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In vitro growth and differentiation of mammalian sensory hair cell progenitors: A requirement for EGF and periotic mesenchyme
Abstract
The sensory hair cells and supporting cells of the organ of Corti are generated by a precise program of coordinated cell division and differentiation. Since no regeneration occurs in the mature organ of Corti, loss of hair cells leads to deafness. To investigate the molecular basis of hair cell differentiation and their lack of regeneration, we have established a dissociated cell culture system in which sensory hair cells and supporting cells can be generated from mitotic precursors. By incorporating a Math1-GFP transgene expressed exclusively in hair cells, we have used this system to characterize the conditions required for the growth and differentiation of hair cells in culture. These conditions include a requirement for epidermal growth factor, as well as the presence of periotic mesenchymal cells. Lastly, we show that early postnatal cochlear tissue also contains cells that can divide and generate new sensory hair cells in vitro.
... Cochlear ducts from E12.0 or E13.5 wild-type (WT) mice were dissociated and infected with adenoviruses expressing either Atoh1 (Ad-Atoh1-RFP) or a control RFP-reporter construct (Ad-RFP). We used this dissociated culture system as, unlike intact organ cultures, dissociated cochlear cells have only a limited capacity to spontaneously differentiate as hair cell (38). Consistent with these earlier observations, only a few Pou4f3-positive hair cells were detected in either E12.0 or E13.5 RFP-controls after 3 d in culture, despite 80 to 90% infection efficiency ( Fig. 1 D and E and SI Appendix, Fig. S2). ...
... and In Vivo. To directly test if SoxC transcription factors can promote Atoh1 expression and sensory differentiation, we overexpressed either Sox4 or Sox11 in the dissociated E12.5 progenitor cells not capable of differentiating toward hair cells spontaneously (Fig. 1) (38). Isolated from the Atoh1-GFP reporter mice, the E12.5 cochlea progenitor cells were infected with adenoviruses containing RFP, Sox2-RFP, Sox4-RFP, or Sox11-RFP coding sequences, and hair cell formation was analyzed 3 d later. ...
The auditory organ of Corti is comprised of only two major cell types-the mechanosensory hair cells and their associated supporting cells-both specified from a single pool of prosensory progenitors in the cochlear duct. Here, we show that competence to respond to Atoh1, a transcriptional master regulator necessary and sufficient for induction of mechanosensory hair cells, is established in the prosensory progenitors between E12.0 and 13.5. The transition to the competent state is rapid and is associated with extensive remodeling of the epigenetic landscape controlled by the SoxC group of transcription factors. Conditional loss of Sox4 and Sox11-the two homologous family members transiently expressed in the inner ear at the time of competence establishment-blocks the ability of prosensory progenitors to differentiate as hair cells. Mechanistically, we show that Sox4 binds to and establishes accessibility of early sensory lineage-specific regulatory elements, including ones associated with Atoh1 and its direct downstream targets. Consistent with these observations, overexpression of Sox4 or Sox11 prior to developmental establishment of competence precociously induces hair cell differentiation in the cochlear progenitors. Further, reintroducing Sox4 or Sox11 expression restores the ability of postnatal supporting cells to differentiate as hair cells in vitro and in vivo. Our findings demonstrate the pivotal role of SoxC family members as agents of epigenetic and transcriptional changes necessary for establishing competence for sensory receptor differentiation in the inner ear.
... However, studies have identified stem/progenitor hair cells in the neonatal and adult inner ear [8][9][10][11][12]. Furthermore, studies have indicated that progenitor cells can be induced to differentiate into hair cells [8,[13][14][15][16][17][18][19], and the restoration of hair cells can restore auditory function [1,2,[20][21][22]. ...
... In another study, White et al. [15] showed that supporting cell proliferative capacity that is agedependent is related to downregulation of p27kip1. Interestingly, Doetzlhofer et al. [14] showed that both growth factor and supporting periotic mesenchyme cells were required for inner hair cell differentiation. ...
Background: Cochlear sensory epithelium-derived progenitor cells initially give rise to compact solid/round spheres. These compact solid/round spheres then gradually convert into irregular and partially hollow spheres, which then ultimately transform into large hollow spheres. The purpose of this study was to observe the differentiation of cochlear sensory epithelium-derived progenitor cells into spheres, and determine factors necessary for their development into hair cells.
Methods: Cochlear epithelial sheets from postnatal day 1 C57BL/6 mice were dissociated and sphere cells were cultured. The morphological changes of the spheres were observed, and the different types of sphere cells were examined for their ability to differentiate into hair cell-like cells.
Results: Solid spheres formed first, and then gradually transformed into hollow spheres over approximately 260 hours. Adherent culture and Transwell culture assays, and immunohistochemistry staining revealed that neither solid nor hollow sphere cells alone could differentiate into mature hair cells. Solid sphere cells, however, were able to differentiate into mature hair cells when co-cultured with p27kip1-positive hollow sphere cells. Direct contact of the cells was necessary for the differentiation of the solid sphere cells into mature hair cell-like cells.
Conclusions: Cochlear sensory epithelium-derived progenitor cells require specific conditions to differentiate into mature hair cells.
... To this end, progenitor cells at day 13 of differentiation were switched to a culture medium containing RA/EGF, modulators of two pathways that are active during inner ear development. The RA has pleiotropic functions during embryogenesis and has been found to expand otic competence within posterior placode during inner ear development [44][45], while EGF participates towards regulating the in vitro production of HCs in embryonic and early postnatal inner ears [46][47]. Of interest, a synergistic interaction between RA and EGF has been found to induce the differentiation of HC-like cells from hESCs [17]. ...
... This observation fits with the roles of EGF and retinoid pathways in inner ear development and with a previous study on hESCs [17]. RA has also been shown to regulate otic vesicle formation in vivo [44][45], whereas EGF ligands i.e., EGF/TGF-alpha promote proliferation and/or maintenance of inner ear progenitor cells in vitro [46][47]. In contrast, for otic progenitors maintained 7 days in vitro under Notch inhibition, we observed a significant increase in the relative gene expression of both ATOTH1 and MYO7A as compared to their levels in age-matched RA/ EGF-treated cultures. ...
The inner ear represents a promising system to develop cell-based therapies from human induced pluripotent stem cells (hiPSCs). In the developing ear, Notch signaling plays multiple roles in otic region specification and for cell fate determination. Optimizing hiPSC induction for the generation of appropriate numbers of otic progenitors and derivatives, such as hair cells, may provide an unlimited supply of cells for research and cell-based therapy. In this study, we used monolayer cultures, otic-inducing agents, Notch modulation, and marker expression to track early and otic sensory lineages during hiPSC differentiation. Otic/placodal progenitors were derived from hiPSC cultures in medium supplemented with FGF3/FGF10 for 13 days. These progenitor cells were then treated for 7 days with retinoic acid (RA) and epidermal growth factor (EGF) or a Notch inhibitor. The differentiated cultures were analyzed in parallel by qPCR and immunocytochemistry. After the 13 day induction, hiPSC-derived cells displayed an upregulated expression of a panel of otic/placodal markers. Strikingly, a subset of these induced progenitor cells displayed key-otic sensory markers, the percentage of which was increased in cultures under Notch inhibition as compared to RA/EGF-treated cultures. Our results show that modulating Notch pathway during in vitro differentiation of hiPSC-derived otic/placodal progenitors is a valuable strategy to promote the expression of human otic sensory lineage genes.
... Lou et al. (2007) demonstrated that such stem cells contributed in the regeneration of damaged cells in the rat. Likewise, recovery of hair cells in vitro have been demonstrated in mice by application of fetal cochlea stem cells (Shi et al. 2012;Savary et al. 2007;Oshima et al. 2007;White et al. 2006;Doetzlhofer et al. 2004). Also, embryonic and neural stem cells have been used to experimentally treat deafness in mice (Parker et al. 2007;Corrales et al. 2006). ...
... Similar results were previously obtained by Diensthuber et al. (2009) for cultures of cochlear epithelial cells of different mice lineages (BALB/c and Math-1/nuclear green fluorescence protein (nGFP) transgenic mice) using similar culture medium. Media containing DMEM-High glucose and DMEM-F12 (1:1) have been proven to be successful for culturing rodent cochlea cells and were suitable for the applied purposes (Chao et al. 2013;White et al. 2012;Diensthuber et al. 2009;Yerukhimovich et al. 2007;Savary et al. 2007;Martinez-Monedero and Edge 2007;Doetzlhofer et al. 2004;Li et al. 2003). In our adherent cultures, cochlear epithelial cells have typical fibroblast-like morphology, as observed for other mesenchymal stem cell cultures, including multipotentiality (Favaron et al. 2014;Chao et al. 2013;Wenceslau et al. 2012). ...
Hearing loss caused by the damage of cochlea sensory cells or neurons is a common human disease, but also affects dogs and other animals. To test their progenitor nature as potential value for future therapies, we characterized cells derived from the cochlear epithelium in dog fetuses. In total, 8 fetuses of 35–40 days of gestation, derived from castration campaigns, were investigated. Cells were analysed by the MTT colorimetric assay and in regard to cell cycle, differentiation capacities, immunophenotypes and qPCR analysis. In culture, cells had a fibroblast-like morphology. Phenotypic immunocharacterization showed positive staining for mesenchymal stem cell and pluripotency markers and were negative for hematopoietic cell markers. Cells possessed differentiation capacity for the three main cell lineages: osteogenic, adipogenic and chondrogenic, altogether indicating their nature as mesenchymal stem cells. Thus, cells derived from fetal cochlear tissues indeed may provide valuable sources of progenitor cells for cell therapy of canine deafness and other diseases.
