Auditory Hair Cell Regeneration Research Articles

COCHLEAR HAIR CELL REGENERATION BASED ON STEM CELLS: A SYSTEMATIC REVIEW

IntroductionAccording to the World Health Organization (WHO), by 2050 at least 700 million people will need access to hearing care and hearing rehabilitation services. The search for cell or gene therapies has been intensifying, and stem cell therapy looks a promising candidate to support hearing regeneration and reduce these numbers. The aim of this study is to provide an overview of current advances in stem cellbased therapies for cochlear hair cell regeneration and the processes being developed for future applicability.Material and methodsIdentification and review of all articles in the databases PubMed, Web of Science, and PLoS One using the terms <i>stem cell</i>, <i>auditory hair cell regeneration</i>, and <i>mammalian</i> during February 2023 and following the PRISMA guidelines.Results50 articles were obtained, published between 2003 and 2022 and were systematically analyzed. The current research quantity is limited and further studies are needed, particularly in human tissue.ConclusionsThe simultaneous use of cell therapy and gene therapy may lead to more promising results. Moreover, advances in cochlear hair cell regeneration with stem cells suggest there is a realistic potential to make the technique a useful future therapy.

An essential signaling cascade for avian auditory hair cell regeneration

Hearing loss is a chronic disease affecting millions of people worldwide, yet no restorative treatment options are available. Although non-mammalian species can regenerate their auditory sensory hair cells, mammals cannot. Birds retain facultative stem cells known as supporting cells that engage in proliferative regeneration when surrounding hair cells die. Here, we investigated gene expression changes in chicken supporting cells during auditory hair cell death. This identified a pathway involving the receptor F2RL1, HBEGF, EGFR, and ERK signaling. We propose a cascade starting with the proteolytic activation of F2RL1, followed by matrix-metalloprotease-mediated HBEGF shedding, and culminating in EGFR-mediated ERK signaling. Each component of this cascade is essential for supporting cell S-phase entry invivo and is integral for hair cell regeneration. Furthermore, STAT3-phosphorylation converges with this signaling toward upregulation of transcription factors ATF3, FOSL2, and CREM. Our findings could provide a basis for designing treatments for hearing and balance disorders.

Stem Cell-Based Therapies for Auditory Hair Cell Regeneration in the Treatment of Hearing Loss.

The incidence and prevalence of hearing loss is increasing globally at an accelerated pace. Hair cells represent the sensory receptors of auditory and vestibular systems. Hair cell absence, loss or degeneration due to congenital diseases, trauma, toxicity, infection or advancing age, results in disabling hearing loss. Regenerative medicine approaches consisting in stem cell-based hair cell rescue or regeneration, gene therapy, as well as cell and tissue engineering are expected to dramatically improve the therapeutic arsenal available for addressing hearing loss. Current strategies that are using different stem cell types to rescue or to induce hair cell proliferation and regeneration are presented. Gene and cell therapy methods that modulates transdifferentiation of surrounding cell types into hair cells are presented, together with their specific advantages and limitations. Several modalities for improving therapeutic targeting to the inner ear such as nanoparticle-mediated cell and gene delivery are introduced. Further steps in building more relevant high-throughput models for testing novel drugs and advanced therapies are proposed as a modality to accelerate translation to clinical settings.

Avian auditory hair cell regeneration is accompanied by JAK/STAT-dependent expression of immune-related genes in supporting cells.

The avian hearing organ is the basilar papilla that, in sharp contrast to the mammalian cochlea, can regenerate sensory hair cells and thereby recover from deafness within weeks. The mechanisms that trigger, sustain and terminate the regenerative response in vivo are largely unknown. Here, we profile the changes in gene expression in the chicken basilar papilla after aminoglycoside antibiotic-induced hair cell loss using RNA-sequencing. We identified changes in gene expression of a group of immune-related genes and confirmed with single-cell RNA-sequencing that these changes occur in supporting cells. In situ hybridization was used to further validate these findings. We determined that the JAK/STAT signaling pathway is essential for upregulation of the damage-response genes in supporting cells during the second day after induction of hair cell loss. Four days after ototoxic damage, we identified newly regenerated, nascent auditory hair cells that express genes linked to termination of the JAK/STAT signaling response. The robust, transient expression of immune-related genes in supporting cells suggests a potential functional involvement of JAK/STAT signaling in sensory hair cell regeneration.

Vestibular and Auditory Hair Cell Regeneration Following Targeted Ablation of Hair Cells With Diphtheria Toxin in Zebrafish.

Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turnover during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However, in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo. This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 days post fertilization (dpf) zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.

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.

MicroRNA‑183 is involved in the differentiation and regeneration of Notch signaling‑prohibited hair cells from mouse cochlea.

