Hair cell regeneration in the avian auditory epithelium

Regeneration of sensory hair cells in the mature avian inner ear was first described just over 20 years ago. Since then, it has been shown that many other non-mammalian species either continually produce new hair cells or regenerate them in response to trauma. However, mammals exhibit limited hair cell regeneration, particularly in the auditory epithelium. In birds and other non-mammals, regenerated hair cells arise from adjacent non-sensory (supporting) cells. Hair cell regeneration was initially described as a proliferative response whereby supporting cells re-enter the mitotic cycle, forming daughter cells that differentiate into either hair cells or supporting cells and thereby restore cytoarchitecture and function in the sensory epithelium. However, further analyses of the avian auditory epithelium (and amphibian vestibular epithelium) revealed a second regenerative mechanism, direct transdifferentiation, during which supporting cells change their gene expression and convert into hair cells without dividing. In the chicken auditory epithelium, these two distinct mechanisms show unique spatial and temporal patterns, suggesting they are differentially regulated. Current efforts are aimed at identifying signals that maintain supporting cells in a quiescent state or direct them to undergo direct transdifferentiation or cell division. Here, we review current knowledge about supporting cell properties and discuss candidate signaling molecules for regulating supporting cell behavior, in quiescence and after damage. While significant advances have been made in understanding regeneration in non-mammals over the last 20 years, we have yet to determine why the mammalian auditory epithelium lacks the ability to regenerate hair cells spontaneously and whether it is even capable of significant regeneration under additional circumstances. The continued study of mechanisms controlling regeneration in the avian auditory epithelium may lead to strategies for inducing significant and functional regeneration in mammals.     

Defining the cellular environment in the organ of Corti following extensive hair cell loss: a basis for future sensory cell replacement in the Cochlea

Background

Following the loss of hair cells from the mammalian cochlea, the sensory epithelium repairs to close the lesions but no new hair cells arise and hearing impairment ensues. For any cell replacement strategy to be successful, the cellular environment of the injured tissue has to be able to nurture new hair cells. This study defines characteristics of the auditory sensory epithelium after hair cell loss.

Methodology/Principal Findings

Studies were conducted in C57BL/6 and CBA/Ca mice. Treatment with an aminoglycoside-diuretic combination produced loss of all outer hair cells within 48 hours in both strains. The subsequent progressive tissue re-organisation was examined using immunohistochemistry and electron microscopy. There was no evidence of significant de-differentiation of the specialised columnar supporting cells. Kir4.1 was down regulated but KCC4, GLAST, microtubule bundles, connexin expression patterns and pathways of intercellular communication were retained. The columnar supporting cells became covered with non-specialised cells migrating from the outermost region of the organ of Corti. Eventually non-specialised, flat cells replaced the columnar epithelium. Flat epithelium developed in distributed patches interrupting regions of columnar epithelium formed of differentiated supporting cells. Formation of the flat epithelium was initiated within a few weeks post-treatment in C57BL/6 mice but not for several months in CBA/Ca''s, suggesting genetic background influences the rate of re-organisation.

Conclusions/Significance

The lack of dedifferentiation amongst supporting cells and their replacement by cells from the outer side of the organ of Corti are factors that may need to be considered in any attempt to promote endogenous hair cell regeneration. The variability of the cellular environment along an individual cochlea arising from patch-like generation of flat epithelium, and the possible variability between individuals resulting from genetic influences on the rate at which remodelling occurs may pose challenges to devising the appropriate regenerative therapy for a deaf patient.     