... In the inner ear, stem cells were used in experiments to differentiate into hair cells and regenerate auditory neurons. In the labyrinth, stem cells are used by experimentation with the hope that they would someday turn out to be hair cells and audile neurons, foetal dorsal root neural cells [33,34], neural ancestor cells [35,36], labyrinth stem or progenitor cells [37][38][39], immortalised audile formative cell cells [40,41], embryonic stem cells and their derived somatic cell cells [34,37,42], and additionally as marrow stromal cells treated with sonic hedgehog and retinoic acid [43]. ...
Background
Damage to the inner ear or cochlear nerve results in sensorineural hearing loss (SNHL), which is typically persistent deafness. SNHL can range in severity from mild to profound. The shape of the audiogram is used to categorise it as high-frequency hearing loss, low-frequency, flat, peaked, or notched. Pure tone audiometry can be used to diagnose SNHL.
Objective
To summarise the recent updates in the usage of stem cells in sensory neural hearing loss (SNHL).
Methods
Published studies about using stem cell therapy in ENT practice through comprehensive PubMed, EKG, and Google Scholar search (from 2010 to 2022). Including studies in English, experimental studies, and studies that discuss the application of regenerative medicine in SNHL.
Results
Progenitor stem cells may be employed to repair damaged cells and restore sensorineural hearing function, according to 36 of the publications. The majority of these articles—about 90%—discussed animal model-based experimental investigations; the remaining 10% were clinical trials.
Conclusion
The application of stem cells in the treatment of SNHL will be a significant step in the future since it will change the way that patients are now treated in the hopes of regaining their hearing. The application to the clinical setting is still in its early stage, although a number of encouraging researches illustrate how progenitor stem cells differentiate into sensorineural cells.
... A direct response requiring precise interpretation of the BMP4 gradient seems unlikely, suggesting instead that Lrrn1 expression is regulated through establishment of the medial-lateral cochlear axis. Finally, as noted by Basch et al. (2016), differentiation of the OC requires inductive signals from mesenchymal cells located on the opposite side of the cochlear basilar membrane (Montcouquiol and Kelley, 2003;Doetzlhofer et al., 2004). These yet unidentified inductive signals could also play a role in regulation of Lrrn1 expression. ...
One of the most striking aspects of the sensory epithelium of the mammalian cochlea, the organ of Corti, is the presence of precise boundaries between sensory and non-sensory cells at its medial and lateral edges. A particular example of this precision is the single row of inner hair cells and associated supporting cells along the medial (neural) boundary. Despite the regularity of this boundary, the developmental processes and genetic factors that contribute to its specification are poorly understood. In this study we demonstrate that Leucine Rich Repeat Neuronal 1 ( Lrrn1 ), which codes for a single-pass, transmembrane protein, is expressed prior to the development of the mouse organ of Corti in the row of cells that will form its medial border. Deletion of Lrrn1 in mice of mixed sex leads to disruptions in boundary formation that manifest as ectopic inner hair cells and supporting cells. Genetic and pharmacological manipulations demonstrate that Lrrn1 interacts with the Notch signaling pathway and strongly suggest that Lrrn1 normally acts to enhance Notch signaling across the medial boundary. This interaction is required to promote formation of the row of inner hair cells and suppress the conversion of adjacent non-sensory cells into hair cells and supporting cells. These results identify Lrrn1 as an important regulator of boundary formation and cellular patterning during development of the organ of Corti.
SIGNIFICANCE STATEMENT:
Patterning of the developing mammalian cochlea into distinct sensory and non-sensory regions and the specification of multiple different cell fates within those regions are critical for proper auditory function. Here, we report that the transmembrane protein LRRN1 is expressed along the sharp medial boundary between the single row of mechanosensory inner hair cells and adjacent non-sensory cells. Formation of this boundary is mediated in part by Notch signaling, and loss of Lrrn1 leads to disruptions in boundary formation similar to those caused by a reduction in Notch activity, suggesting that LRRN1 likely acts to enhance Notch signaling. Greater understanding of sensory/non-sensory cell fate decisions in the cochlea will help inform the development of regenerative strategies aimed at restoring auditory function.
... 79 Also the interactions between JAG1, JAG2, or DLL1 with NOTCH1, NOTCH2, or NOTCH3 between these same cell types are known mechanisms by which NOTCH signaling contributes to hair cell formation. [80][81][82][83] Additionally, the POM, a crucial component for hair cell differentiation, 84 demonstrates communication with hair cells and supportive cells, highlighting the role of BMP signaling, in interacting to known receptors within the sensory epithelium. 85 These ligand-receptor interactions of the NECTIN, NOTCH, and BMP pathways could be validated in the fetal inner ear data ( Figure 7A). ...
Inner ear disorders are among the most common congenital abnormalities; however, current tissue culture models lack the cell type diversity to study these disorders and normal otic development. Here, we demonstrate the robustness of human pluripotent stem cell-derived inner ear organoids (IEOs) and evaluate cell type heterogeneity by single-cell transcriptomics. To validate our findings, we construct a single-cell atlas of human fetal and adult inner ear tissue. Our study identifies various cell types in the IEOs including periotic mesenchyme, type I and type II vestibular hair cells, and developing vestibular and cochlear epithelium. Many genes linked to congenital inner ear dysfunction are confirmed to be expressed in these cell types. Additional cell-cell communication analysis within IEOs and fetal tissue highlights the role of endothelial cells on the developing sensory epithelium. These findings provide insights into this organoid model and its potential applications in studying inner ear development and disorders.
... In the latter, it is preferentially expressed in the cochlear duct which houses the sensory epithelium (Supplementary Figures S6). Furthermore, Map7d2 expression is upregulated when otic progenitor (iMOP) cells are cultured in EGF, a growth factor important in the growth and differentiation of hair cells (Doetzlhofer et al., 2004). This suggests Map7d2 may have a potential role in hair cell growth and differentiation. ...
Age-related (AR) hearing loss (HL) is the most common sensory impairment with heritability of 55%. The aim of this study was to identify genetic variants on chromosome X associated with ARHL through the analysis of data obtained from the UK Biobank. We performed association analysis between self-reported measures of HL and genotyped and imputed variants on chromosome X from ∼460,000 white Europeans. We identified three loci associated with ARHL with a genome-wide significance level (p < 5 × 10⁻⁸), ZNF185 (rs186256023, p = 4.9 × 10⁻¹⁰) and MAP7D2 (rs4370706, p = 2.3 × 10⁻⁸) in combined analysis of males and females, and LOC101928437 (rs138497700, p = 8.9 × 10⁻⁹) in the sex-stratified analysis of males. In-silico mRNA expression analysis showed MAP7D2 and ZNF185 are expressed in mice and adult human inner ear tissues, particularly in the inner hair cells. We estimated that only a small amount of variation of ARHL, 0.4%, is explained by variants on the X chromosome. This study suggests that although there are likely a few genes contributing to ARHL on the X chromosome, the role that the X chromosome plays in the etiology of ARHL may be limited.
... Single-cell in vitro studies suggested that precursor cell differentiation into hair cells requires the POM, as its presence in the culture system induces cell growth and proliferation (Doetzlhofer et al., 2004). However, the absence of the typical stereoscopic microenvironment and related regulatory factors prevents cells from completely expressing the necessary gene products required for differentiation, including factors involved in Notch signaling. ...
Objective
To investigate the role of the periotic mesenchyme (POM) in the development of sensory cells of developing auditory epithelium.
Methods
Developing auditory epithelium with or without periotic mesenchyme was isolated from mice at embryonic days 11.5 (E11.5), E12.5 and E13.5, respectively, and cultured in vitro to an equivalent of E18.5’s epithelium in vivo. Then, the explants were co-stained with antibodies targeting myosin VIIA, Sox2 and BrdU.
Results
More hair cells in E11.5+7 DIV, E12.5+6 DIV and E13.5+5 DIV auditory epithelia were found upon culture with POM (225.90±62.44, 476.94±100.81, and 1386.60±202.38, respectively) compared with the non-POM group (68.17±23.74, 205.00±44.23, and 1266.80±38.84, respectively). Moreover, regardless of developmental stage, the mesenchymal tissue increased the amount of cochlear sensory cells as well as the ratio of differentiated hair cells to total sensory cells.
Conclusions
The periotic mesenchyme promotes the development of cochlear sensory cells, and its effect depends on the developmental stage of the auditory epithelium.
... Chromatin accessibility changes at specific hair cell enhancers for Pou4f3, Mreg, Rasd2 and Barhl1 are highlighted in grey boxes. (Doetzlhofer et al., 2004;White et al., 2006). To determine if iHCs are capable of integrating appropriately into these sensory epithelial-like structures which contain both primary hair cells and supporting cells, we FACS-purified Atoh1-nGFP+ iHCs and mixed them with dissociated primary embryonic (E13.5) ...
The mechanoreceptive sensory hair cells in the inner ear are selectively vulnerable to numerous genetic and environmental insults. In mammals, hair cells lack regenerative capacity, and their death leads to permanent hearing loss and vestibular dysfunction. Their paucity and inaccessibility has limited the search for otoprotective and regenerative strategies. Growing hair cells in vitro would provide a route to overcome this experimental bottleneck. We report a combination of four transcription factors (Six1, Atoh1, Pou4f3, and Gfi1) that can convert mouse embryonic fibroblasts, adult tail-tip fibroblasts and postnatal supporting cells into induced hair cell-like cells (iHCs). iHCs exhibit hair cell-like morphology, transcriptomic and epigenetic profiles, electrophysiological properties, mechanosensory channel expression, and vulnerability to ototoxin in a high-content phenotypic screening system. Thus, direct reprogramming provides a platform to identify causes and treatments for hair cell loss, and may help identify future gene therapy approaches for restoring hearing.