Auditory hair cell regeneration following injury is critical to hearing restoration. The Notch signaling pathway participates in the regulation of inner ear development and cell differentiation. Recent evidence suggests that microRNA (miR)-183 has a similar role in the inner ear. However, it is unclear how Notch signaling functions in hair cell regeneration in mammals and if there is cross-talk between Notch signaling and miR-183. The present study used a gentamicin-induced cochlear injury mouse model. Gentamicin-induced damage of the hair cells activated the Notch signaling pathway and downregulated miR-183 expression. Notch signaling inhibition by the γ-secretase inhibitor, 24-diamino-5-phenylthiazole (DAPT), attenuated gentamicin-induced hair cell loss and reversed the downregulation of miR-183 expression. Further investigation revealed that the novel hair cells produced, induced by DAPT, were derived from transdifferentiated supporting cells. Additionally, myosin VI-positive hair cell numbers were increased by Notch signaling inhibition in in vitro experiments with cultured neonatal mouse inner ear precursor cells. This effect was reversed by miR-183 inhibition. These findings indicate that the Notch signaling pathway served a repressing role during the regeneration of hair cells. Inhibiting this signal improved hair cell regeneration in the gentamicin-damaged cochlear model. miR-183 was demonstrated to be involved in hair cell differentiation and regeneration, and was required for the differentiation of the Notch-inhibited hair cells.

Regulation of cell proliferation and apoptosis by growth hormone during zebrafish auditory hair cell regeneration

Background In order to develop treatments or preventive measures for auditory hair cell loss, an understanding of both the process of auditory hair cell regeneration and factors that influence this process, is needed. Our previous microarray analysis showed that growth hormone (GH) was significantly upregulated during zebrafish auditory hair cell regeneration, coupled with cell proliferation [1,2]. We further tested the effects of GH on zebrafish auditory hair cell regeneration by injecting GH after sound exposure and found that GH can efficiently promote post-trauma auditory hair cell regeneration, which may be achieved through stimulating proliferation and suppressing apoptosis [3]. In the current study, we used Next Generation Sequencing (NGS) to examine the possible GH pathways involved in zebrafish auditory hair cell regeneration.

Atoh1 expression and function during auditory hair cell regeneration in post-hatch chickens

Loss of hair cells in humans leads to irreversible hearing deficits, since auditory hair cells are not replaced. In contrast, hair cells are regenerated in the auditory epithelium of mature birds after damage by non-sensory supporting cells that transdifferentiate into hair cells by mitotic and/or non-mitotic mechanisms. Factors controlling these processes are poorly understood. The basic helix-loop-helix transcription factor ATOH1 is both necessary and sufficient for developmental hair cell differentiation, but it is unclear if it plays the same role in the mitotic and non-mitotic pathways in hair cell regeneration. We examined Atoh1 expression and function during hair cell regeneration in chickens. Atoh1 transcripts were increased in many supporting cells in the damaged auditory epithelium shortly after ototoxin administration and later became restricted to differentiating hair cells. Fate-mapping in vitro using an Atoh1 enhancer reporter demonstrated that only 56% of the supporting cells that spontaneously upregulate Atoh1 enhancer activity after damage acquired the hair cell fate. Inhibition of notch signaling using a gamma secretase antagonist stimulated an increase in Atoh1 reporter activity and induced a higher proportion of supporting cells with Atoh1 activity (73%) to differentiate as hair cells. Forced overexpression of Atoh1 in supporting cells triggered 66% of them to acquire the hair cell fate and nearly tripled their likelihood of cell cycle entry. These findings demonstrate that Atoh1 is broadly upregulated in supporting cells after damage, but a substantial proportion of supporting cells with Atoh1 activation fails to acquire hair cell features, in part due to gamma secretase-dependent activities.

Growth Hormone Promotes Hair Cell Regeneration in the Zebrafish (Danio rerio) Inner Ear following Acoustic Trauma

