Regeneration of hair cells in the mammalian vestibular system

Abstract

Hair cells regenerate throughout the lifetime of non-mammalian vertebrates, allowing these animals to recover from hearing and balance deficits. Such regeneration does not occur efficiently in humans and other mammals. Thus, balance deficits become permanent and is a common sensory disorder all over the world. Since Forge and Warchol discovered the limited spontaneous regeneration of vestibular hair cells after gentamicin-induced damage in mature mammals, significant efforts have been exerted to trace the origin of the limited vestibular regeneration in mammals after hair cell loss. Moreover, recently many strategies have been developed to promote the hair cell regeneration and subsequent functional recovery of the vestibular system, including manipulating the Wnt, Notch and Atoh1. This article provides an overview of the recent advances in hair cell regeneration in mammalian vestibular epithelia. Furthermore, this review highlights the current limitations of hair cell regeneration and provides the possible solutions to regenerate functional hair cells and to partially restore vestibular function.

Keywords

utricle / hair cell / regeneration / Atoh1 / Notch / Wnt

Introduction

Hair cells in the vestibular system of the inner ear provide signals regarding the movements and the tilting of the head to drive the reflexes controlling the eye, head, and body positions; however, these cells are vulnerable to aging, disease, and trauma [ 1]. The clinical use of aminoglycoside antibiotics and certain chemotherapeutic agents, such as cisplatin, can also cause hair cell loss [ 2– 4]. Chronic bilateral loss of vestibular signals can cause oscillopsia, blurry vision, distorted perception of self-orientation, imbalance, gait ataxia, and abnormal posture. In contrast to the production of hair cells throughout the lifetime of many non-mammalian vertebrates, such as fishes, amphibians, reptiles, and birds, limited spontaneous hair cell regeneration occurs in the vestibular system of postnatal mammals [ 5– 8].

The vestibular system contains multiple sensory organs, including utricular and saccular maculae for sensing of linear acceleration and cristae for detection of head rotation [ 9]. Each of these sensory organs is composed of supporting cells and mechanotransducing hair cells. Two types of vestibular hair cells exist, namely, types I and II, which are distinguishable primarily by their postsynaptic contacts established by their vestibular afferent fibers [ 10]. Types I and II hair cells demonstrate differential ototoxic sensitivity and regenerative capacity. In addition, type I hair cells are more sensitive to gentamicin toxicity than type II hair cells, and the former display weaker regenerative capacity [ 11]. Considering that no artificial devices can be used as an alternative solution for vestibular dysfunction, the recovery of vestibular dysfunction primarily depends on the compensation of central vestibular function rather than partial recovery of the structure and function of the vestibular sensory epithelium itself. Therefore, the regeneration of vestibular hair cells is the only possible solution to recover lost vestibular function.

The hair cells and supporting cells in the mammalian cochlea are highly specialized, and these cells have no direct counterparts in the auditory organs of other vertebrates. This high degree of differentiation and specialization suggests that regeneration is limited in the mammalian cochlea. By contrast, the hair cells and supporting cells in the vestibular system of mammals are similar to their counterparts in birds, fishes, and amphibians. This similarity suggests that hair cells are more likely to regenerate in the mammalian vestibular epithelium.

Discovery of potential for cell proliferation and self-repair of balance epithelia in mammals

Until the 1990s, the general view indicated that hair cell loss in mammalian inner ear is irreversible. However, in 1993, Forge identified many cells at different stages of development with immature hair bundles in the utricle of guinea pigs, following hair cell death caused by aminoglycoside gentamicin, and this finding provided the first piece of evidence indicating that some degree of hair cell regeneration possibly occurs in vivo in mature mammalian inner ear [ 12]. Warchol also observed that new cells in the damaged vestibular sensory epithelium of mature guinea pigs and adult humans in vitro display some characteristics of immature hair cells [ 13]. Although Warchol reported that regenerative proliferation of supporting cells occurs in the utricles of mature mammals in response to death of sensory hair cells, they did not provide a convincing evidence demonstrating that DNA labeling, which occurs at a low frequency in vitro, causes the apparent recovery of hair cell apical surfaces observed in vivo. In 1995, Rubel used ³H-thymidine labeling to show that the direct transformation from supporting cells into hair cells and the regenerative proliferation of supporting cells are possibly the two major factors causing hair cell regeneration in the mammalian vestibular system, following ototoxic aminoglycoside treatment [ 14]. The discovery of the potential of cells in the vestibular epithelia to proliferate and transform was inspiring, although subsequent experiments showed the limited capacity of the vestibular sensory epithelium for self-repair after damage. The capacity of supporting cells to proliferate and transform declines sharply after birth, and only nominal regenerative proliferation occurs to recover the damaged vestibular epithelia in adult mammals. The recovery of unilateral dysfunction in the vestibular system caused by hair cell loss mainly relies on the adjustment of the central nervous system instead of hair cell regeneration, although such adjustments are hardly possible for the recovery of bilateral dysfunction in the vestibular system. To induce hair cell regeneration in the vestibular epithelia of mammals for functional recovery, researchers have exerted considerable efforts to improve the efficiency of regenerative proliferation and transformation following hair cell loss.