Hair cell regeneration in the chick inner ear following acoustic trauma: ultrastructural and immunohistochemical studies

The regeneration of hair cells in the chick inner ear following acoustic trauma was examined using transmission electron microscopy. In addition, the localization of proliferation cell nuclear antigen (PCNA) and basic fibroblast growth factor (b-FGF) was demonstrated immunohistochemically. The auditory sensory epithelium of the normal chick consists of short and tall hair cells and supporting cells. Immediately after noise exposure to a 1500-Hz pure tone at a sound pressure level of 120 decibels for 48 h, all the short hair cells disappeared in the middle region of the auditory epithelium. Twelve hours to 1 day after exposure, mitotic cells, binucleate cells and PCNA-positive supporting cells were observed, and b-FGF immunoreactivity was shown in the supporting cells and glial cells near the habenula perforata. Spindle-shaped hair cells with immature stereocilia and a kinocilium appeared 3 days after exposure; these cells had synaptic connections with the newly developed nerve endings. The spindle-shaped hair cell is considered to be a transitional cell in the lineage of the supporting cell to the mature short hair cell. These results indicate that, after acoustic trauma, the supporting cells divide and differentiate into new short hair cells via spindle-shaped hair cells. Furthermore, it is suggested that b-FGF is related to the proliferation of the supporting cells and the extension of the nerve fibers.     

Cell Division and Maintenance of Epithelial Integrity in the Deafened Auditory Epithelium

Quiescence is among the hallmarks of the sensory epithelium of the cochlea. When auditory sensory cells (hair cells) degenerate they are not replaced, and therefore hearing loss is permanent. Cochlear hair cells are susceptible to several types of lesions, including aminoglycoside antibiotics. The application of the aminoglycoside neomycin in the inner ear mimics cases of severe hair cell loss and leads to collapse of the cochlear epithelium. We now report that in mature guinea pig cochleae injected with neomycin, the remaining non-sensory cells undergo a robust proliferative response. p27Kip1, an inhibitor of cell cycle in the cochlea, was present in non-dividing cells and absent during mitosis. Dividing cells retained their tight junction complexes and maintained the structural confluence of the auditory epithelium during cell division. The plane of mitosis was invariably parallel to the luminal surface. These results indicate that the flat epithelium of the cochlea can down-regulate p27Kip1 and divide after a severe lesion and suggest that the cell divisions assist in maintaining the epithelial confluence throughout the cochlea. Presence of mitosis in the tissue presents therapeutic opportunities for gene transfer and stem cells therapies.     

The epidermal stem cell: an overview

The epidermis, the intestinal epithelium and the bone marrow are constantly renewed. Once a terminally differentiated cell has fulfilled its function, it is eliminated. Thus, new differentiated cells need to be constantly produced by the proliferative compartment to ensure the function of the tissue. As this process continues throughout a lifetime, cells must exist with a large capacity for proliferation within each of these tissues. These cells must also be the depository of all the information necessary for suitable differentiation to occur. This cell population which is qualified as the stem population, has attracted, in recent years, considerable attention not only because of its role during development, but also because of its potential sensitivity to radiations and carcinogenesis and to antineoplastic drugs. The epidermis, which is a stratified and squamous epithelium, has appendages which developed from the primitive epidermis during embryonic life. These appendages are also renewed during the adult lifetime, as illustrated by hair growth. The epidermis proves to be a unique model with which the development and the renewal of a stratified epithelium can be studied.     

Stemness of the organ of Corti relates to the epigenetic status of Sox2 enhancers

In the adult mammalian auditory epithelium, the organ of Corti, loss of sensory hair cells results in permanent hearing loss. The underlying cause for the lack of regenerative response is the depletion of otic progenitors in the cell pool of the sensory epithelium. Here, we show that an increase in the sequence-specific methylation of the otic Sox2 enhancers NOP1 and NOP2 is correlated with a reduced self-renewal potential in vivo and in vitro; additionally, the degree of methylation of NOP1 and NOP2 is correlated with the dedifferentiation potential of postmitotic supporting cells into otic stem cells. Thus, the stemness the organ of Corti is related to the epigenetic status of the otic Sox2 enhancers. These observations validate the continued exploration of treatment strategies for dedifferentiating or reprogramming of differentiated supporting cells into progenitors to regenerate the damaged organ of Corti.     