BackgroundPrevious microarray analysis showed that growth hormone (GH) was significantly upregulated following acoustic trauma in the zebrafish (Danio rerio) ear suggesting that GH may play an important role in the process of auditory hair cell regeneration. Our objective was to examine the effects of exogenous and endogenous GH on zebrafish inner ear epithelia following acoustic trauma.Methodology/Principal FindingsWe induced auditory hair cell damage by exposing zebrafish to acoustic overstimulation. Fish were then injected intraperitoneally with either carp GH or buffer, and placed in a recovery tank for either one or two days. Phalloidin-, bromodeoxyuridine (BrdU)-, and TUNEL-labeling were used to examine hair cell densities, cell proliferation, and apoptosis, respectively. Two days post-trauma, saccular hair cell densities in GH-treated fish were similar to that of baseline controls, whereas buffer-injected fish showed significantly reduced densities of hair cell bundles. Cell proliferation was greater and apoptosis reduced in the saccules, lagenae, and utricles of GH-treated fish one day following trauma compared to controls. Fluorescent in situ hybridization (FISH) was used to examine the localization of GH mRNA in the zebrafish ear. At one day post-trauma, GH mRNA expression appeared to be localized perinuclearly around erythrocytes in the blood vessels of the inner ear epithelia. In order to examine the effects of endogenous GH on the process of cell proliferation in the ear, a GH antagonist was injected into zebrafish immediately following acoustic trauma, resulting in significantly decreased cell proliferation one day post-trauma in all three zebrafish inner ear end organs.Conclusions/SignificanceOur results show that exogenous GH promotes post-trauma auditory hair cell regeneration in the zebrafish ear through stimulating proliferation and suppressing apoptosis, and that endogenous GH signals are present in the zebrafish ear during the process of auditory hair cell regeneration.

Growth hormone induces proliferation in the zebrafish inner ear

Background While acoustic trauma results in a loss of hair cells in the ears of fishes, they have the ability to recover their hair cells and hearing sensitivity within a few weeks [1]. Our previous microarray and RT-PCR analysis of sound-exposed zebrafish (Danio rerio) ears, showed that growth hormone (GH) was significantly upregulated during zebrafish auditory tissue cell proliferation and hair cell regeneration [2]. This upregulation was greatest two days following acoustic trauma, coincident with an increase in cell proliferation [3]. In order to better understand the role of GH in the regenerative abilities of the zebrafish ear, we performed two GH-injection experiments.

Auditory hair cell regeneration in teleost fishes: A review.

Exposure to high levels of sound can cause damage to the auditory hair cells of many organisms, including fishes, resulting in hearing deficits. While such exposure can lead to permanent hearing loss in mammals, other phylogenetically older nonmammalian vertebrates, such as birds, reptiles, amphibians, and fishes, can spontaneously regenerate hair cells in both the vestibular and auditory portions of the inner ear following trauma. The general mechanism of hair cell regeneration is currently being examined in a number of different taxa, but data are limited on hair cell regeneration in the inner ear of fishes. This presentation will summarize the few studies that have examined auditory hair cell regeneration in teleost fishes and compare the results reported for fishes to those reported for other species. While understanding the process of hair cell regeneration in fishes has biomedical relevance, this knowledge could also benefit marine managers attempting to mitigate the effects of anthropogenic noise on fishes. [Work supported by NIH P20 RR16481.]

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.

Microarray analysis and quantitative real-time PCR validation of gene expression during auditory hair cell regeneration in zebrafish (Danio rerio)

Background Fishes are capable of regenerating sensory hair cells in the inner ear after acoustic trauma [1]. We recently established the time course for auditory hair cell regeneration in zebrafish. Immediately following acoustic overexposure, zebrafish saccules exhibited significant hair cell loss, but recovered to pretreatment levels at 14 days post-sound exposure [2]. Cell proliferation peaked two days postsound exposure (dpse), so we examined gene expression patterns before, during, and after this time to find genes associated with this proliferation and subsequent hair cell regeneration.

Cell proliferation follows acoustically-induced hair cell bundle loss in the zebrafish saccule

Fishes are capable of regenerating sensory hair cells in the inner ear after acoustic trauma. However, a time course of auditory hair cell regeneration has not been established for zebrafish. Adult zebrafish ( Danio rerio) were exposed to a 100 Hz pure tone at 179 dB re 1 μPa RMS for 36 h and then allowed to recover for 0–14 days before morphological analysis. Hair cell bundle loss and recovery were determined using phalloidin to visualize hair bundles. Cell proliferation was quantified through bromodeoxyuridine (BrdU) labeling. Immediately following sound exposure, zebrafish saccules exhibited significant hair bundle damage (e.g., splayed, broken, and missing stereocilia) and loss (i.e., missing bundles and lesions in the epithelia) in the caudal region. Hair bundle counts increased over the course of the experiment, reaching pre-treatment levels at 14 days post-sound exposure (dpse). Low levels of proliferation were observed in untreated controls, indicating that some cells of the zebrafish saccule are mitotically active in the absence of a damaging event. In sound-exposed fish, cell proliferation peaked two dpse in the caudal region, and to a lesser extent in the rostral region. This proliferation was followed by an increase in numbers of cuticular plates with rudimentary stereocilia and immature-like hair bundles at 7 and 14 dpse, suggesting that at least some of the saccular cell proliferation resulted in newly formed hair cells. This study establishes a time course of hair cell bundle regeneration in the zebrafish inner ear and demonstrates that cell proliferation is associated with the regenerative process.

