Regeneration Of Auditory Hair Cells Research Articles

Progress of research on the role of Atoh1 gene in the regeneration of mammalian auditory hair cells

Atoh1 gene encodes a helix-loop-helix transcription factor which is involved in the generation and differentiation of mammalian auditory hair cells and supporting cells, and regulation of the proliferation of cochlear cells, therefore plays an important role in the pathogenesis and recovery of sensorineural deafness. This study reviews the progress of the Atoh1 gene in hair cell regeneration, with the aim of providing a reference for the study of hair cell regeneration gene therapy for sensorineural deafness.

Future Pharmacotherapy for Sensorineural Hearing Loss by Protection and Regeneration of Auditory Hair Cells.

Sensorineural hearing loss has been a global burden of diseases for decades. However, according to recent progress in experimental studies on hair cell regeneration and protection, clinical trials of pharmacotherapy for sensorineural hearing loss have rapidly progressed. In this review, we focus on recent clinical trials for hair cell protection and regeneration and outline mechanisms based on associated experimental studies. Outcomes of recent clinical trials provided valuable data regarding the safety and tolerability of intra-cochlear and intra-tympanic applications as drug delivery methods. Recent findings in molecular mechanisms of hair cell regeneration suggested the realization of regenerative medicine for sensorineural hearing loss in the near future.

Hearing Recovery Induced by DNA Demethylation in a Chemically Deafened Adult Mouse Model.

Functional hair cell regeneration in the adult mammalian inner ear remains challenging. This study aimed to study the function of new hair cells induced by a DNA demethylating agent 5-azacytidine. Adult mice were deafened chemically, followed by injection of 5-azacytidine or vehicle into the inner ear. Functionality of regenerated hair cells was evaluated by expression of hair cell proteins, auditory brainstem response (ABR), and distortion-product otoacoustic emission (DPOAE) tests for 6 weeks. In the vehicle-treated group, no cells expressed the hair cell-specific protein myosin VIIa in the cochlea, whereas numerous myosin VIIa-expressing cells were found in the 5-azacytidine-treated cochlea, suggesting the regeneration of auditory hair cells. Moreover, regenerated hair cells were co-labeled with functional proteins espin and prestin. Expression of ribbon synapse proteins suggested synapse formation between new hair cells and neurons. In hearing tests, progressive improvements in ABR [5–30 dB sound pressure level (SPL)] and DPOAE (5–20 dB) thresholds were observed in 5-azacytidine-treated mice. In vehicle-treated mice, there were <5 dB threshold changes in hearing tests. This study demonstrated the ability of 5-azacytidine to promote the functional regeneration of auditory hair cells in a mature mouse model via DNA demethylation, which may provide insights into hearing regeneration using an epigenetic approach.

Research Progress on the Mechanism of Cochlear Hair Cell Regeneration.

Mammalian inner ear hair cells do not have the ability to spontaneously regenerate, so their irreversible damage is the main cause of sensorineural hearing loss. The damage and loss of hair cells are mainly caused by factors such as aging, infection, genetic factors, hypoxia, autoimmune diseases, ototoxic drugs, or noise exposure. In recent years, research on the regeneration and functional recovery of mammalian auditory hair cells has attracted more and more attention in the field of auditory research. How to regenerate and protect hair cells or auditory neurons through biological methods and rebuild auditory circuits and functions are key scientific issues that need to be resolved in this field. This review mainly summarizes and discusses the recent research progress in gene therapy and molecular mechanisms related to hair cell regeneration in the field of sensorineural hearing loss.

Insights into the molecular mechanisms regulating mammalian hair cell regeneration.