Factors enhancing the regenerative proliferation of vestibular epithelia in mammals

During embryonic development of the inner ear, growth factors and other extracellular signals regulate many events, including induction of the ear, specific induction of sensory epithelia, proliferation of progenitors, differentiation of hair cells and supporting cells, and attraction and maintenance of innervation [ 15]. Therefore, normal growth factors or other extracellular signals possibly promote proliferation and differentiation, which are required to replace dead hair cells and restore hearing and balance. The expression of growth factor receptors slightly varies between cultured sensory epithelia with or without damage [ 16], and transforming growth factor a (TGF-a), epidermal growth factor (EGF), insulin, and insulin-like growth factors (IGF-1) can all enhance proliferation in cultured mammalian vestibular sensory epithelia [ 1, 17– 19]. Insulin enhances proliferation in undamaged maculae by interacting with either TGF-a or EGF [ 17, 20]. In addition, perilymphatic injection of TGF-a with insulin can induce cell proliferation in both utricular sensory and extrasensory epithelia of adult rats [ 21, 22] compared with injection of TGF-a or insulin alone [ 20]. Retinoic acid (RA) can also increase the ability of vestibular hair cells to regenerate in cultured ototoxic-poisoned utricle explants from mice [ 22].

Brain-derived neurotropic factor (BDNF) protects the vestibular neurons from ototoxins [ 21] and plays important roles during the development of type I hair cells in vestibular epithelia [ 22]. When combined with growth factor mixture consisting of TGF-a, IGF-1, and RA, BDNF can significantly enhance the renewal of hair cells and the differentiation of type I vestibular hair cells following hair cell loss in vestibular sensory epithelia [ 22].

Glial growth factor 2 (rhGGF2) is a secreted member of a family of factors encoded by alternatively spliced transcripts from the neuregulin gene [ 23]; rhGGF2 can enhance the proliferation of supporting cells after hair cell death in vestibular epithelia in neonates [ 24]. Mechanistic studies have shown that phosphatidylinositol-3-kinase, mammalian target of rapamycin, protein kinase C (PKC), and mitogen-activated protein kinase, as well as increased intracellular calcium, are possibly involved in intracellular signaling cascades that trigger the proliferation of supporting cells in mammalian balance epithelia in vitro [ 25].

The second messenger cAMP is speculated to be a key mediator for regenerative proliferation in the hearing organ of birds [ 26]. Montcouquiol reported that brief exposures to forskolin or a cAMP analog can induce proliferation in neonatal rat utricles in vitro by increasing the number of growth factor receptors in plasma membrane [ 27].

Although many growth factors and extracellular signals have already been identified as promoters of proliferation and hair cell regeneration in vestibular sensory epithelia in mammals, regenerative proliferation remains limited and decreases with age. The factors inhibiting the proliferation and transformation of supporting cells during maturation of vestibular epithelia were investigated to regulate regeneration.