Microarray analysis of gene expression during auditory hair cell regeneration in zebrafish (Danio rerio)

Background Fishes are capable of regenerating sensory hair cells in the inner ear after overexposure to sound [1]. However, the time course and gene expression of auditory hair cell regeneration has not been established for zebrafish (Danio rerio). This study establishes a time course of cell proliferation associated with hair cell regeneration in the zebrafish inner ear and examines gene expression patterns exhibited during this process.

CProx1 immunoreactivity distinguishes progenitor cells and predicts hair cell fate during avian hair cell regeneration.

In birds, mature sensory hair cells are regenerated continually in vestibular epithelia and after damage in the auditory basilar papilla. Molecular mechanisms governing the cellular processes associated with hair cell regeneration are poorly understood. Transcription factors are critical regulators of cell proliferation and differentiation in developing tissues. We examined immunoreactivity for cProx1 during both ongoing and damage-induced hair cell regeneration in chickens. Homologues of this divergent homeobox transcription factor are required for cell cycle withdrawal and differentiation in several vertebrate and invertebrate tissues. In the mitotically quiescent basilar papilla, a population of resting progenitor cells (supporting cells) shows faint nuclear immunoreactivity for cProx1. When auditory hair cell regeneration is triggered by experimental damage, nuclear cProx1 immunolabel is highly elevated in approximately 50% of dividing progenitor cells. Shortly after cytokinesis, all sibling pairs show symmetric patterns of nuclear cProx1 labeling, but pairs with asymmetric labeling emerge shortly thereafter. Strongly immunoreactive cells acquire the hair cell fate, whereas cells with low nuclear immunoreactivity differentiate as supporting cells. By contrast, cProx1 is not detected in any dividing progenitor cells during ongoing regeneration in the utricle. However, nuclear cProx1 immunoreactivity becomes asymmetric in postmitotic sibling cells, and as in the basilar papilla, cells with elevated cProx1 label differentiate as hair cells. In conclusion, cProx1 immunolabeling varies across sensory epithelial progenitors and distinguishes early differentiating hair cells from supporting cells. cProx1 may regulate the proliferative or differentiative capacities of progenitor cells and specify hair cell fate in postmitotic cells during avian hair cell regeneration.

Cellular studies of auditory hair cell regeneration in birds.

A decade ago it was discovered that mature birds are able to regenerate hair cells, the receptors for auditory perception. This surprising finding generated hope in the field of auditory neuroscience that new hair cells someday may be coaxed to form in another class of warm-blooded vertebrates, mammals. We have made considerable progress toward understanding some cellular and molecular events that lead to hair cell regeneration in birds. This review discusses our current understanding of avian hair cell regeneration, with some comparisons to other vertebrate classes and other regenerative systems.

Regenerative Biology of Hearing: Taking the Brakes Off the Cell Cycle Engine

Hearing impairment is often caused by loss of auditory sensory hair cells which is irreversible in current clinical practice. The phenomenon of auditory hair cell regeneration including the recovery of hearing function is known for most vertebrates including birds. However mammals, including humans have not demonstrated the ability to regenerate lost hair cells most likely due to a lack of proliferative capacity in their sensory epithelia. Recent insights into cell cycle regulation in the mammalian hearing organ may provide a promising basis for inducing hair cell regeneration in mammals.

Temporal, spatial, and morphologic features of hair cell regeneration in the avian basilar papilla.

Hair cell-selective antibodies were used in combination with the nucleotide bromode-oxyuridine (BrdU) to examine the temporal, spatial, and morphologic progression of auditory hair cell regeneration in chicks after a single gentamicin injection. New hair cells are first identifiable with an antibody to class III beta (beta) tubulin (TuJ1) by 14 hours after BrdU incorporation, but progenitor cells in S phase and M phase are TuJ1-negative. TuJ1 labeling reveals that new hair cells are first detected at 3 days after gentamicin, in the base, and the emergence and maturation of regenerating hair cells spreads apically over time. Differentiation of regenerating hair cells consists of a progressive series of morphologic changes. During early differentiation (14 hours to 1 day after BrdU), regenerating hair cells are round or fusiform and remain near the lumen, where they are generated. During intermediate differentiation (2-4 days after BrdU), regenerating hair cells resemble support cells; their somata are elongated, their nuclei are in the support cell layer, and they appear to contact both the lumenal surface and the basal lamina. The 275-kDa hair cell antigen is first expressed in regenerating hair cells during this period. During late differentiation (7 days after BrdU and later), TuJ1-positive cells acquire the globose shape of mature hair cells. Labeling with antibodies to hair cell antigen, calmodulin, and ribosomal RNA confirms this morphologic progression. Examination of sister cells born at 3 days post-gentamicin reveals that there is equal likelihood that they will assume the hair cell or support cell fate (i.e., both asymmetric and symmetric differentiation occur).