Merkel cells of the epidermis—is unable to access much of its target enhancer network in the progenitor populations of either cell type when it first appears, imposing a block to further differentiation. This block is overcome by a feed-forward mechanism in which ATOH1 first stimulates expression of POU4F3, which subsequently acts as a pioneer factor to provide access to closed ATOH1 enhancers, allowing hair cell and Merkel cell differentiation to proceed. Our analysis also indicates the presence of both shared and divergent ATOH1/POU4F3-dependent enhancer networks in hair cells and Merkel cells. These cells share a deep developmental lineage relationship, deriving from their common epidermal origin, and suggesting that this feed-forward mechanism preceded the evolutionary divergence of these very different mechanoreceptive cell types.

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The mammalian cochlea loses its ability to regenerate new hair cells prior to the onset of hearing. In contrast, the adult vestibular system can produce new hair cells in response to damage, or by reprogramming of supporting cells with the hair cell transcription factor Atoh1. We used RNA-seq and ATAC-seq to probe the transcriptional and epigenetic responses of utricle supporting cells to damage and Atoh1 transduction. We show that the regenerative response of the utricle correlates with a more accessible chromatin structure in utricle supporting cells compared to their cochlear counterparts. We also provide evidence that Atoh1 transduction of supporting cells is able to promote increased transcriptional accessibility of some hair cell genes. Our study offers a possible explanation for regenerative differences between sensory organs of the inner ear, but shows that additional factors to Atoh1 may be required for optimal reprogramming of hair cell fate.

Aminoglycoside antibiotics are “the drug of choice” for treating many bacterial infections, but their administration results in hearing loss in up to one fourth of the patients who receive them. Several biochemical pathways have been implicated in aminoglycoside antibiotic ototoxicity; however, little is known about how hair cells respond to aminoglycoside antibiotics at the transcriptome level. Here we have investigated the genome-wide response to the aminoglycoside antibiotic gentamicin. Using organotypic cultures of the perinatal organ of Corti, we performed RNA sequencing using cDNA libraries obtained from FACS-purified hair cells. Within 3 h of gentamicin treatment, the messenger RNA level of more than three thousand genes in hair cells changed significantly. Bioinformatic analysis of these changes highlighted several known signal transduction pathways, including the JNK pathway and the NF-κB pathway, in addition to genes involved in the stress response, apoptosis, cell cycle control, and DNA damage repair. In contrast, only 698 genes, mainly involved in cell cycle and metabolite biosynthetic processes, were significantly affected in the non-hair cell population. The gene expression profiles of hair cells in response to gentamicin share a considerable similarity with those previously observed in gentamicin-induced nephrotoxicity. Our findings suggest that previously observed early responses to gentamicin in hair cells in specific signaling pathways are reflected in changes in gene expression. Additionally, the observed changes in gene expression of cell cycle regulatory genes indicate a disruption of the postmitotic state, which may suggest an alternate pathway regulating gentamicin-induced apoptotic hair cell death. This work provides a more comprehensive view of aminoglycoside antibiotic ototoxicity, and thus contributes to identifying potential pathways or therapeutic targets to alleviate this important side effect of aminoglycoside antibiotics.

Neuropilin-1 (Nrp1) encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific Nrp1 conditional knockout (CKO) mouse line because Nrp1 constitutive knockouts are embryonic lethal. In situ hybridization demonstrated weak Nrp1 mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, Nrp1 CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old Nrp1 CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old Nrp1 CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of Nrp1 in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of Nrp1 show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons in vitro. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that Nrp1 may play a role in ARHL.

Precise control of organ growth and patterning is executed through a balanced regulation of progenitor self-renewal and differentiation. In the auditory sensory epithelium—the organ of Corti—progenitor cells exit the cell cycle in a coordinated wave between E12.5 and E14.5 before the initiation of sensory receptor cell differentiation, making it a unique system for studying the molecular mechanisms controlling the switch between proliferation and differentiation. Here we identify the Yap/Tead complex as a key regulator of the self-renewal gene network in organ of Corti progenitor cells. We show that Tead transcription factors bind directly to the putative regulatory elements of many stemness- and cell cycle-related genes. We also show that the Tead coactivator protein, Yap, is degraded specifically in the Sox2-positive domain of the cochlear duct, resulting in down-regulation of Tead gene targets. Further, conditional loss of theYapgene in the inner ear results in the formation of significantly smaller auditory and vestibular sensory epithelia, while conditional overexpression of a constitutively active version ofYap,Yap5SA, is sufficient to prevent cell cycle exit and to prolong sensory tissue growth. We also show that viral gene delivery ofYap5SAin the postnatal inner ear sensory epithelia in vivo drives cell cycle reentry after hair cell loss. Taken together, these data highlight the key role of the Yap/Tead transcription factor complex in maintaining inner ear progenitors during development, and suggest new strategies to induce sensory cell regeneration.

3D heterogeneous and anisotropic scaffolds that approximate native heart valve tissues are indispensable for the successful construction of tissue engineered heart valves (TEHVs). In this study, novel tri-layered and gel-like nanofibrous scaffolds, consisting of poly(lactic-co-glycolic) acid (PLGA) and poly(aspartic acid) (PASP), are fabricated by a combination of positive/negative conjugate electrospinning and bioactive hydrogel post-processing. The nanofibrous PLGA-PASP scaffolds present tri-layered structures, resulting in anisotropic mechanical properties that are comparable with native heart valve leaflets. Biological tests show that nanofibrous PLGA-PASP scaffolds with high PASP ratios significantly promote the proliferation and collagen and glycosaminoglycans (GAGs) secretions of human aortic valvular interstitial cells (HAVICs), compared to PLGA scaffolds. Importantly, the nanofibrous PLGA-PASP scaffolds are found to effectively inhibit the osteogenic differentiation of HAVICs. Two types of porcine VICs, from young and adult age groups, are further seeded onto the PLGA-PASP scaffolds. The adult VICs secrete higher amounts of collagens and GAGs and undergo a significantly higher level of osteogenic differentiation than young VICs. RNA sequencing analysis indicates that age has a pivotal effect on the VIC behaviors. This study provides important guidance and a reference for the design and development of 3D tri-layered, gel-like nanofibrous PLGA-PASP scaffolds for TEHV applications.