Factors inhibiting hair cell regeneration in vestibular epithelia in mammals

Nearly 50% of hair cells in adult mice arise in the vestibular organs during the first 2 weeks after birth; they arise either through progressive differentiation of cells generated before birth or through the differentiation of new cells from the proliferation of progenitors progressing up to the S-phase after birth. These processes decline sharply after birth, and this decline coincides with the unique maturation of sensory epithelium in mammals [ 28]. After hair cell death caused by neomycin treatment in vitro or resulting from the expression of inducible diphtheria toxin fragment A in hair cells in vivo, increased proliferation and significant mitotic hair cell replacement can only be identified in neonatal mouse utricles, and factors limiting regeneration increased significantly in vivo during postnatal period [ 6]. Moreover, rhGGF2 or insulin can induce significant supporting cell proliferation responses even during short-term cultures in the vestibular epithelia of neonates, although all these responses progressively decline during the first 2 weeks of postnatal maturation [ 24]. The decline in the capacity for cell proliferation during the early postnatal stages is apparently responsible for the limited hair cell regeneration, which is unique to mammals, and further identification of the mechanisms underlying the maturation of vestibular epithelia might lead to the development of new methods to induce hair cell regeneration.

Lost plasticity of supporting cells as a brake for hair cell regeneration

The general repair process for damaged epithelium includes cell spreading, translocation, and proliferation. These processes are well-defined events occurring sequentially. Spreading, translocation, and proliferation are mediated by cell cytoskeleton-F-actin microfilaments, microtubules, and associated centrosomes. In the vestibular sensory epithelium, the distribution of a6 integrin and b4 integrin in supporting cells associated with cytoskeletal organization and anchorage changes during the early postnatal stages. Activation of PKC to modify the cytoskeletons or the anchorage of supporting cells can significantly increase the cellular spreading and entry into the S-phase in epithelia at 6 days postnatally (P), suggesting that the unique cytological characteristics of the skeletal organization and the lost plasticity of the supporting cells directly inhibit the regeneration or eliminate a pool of progenitor cells [ 29]. However, Meyers observed that the supporting cells in vestibular epithelia displaying excision lesions remained compact and columnar, as well as contained significantly stouter cortical actin belts than those found in embryonic sensory epithelia [ 30]. Burns also found that the circumferential bands of F-actin bracketing the apical junctions between the supporting cells grow much thicker as mice mature postnatally, whereas the F-actin bands remain thin in chickens from hatching through adulthood [ 31] and in other species that can spontaneously regenerate hair cells [ 32]. The uniquely thick F-actin bands at the junctions of mature supporting cells in mammals are exceptionally stable, possibly restricting the dynamic repair responses in the mammalian vestibular epithelia [ 33].

Overall, the results suggest that cytoskeletal changes underlie the age-related loss of proliferation in mammalian ears by limiting the capacity of mature supporting cells to change their shape, thereby limiting cell proliferation and hair cell replacement. Exogenous stimulation with lysophosphatidic acid can overcome this inhibition and promote supporting cell expansion and lesion closure [ 30].

In epithelial tumors, the expression of adhesion molecules is correlated with cell proliferation, invasiveness, and malignant progression. As a prominent component of adherence junctions in many epithelia, E-cadherin of the vestibular epithelia is presumed to play an important role in maintaining the normal structure of vestibular sensory epithelia in mammals [ 34, 35]. E-cadherin expression increases by more than six-fold in mice from P0 to P7, coinciding with the reduced proliferation and replacement capacity. With pharmacological inhibition of glycogen synthase kinase-3 in cultured utricular epithelium, downregulation of E-cadherin was detected accompanied with increased cell proliferation across a range of postnatal ages [ 36]. Moreover, following treatment of cultured utricle obtained from young mice with g-secretase inhibitors (GSIs), E-cadherin was internalized in striolar region followed by conversion of supporting cells into hair cells [ 37]. Low levels of E-cadherin possibly contribute to weak cell-cell junctions, which can facilitate proliferation or transdifferentiation occurring after the changes in cell shape of utricular epithelia.

Spontaneous proliferation of supporting cells only occurs after hair cell loss and is localized in the damaged region of the utricle [ 38, 39], further confirming that disruption of intercellular junction adhesions or the loss of function between cell-cell contacts possibly trigger regenerative proliferation. Thus, the integrity of sensory epithelium may serve as a negative regulator for proliferation of supporting cells and extrusion of damaged hair cells.

All the available lines of evidence show that the lost plasticity of supporting cells and the lost capacity for proliferation of vestibular epithelia are possibly caused partly by the changes in the cytoskeleton, as well as the strengthened cellular junctions during postnatal maturation.