... Chromatin accessibility changes at specific hair cell enhancers for Pou4f3, Mreg, Rasd2 and Barhl1 are highlighted in grey boxes. (Doetzlhofer et al., 2004;White et al., 2006). To determine if iHCs are capable of integrating appropriately into these sensory epithelial-like structures which contain both primary hair cells and supporting cells, we FACS-purified Atoh1-nGFP+ iHCs and mixed them with dissociated primary embryonic (E13.5) ...
The mechanoreceptive sensory hair cells in the inner ear are selectively vulnerable to numerous genetic and environmental insults. In mammals, hair cells lack regenerative capacity, and their death leads to permanent hearing loss and vestibular dysfunction. Their paucity and inaccessibility has limited the search for otoprotective and regenerative strategies. Growing hair cells in vitro would provide a route to overcome this experimental bottleneck. We report a combination of four transcription factors (Six1, Atoh1, Pou4f3, and Gfi1) that can convert mouse embryonic fibroblasts, adult tail-tip fibroblasts and postnatal supporting cells into induced hair cell-like cells (iHCs). iHCs exhibit hair cell-like morphology, transcriptomic and epigenetic profiles, electrophysiological properties, mechanosensory channel expression, and vulnerability to ototoxin in a high-content phenotypic screening system. Thus, direct reprogramming provides a platform to identify causes and treatments for hair cell loss, and may help identify future gene therapy approaches for restoring hearing.
... Chromatin accessibility changes at specific hair cell enhancers for Pou4f3, Mreg, Rasd2 and Barhl1 are highlighted in grey boxes. (Doetzlhofer et al., 2004;White et al., 2006). To determine if iHCs are capable of integrating appropriately into these sensory epithelial-like structures which contain both primary hair cells and supporting cells, we FACS-purified Atoh1-nGFP+ iHCs and mixed them with dissociated primary embryonic (E13.5) ...
The mechanoreceptive sensory hair cells in the inner ear are selectively vulnerable to numerous genetic and environmental insults. In mammals, hair cells lack regenerative capacity, and their death leads to permanent hearing loss and vestibular dysfunction. Their paucity and inaccessibility has limited the search for otoprotective and regenerative strategies. Growing hair cells in vitro would provide a route to overcome this experimental bottleneck. We report a combination of four transcription factors (Six1, Atoh1, Pou4f3, and Gfi1) that can convert mouse embryonic fibroblasts, adult tail-tip fibroblasts and postnatal supporting cells into induced hair cell-like cells (iHCs). iHCs exhibit hair cell-like morphology, transcriptomic and epigenetic profiles, electrophysiological properties, mechanosensory channel expression, and vulnerability to ototoxin in a high-content phenotypic screening system. Thus, direct reprogramming provides a platform to identify causes and treatments for hair cell loss, and may help identify future gene therapy approaches for restoring hearing.
... Ligands and receptors from the epidermal growth factor (EGF) family were linked to cell proliferation in auditory cells [11,12]. EGF promotes hair cell differentiation from dissociated embryonic cochlear precursors [13] and promotes the proliferation of dissociated mouse cochlear supporting cells [14]. Additionally, other studies have implicated that the downstream signaling molecules of the epidermal growth factor receptor (EGFR) signaling pathway, such as phosphoinositide 3-kinase (PI3K)-AKT, could contribute to cell proliferation and survival [15][16][17]. ...
Glucose metabolism is an important metabolic pathway in the auditory system. Chronic alcohol exposure can cause metabolic dysfunction in auditory cells during hearing loss. While alcohol exposure has been linked to hearing loss, the mechanism by which impaired glycolysis promotes cytotoxicity and cell death in auditory cells remains unclear. Here, we show that the inhibition of epidermal growth factor receptor (EGFR)-induced glycolysis is a critical mechanism for alcohol exposure-induced apoptosis in HEI-OC1 cells. The cytotoxicity via apoptosis was significantly increased by alcohol exposure in HEI-OC1 cells. The glycolytic activity and the levels of hexokinase 1 (HK1) were significantly suppressed by alcohol exposure in HEI-OC1 cells. Mechanistic studies showed that the levels of EGFR and AKT phosphorylation were reduced by alcohol exposure in HEI-OC1 cells. Notably, HK1 expression and glycolytic activity was suppressed by EGFR inhibition in HEI-OC1 cells. These results suggest that impaired glycolysis promotes alcohol exposure-induced apoptosis in HEI-OC1 cells via the inhibition of EGFR signaling.
... It is known that neurotrophic factors are important for hair cell development [30]. Survival of Mouse Embryonic Stem Cells in the Scala Media factor (EGF) may be required to induce formation of new hair cells [31]. The formation of new hair cells also requires expression of Math1, a transcription factor downstream in the EGF pathway essential for hair cell development [32,33]. ...
Genetical, ageing, excessive noise and certain antibiotics are account for majority of permanent hearing loss in humans. Hearing loss is caused by dysfunction of the sensory epithelium (the organ of Corti) within the inner ear (cochlea). It is associated with irreversible loss of sensory hair cells and spiral ganglion neurons.Inner ear hair cells are specialized mechanoreceptors converts mechanical stimuli into neural information for transmission to the brain. Several areas of research have addressed the treatment of hearing loss. There are evidences for regeneration of sensory hair cells in non-mammalian species; however, studies in mammals have failed to find evidence of cochlear hair cells regeneration.Stem cell regenerative medicine is the current focus on cure for deafness while through regeneration of specific cell types from different sources of stem cells.This review deled present stem cell regenerative therapy promising outcome on clinical application.
... In other epithelial organs, members of the epidermal growth factor receptor (EGFR) family can be activated through tissue stretch damage to induce proliferation (Vermeer et al., 2003), making this signaling pathway an attractive candidate for sensing acoustic injury in the cochlea. In vitro, EGF promotes hair cell differentiation from dissociated embryonic cochlear precursors (Doetzlhofer, White, Johnson, Segil, & Groves, 2004) and promotes the proliferation of dissociated mouse cochlear supporting cells (White, Stone, Groves, & Segil, 2012). Moreover, the avian auditory supporting cells require signaling from the EGFR family for proliferation in regeneration experiments in vitro (White et al., 2012). ...
In mammals, cochlear hair cells are not regenerated once they are lost, leading to permanent hearing deficits. In other vertebrates, the adjacent supporting cells act as a stem cell compartment, in that they both proliferate and differentiate into de novo auditory hair cells. Although there is evidence that mammalian cochlear supporting cells can differentiate into new hair cells, the signals that regulate this process are poorly characterized. We hypothesize that signaling from the epidermal growth factor receptor (EGFR) family may play a role in cochlear regeneration. We focus on one such member, ERBB2, and report the effects of expressing a constitutively active ERBB2 receptor in neonatal mouse cochlear supporting cells, using viruses and transgenic expression. Lineage tracing with fluorescent reporter proteins was used to determine the relationships between cells with active ERBB2 signaling and cells that divided or differentiated into hair cells. In vitro, individual supporting cells harboring a constitutively active ERBB2 receptor appeared to signal to their neighboring supporting cells, inducing them to down‐regulate a supporting cell marker and to proliferate. In vivo, we found supernumerary hair cell‐like cells near supporting cells that expressed ERBB2 receptors. Both supporting cell proliferation and hair cell differentiation were largely reproduced in vitro using small molecules that we show also activate ERBB2. Our data suggests that signaling from the receptor tyrosine kinase ERBB2 can drive the activation of secondary signaling pathways to regulate regeneration, suggesting a new model where an interplay of cell signaling regulates regeneration by endogenous stem‐like cells.
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... Early studies of inner ear development involving grafting and explant cultures of mesenchyme-free otocysts showed that the POM is necessary for proper inner ear development (see Anniko and Schacht, 1984;Doetzlhofer et al., 2004;Miura et al., 2004;Montcouquiol and Kelley, 2003;Swanson et al., 1990). However, the extent to which the POM affects epithelial differentiation and morphogenesis was unclear, as results from different studies were not always consistent with one another. ...
The vertebrate skull is a complex structure housing the brain and specialized sensory organs, including the eye, the inner ear, and the olfactory system. The close association between bones of the skull and the sensory organs they encase has posed interesting developmental questions about how the tissues scale with one another. Mechanisms that regulate morphogenesis of the skull are hypothesized to originate in part from the encased neurosensory organs. Conversely, the developing skull is hypothesized to regulate the growth of neurosensory organs, through mechanical forces or molecular signaling. Here, we review studies of epithelial–mesenchymal interactions during inner ear and olfactory system development that may coordinate the growth of the two sensory organs with their surrounding bone. We highlight recent progress in the field and provide evidence that mechanical forces arising from bone growth may affect olfactory epithelium development. Developmental Dynamics 248:88–97, 2019. © 2018 Wiley Periodicals, Inc.
... It is known that neurotrophic factors are important for hair cell development [30]. Survival of Mouse Embryonic Stem Cells in the Scala Media factor (EGF) may be required to induce formation of new hair cells [31]. The formation of new hair cells also requires expression of Math1, a transcription factor downstream in the EGF pathway essential for hair cell development [32,33]. ...
