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.     

Profiling drug-induced cell death pathways in the zebrafish lateral line

Programmed cell death (PCD) is an important process in development and disease, as it allows the body to rid itself of unwanted or damaged cells. However, PCD pathways can also be activated in otherwise healthy cells. One such case occurs in sensory hair cells of the inner ear following exposure to ototoxic drugs, resulting in hearing loss and/or balance disorders. The intracellular pathways that determine if hair cells die or survive following this or other ototoxic challenges are incompletely understood. We use the larval zebrafish lateral line, an external hair cell-bearing sensory system, as a platform for profiling cell death pathways activated in response to ototoxic stimuli. In this report the importance of each pathway was assessed by screening a custom cell death inhibitor library for instances when pathway inhibition protected hair cells from the aminoglycosides neomycin or gentamicin, or the chemotherapy agent cisplatin. This screen revealed that each ototoxin likely activated a distinct subset of possible cell death pathways. For example, the proteasome inhibitor Z-LLF-CHO protected hair cells from either aminoglycoside or from cisplatin, while d-methionine, an antioxidant, protected hair cells from gentamicin or cisplatin but not from neomycin toxicity. The calpain inhibitor leupeptin primarily protected hair cells from neomycin, as did a Bax channel blocker. Neither caspase inhibition nor protein synthesis inhibition altered the progression of hair cell death. Taken together, these results suggest that ototoxin-treated hair cells die via multiple processes that form an interactive network of cell death signaling cascades.     

Basic fibroblast growth factor (FGF-2) protects rat cochlear hair cells in organotypical culture from aminoglycoside injury

Given the evidence that basic fibroblast growth factor (FGF-2) can protect neural and retinal cells from degeneration, we evaluated the potential of this growth factor to protect sensory cells in the inner ear. When sensory cells of the organ of Corti are exposed to aminoglycoside antibiotics such as neomycin either in vivo or in vitro, significant ototoxicity is observed. The in vitro cytotoxic effects of neomycin are dose and time dependent. In neonatal rat organ of Corti cultures, complete inner and outer hair cell destruction is observed at high (mM) concentrations of neomycin while inner hair cell survival and severely damaged outer hair cells are noted at moderate (μM) concentrations, with a maximal effect observed after 2 days of culture. Approximately 50% of cochlear outer hair cells are lost at a dose of 35 μM neomycin, and most surviving cells show disorganized stereocilia. Inner hair cells show primarily disorganization of their stereocilia. A significant protective effect is observed when the organ of Corti is pre-treated with FGF-2 (500 ng/ml) for 48 hours, and then FGF-2 is included with neomycin in the culture medium. A greater extent of outer hair cell survival and a significant decrease in stereociliary damage are noted with FGF-2. However, disorganization of inner hair cell stereocilia is unaffected by FGF-2. The protective effect of FGF-2 is specific, since interleukin-1B, nerve growth factor, tumor necrosis factor, and epidermal growth factor are ineffective, while retinoic acid and transforming growth factor alpha show only a moderate protective effect. These results confirm the potential of molecules like FGF-2 for preventing cell death due to a variety of causes. © 1996 Wiley-Liss, Inc.     

Overexpression of Bcl‐2 prevents neomycin‐induced hair cell death and caspase‐9 activation in the adult mouse utricle in vitro

Mechanosensory hair cells of the inner ear are especially sensitive to death induced by exposure to aminoglycoside antibiotics. This aminoglycoside‐induced hair cell death involves activation of an intrinsic program of cellular suicide. Aminoglycoside‐induced hair cell death can be prevented by broad‐spectrum inhibition of caspases, a family of proteases that mediate apoptotic and programmed cell death in a wide variety of systems. More specifically, aminoglycoside‐induced hair cell death requires activation of caspase‐9. Caspase‐9 activation requires release of mitochondrial cytochrome c into the cytoplasm, indicating that aminoglycoside‐induced hair cell death is mediated by the mitochondrial (or “intrinsic”) cell death pathway. The Bcl‐2 family of pro‐apoptotic and anti‐apoptotic proteins are important upstream regulators of the mitochondrial apoptotic pathway. Bcl‐2 is an anti‐apoptotic protein that localizes to the mitochondria and promotes cell survival by preventing cytochrome c release. Here we have utilized transgenic mice that overexpress Bcl‐2 to examine the role of Bcl‐2 in neomycin‐induced hair cell death. Overexpression of Bcl‐2 significantly increased hair cell survival following neomycin exposure in organotypic cultures of the adult mouse utricle. Furthermore, Bcl‐2 overexpression prevented neomycin‐induced activation of caspase‐9 in hair cells. These results suggest that the expression level of Bcl‐2 has important effects on the pathway(s) important for the regulation of aminoglycoside‐induced hair cell death. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 89–100, 2004     

Dimethyl Sulfoxide (DMSO) Exacerbates Cisplatin-induced Sensory Hair Cell Death in Zebrafish (Danio rerio)

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO.     