Disappearance of progenitor cells as another brake for hair cell regeneration

Li and colleagues have demonstrated that the adult vestibular sensory epithelia of mice harbor stem cells can promote self-renewal and give rise to different cell types of the inner ear [ 40]. Compared with the number of pluripotent cells in neonatal mice, those in the adult vestibular epithelia significantly decreased, and this finding provides a reasonable explanation for the limited capacity of proliferation and regeneration in adult mammalian utricle following hair cell loss. This result suggests that eliminating an essential but yet to be identified pool of stem cells serves as another brake for hair cell regeneration.

The progenitors of the neonatal mouse cochlea were recently identified as Wnt-responsive leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5⁺) cells, which can regenerate hair cells under certain conditions [ 41– 45]. Although Lgr5 is no longer expressed in the vestibular epithelia after birth, Lgr5⁺ supporting cells can still be recruited into the striolar region of the neonatal mouse utricle by treating the mice with neomycin for 24 h; these cells can regenerate new hair cells through both mitotic regeneration and direct transdifferentiation [ 46]. Isolation by flow cytometry has revealed that the damage-activated Lgr5⁺ supporting cells can self-renew to form spheres and can transdifferentiate into hair cells; these Lgr5⁺ cells are regarded as hair cell progenitors in neonatal mouse utricle [ 47]. Given that the damage-recruited Lgr5⁺ cells were undetectable beyond P15, the elimination of an essential progenitor pool for hair cell regeneration during maturation of sensory epithelia is suggested to serve as another brake for hair cell regeneration and subsequent functional recovery [ 46]. Further dissection of the mechanism underlying the disappearance and recruitment of Lgr5⁺ cells during development and regeneration in mammalian utricles might be useful to regulate mammalian hair cell regeneration.

Hair cell regeneration in adult mouse utricles

The capacity of supporting cells in mammalian utricles to proliferate and regenerate declines significantly during the first couple of weeks after birth, and regulation of these processes is controlled by multiple signaling pathways. Significant efforts have been made to investigate the mechanisms underlying the limited spontaneous regeneration in adult vestibular sensory epithelia, and some strategies have been developed to overcome the brakes for hair cell regeneration in adult mammalian utricles and ultimately increase the vestibular functional recovery.

Studies have reported that hair cells containing the immature bundle, which spontaneously re-emerge in mature rodent utricles following hair cell loss induced by ototoxic drugs in vivo [ 12, 38, 48]. This phenomenon is possibly caused by the regeneration of hair cells or the recovery of damaged hair cells. The reduction in number of supporting cells, coinciding with the replacement of hair cells in guinea pig utricles [ 48], suggests that the direct transdifferentiation from supporting cells into hair cells is the main source of hair cell replacement in adult utricles displaying ototoxin-induced hair cell loss. In adult Pou4f3^+/DTR mice, the vestibular hair cells can be completely and specifically killed by injection with diphtheria toxin (DT) in vivo. In addition, Golub et al. observed that the number of hair cells gradually increased within 60 days post-DT, which quantitatively and spatially coincided with the significant decrease in the number of supporting cells. Furthermore, they observed upregulated atonal homolog 1(Atoh1) in supporting cells prior to the significant increase in vivo in the number of hair cells without cell division. This finding strongly supports the idea indicating that direct transdifferentiation from supporting cells into hair cells is the main source of hair cell replacement after DT-induced hair cell loss in vivo [ 49].

Although spontaneous hair cell replacement in adult utricles has been identified by different groups, the number of regenerated hair cells is limited and is accompanied by a decrease in the number of supporting cells. This phenomenon presents a great challenge for the functional recovery of the vestibular system.

Hair cell regeneration through re-expression of Atoh1

Atoh1 is one of the most important transcription factors involved in the development of the inner ear; Atoh1 is both sufficient and necessary for hair cell formation, as well as a promising gene target for inducing hair cell generation in mammals [ 50, 51]. Extra hair cells can be generated by overexpressing Atoh1 in the greater epithelial ridge region of the neonatal mouse cochlea [ 51]. Shou and colleagues showed that a large number of hair cells can be produced in the utricular maculae of a cultured adult rat with or without hair cell loss caused by adenovirus-mediated overexpression of Hath1, a human atonal homolog; they also claimed that proliferation was not involved in the generation of new hair cells via Hath1 overexpression [ 52]. Their results suggest that mature mammalian vestibular epithelia are potentially competent to produce new hair cells under the driving force of Atoh1.