Genetical, ageing, excessive noise and certain antibiotics are account for majority of permanent hearing loss in humans. Hearing loss is caused by dysfunction of the sensory epithelium (the organ of Corti) within the inner ear (cochlea). It is associated with irreversible loss of sensory hair cells and spiral ganglion neurons. Inner ear hair cells are specialized mechanoreceptors converts mechanical stimuli into neural information for transmission to the brain. Several areas of research have addressed the treatment of hearing loss. There are evidences for regeneration of sensory hair cells in non-mammalian species; however, studies in mammals have failed to find evidence of cochlear hair cells regeneration. Stem cell regenerative medicine is the current focus on cure for deafness while through regeneration of specific cell types from different sources of stem cells. This review deled present stem cell regenerative therapy promising outcome on clinical application.
... Previous studies have demonstrated that EGF and retinoic acid (RA) improve the differentiation of HC progenitors and the induction of Myosin VIIA protein [43,44,45]. Furthermore, Costa et al. [7] observed that addition of RA to GPA-expressing mESC cultures enhanced the efficiency of their programming protocol. ...
Hearing loss is the most common sensorineural disorder, affecting over 5% of the population worldwide. Its most frequent cause is the loss of hair cells (HCs), the mechanosensory receptors of the cochlea. HCs transduce incoming sounds into electrical signals that activate auditory neurons, which in turn send this information to the brain. Although some spontaneous HC regeneration has been observed in neonatal mammals, the very small pool of putative progenitor cells that have been identified in the adult mammalian cochlea is not able to replace the damaged HCs, making any hearing impairment permanent. To date, guided differentiation of human cells to HC-like cells has only been achieved using either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, use of such cell types suffers from a number of important disadvantages, such as the risk of tumourigenicity if transplanted into the host´s tissue. We have obtained cells expressing hair cell markers from cultures of human fibroblasts by overexpression of GFI1, Pou4f3 and ATOH1 (GPA), three genes that are known to play a critical role in the development of HCs. Immunocytochemical, qPCR and RNAseq analyses demonstrate the expression of genes typically expressed by HCs in the transdifferentiated cells. Our protocol represents a much faster approach than the methods applied to ESCs and iPSCs and validates the combination of GPA as a set of genes whose activation leads to the direct conversion of human somatic cells towards the hair cell lineage. Our observations are expected to contribute to the development of future therapies aimed at the regeneration of the auditory organ and the restoration of hearing.
... Although NEUROG1 is expressed in delaminating neural precursors of the inner ear (Matei et al., 2005), its function in regulating proliferation has not been described (Doetzlhofer et al., 2004;Nagtegaal et al., 2015). Here we show that NEUROG1 contributes to cellular proliferation in otic progenitors by regulating CDK2 levels. ...
... Although NEUROG1 is expressed in delaminating neural precursors of the inner ear (Matei et al., 2005), its function in regulating proliferation has not been described (Doetzlhofer et al., 2004;Nagtegaal et al., 2015). Here we show that NEUROG1 contributes to cellular proliferation in otic progenitors by regulating CDK2 levels. ...
Loss of spiral ganglion neurons (SGNs) significantly contributes to hearing loss. Otic progenitor cell transplantation is a potential strategy to replace lost SGNs. Understanding how key transcription factors promote SGN differentiation in otic progenitors accelerates efforts for replacement therapies. A pro-neural transcription factor, Neurogenin1 (Neurog1), is essential for SGN development. Using an immortalized multipotent otic progenitor (iMOP) cell line that can self-renew and differentiate into otic neurons, NEUROG1 was enriched at the promoter of cyclin-dependent kinase 2 (Cdk2) and neurogenic differentiation 1 (NeuroD1) genes. Changes in H3K9ac and H3K9me3 deposition at the Cdk2 and NeuroD1 promoters suggested epigenetic regulation during iMOP proliferation and differentiation. In self-renewing iMOP cells, overexpression of NEUROG1 increased CDK2 to drive proliferation, while knockdown of NEUROG1 decreased CDK2 and reduced proliferation. In iMOP-derived neurons, overexpression of NEUROG1 accelerated acquisition of neuronal morphology, while knockdown of NEUROG1 prevented differentiation. Our findings suggest that NEUROG1 can promote proliferation or neuronal differentiation.
... For example, proliferation is blocked by downregulation of Wnt signaling in the developing murine inner ear (Jacques et al., 2012;Chai et al., 2012). ErbB signaling is also involved, for EGF and heregulin enhance supporting-cell proliferation in vitro and inhibition of the EGFR pathway arrests mitotic activity in sensory epithelia (Zheng et al., 1999;Hume et al., 2003;Doetzlhofer et al., 2004;White et al., 2012). We have recently demonstrated that SoxC transcription factors play an important role and that reactivation of their expression in the adult utricle elicits supporting-cell proliferation and the production of hair cells (Gnedeva and Hudspeth, 2015). ...
Dysfunctions of hearing and balance are often irreversible in mammals owing to the inability of cells in the inner ear to proliferate and replace lost sensory receptors. To determine the molecular basis of this deficiency we have investigated the dynamics of growth and cellular proliferation in a murine vestibular organ, the utricle. Based on this analysis, we have created a theoretical model that captures the key features of the organ’s morphogenesis. Our experimental data and model demonstrate that an elastic force opposes growth of the utricular sensory epithelium during development, confines cellular proliferation to the organ’s periphery, and eventually arrests its growth. We find that an increase in cellular density and the subsequent degradation of the transcriptional cofactor Yap underlie this process. A reduction in mechanical constraints results in accumulation and nuclear translocation of Yap, which triggers proliferation and restores the utricle’s growth; interfering with Yap’s activity reverses this effect.
DOI:http://dx.doi.org/10.7554/eLife.25681.001
... For example trypsin, several studies had reported to apply trypsin in cochlear progenitor cell culture Diensthuber et al. 2009;Lou et al. 2014a, b). Doetzlhofer successfully isolated cochlear progenitor cells by using elastase, collagenase, and trypsin to digest basilar membrane, following several minutes of trituration (Doetzlhofer et al. 2004). Thermolysin was applied to separate epithelium from basilar membrane, which loosens the connection between epithelium and mesenthymal tissue below, then pure epithelial cells can be harvested by following mechanical separation under high-powered microscope (Zhang et al. 2007). ...
The high incidence of hearing loss in human combined with the lack of hair cell regeneration in mammalian cochleae had got the attention to manipulate stem/progenitor cells to participate in hair cell regeneration for years. Cochlear progenitor cells are considered as the best candidate for hair cell regeneration. However, there is not any effective and feasible way to separate hair cell progenitors from rat cochleae, yet. In this study, we tried to isolate single epithelial cells from rat basilar membrane by combinatorial enzymatic digestion with thermolysin and collagenase type I. The results showed that the harvested single cells gave rise to otospheres with features of stem cells and could be induced to differentiate into hair cells. Significantly, more otospheres of epithelial origin were obtained by digesting with thermolysin and collagenase type I. The combinatorial enzymatic digestion would be a potential method for hair cell progenitor isolation and culture with broad applications.
... In addition, these spheres differentiated into various cell types including hair cells, supporting cells, and neurons. Using immunocytochemistry, these spheres were shown to be positive for stem cell markers nestin (4)(5)(6)(7) and Sox2 (7,8). However, it remains unclear whether these progenitor/stem cells can serve as a source for cell replacement therapy of injured cochleas. ...
In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.
... Therefore, we attempted to find a new solution. Both EGF and IGF-1 are potently mitogenic factors, confirmed as necessary growth factors for the development and differentiation of the inner ear cells [22][23][24][25]. These studies enlightened us to select the combination of EGF and IGF-1 to induce MSCs, and the result shows that we obtained similar OPs that co-express PAX8 and SOX2. ...
Background:
Previous studies have shown that BMP4 may play an important part in the development of auditory neurons (ANs), which are degenerated in sensorineural hearing loss. However, whether BMP4 can promote sensory fate specification from mesenchymal stromal cells (MSCs) is unknown so far.
Methods:
MSCs isolated from Sprague-Dawley (SD) rats were confirmed by expression of MSC markers using flow cytometry and adipogenesis/osteogenesis using differentiation assays. MSCs treated with a complex of neurotrophic factors (BMP4 group and non-BMP4 group) were induced into auditory neuron-like cells, then the differences between the two groups were analyzed in morphological observation, cell growth curve, qRT-PCR, and immunofluorescence.
Results:
Flow cytometric analysis showed that the isolated cells expressed typical MSC surface markers. After adipogenic and osteogenic induction, the cells were stained by oil red O and Alizarin Red. The neuronal induced cells were in the growth plateau and had special forms of neurons. In the presence of BMP4, the inner ear genes NF-M, Neurog1, GluR4, NeuroD, Calretinin, NeuN, Tau, and GATA3 were up-regulated in MSCs.
Conclusions:
MSCs have the capacity to differentiate into auditory neuron-like cells in vitro. As an effective inducer, BMP4 may play a key role in transdifferentiation.
... The EGFR pathway is a crucial pathway in mammalian hair cell regeneration. Hence, EGF is required for the growth and differentiation of hair cells in vitro (Doetzlhofer et al., 2004;Hartman et al., 2010). In previous studies, De Silva et al. (2006) induced the differentiation of murine ES cells into the hair cell lineage in the presence of EGF. ...