To give an overview of recent advances in mammalian auditory hair cell regeneration. Supporting cells act as progenitors to regenerate hair cells in the prehearing mammalian cochlea but not in the mature cochlea. To overcome this developmental obstacle, manipulation of multiple genes and intracellular pathways has been investigated, which has obtained promising data. This review focuses on recent advances in auditory hair cell regeneration, including synergic gene regulation associated with Atoh1 and Notch signaling, epigenetics, and functional recovery of regenerated hair cells. Co-manipulation of genes critical for hair cell development and cell cycle re-entry, including Atoh1, Isl1, Pou4f3, Gata3, Gfi1, P27kip1, RB, Myc, and Notch-signaling genes, has generated hair cell-like cells in the adult cochlea both in vitro and in vivo. The epigenetic mechanism has been studied in hair cell development and regeneration. Regeneration of hair cell function has a very limited progress, which lacks in-vitro and in-vivo electrophysiology data. Regeneration of adult auditory hair cells remains a major challenge. Manipulation of multiple genes and pathways together with epigenetic regulation might potentially regenerate functional hair cells in the adult mammalian cochlea.

Generation of Cochlear Hair Cells from Sox2 Positive Supporting Cells via DNA Demethylation

Regeneration of auditory hair cells in adult mammals is challenging. It is also difficult to track the sources of regenerated hair cells, especially in vivo. Previous paper found newly generated hair cells in deafened mouse by injecting a DNA methyltransferase inhibitor 5-azacytidine into the inner ear. This paper aims to investigate the cell sources of new hair cells. Transgenic mice with enhanced green fluorescent protein (EGFP) expression controlled by the Sox2 gene were used in the study. A combination of kanamycin and furosemide was applied to deafen adult mice, which received 4 mM 5-azacytidine injection into the inner ear three days later. Mice were followed for 3, 5, 7 and 14 days after surgery to track hair cell regeneration. Immunostaining of Myosin VIIa and EGFP signals were used to track the fate of Sox2-expressing supporting cells. The results show that (i) expression of EGFP in the transgenic mice colocalized the supporting cells in the organ of Corti, and (ii) the cell source of regenerated hair cells following 5-azacytidine treatment may be supporting cells during 5–7 days post 5-azacytidine injection. In conclusion, 5-azacytidine may promote the conversion of supporting cells to hair cells in chemically deafened adult mice.

Vascular endothelial growth factor is required for regeneration of auditory hair cells in the avian inner ear

Hair cells in the auditory organ of the vertebrate inner ear are the sensory receptors that convert acoustic stimuli into electrical signals that are conveyed along the auditory nerve to the brainstem. Hair cells are highly susceptible to ototoxic drugs, infection, and acoustic trauma, which can cause cellular degeneration. In mammals, hair cells that are lost after damage are not replaced, leading to permanent hearing impairments. By contrast, supporting cells in birds and other non-mammalian vertebrates regenerate hair cells after damage, which restores hearing function. The cellular mechanisms that regulate hair cell regeneration are not well understood. We investigated the role of vascular endothelial growth factor (VEGF) during regeneration of auditory hair cells in chickens after ototoxic injury. Using RNA-Seq, immunolabeling, and in situ hybridization, we found that VEGFA, VEGFC, VEGFR1, VEGFR2, and VEGFR3 were expressed in the auditory epithelium, with VEGFA expressed in hair cells and VEGFR1 and VEGFR2 expressed in supporting cells. Using organotypic cultures of the chicken cochlear duct, we found that blocking VEGF receptor activity during hair cell injury reduced supporting cell proliferation as well as the numbers of regenerated hair cells. By contrast, addition of recombinant human VEGFA to organ cultures caused an increase in both supporting cell division and hair cell regeneration. VEGF’s effects on supporting cells were preserved in isolated supporting cell cultures, indicating that VEGF can act directly upon supporting cells. These observations demonstrate a heretofore uncharacterized function for VEGF signaling as a critical positive regulator of hair cell regeneration in the avian inner ear.

Recent Advancements in the Regeneration of Auditory Hair Cells and Hearing Restoration.

Neurosensory responses of hearing and balance are mediated by receptors in specialized neuroepithelial sensory cells. Any disruption of the biochemical and molecular pathways that facilitate these responses can result in severe deficits, including hearing loss and vestibular dysfunction. Hearing is affected by both environmental and genetic factors, with impairment of auditory function being the most common neurosensory disorder affecting 1 in 500 newborns, as well as having an impact on the majority of elderly population. Damage to auditory sensory cells is not reversible, and if sufficient damage and cell death have taken place, the resultant deficit may lead to permanent deafness. Cochlear implants are considered to be one of the most successful and consistent treatments for deaf patients, but only offer limited recovery at the expense of loss of residual hearing. Recently there has been an increased interest in the auditory research community to explore the regeneration of mammalian auditory hair cells and restoration of their function. In this review article, we examine a variety of recent therapies, including genetic, stem cell and molecular therapies as well as discussing progress being made in genome editing strategies as applied to the restoration of hearing function.