Hair cells without supporting cells: further studies in the ear of the zebrafish mind bomb mutant

Each sensory hair cell in the ear is normally surrounded by supporting cells, which separate it from the next hair cell. In the mind bomb mutant, as a result of a failure of lateral inhibition, cells that would normally become supporting cells differentiate as hair cells instead, creating sensory patches that consist of hair cells only. This provides a unique opportunity to pinpoint the functions for which supporting cells are required in normal hair cell development. We find that hair cells in the mutant develop an essentially normal cytoskeleton, with a correctly structured hair bundle and well-defined planar polarity, and form apical junctional complexes with one another in standard epithelial fashion. They fail, however, to form a basal lamina or to adhere properly to the adjacent non-sensory epithelial cells, which overgrow them. The hair cells are eventually expelled from the ear epithelium into the underlying mesenchyme, losing their hair bundles in the process. It is not clear whether they undergo apoptosis: many cells staining strongly with the TUNEL procedure are seen but do not appear apoptotic by other criteria. Supporting cells, therefore, are needed to hold hair cells in the otic epithelium and, perhaps, to keep them alive, but are not needed for the construction of normal hair bundles or to give the hair bundles a predictable polarity. Moreover, supporting cells are not absolutely required as a source of materials for otoliths, which, though small and deformed, still develop in their absence.     

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) ^1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions ^{7, 8}. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium ^{9, 10}. Supporting cells are also important mediators of hair cell survival and death ¹¹. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.     

The lateral line canal sensory organs of the Epaulette Shark (Hemiscyllium ocellatum)

Examination of the lateral line canals in the Epaulette Shark reveals a much more differentiated sensory system than previously reported from any elasmobranch. Two main types of lateral line canals are found. In one type rounded patches of sensory epithelia are separated by elevations of the canal floor. The other type is a straight canal without restrictions and with an almost continuous sensory epithelium. In addition, we found epithelia (type A) with very long apical microvilli on the supporting cells. These microvilli reach beyond the stereovilli of the hair cells. Another type (B) of sensory epithelium has short microvilli on the supporting cells. In this latter type of epithelium the stereovilli of the hair cells are comparatively tall and reach out beyond the supporting cell microvilli.
  New hair cells are found widely in both types of sensory epithelia. These always occur as single cells, unlike those described in teleost lateral line canal sensory epithelia where new hair cells seem to form in pairs. Dying hair cells are also widespread, indicating a continuous turnover of hair cells.     

Specification and loss of melanocyte stem cells

The melanocyte stem cells of the hair follicle provide an attractive system for the study of stem cells. The stem cells exist in an anatomically defined niche clearly separated from their differentiated progeny, differentiated progeny can be selected against by treatment with an inhibitor of Stem Cell Factor signaling, perturbations which affect survival and differentiation have clearly visible pigmentation phenotypes, and genetic mutations can impair or completely abolish this lineage without lethality. In mice several coat color mutants have been shown to have impaired specification or survival of melanocyte stem cells. Furthermore understanding of the normal regulation and behaviors of melanocytes and melanocyte stem cells will allow development of better strategies for cancer treatment. This article will review the discovery and behaviors of melanoctye stem cells as well as some aspects of melanocyte biology.     

Sox2 is required for maintenance and regeneration, but not initial development, of hair cells in the zebrafish inner ear

Sox2 has been variously implicated in maintenance of pluripotent stem cells or, alternatively, early stages of cell differentiation, depending on context. In the developing inner ear, Sox2 initially marks all cells in the nascent sensory epithelium and, in mouse, is required for sensory epithelium formation. Sox2 is eventually downregulated in hair cells but is maintained in support cells, the functional significance of which is unknown. Here we describe regulation and function of sox2 in the zebrafish inner ear. Expression of sox2 begins after the onset of sensory epithelium development and is regulated by Atoh1a/b, Fgf and Notch. Knockdown of sox2 does not prevent hair cell production, but the rate of accumulation is reduced due to sporadic death of differentiated hair cells. We next tested the capacity for hair cell regeneration following laser ablation of mature brn3c:gfp-labeled hair cells. In control embryos, regeneration of lost hair cells begins by 12 h post-ablation and involves transdifferentiation of support cells rather than asymmetric cell division. In contrast, regeneration does not occur in sox2-depleted embryos. These data show that zebrafish sox2 is required for hair cell survival, as well as for transdifferentiation of support cells into hair cells during regeneration.     