Sensorineural hearing loss and vestibular dysfunction have become the most common forms of sensory defects. Stem cell-based therapeutic strategies for curing hearing loss are being developed. Several attempts to develop hair cells by using chicken utricle stromal cells as feeder cells have resulted in phenotypic conversion of stem cells into inner ear hair-cell-like cells. Here, we induced the differentiation of human embryonic stem cells (hESCs) into otic epithelial progenitors (OEPs), and further induced the differentiation of OEPs into hair-cell-like cells using different substrates. Our results showed that OEPs cultured on the chicken utricle stromal cells with the induction medium could differentiate into hair-cell-like cells with stereociliary bundles. Co-culture with stromal cells, however, may be problematic for subsequent examination of the induced hair-cell-like cells. In order to avoid the interference from stromal cells, we cultured OEPs on laminin with different induction media and examined the effects of the induction medium on the differentiation potentials of OEPs into hair-cell-like cells. The results revealed that the culture of OEPs on laminin with the conditioned medium from chicken utricle stromal cells supplemented with EGF and all-trans retinoic acid (RA) could promote the organization of cells into epithelial clusters displaying hair-cell-like cells with stereociliary bundles. These cells also displayed the expected electrophysiological properties.
... In addition, epidermal growth factor (EGF) signaling specifies some of this group of cells (Okabe, M. and Okano, H., 1997). This has parallels with the development of vertebrate hair cells: EGF is required for the survival and proliferation of mouse hair cells (Doetzlhofer, A. et al., 2004). The concerted action of dpp, wingless, and EGF causes a group of cells within the Drosophila ectoderm to become specified as proneural. ...
Molecular, morphological, and some physiological evidence suggest that vertebrate hair cells arose as one lineage that descended from mechanoreceptive cells that originated in the earliest animals with true tissues, the cnidarians. The ancestry of these cells can be traced into the different evolutionary lineages, for example, not only to the modern relatives of insects and octopuses, but also through the deuterostome chordate relatives of vertebrates to the earliest fish. Undoubtedly, chordate ciliated mechanoreceptors shared a common origin. Ciliated mechanoreceptors are, however, also found in basal eumetazoans and in the protostomes and share in all cases substantial similarities but numerous differences. The most primitive arrangement was likely that of a microtubule-based cilium surrounded by actin-based microvilli. In the protostome lineages, microvilli have been lost, whereas in the deuterostome chordates, the microvilli play an increasingly important role in mechanotransduction. Despite such differences, many of the genetic pathways that lead to the development of the ciliated mechanotransducers (and that have been studied in chordates and in the protostomes Caenorhabditis elegans and Drosophila) are highly conserved, suggesting that these important transducers of sensory information have a very ancient origin.
... Cochleas were cultured using a modified otic organ culture system first described by Van de Water and Ruben (1971) and chemicallydefined BGJb medium (Invitrogen Corporation, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS), 0.5 mg ascorbic acid/ml and 50 units/ml penicillin/streptomycin (Invitrogen Corporation), pH 7.2 as previously described (Melnick et al., 2006(Melnick et al., , 2011. We cultured cochlear ducts with surrounding periotic mesenchyme since prior studies have demonstrated cochlear morphogenesis and hair cell differentiation are dependent on a complex series of interactions between otic epithelia and periotic mesenchyme (e.g., Van de Water and Represa, 1991;Doetzlhofer et al., 2004;Xu et al., 2007) and mCMV appears to primarily infect embryonic mesenchyme (Melnick et al., 2006;Jaskoll et al., 2008aJaskoll et al., , 2008bMelnick and Jaskoll, 2013). For mCMV infection, cochleas were incubated with 1 × 10 −5 plaque-forming units (PFU)/ml of lacZ-tagged mCMV RM427+ in BGJb on day 0 for 24 hours and then cultured in virus-free media for a total culture period of 6 (E15 + 6) or 9 (E15 + 9) days; controls consisted of cochleas cultured in control medium for the entire period. ...
Congenital cytomegalovirus infection is the major nongenetic cause of sensorineural hearing loss at birth and beyond. Among other pathologies, there is a striking dysplasia/hyperplasia of organ of Corti hair and supporting cells.
Using an in vitro embryonic mouse model of cytomegalovirus-induced cochlear teratogenesis that mimics the known human pathology, and functional signaling network modeling, we tested the hypothesis that cytomegalovirus disrupts the highly ordered organ of Corti hair and supporting cells pattern by dysregulating Notch and Fgfr3, their cognate ligands and downstream effectors.
Several novel emergent properties of the critical lateral inhibition subnetwork became apparent. The subnetwork has classic small-world properties such as short paths between most gene pairs, few long-distance links, and considerable clustering. Concomitantly, the calculated probability that our specific gene expression dataset is from dysplastic organs of Corti is highly significant (p < 1 × 10(-12) ). Furthermore, we determined that the subnetwork has a highly heterogeneous scale-free topology in which the highly linked genes (hubs), Notch and Fgfr3, play a central role in mediating interactions among the less linked genes.
This phenomenon has important biologic and therapeutic implications. Birth Defects Research (Part A), 2015. © 2015 Wiley Periodicals, Inc.
© 2015 Wiley Periodicals, Inc.
... FGF is a key player in processes of proliferation and differentiation of a wide variety of cells and tissues (21,22). EGF is the founding member of the EGF-family of proteins and is a growth factor that stimulates proliferation and differentiation by binding to its receptor EGFR (21,23,24). OSM is a pleiotropic cytokine that belongs to the interleukin 6 (IL-6) group of cytokines (25) which closely resembles leukaemia inhibitory factor (LIF) in both structure and function, and has been proven to be important in liver development (26)(27)(28). ...
End-stage liver disease can be the termination of acute or chronic liver diseases, with manifestations of liver failure; transplantation is currently an effective treatment for these. However, transplantation is severely limited due to the serious lack of donors, expense, graft rejection and requirement of long-term immunosuppression. Mesenchymal stem cells (MSCs) have attracted considerable attention as therapeutic tools as they can be obtained with relative ease and expanded in culture, along with features of self-renewal and multidirectional differentiation. Many scientific groups have sought to use MSCs differentiating into functional hepatocytes to be used in cell transplantation with liver tissue engineering to repair diseased organs. In most of the literature, hepatocyte differentiation refers to use of various additional growth factors and cytokines, such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), oncostatin M (OSM) and more, and most are involved in signalling pathway regulation and cell–cell/cell–matrix interactions. Signalling pathways have been shown to play critical roles in embryonic development, tumourigenesis, tumour progression, apoptosis and cell-fate determination. However, mechanisms of MSCs differentiating into hepatocytes, particularly signalling pathways involved, have not as yet been completely illustrated. In this review, we have focused on progress of signalling pathways associated with mesenchymal stem cells differentiating into hepatocytes along with the stepwise differentiation procedure.
... The cellular environment is important for regulating the iMOP cell phenotype. In vitro, hair cell differentiation after cell-cycle arrest requires additional, as yet unidentified cues provided by neighboring otic cells (Doetzlhofer et al., 2004;Oshima et al., 2010). To determine whether iMOP cells can become hair cells when provided with cues from the inner ear, we engrafted iMOP cells into developing chicken otocysts. ...
Sensorineural hearing loss is caused by the loss of sensory hair cells and neurons of the inner ear. Once lost, these cell types are not replaced. Two genes expressed in the developing inner ear are c-Myc and Sox2. We created immortalized multipotent otic progenitor (iMOP) cells, a fate-restricted cell type, by transient expression of C-MYC in SOX2-expressing otic progenitor cells. This activated the endogenous C-MYC and amplified existing SOX2-dependent transcripts to promote self-renewal. RNA-seq and ChIP-seq analyses revealed that C-MYC and SOX2 occupy over 85% of the same promoters. C-MYC and SOX2 target genes include cyclin-dependent kinases that regulate cell-cycle progression. iMOP cells continually divide but retain the ability to differentiate into functional hair cells and neurons. We propose that SOX2 and C-MYC regulate cell-cycle progression of these cells and that downregulation of C-MYC expression after growth factor withdrawal serves as a molecular switch for differentiation.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
... To confirm cell purity, we immunostained cells immediately post-sort and found that the Axin2 hi cells contained over 93% β-galactosidasepositive cells, 0% myosin 7a-positive hair cells, 0% Prox1-positive supporting cells, 75% Brn4-positive cells and <0.1% other supporting cell types (Sox2-, Jag1-or p27 Kip1 -positive; n=3 sorts We first compared the proliferative capacity of Axin2 hi and Axin2 lo cells using a colony formation assay: cells were seeded in serum-free, N2/B27-supplemented media onto feeder cells harvested from embryonic chicken utricles (supplementary material Fig. S4G). The use of embryonic otic mesenchymal tissues and deprivation of growth factors has been shown to promote differentiation of inner ear progenitor cells Doetzlhofer et al., 2004;Oshima et al., 2010;Sinkkonen et al., 2011;White et al., 2006). After 10 days in vitro, all newly generated colonies were immunopositive for the epithelial marker cytokeratin. ...