In Vivo Generation of Immature Inner Hair Cells in Neonatal Mouse Cochleae by Ectopic Atoh1 Expression

Regeneration of auditory hair cells (HCs) is a promising approach to restore hearing. Recent studies have demonstrated that induced pluripotent stem cells/embryonic stem cells or supporting cells (SCs) adjacent to HCs can be converted to adopt the HC fate. However, little is known about whether new HCs are characteristic of outer or inner HCs. Here, we showed in vivo conversion of 2 subtypes of SCs, inner border cells (IBs) and inner phalangeal cells (IPhs), to the inner HC (IHC) fate. This was achieved by ectopically activating Atoh1, a transcription factor necessary for HC fate, in IBs/IPhs at birth. Atoh1+ IBs/IPhs first turned on Pou4f3, another HC transcription factor, before expressing 8 HC markers. The conversion rate gradually increased from ∼2.4% at 1 week of age to ∼17.8% in adult. Interestingly, new HCs exhibited IHC characteristics such as straight line–shaped stereociliary bundles, expression of Fgf8 and otoferlin, and presence of larger outward currents than those of outer HCs. However, new HCs lacked the terminal differentiation IHC marker vGlut3, exhibited reduced density of presynaptic Cbtp2 puncta that had little postsynaptic GluR2 specialization, and displayed immature IHC outward currents. Our results demonstrate that the conversion rate of IBs/IPhs in vivo by Atoh1 ectopic expression into the IHC fate was higher and faster and the conversion was more complete than that of the 2 other SC subtypes underneath the outer HCs; however, these new IHCs are arrested before terminal differentiation. Thus, IBs/IPhs are good candidates to regenerate IHCs in vivo.

The Transcriptomics to Proteomics of Hair Cell Regeneration: Looking for a Hair Cell in a Haystack

Mature mammals exhibit very limited capacity for regeneration of auditory hair cells, while all non-mammalian vertebrates examined can regenerate them. In an effort to find therapeutic targets for deafness and balance disorders, scientists have examined gene expression patterns in auditory tissues under different developmental and experimental conditions. Microarray technology has allowed the large-scale study of gene expression profiles (transcriptomics) at whole-genome levels, but since mRNA expression does not necessarily correlate with protein expression, other methods, such as microRNA analysis and proteomics, are needed to better understand the process of hair cell regeneration. These technologies and some of the results of them are discussed in this review. Although there is a considerable amount of variability found between studies owing to different species, tissues and treatments, there is some concordance between cellular pathways important for hair cell regeneration. Since gene expression and proteomics data is now commonly submitted to centralized online databases, meta-analyses of these data may provide a better picture of pathways that are common to the process of hair cell regeneration and lead to potential therapeutics. Indeed, some of the proteins found to be regulated in the inner ear of animal models (e.g., IGF-1) have now gone through human clinical trials.

Supporting Cell Division Is Not Required for Regeneration of Auditory Hair Cells After Ototoxic Injury In Vitro

In chickens, nonsensory supporting cells divide and regenerate auditory hair cells after injury. Anatomical evidence suggests that supporting cells can also transdifferentiate into hair cells without dividing. In this study, we characterized an organ culture model to study auditory hair cell regeneration, and we used these cultures to test if direct transdifferentiation alone can lead to significant hair cell regeneration. Control cultures (organs from posthatch chickens maintained without streptomycin) showed complete hair cell loss in the proximal (high-frequency) region by 5 days. In contrast, a 2-day treatment with streptomycin induced loss of hair cells from all regions by 3 days. Hair cell regeneration proceeded in culture, with the time course of supporting cell division and hair cell differentiation generally resembling in vivo patterns. The degree of supporting cell division depended upon the presence of streptomycin, the epithelial region, the type of culture media, and serum concentration. On average, 87% of the regenerated hair cells lacked the cell division marker BrdU despite its continuous presence, suggesting that most hair cells were regenerated via direct transdifferentiation. Addition of the DNA polymerase inhibitor aphidicolin to culture media prevented supporting cell division, but numerous hair cells were regenerated nonetheless. These hair cells showed signs of functional maturation, including stereociliary bundles and rapid uptake of FM1-43. These observations demonstrate that direct transdifferentiation is a significant mechanism of hair cell regeneration in the chicken auditory after streptomycin damage in vitro.