干细胞治疗感音神经性耳聋

治疗内耳疾病的主要困难之一是找到耳蜗毛细胞或者螺旋神经元丢失所导致的听力损失的治疗方法。本文讨论使用干细胞替代感觉细胞丢失为目的的几个治疗策略。作者最近在成年内耳中发现了可以分化为毛细胞的干细胞,发现了胚胎干细胞可在体外转化为毛细胞并表达毛细胞标记物。在动物模型中,成年内耳干细胞、神经干细胞和胚胎干细胞来源的前体细胞可分化成为毛细胞和神经细胞。本文将讨论使用干细胞再生损伤毛细胞的不同方法,介绍几种可行的动物模型,并讨论发展基于干细胞的细胞替代疗法治疗内耳损伤中存在的困难。     

Stem cells as therapy for hearing loss

One of the greatest challenges in the treatment of inner-ear disorders is to find a cure for the hearing loss that is caused by the loss of cochlear hair cells or spiral ganglion neurons. The recent discovery of stem cells in the adult inner ear that are capable of differentiating into hair cells, as well as the finding that embryonic stem cells can be converted into hair cells, raise hope for the future development of stem-cell-based treatment regimens. Here, we propose different approaches for using stem cells to regenerate the damaged inner ear and we describe the potential obstacles that translational approaches must overcome for the development of stem-cell-based cell-replacement therapies for the damaged inner ear.     

Isolation and culture of hair cell progenitors from postnatal rat cochleae

Cochlear hair cells are a terminally differentiated cell population that is crucial for hearing. Although recent work suggests that there are hair cell progenitors in postnatal mammalian cochleae, isolation and culture of pure hair cell progenitors from a well-defined cochlear area have not been reported. Here we present an experimental method that allows isolation and culture of hair cell progenitors from postnatal rat cochleae. These progenitor cells are isolated from the lesser epithelial ridge (LER, or outer spiral sulcus cell) area of pre-plated neonatal rat cochlear segments. They express the same markers as LER cells in vivo, including ZO1, Islet1, Hes1, and Hes5. When these cells are induced to express Hath1, they show the potential to differentiate into hair cell-like cells. Interestingly, these cells can be lifted from monolayer cultures and maintained in aggregate cultures in which spheres can be formed. Hair cell progenitors in the spheres display their proliferating capability and express only epithelial markers. Furthermore, when these spheres are mixed with dissociated mesenchymal cells prepared from postnatal rat utricular whole mounts, and replated onto a collagen substratum, the epithelial progenitor cells are able to differentiate into cells expressing markers of hair cells and supporting cells in epithelial islands, which mirrors the inner ear sensory epithelium in vivo. Successful isolation and culture of hair cell progenitors from the mammalian cochlea will facilitate studies on gene expression profiling and mechanism of differentiation/regeneration of hair cells, which are crucial for repairing hearing loss.     

Stem cells for the replacement of inner ear neurons and hair cells

Stem cells in the nervous system have some capacity to restore damaged tissue. Proliferation of stem cells endows them with self-renewal ability and accounts for in vitro formation of neurospheres, clonally derived colonies of floating cells. However, damage to the nervous system is not readily repaired, suggesting that the stem cells do not provide an easily recruited source of cells for regeneration. The vestibular and auditory organs, despite their limited ability to replace damaged cells, appear to contain cells with stem cell properties. These inner ear stem cells, identified by neurosphere formation and by their expression of markers of inner ear progenitors, can differentiate to hair cells and neurons. Differentiated cells obtained from inner ear stem cells expressed sensory neuron markers and, after co-culture with the organ of Corti, grew processes that extended to hair cells. The neurons expressed synaptic vesicle markers at points of contact with hair cells. Exogenous stem cells have also been used for hair cell and neuron replacement. Embryonic stem cells are one potential source of both hair cells and sensory neurons. Neural progenitors made from embryonic stem cells, transplanted into the inner ear of gerbils that had been de-afferented by treatment with a toxin, differentiated into cells that expressed neuronal markers and grew processes both peripherally into the organ of Corti and centrally. The regrowth of these neurons suggests that it may be possible to replace auditory neurons that have degenerated with neurons that restore auditory function by regenerating connections to hair cells.     