Permanent hearing loss is caused by the irreversible damage of cochlear sensory hair cells and nonsensory supporting cells. In the postnatal cochlea, the sensory epithelium is terminally differentiated, whereas tympanic border cells (TBCs) beneath the sensory epithelium are proliferative. The functions of TBCs are poorly characterized. Using an Axin2(lacZ) Wnt reporter mouse, we found transient but robust Wnt signaling and proliferation in TBCs during the first 3 postnatal weeks, when the number of TBCs decreases. In vivo lineage tracing shows that a subset of hair cells and supporting cells is derived postnatally from Axin2-expressing TBCs. In cochlear explants, Wnt agonists stimulated the proliferation of TBCs, whereas Wnt inhibitors suppressed it. In addition, purified Axin2(lacZ) cells were clonogenic and self-renewing in culture in a Wnt-dependent manner, and were able to differentiate into hair cell-like and supporting cell-like cells. Taken together, our data indicate that Axin2-positive TBCs are Wnt responsive and can act as precursors to sensory epithelial cells in the postnatal cochlea.
... In addition, epidermal growth factor (EGF) signaling specifies some of this group of cells (Okabe, M. and Okano, H., 1997). This has parallels with the development of vertebrate hair cells: EGF is required for the survival and proliferation of mouse hair cells (Doetzlhofer, A. et al., 2004). The concerted action of dpp, wingless, and EGF causes a group of cells within the Drosophila ectoderm to become specified as proneural. ...
... Cochleas were cultured using a modified otic organ culture system first described by Van de Water and Ruben (1971) and chemically defined BGJb medium (Invitrogen Corporation, Carlsbad, CA) supplemented with 10% fetal calf serum, 0.5 mg ascorbic acid/mL, and 50 units/mL penicillin/streptomycin (Invitrogen Corporation), pH 7.2, as previously described (Melnick et al., 2006). We cultured the cochlear duct with surrounding periotic mesenchyme because prior studies have demonstrated cochlear morphogenesis and hair cell differentiation are dependent on a complex series of interactions between otic epithelia and periotic mesenchyme (e.g., Van de Water and Represa, 1991;Doetzlhofer et al., 2004;Xu et al., 2007) and CMV seems to primarily infect embryonic mesenchyme (Melnick et al., 2006;Jaskoll et al., 2008a;Jaskoll et al., 2008b). For mCMV infection, cochleas were incubated with 1 × 10 −5 plaque-forming units (PFU)/mL of lacZ-tagged mCMV RM427+ in BGJb on day 0 for 24 hours and then cultured in virus-free media; controls consisted of cochleas cultured in control medium for the entire period. ...
Congenital human cytomegalovirus (CMV) infection is the leading nongenetic etiology of sensorineural hearing loss (SNHL) at birth and prelingual SNHL not expressed at birth. The paucity of temporal bone autopsy specimens from infants with congenital CMV infection has hindered the critical correlation of histopathology with pathogenesis. Here, we present an in vitro embryonic mouse model of CMV-infected cochleas that mimics the human sites of viral infection and associated pathology. There is a striking dysplasia/hyperplasia in mouse CMV-infected cochlear epithelium and mesenchyme, including organ of Corti hair and supporting cells and stria vascularis. This is concomitant with significant dysregulation of p19, p21, p27, and Pcna gene expression, as well as proliferating cell nuclear antigen (PCNA) protein expression. Other pathologies similar to those arising from known deafness gene mutations include downregulation of KCNQ1 protein expression in the stria vascularis, as well as hypoplastic and dysmorphic melanocytes. Thus, this model provides a relevant and reliable platform within which the detailed cell and molecular biology of CMV-induced deafness may be studied. Birth Defects Research (Part A), 2012. © 2012 Wiley Periodicals, Inc.
... This pattern of expression suggests the loss of EGFR signaling as an important factor in the age-related decline in regenerative potential. Indeed, many studies in neonatal rodent cochleae have demonstrated a pronounced effect of EGF signaling to promote proliferation and the production of supernumerary HCs Doetzlhofer et al., 2004;Lefebvre et al., 2000;Malgrange et al., 2002;White et al., 2012), and EGF signaling has been shown to promote SC proliferation in the regenerating chick BP (White et al., 2012). Thus, the data presented suggest a role for EGFR in HC regeneration, and hint that existing models of induced SC proliferation may be improved at later ages via the reinstatement or enhancement of EGFR signaling. ...
The organ of Corti in the mammalian inner ear is comprised of mechanosensory hair cells (HCs) and nonsensory supporting cells (SCs), both of which are believed to be terminally post-mitotic beyond late embryonic ages. Consequently, regeneration of HCs and SCs does not occur naturally in the adult mammalian cochlea, though recent evidence suggests that these cells may not be completely or irreversibly quiescent in at earlier postnatal ages. Furthermore, regenerative processes can be induced by genetic and pharmacological manipulations, but, more and more reports suggest that regenerative potential declines as the organ of Corti continues to age. In numerous mammalian systems, such effects of aging on regenerative potential are well established. However, in the cochlea, the problem of regeneration has not been traditionally viewed as one of aging. This is an important consideration as current models are unable to elicit widespread regeneration or full recovery of function at adult ages yet regenerative therapies will need to be developed specifically for adult populations. Still, the advent of gene targeting and other genetic manipulations has established mice as critically important models for the study of cochlear development and HC regeneration and suggests that auditory HC regeneration in adult mammals may indeed be possible. Thus, this review will focus on the pursuit of regeneration in the postnatal and adult mouse cochlea and highlight processes that occur during postnatal development, maturation, and aging that could contribute to an age-related decline in regenerative potential. Second, we will draw upon the wealth of knowledge pertaining to age related senescence in tissues outside of the ear to synthesize new insights and potentially guide future research aimed at promoting HC regeneration in the adult cochlea.
... Thus, our finding of an EGF-induced hollow otic sphere phenotype with a reduced self-renewal potential [27] and the reduced self-renewal potential of NSCs of the SVZ [50] indicate that self-renewal potential in both multipotent stem cell types is negatively regulated by EGF supplementation. In addition, EGF has been also shown to induce differentiation of cochlear hair cells from dividing progenitor cells from the embryonic developmental stage E13.5 which were directly isolated and plated as epithelial island without going through sphere formation [51]. It has been previously reported that otic sphere formation is related to a gain in developmental potential, with transcriptional and translational changes that are indicative of dedifferentiation [2,4]. ...
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.
Our previous study reported neural stem cells (NSCs) in the auditory cortex (AC) of postnatal day 3 (P3) mice in vitro. It is unclear whether AC-NSCs exist in vivo. This study aims to determine the presence and changes of AC-NSCs during postnatal development and maturation both in vitro and in vivo. P3, postnatal day 14 (P14), 2-month-old (2M), and 4-month-old (4M) mouse brain tissues were fixed and cryosectioned for NSC marker immunostaining. In vitro, P3, P14, and 2M AC tissues were dissected and cultured in suspension to study NSCs. NSC proliferation was examined by EdU incorporation and cell doubling time assays in vitro. The results show that Nestin and Sox2 double expressing NSCs were observed in the AC area from P3 to 4M in vivo, in which the number of NSCs remarkably reduced with age. In vitro, the neurosphere forming capability, cell proliferation, and percentage of Nestin and Sox2 double expressing NSCs significantly diminished with age. These results suggest that AC-NSCs exist in the mouse AC area both in vitro and in vivo, and the percentage of AC-NSCs decreases during postnatal development and maturation. The results may provide important cues for the future research of the central auditory system.
An improved understanding of the mechanisms underlying genetic, acquired and “mixed” deafness has driven development of new interventions and treatments as well as refinement of previous approaches to prevent hearing loss and restore hearing after deafness. Hearing loss induced by cochlear damage from overstimulation, ototoxic drugs, trauma, infections, and/or aging can be reduced by reducing oxidative stress to the cochlea with antioxidants and enhancing endogenous protective systems with agents such as neurotrophic factors and heat shock proteins (HSPs). Genetic modification has now been successful in animal models and may become clinically feasible, with the development of safe and effective vectors to treat genetic hearing loss as well as induce regeneration of hair cells. Placement of exogenous stem cells and transdifferentiation of endogenous cells is being developed as a strategy to replace lost hair cells or auditory. Survival and growth factors can be applied to the cochlea to improve auditory nerve survival following deafness and to induce the regeneration of its peripheral processes. Major advances in region specific delivery of therapeutics to the cochlea to allow these interventions include gene transfer, microcannulation, and biomaterials placed into the middle ear or directly into the cochlea. Auditory prostheses are being refined, with significant improvements occurring in cochlear prostheses as well as in development of central auditory prostheses. Prostheses can also be engineering for the application of therapeutics through microchannels. Biomaterial on prostheses can not only improve function and histocompatibility but also provide a means for delivering therapeutics. Artificial cochleae capable of transforming sound-induced movement of cochlear fluids into electrical signals are also being developed. The strategies for tissue engineering interventions based on recent scientific discoveries are the focus of this chapter.
Auditory signals are processed in multiple central nervous system structures, including the auditory cortex (AC). Development of stem cell biology provides the opportunity to identify neural stem cells (NSCs) in the central nervous system. However, it is unclear whether NSCs exist in the AC. The aim of this study is to determine the existence of NSCs in the postnatal mouse AC. To accomplish this aim, postnatal mouse AC tissues were dissected and dissociated into singular cells and small cell clumps, which were suspended in the culture medium to observe neurosphere formation. The spheres were examined by quantitative real-time polymerase chain reaction and immunofluorescence to determine expression of NSC genes and proteins. In addition, AC-spheres were cultured in the presence or absence of astrocyte-conditioned medium (ACM) to study neural differentiation. The results show that AC-derived cells were able to proliferate to form neurospheres, which expressed multiple NSC genes and proteins, including SOX2 and NESTIN. AC-derived NSCs (AC-NSCs) differentiated into cells expressing neuronal and glial cell markers. However, the neuronal generation rate is low in the culture medium containing nerve growth factor, ∼8%. To stimulate neuronal generation, AC-NSCs were cultured in the culture medium containing ACM. In the presence of ACM, ∼29% AC-NSCs differentiated into cells expressing neuronal marker class III β-tubulin (TUJ1). It was observed that the length of neurites of AC-NSC-derived neurons in the ACM group was significantly longer than that of the control group. In addition, synaptic protein immunostaining showed significantly higher expression of synaptic proteins in the ACM group. These results suggest that ACM is able to stimulate neuronal differentiation, extension of neurites, and expression of synaptic proteins. Identifying AC-NSCs and determining effects of ACM on NSC differentiation will be important for the auditory research and other neural systems.