Symposium Overview

INTERNATIONAL SYMPOSIUM ON OLFACTION AND TASTE Symposium Overview Stem Cells in Sensory Epithelium Development and Regeneration Linda A. Barlow a and Anne L. Calof b a b Department of Cell and Developmental Biology and the Rocky Mountain Taste & Smell Center, University of Colorado Denver, School of Medicine, Aurora, Colorado, USA Department of Anatomy & Neurobiology and the Center for Complex Biological Systems, University of California, Irvine, California, USA Many adult tissues are renewed throughout life via processes that are either known or thought to rely on a population of tissue-specific stem cells. For example, studies of both skin and brain have identified local stem cells that have complex interactions with their niche environ- ment, leading to regulated cell genesis. The senses of both smell and taste are well known to be mediated by sensory epithelia that are also continually replaced, although the mecha- nisms that govern renewal are less well under- stood. In this symposium, we showcased some recent advances in development of the taste and olfactory periphery, with the hope of shed- ding light on potential mechanisms governing adult renewal of these sensory epithelia. These chemical senses presentations were bookended by talks pertaining to regeneration and mainte- nance of auditory and skin epithelia in adults. The purpose here was to highlight potential parallels in our understanding of molecular and cellular mechanisms regulating auditory hair cells and skin with cell renewal in chemosen- sory epithelia. Neil Segil (House Ear Institute, Los Ange- les) began with an overview of his work on the development and potential for regeneration of auditory hair cells. In vertebrates, hair cells of the inner ear are delicate mechanoreceptors that transduce pressure waves into electrical sig- nals interpreted by the nervous system. In many species, including birds, amphibians, and fishes, these hair cells can be generated in the event of injury or to accommodate growth; however, mammals appear to have lost this regenerative capacity during evolution. Thus, the mecha- nisms that restrict regeneration in mammals are of great interest to both developmental neu- robiologists and clinicians. One exciting idea explored by Dr. Segil is that under certain con- ditions mammalian auditory epithelium may be able to regenerate. Specifically, as is the case in birds, a population of mammalian auditory support cells, located adjacent to hair cells, may be able to reactivate the potential to give rise to new hair cells by altering levels of key cell cycle inhibitors and through alteration of signaling through the Notch pathway. After this introduction to key concepts in sen- sory epithelium differentiation, Shoba Thiru- mangalathu (University of Colorado Denver, School of Medicine) discussed development of the taste epithelium, focusing on her stud- ies to identify the embryonic cell population that gives rise to adult taste buds. She em- ployed inducible and tissue-specific molecular genetic mouse models to label the developing tongue epithelium as the first taste structures— the taste placodes—form, and to follow these cells and their progeny through to adult stages. Although it has long been assumed that taste placodes represent taste papillae, which in turn Address for correspondence: Linda A. Barlow, University of Colorado, Denver, School of Medicine, Dept of cell & Developmental Biology, Mail Stop 8108, 12801 East 17th Avenue, Room L18-11121, Aurora, CO 80045. Voice: 303/724-3438; fax: 303/724-3420. linda.barlow@ ucdenver.edu International Symposium on Olfaction and Taste: Ann. N.Y. Acad. Sci. 1170: 5–6 (2009). c 2009 New York Academy of Sciences. doi: 10.1111/j.1749-6632.2009.04108.x

Xenografted fetal dorsal root ganglion, embryonic stem cell and adult neural stem cell survival following implantation into the adult vestibulocochlear nerve