Presynaptic terminals in hyaline cells of normal and overstimulated chick inner ears

Hyaline cells are non-sensory epithelial cells of the vibrating part of the basilar membrane of chicks; they receive an extensive efferent innervation. Although these anatomical features suggest roles in auditory transduction, very little is known about the function of these cells. One possible way to understand function is by lesion experiments. We used synapsin-specific antibodies to study changes that occur in the pattern of efferent innervation in hyaline cells after lesion of the sensory epithelium induced by acoustic overstimulation. We found only small changes in hyaline cells after such trauma. These included a small increase in size and a small decrease in density of nerve terminals on hyaline cells. This suggests that hyaline cells and their nerve terminals are less susceptible to acoustic trauma than hair cells. Using neurofilament-specific antibodies we found little or no trauma-induced change in the density of nerve fibres that cross the basilar papilla and reach the hyaline cell region. This finding suggested that trauma to the hair cells does not necessarily lead to changes in the efferent fibres that cross the papilla and extend into the hyaline cell region. Using the trauma and the morphological parameters studied here, it appears that a moderate lesion in the hair cell region in the avian inner ear does not influence the hyaline cells or their innervation.     

Cell cycle regulation in hair cell development and regeneration in the mouse cochlea

Cell cycle inhibitors play important roles in the development of mammalian cochleae. Loss of function of those factors in mice at various developmental stages results in distinct phenotypes characterized by overproduction or loss of cochlear sensory cells. Our recent study showed that acute deletion of the retinoblastoma protein (Rb) induces rapid cell cycle reentry and subsequent loss of postnatal cochlear hair cells in mice. Clearly, these regulators play multiple roles in cell cycle exit and differentiation of hair cell and supporting cell progenitors. They are also crucial in maintenance of postmitotic states and survival of differentiated hair cells and supporting cells. In mammals, lost hair cells cannot be spontaneously replaced, leading to permanent deafness. However, lower vertebrates such as birds and fish can naturally regenerate damaged hair cells from the underlying supporting cells through proliferation and transdifferentiation. Thus, manipulating cell cycle inhibitors in mammalian cochleae could provide a new avenue to restore hearing in deaf people caused by a variety of genetic mutations and environmental insults.     

Ultrastructure of the putative stem cell niche in rat mammary epithelium

There is now strong evidence that the stem cells of many tissues reside in specialized structures termed niches. The stem cell niche functions to house and regulate symmetric and asymmetric mitosis of stem cells in mammalian skin, mouse and human bone marrow, mouse brain, gut, and hair follicle, and Drosophila ovary and testis. This regulation is effected through the action of various signaling pathways such as Notch, Hedgehog, Wnt and others. The hormones of the estrous cycle, pregnancy and lactation that initiate growth in mouse mammary epithelium appear to act at a paracrine level to regulate mitosis through Notch receptors. Previous work has established that the putative stem cells of the mammary epithelium in several animal species reside near the basement membrane and never make contact with the ductal lumen. We show that these putative stem cells are found in anatomically specialized places created by the cytoplasmic extensions and modifications of neighboring differentiated cells. Such specializations may help to regulate stem cell activity by modulating molecular traffic to putative stem cells and contact with signaling molecules in the basement membrane. The histological characteristics of these putative niches vary as to the kinds of relationships the cells can have with the basement membrane and neighboring cells and as to how many stem or progenitor cells they may contain. This suggests a plasticity that may be relevant to the response of niches to tissue demands, such as wound healing, the periodic growth and regression of mammary epithelium, the process of mammary tumorigenesis therapeutic strategies for breast cancer.