As the irreversibility of hair cell loss in mammals is among the main reasons giving rise to permanent hearing loss, studies have been focused on the development of biological technologies to generate new hair cells as a means of replacing lost hair cells. Bone marrow-derived mesenchymal stem cells (BMSCs) possess the capacity to differentiate into hair cell-like cells, and may find applications in the regeneration of mammalian cochlear hair cells. In order to efficiently induce BMSCs into hair cells, alginate microcapsules co-delivering rat bone marrow-derived mesenchymal stem cells (rBMSCs) and anti-EGF monoclonal antibody (mAb) were developed to examine the feasibility of differentiating rBMSC into hair cell-likes cells in vitro and in vivo by taking advantage of epidermal growth factor (EGF) ligands. In vitro analysis showed that anti-EGF mAbs bonded with exogenous EGF ligands activated the EGF receptors on rBMSCs, enhancing the expression of myosin 7a (a hair cell marker) and Notch1 (supporting cell marker) via the EGFR/Ras/Raf/ERK1/2 signal pathway. In in vivo experiments, alginate microcapsules loaded with rBMSCs (2 × 10⁶ cells per microcapsule) together with Iso mAbs or anti-EGF mAbs were grafted into guinea pig cochlea. After 6 weeks of treatment, immunofluorescence analyses indicated that the rBMSCs embedded into anti-EGF microcapsules were more efficiently transformed into hair cells compared with the group with Iso mAb microcapsules and displayed an ordered arrangement in Reissner's membranes. The results highlighted the significance of engineering the microenvironment of stem cells for hair cell differentiation, and particularly the advantage of hair cell differentiation of rBMSCs by recruiting host EGF ligands via tethered mAbs. In conclusion, this strategy of co-embedding rBMSCs and anti-EGF mAb in alginate microcapsules is a promising modality for the regeneration of hair cell-like cells.
Dental stem cells (DSCs) can generate hepatic-like cells and represent a promising therapeutic approach for the treatment of patients suffering from liver disease such as cirrhosis and acute hepatic injury (AHF). A range of studies showed successful differentiation of DSCs into hepatocyte-like cells, demonstrating that the dental stem/progenitor cells of dental tissue. The progenitor cells isolated from dental pulp (DPSCs) and exfoliated deciduous teeth (SHEDs), under specific stimuli, are able to express hepatic specific markers, and most importantly, can engraft functionally into murine and rat models of liver injury. Furthermore, other reports demonstrated that DSCs can be reprogrammed into induced pluripotent stem cells (DP-iPSCs), and then successfully differentiated into hepatocyte-like cells (DP-iPSC-Heps) having characteristics of mature hepatocytes. The engraftment of DP-iPSC-Heps in immunocompromised AHF mouse model can be improved by the additional use of scaffolds that release hepatocyte growth factor (HGF) or liver-specific microRNA122 (miR122). This chapter shows that DSCs are a good candidate to generate hepatocyte-like cells and dental pulp-derived iPSCs-differentiated hepatocytes (DP-iPSCs-Heps) and demonstrates that they could be considered an optimal cell source for liver cell therapy.
Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27(KIP)) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal β-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.
Hair cells with stereocilia are the sensory receptors of the inner ear that are located in the organ of corti of the cochlea, involved in detecting sound, and are connected with the nerve fibers that stretch from the inner ear to the brain. These hair cells convert sound information to electric signals, which are then sent to the higher brain centers. In humans, hair cell damage results in permanent hearing impairment; whereas, birds have the capacity to rebuild a damaged inner ear and various studies have demonstrated hair cell regeneration. Recently, stem cell research has triggered many programmes around the world to examine the possibility for regenerating the hair cells from embryonic/adult stem cells. The important spin-off of the research would be to find signaling mechanisms that regulate hair cell regeneration to restore nearly normal hearing in humans. The current review focuses on stem cell-based therapy with particular emphasis to summarize the recent progress.
The use of brain signals for interaction and for controlling robots and prosthetic devices is a rapidly emerging field of multidisciplinary research called brain–machine interfaces (BMI), or brain–computer interfaces (BCI), which has seen impressive achievements in recent years. A BMI monitors the user’s brain activity, extracts specific features from the brain signals that reflect the intent of the subject, and translates their intentions into actions – such as closing the prosthetic hand – without the activity of any muscle or peripheral nerve. Adaptation is a key component of a BMI, because, on the one hand, users must learn to modulate their brainwaves so as to generate distinct brain patterns, while, on the other hand, machine learning techniques need to discover the individual brain patterns characterizing the mental tasks executed by the user. This chapter provides an introduction to the field of BMI, with a particular focus on principles for reliable and long-term operation of neuroprostheses. BMI offers a natural way to aid patients with severe neuromuscular disabilities, and also opens up new possibilities in human–machine interaction for able-bodied people. This chapter reviews the different kinds of brain signals that can be used as input for a BMI and discusses a series of principles to build efficient BMIs that are independent of the particular signal of choice. These principles concern the nature of electrical brain correlates more suitable to control neuroprosthetic devices and to promote motor rehabilitation, the use of machine learning techniques, and the design of context-aware BMIs. The authors conclude by discussing some future research directions in the field of BMI.
Microenvironment and cell-cell interactions play an important role during embryogenesis and are required for the stemness and differentiation of stem cells. The inner-ear sensory epithelium, containing hair cells and supporting cells, is derived from the stem cells within the otic vesicle at early embryonic stages. However, whether or not such microenvironment or cell-cell interactions within the embryonic otic tissue have the capacity to regulate the proliferation and differentiation of stem cells and to autonomously reassemble the cells into epithelial structures is unknown. Here, we report that on enzymatic digestion and dissociation to harvest all the single cells from 13.5-day-old rat embryonic (E13.5) inner-ear tissue as well as on implantation of these cells under renal capsules; the dissociated cells are able to reassemble themselves to form epithelial structures as early as 7 days after implantation. By 25 days after implantation, more mature epithelial structures are formed. Immunostaining with cell-type-specific markers reveals that hair cells and supporting cells are not only formed, but are also well aligned with the hair cells located in the apical layer surrounded by the supporting cells. These findings suggest that microenvironment and cell-cell interactions within the embryonic inner-ear tissue have the autonomous signals to induce the formation of sensory epithelial structures. This method may also provide a useful system to study the potential of stem cells to differentiate into hair cells in vivo.
Notch signalling pathway plays an essential role in the development of cochlea, which inhibits the proliferation of hair cells. Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea, which presents strong antioxidant activation and has been applied for anti-cancer and anti-inflammatory. In this study, we treated the cochlear explant cultures with EGCG and found that EGCG can protect cochlear hair cells from ototoxic drug gentamicin. We demonstrated that EGCG could down-regulate the expression of Notch signalling pathway target genes, such as Hes1, Hes5, Hey1 and Hey5. However, the Notch pathway ligands such as Delta1, Jag1 and Jag2 were not affected by EGCG. To further illustrate the mechanism of recover cochlear hair cells, we demonstrated that EGCG inhibited the activity of γ-secrectase to suppress Notch signalling pathway and promoted the proliferation and regeneration of hair cells in the damaged cochlea. Our results suggest for the first time the role of EGCG as an inhibitor of the Notch signalling pathway, and support its potential value in hearing-impaired recovery in clinical therapy.
Sensory hair cells (HC) of the inner ear are susceptible to damage from a variety of sources including ageing, genetic defects, noise or chemotherapeutic drugs. As the adult mammalian cochlea lacks regenerative capacity, the consequence of this damage in humans is permanent and results in hearing loss. Since the discovery that hair cells can regenerate in birds, a wide range of studies have been designed in order to understand this process. At the same time efforts have been made to identify the steps in mammalian hair cell development. The aim of this paper is to re-examine recent research on mammalian HC development and avian HC regeneration, as this process could help in understanding possible future directions and targets of mammalian inner ear regeneration.
In the management of sensorineural hearing loss, effective therapy for degenerated hair cells, third order neurons, ganglions, dendrites and synaptic areas of the vestibulo-cochleo-cerebral pathway remains an enigma. Transplantation of stem and progenitor cells appears to be an emerging potential solution, and is the focus of this review.
To review recent developments in the management of sensorineural hearing loss in the field of stem cell research.
A systematic review of the English language literature included all experimental and non-experimental studies with a Jadad score of three or more, published between 2000 and 2010 and included in the following databases: Cochrane Library Ear, Nose and Throat Disorders; Medline; Google Scholar; Hinari; and the Online Library of Toronto University.
Of the 455 and 29 600 articles identified from Medline and Google Scholar, respectively, 48 met the inclusion criteria. These were independently reviewed and jointly analysed.
Although there is not yet any evidence from successful human studies, stem cell and 'alternative stem cell' technology seems to represent the future of sensorineural hearing loss management.