After delivering Atoh1 into the inner ear through an adenoviral vector, hair cell regeneration was observed after hair cell loss was induced by the ototoxic drugs neomycin or IDPN in the adult vestibular sensory epithelia in vivo, and some functions of the vestibular system were recovered [ 53– 55]. Despite the significant in vivo recovery of sensory cells in these experiments, the overall epithelial thickness and the total number of supporting cells decreased, suggesting that the replacement of vestibular hair cells by Atoh1 gene transfer results from the conversion of existing supporting cells into sensory cells. Further investigation on the cell types and innervation has shown that the majority of the regenerated hair cells were type I hair cells that displayed complete calyx-shaped synaptic linkages with afferent nerve fibers; however, neither the number nor the appearance of the hair cells was normal [ 56].

Gao [ 57] recently observed an age-dependent hair cell generation induced by Atoh1 overexpression in mammalian utricles in an inducible transgenic mouse model. The efficacy of hair cell induction declined as the animals aged, and this phenomenon is possibly caused partially by the limitation of this mouse model, in which Atoh1 induction is not strictly limited to the inner ear epithelia. Moreover, the intact sensory epithelia without hair cell loss is another possible inhibitory factor for hair cell generation. Gao also observed a significant increase in mitotic events following Atoh1 induction in the sensory region of the utricle, resulting in an apparent increase in cell numbers or in thickening of the sensory epithelium, suggesting another advantage of the induced expression of Atoh1 in hair cell regeneration [ 57]. Another transgenic mouse model in which Atoh1 can be specifically expressed in the inner ear or in supporting cells following hair cell loss in adult utricles should be employed in studies on hair cell regeneration.

Hair cell regeneration through regulation of Notch signaling

Notch signaling plays dual developmental roles in the inner ear. First, Notch signaling occurs in early otic development and defines the prosensory domains that will develop into the six sensory organs of the inner ear [ 58]. Second, in later developmental stages, Notch signaling establishes the mosaic-like pattern of the mechanosensory hair cells and their surrounding supporting cells that develop in the second postnatal week through the well-characterized lateral inhibition [ 59]. The Notch pathway is apparently altered shortly after vestibular epithelial injury, resulting in limited hair cell regeneration in mammals [ 60]. Two different strategies are possibly involved in hair cell regeneration in the inner ear. One possible strategy is the activation of Notch signaling to promote formation of ectopic sensory regions in normal non-sensory regions in the inner ear, and the other strategy is the inhibition of Notch signaling to disrupt lateral inhibition, allowing supporting cells to transdifferentiate into hair cells.

Although ectopic hair cells were observed in mouse utricles upon overactivation of the Notch1 intracellular domain (NICD) at the embryonic stage in vivo, no similar results were observed by activating NICD after birth, demonstrating an age-dependent decrease in responsiveness of inner ear non-sensory cells to NICD activation. This finding suggests that other signals apart from Notch1 signaling are needed to maintain the ability of non-sensory utricular cells to react to NICD through adulthood.

New hair cells can be induced by a GSI in the noise-damaged cochlea in an adult mouse, resulting in a partial recovery of hearing [ 61]. In utricle, Notch signaling is required to maintain the striolar supporting cell phenotype up to the second postnatal week and plays a role in regeneration after damage [ 37, 60, 62, 63].

The reduction in Notch pathway activity in adult mouse vestibular epithelia in vitro by inhibiting g-secretase or other enzymes required for Notch activity results in a non-mitotic increase in hair cells, which is limited to the striolar/juxtastriolar region [ 62]. The limited number and the restricted location of the new hair cells indicate the limited potential for functional recovery of the vestibular system by inhibiting Notch for hair cell regeneration in adult vestibular epithelia.

Hair cell regeneration by promoting mitosis in supporting cells

Proliferative regeneration is a more attractive strategy for therapeutic purposes because restoration of vestibular function in the damaged inner ear requires the concomitant replacement of both hair cells and supporting cells [ 64]. Given that spontaneous re-entry into the cell cycle is very rare even after ototoxin-induced hair cell loss in adult vestibular epithelia, some strategies have been developed to promote proliferative regeneration of hair cells.