Sensorineural hearing loss is a disabling condition. In the post-embryonic and adult mammalian inner ear, the regeneration of auditory hair cells, spiral ganglion neurons or their axons does not occur naturally. This decrease in excitable neurons limits the success of auditory rehabilitation. Allografts and xenografts have shown promise in the treatment of a variety of neurological diseases. Fetal dorsal root ganglion (DRG) neurons can extend functional connections in the rat spinal cord. Embryonic stem cells (ES cells) and adult neural stem cells (ANSC) have the potential to differentiate into neurons. We have implanted embryonic days (E) 13–16 fetal mouse DRGs from transgenic mouse lines that express Enhanced Green Fluorescent Protein (EGFP) or lacZ reporter genes, EGFP-expressing ES cells or lacZ-expressing ANSC into the injured vestibulocochlear nerve of adult rats and guinea pigs. Survival of the implants was assessed 2 to 4 weeks postoperatively. For further evaluation of the differentiation of the implanted ES-cells, we double labeled with the mouse-specific neuronal antibody Thy 1.2. The rats implanted with EGFP- or lacZ-expressing DRGs showed labeled DRGs after sacrifice. In addition, EGFP-positive nerve fibers were seen growing within the proximal nerve. The results from the EGFP ES cells and lacZ ANSC revealed reporter-expressing cells at the site of injection in the vestibulocochlear nerve of the host rats and guinea pigs but also within the brain stem. Thy 1.2 profiles were seen among the EGFP ES cells within the 8th cranial nerve. The findings of this study indicate that the vestibulocochlear nerve of adult rats and guinea pigs will support xenotransplants of embryonic DRG, ES cells and ANSC. This may have future clinical applicability in recreating a neuronal conduit following neuronal injury between the inner ear and the central nervous system (CNS).

Vocal Memory and Learning in Adult Bengalese Finches with Regenerated Hair Cells

Critical learning periods are common in vertebrate development. In many birds, song learning is limited by a critical period; juveniles copy songs from adult birds by forming memories of those songs during a restricted developmental period and then using auditory feedback to practice their own vocalizations. Adult songs are stable over time regardless of exposure to other birds, but auditory feedback is required for the maintenance of stable adult song. A technique was developed to reversibly deafen Bengalese Finches by destruction and regeneration of inner ear auditory hair cells. With this approach, we asked two questions about the plasticity of song information stored in the adult brain. First, do adult birds store memories or "templates" of their songs that exist independent of auditory reinforcement? Such memories could be used to control vocal output by acting as fixed models of song to which ongoing vocalizations are matched. Second, can adult song learning, which does not normally occur in this species, be induced by removing and then restoring hearing? Studying changes in adult song behavior during hair cell loss and regeneration revealed two findings: (1) adult birds store memories or templates of their songs that exist independent of auditory input and can be used to restore normal vocal behavior when hearing is restored; (2) under experimental circumstances, adult birds can be induced to acquire song material from other birds. Results suggest that, in Bengalese Finches, the degree of behavioral and neural plasticity in juvenile and adult birds may be less distinct that previously thought.

Recent insights into regeneration of auditory and vestibular hair cells.

Advances in hair cell regeneration are progressing at a rapid rate. This review will highlight and critique recent attempts to understand some of the cellular and molecular mechanisms underlying hair cell regeneration in non-mammalian vertebrates and efforts to induce regeneration in the mammalian inner ear sensory epithelium.

Retinoic acid stimulates regeneration of mammalian auditory hair cells.

Sensorineural hearing loss resulting from the loss of auditory hair cells is thought to be irreversible in mammals. This study provides evidence that retinoic acid can stimulate the regeneration in vitro of mammalian auditory hair cells in ototoxic-poisoned organ of Corti explants in the rat. In contrast, treatment with retinoic acid does not stimulate the formation of extra hair cells in control cultures of Corti's organ. Retinoic acid-stimulated hair cell regeneration can be blocked by cytosine arabinoside, which suggests that a period of mitosis is required for the regeneration of auditory hair cells in this system. These results provide hope for a recovery of hearing function in mammals after auditory hair cell damage.