Cell Cycle,Differentiation and Regeneration: Where to Begin?

Hair cells, the sensory cells of inner ear, perform essential functions in hearing and balance. However, mammalian hair cells, like most of the CNS neurons, lack the capacity to regenerate. This is in sharp contrast to lower vertebrates in which hair cell regeneration occurs spontaneously through cell division of supporting cells, which leads to hearing restoration. It is believed that the lack of regeneration in mammals is, to a large degree, due to the block of cell cycle re-entry imposed by negative cell growth genes in the inner ear. Recent studies have identified retinoblastoma gene, a well-known tumor suppressor, as the key gene involved in cell cycle exit of inner ear sensory cells. In the inner ear of pRb conditional knockout mice, hair cells undergo continuous cell division, and at the same time differentiate and become functional. Cell division continues in early postnatal cochlea and adult vestibule. Remarkably, the vestibular hair cells without pRb survive, and function at both the cellular and system levels. The time course and effects of pRb inhibition shows that there is a separation between the roles of pRb in cell cycle exit, and subsequent maturation and apoptosis. Those studies reveal distinctly different roles of pRb in the cochlear and vestibular sensory epithelia. The review discusses additional areas to be studied for regeneration of mature hair cells, and highlights the importance of transient and reversible block of pRb function as one of the routes to be explored for regeneration.     

Lineage Analysis of the Late Otocyst Stage Mouse Inner Ear by Transuterine Microinjection of A Retroviral Vector Encoding Alkaline Phosphatase and an Oligonucleotide Library

The mammalian inner ear subserves the special senses of hearing and balance. The auditory and vestibular sensory epithelia consist of mechanically sensitive hair cells and associated supporting cells. Hearing loss and balance dysfunction are most frequently caused by compromise of hair cells and/or their innervating neurons. The development of gene- and cell-based therapeutics will benefit from a thorough understanding of the molecular basis of patterning and cell fate specification in the mammalian inner ear. This includes analyses of cell lineages and cell dispersals across anatomical boundaries (such as sensory versus nonsensory territories). The goal of this study was to conduct retroviral lineage analysis of the embryonic day 11.5(E11.5) mouse otic vesicle. A replication-defective retrovirus encoding human placental alkaline phosphatase (PLAP) and a variable 24-bp oligonucleotide tag was microinjected into the E11.5 mouse otocyst. PLAP-positive cells were microdissected from cryostat sections of the postnatal inner ear and subjected to nested PCR. PLAP-positive cells sharing the same sequence tag were assumed to have arisen from a common progenitor and are clonally related. Thirty five multicellular clones consisting of an average of 3.4 cells per clone were identified in the auditory and vestibular sensory epithelia, ganglia, spiral limbus, and stria vascularis. Vestibular hair cells in the posterior crista were related to one another, their supporting cells, and nonsensory epithelial cells lining the ampulla. In the organ of Corti, outer hair cells were related to a supporting cell type and were tightly clustered. By contrast, spiral ganglion neurons, interdental cells, and Claudius'' cells were related to cells of the same type and could be dispersed over hundreds of microns. These data contribute new information about the developmental potential of mammalian otic precursors in vivo.     

Variations in shape-sensitive restriction points mirror differences in the regeneration capacities of avian and mammalian ears

When inner ear hair cells die, humans and other mammals experience permanent hearing and balance deficits, but non-mammalian vertebrates quickly recover these senses after epithelial supporting cells give rise to replacement hair cells. A postnatal decline in cellular plasticity appears to limit regeneration in mammalian balance organs, where declining proliferation responses are correlated with decreased spreading of supporting cells on artificial and native substrates. By culturing balance epithelia on substrates that differed in flexibility, we assessed spreading effects independent of age, showing a strong correlation between shape change and supporting cell proliferation. Then we made excision wounds in utricles cultured from young and old chickens and mice and compared quantified levels of spreading and proliferation. In utricles from young mice, and both young and old chickens, wounds re-epithelialized in <24 hours, while those in utricles from mature mice took three times longer. More cells changed shape in the fastest healing wounds, which accounted for some differences in the levels of proliferation, but inter-species and age-related differences in shape-sensitive restriction points, i.e., the cellular thresholds for shape changes that promote S-phase, were evident and may be particularly influential in the responses to hair cell losses in vivo.     

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.     