Manipulating the cyclin-dependent kinase inhibitor p27Kip1 can induce supporting cells to re-enter into the cell cycle and generate new hair cells and supporting cells; these findings suggest that supporting cells still demonstrate the capacity to re-enter into mitosis under certain conditions [ 65– 67]. Sage found that differentiated mammalian supporting cells and hair cells can proliferate and give rise to functional hair cells in the absence of retinoblastoma (Rb) tumor-suppressor protein [ 68]. Rb null mice showed complete hair cell loss and profound deafness, suggesting that Rb plays important roles in hair cell maturation and survival [ 69– 71]. Furthermore, even when manipulating cell-cycle-related genes, the proliferative capacity of supporting cells still decreases sharply during the maturation of sensory epithelia [ 67, 68], which provided blocks for hair cell regeneration in adult vestibular epithelia by manipulating the cell-cycle-related genes.

The expression of cyclin D1 is downregulated along with the maturation of supporting cells in mice [ 72]. However, the adenovirus-mediated ectopic cyclin D1 overexpression can induce supporting cells in adult mouse vestibular epithelia to re-enter the cell cycle, and this phenomenon is accompanied by the downregulation of P27kip1 and P21clip1 [ 73]. However, most of the proliferated supporting cells are blocked at the G₂/M boundary or end up with DNA damage and do not contribute to hair cell replacement.

Compared with progenitor cells, the other differentiated supporting cells in the adult vestibular epithelia have very limited self-renew and hair cell regeneration abilities. Therefore, reprogramming differentiated supporting cells into otic progenitors is a potential strategy to restore the regenerative potential of the ear. Burns observed significant proliferation of supporting cells upon overexpression of the degradation-resistant T58A mutant of c-Myc (c-MycT58A) in cultured adult utricles, and few proliferated supporting cells were labeled with the hair cell marker Myo7a [ 74]. The number of proliferated cells decreased after 3 weeks because many of the cycling cells appeared to undergo apoptosis, and very few proliferated cells transformed into hair cells after long-term culture in differentiation-promoting medium. The proliferation of supporting cells caused by induced c-Myc expression possibly inhibits hair cell differentiation, or other signals may be needed to completely differentiate these cells. Moreover, long-term survival of regenerated cells is essential for the functional recovery of the vestibular system, which has not yet been addressed.

Hair cell regeneration by activating progenitors in the vestibular epithelia

The presence of few stem cells rather than a large population of potential progenitors is hypothesized to be involved in the replacement of lost hair cells in mammals and other vertebrates. Thus, another strategy for hair cell regeneration is to identify and expand the pool of proliferative progenitors in the vestibular sensory epithelia.

The Lgr5⁺ cochlear supporting cells, which demonstrate the capacity for self-renewal and hair cell differentiation in vitro, can still be induced to proliferate and give rise to hair cells by overexpressing b-catenin in vivo [ 41– 43, 75]. In mouse utricle, although Lgr5 is no longer expressed in the vestibular epithelia after birth, the Lgr5⁺ supporting cells can be recruited after hair cell loss in the striolar region to regenerate new hair cells through both mitotic regeneration and direct transdifferentiation [ 46]. In addition, activating Wnt signaling in the Lgr5⁺ cells significantly increases the proliferation of these progenitor cells and their subsequent conversion into hair cells [ 46].

The Lgr5⁺ subsets of supporting cells, which are responsive to Wnt signaling, display the properties of progenitor cells. The mechanisms underlying the disappearance and recruitment of Lgr5⁺ cells during development and regeneration in mammalian utricles require further investigation, the result of which might be useful to regulate mammalian hair cell regeneration.

Consistent with previous studies [ 57, 62], the striola, which is located in the central region of the sensory epithelia, is the most conducive site for induction of hair cell formation, indicating the intrinsic properties of the supporting cells in this region that endow them with high competency in hair cell generation and their ability to serve as a pool of progenitors under certain conditions. Determining the difference between supporting cells in the striolar region and supporting cells in the extrastriolar region might provide information critical for the development of new strategies for hair cell regeneration (Fig. 1).

Both hair cell regeneration and subsequent functional recovery of the vestibular system require the coordinated regulation of multiple biological events, including the controllable proliferation of remaining supporting cells, conversion from proliferated supporting cells into hair cells, differentiation and maturation of the subtypes of hair cells, reconstruction of innervation, and long-term survival of regenerated cells. Therefore, multiple pathways and factors are possibly needed for the coordinated regulation of hair cell regeneration. Future studies must investigate the potential of the combination of the factors for proliferation and transdifferentiation as a strategy for hair cell regeneration in the vestibular system.

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