Immune cytokines and dexamethasone influence sensory regeneration in the avian vestibular periphery

Prior studies have shown that macrophages are recruited to sites of hair cell lesions in the avian inner ear in vitro (Warchol, 1997) and in vivo (Bhave et al., 1998). Although the avian ear has a high capacity for sensory regeneration (Oberholtzer & Corwin, 1997; Stone et al., 1998), the role of macrophages in the regenerative process is uncertain. The present study examined the possible influence of macrophages and immune cytokines on regenerative proliferation in the avian utricle, one of the sensory endorgans of the vestibular system. Utricles from post-hatch chicks were placed in organ culture and hair cell lesions were created by incubation in neomycin. The cultures were then maintained for an additional 24–48 hours in vitro, and some cultures were treated with dexamethasone, which inhibits macrophage activation and cytokine production. Following fixation, resident macrophages were identified by immunoreactivity to CD68. Labeled macrophages were present in all specimens and increased numbers of macrophages were observed following neomycin treatment. Regenerative proliferation in dexamethasone-treated specimens was reduced by about 50%, relative to untreated controls. Additional experiments showed that two macrophage secretory products—TGF-α and TNF-α—enhanced the proliferation of utricular supporting cells. The results are consistent with a role for macrophages in hair cell regeneration.     

Hes1 is a negative regulator of inner ear hair cell differentiation

Hair cell fate determination in the inner ear has been shown to be controlled by specific genes. Recent loss-of-function and gain-of-function experiments have demonstrated that Math1, a mouse homolog of the Drosophila gene atonal, is essential for the production of hair cells. To identify genes that may interact with Math1 and inhibit hair cell differentiation, we have focused on Hes1, a mammalian hairy and enhancer of split homolog, which is a negative regulator of neurogenesis. We report here that targeted deletion of Hes1 leads to formation of supernumerary hair cells in the cochlea and utricle of the inner ear. RT-PCR analysis shows that Hes1 is expressed in inner ear during hair cell differentiation and its expression is maintained in adulthood. In situ hybridization with late embryonic inner ear tissue reveals that Hes1 is expressed in supporting cells, but not hair cells, of the vestibular sensory epithelium. In the cochlea, Hes1 is selectively expressed in the greater epithelial ridge and lesser epithelial ridge regions which are adjacent to inner and outer hair cells. Co-transfection experiments in postnatal rat explant cultures show that overexpression of Hes1 prevents hair cell differentiation induced by Math1. Therefore Hes1 can negatively regulate hair cell differentiation by antagonizing Math1. These results suggest that a balance between Math1 and negative regulators such as Hes1 is crucial for the production of an appropriate number of inner ear hair cells.     

Identification of stage-specific markers during differentiation of hair cells from mouse inner ear stem cells or progenitor cells in vitro

The induction of inner ear hair cells from stem cells or progenitor cells in the inner ear proceeds through a committed inner ear sensory progenitor cell stage prior to hair cell differentiation. To increase the efficacy of inducing inner ear hair cell differentiation from the stem cells or progenitor cells, it is essential to identify comprehensive markers for the stem cells/progenitor cells from the inner ear, the committed inner ear sensory progenitor cells and the differentiating hair cells to optimize induction conditions. Here, we report that we efficiently isolated and expanded the stem cells or progenitor cells from postnatal mouse cochleae, and induced the generation of inner ear progenitor cells and subsequent differentiation of hair cells. We profiled the gene expression of the stem cells or progenitor cells, the inner ear progenitor cells, and hair cells using aRNA microarray analysis. The pathway and gene ontology (GO) analysis of differentially expressed genes was performed. Analysis of genes exclusively detected in one particular cellular population revealed 30, 38, and 31 genes specific for inner ear stem cells, inner ear progenitor cells, and hair cells, respectively. We further examined the expression of these genes in vivo and determined that Gdf10+Ccdc121, Tmprss9+Orm1, and Chrna9+Espnl are marker genes specific for inner ear stem cells, inner ear progenitor cells, and differentiating hair cells, respectively. The identification of these marker genes will likely help the effort to increase the efficacy of hair cell induction from the stem cells or progenitor cells.     

The molecular basis of neurosensory cell formation in ear development: a blueprint for hair cell and sensory neuron regeneration?

The inner ear of mammals uses neurosensory cells derived from the embryonic ear for mechanoelectric transduction of vestibular and auditory stimuli (the hair cells) and conducts this information to the brain via sensory neurons. As with most other neurons of mammals, lost hair cells and sensory neurons are not spontaneously replaced and result instead in age-dependent progressive hearing loss. We review the molecular basis of neurosensory development in the mouse ear to provide a blueprint for possible enhancement of therapeutically useful transformation of stem cells into lost neurosensory cells. We identify several readily available adult sources of stem cells that express, like the ectoderm-derived ear, genes known to be essential for ear development. Use of these stem cells combined with molecular insights into neurosensory cell specification and proliferation regulation of the ear, might allow for neurosensory regeneration of mammalian ears in the near future.