Proliferation and transdifferentiaton of supporting cells in the damaged auditory organ of birds lead to robust regeneration of sensory hair cells. In contrast, regeneration of lost auditory hair cells does not occur in deafened mammals, resulting in permanent hearing loss. In spite of this failure of regeneration in mammals, we have previously shown that the perinatal mouse supporting cells harbor a latent potential for cell division. Here we show that in a subset of supporting cells marked by p75, EGFR signaling is required for proliferation, and this requirement is conserved between birds and mammals. Purified p75+ mouse supporting cells express receptors and ligands for the EGF signaling pathway, and their proliferation in culture can be blocked with the EGFR inhibitor AG1478. Similarly, in cultured chicken basilar papillae, supporting cell proliferation in response to hair cell ablation requires EGFR signaling. In addition, we show that EGFR signaling in p75+ mouse supporting cells is required for the down-regulation of the cell cycle inhibitor p27(Kip1) (CDKN1b) to enable cell cycle re-entry. Taken together, our data suggest that a conserved mechanism involving EGFR signaling governs proliferation of auditory supporting cells in birds and mammals and may represent a target for future hair cell regeneration strategies.
Conclusion. Bone marrow mesenchymal stem cells (MSCs) have the ability to differentiate into hair cells, and this method of culturing MSCs provides a useful tool for studies on mammalian cochlear hair cell regeneration. Objective: To investigate a method to induce bone marrow MSCs to differentiate into inner ear hair cells. Methods: Rat bone marrow MSCs were isolated from healthy rats and cultured in vitro. To make sure that the cultured cells were bone marrow MSCs, the expression of MSC markers such as SH2, CD31, CD34, and CD44 genes on the cultured cells was assessed by RT-PCR. Adipogenic cells and osteogenic cells were induced by the differentiation of the cultured cells, respectively, suggesting that the cultured cells have the characteristic of pluripotent differentiation. Then they were induced to differentiate into neural stem cells and hair cell progenitor cells. Immunohistochemistry experiments were carried out to detect the expression of molecular markers. Scanning electron microscope samples were prepared for observation of the morphology of the cells. Results: Rat bone marrow MSCs were successfully isolated, purified, cultured, and identified in vitro. They were also successfully induced to differentiate into neural progenitor cells and then hair cell-like cells that expressed myosin VIIa.
Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults.
In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever
been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of
nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated
cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been
shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al. (1995) Science 267, 1024–1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report
that the cyclin-dependent kinase inhibitor p27Kip1 is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27Kip1-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after
mitosis normally has ceased during embryonic development. This suggests that release from p27Kip1-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important
pathway for inducing hair-cell regeneration in the mammalian hearing organ.
The gene encoding myosin VIIA is responsible for the mouse shaker-1 phenotype, which consists of deafness and balance deficiency related to cochlear and vestibular neuroepithelial defects. In humans, a defective myosin VIIA gene is responsible for Usher syndrome type IB, which associates congenital deafness, vestibular dysfunction and retinitis pigmentosa. In an attempt to progress in the understanding of the function(s) of myosin VIIA, we studied the expression of the myosin VIIA gene during mouse embryonic development. Embryos from day 9 (E9) to E18 were analyzed by in situ hybridization and immunohistofluorescence. The myosin VIIA mRNA and protein were consistently detected in the same embryonic tissues throughout development. Myosin VIIA was first observed in the otic vesicle at E9, and later in a variety of tissues. The olfactory epithelium and the liver express it as early as E10. In the retinal pigment epithelium, choroid plexus, adrenal gland and tongue, expression begins at E12 and in the testis and the adenohypophysis at E13. In the small intestine, kidney and hair follicles of the vibrissae, expression of myosin VIIA starts only at E15. Myosin VIIA expression was observed only in epithelial cell types, most of which possess microvilli or cilia. Interestingly, myosin VIIA expression seems to be concomitant with the appearance of these structures in the epithelial cells, suggesting a role for this myosin in their morphogenesis. The cellular location of myosin VIIA within sensory hair cells and olfactory receptor neurons also argues for a role of this system vesicle trafficking.
We investigated the potential for hair cell regeneration in neonatal rat organs of Corti grown in culture following destruction of hair cells by neomycin toxicity. Replacement hair cells were observed by light and scanning electron microscopy in lesion sites in the cultures treated with transforming growth factor-alpha, epidermal growth factor or a combination of both factors for 5-7 days post-injury. These new cells had morphological characteristics of immature hair cells. Autoradiographic localization of [3H]thymidine-labelled cells on semi-thin sections indicated that these replacement hair cells did not arise through renewed mitotic division, although an important mitotic proliferation was observed outside the area of supporting and sensory cells.
Recovery of hair cells was studied at various times after acoustic trauma in adult quail. An initial loss of hair cells recovered to within 5 percent of the original number of cells. Tritium-labeled thymidine was injected after this acoustic trauma to determine if mitosis played a role in recovery of hair cells. Within 10 days of acoustic trauma, incorporation of [3H]thymidine was seen over the nuclei of hair cells and supporting cells in the region of initial hair cell loss. Thus, hair cell regeneration can occur after embryonic terminal mitosis.
Any loss of cochlear hair cells has been presumed to result in a permanent hearing deficit because the production of these cells normally ceases before birth. However, after acoustic trauma, injured sensory cells in the mature cochlea of the chicken are replaced. New cells appear to be produced by mitosis of supporting cells that survive at the lesion site and do not divide in the absence of trauma. This trauma-induced division of normally postmitotic cells may lead to recovery from profound hearing loss.
Examination of pure-tone acoustic damage in the chick cochlea revealed a significant amount of hair cell recovery over a 10 day period following the exposure. The recovery included both a regeneration of stereociliary bundles to replace those that were lost and a reshuffling of the mosaic pattern of the hair cell surfaces that survived. Ten-day-old chicks were exposed to a 1500 Hz pure tone at 120 dB SPL for 48 h and their cochleae were processed for scanning, transmission and light microscopy at 0 h, 24 h, 48 h, 4 d, 6 d and 10 d after exposure. Immediately after exposure the damaged region exhibited two types of hair cell trauma. The first was a defined area of complete hair cell loss and the second was an area where the hair cells survived but exhibited varying amounts of stereocilia injury. After 48 h of recovery, new hair cells were identifiable in the region of hair cell loss and with time they underwent a progressive maturation of their stereociliary bundles. The surviving hair cells showed a dramatic rearrangement and expansion of their surfaces but exhibited no repair of the damaged stereociliary bundles. These results suggest that the chick cochlea is capable of a significant amount of recovery and regeneration following acoustic trauma.
Regenerative proliferation occurs in the inner-ear sensory epithelial of warm-blooded vertebrates after insult. To determine how this proliferation is controlled in the mature mammalian inner ear, several growth factors were tested for effects on progenitor-cell division in cultured mouse vestibular sensory epithelia. Cell proliferation was induced in the sensory epithelium by transforming growth factor alpha (TGF-alpha) in a dose-dependent manner. Proliferation was also induced by epidermal growth factor (EGF) when supplemented with insulin, but not EGF alone. These observations suggest that stimulation of the EGF receptors by TGF-alpha binding, or EGF (plus insulin) binding, stimulates cell proliferation in the mature mammalian vestibular sensory epithelium.
Calretinin is a calcium-binding protein of the EF-hand family. It has been previously identified in particular cell types of adult guinea pig, rat, and chinchilla inner ear. Development of calretinin immunoreactivity in the mouse inner ear was investigated from embryonic day 13 (E13) to the adult stage. In the adult mouse vestibule, calretinin immunoreactivity was present in the same structures as described for the rat and guinea pig: the population of afferent fibers forming calyx units and a small number of ganglion neurons. The earliest immunoreactivity was found at E17 in vestibular hair cells (VHCs), then, at E19, in afferent fibers entering the sensory epithelia and in rare ganglion neurons. At postnatal day 4 (P4), a few vestibular nerve fibers and ganglion neurons were reactive. From this stage until P14, immunoreactivity developed in the calyx units and disappeared from VHCs. At P14, immunostaining was adult-like. In the adult mouse cochlea, immunoreactivity was present in the same cell populations as described in the rat: the inner hair cells (IHCs) and most of Corti's ganglion neurons. Calretinin immunoreactivity appeared at E19-P0 in IHCs and ganglion neurons of the basal turn. At P1, outer hair cells (OHCs) of the basal turn were positive. Calretinin immunoreactivity then appeared in IHCs, OHCs, and ganglion neurons of the medial turn, then of the apical turn. At P4, all IHCs and OHCs and most of the ganglion neurons were immunostained. Immunoreactivity gradually disappeared from the OHCs starting at P10 and, at P22, only IHCs and ganglion neurons were positive. The sequences of appearance of calretinin were specific to each cell type of the inner ear and paralleled their respective maturation. Calretinin was transiently expressed in VHCs and OHCs.
Sensory hair cells in the cochleae of birds are regenerated after the death of preexisting hair cells caused by acoustic over-stimulation or administration of ototoxic drugs. Regeneration involves renewed proliferation of cells in an epithelium that is otherwise mitotically quiescent. To determine the identity of the first cells that proliferate in response to the death of hair cells and to measure the latency of this proliferative response, we have studied hair-cell regeneration in organ culture. Cochleae from hatchling chicks were placed in culture, and hair cells were killed individually by a laser microbeam. The culture medium was then replaced with a medium that contained a labeled DNA precursor. The treated cochleae were incubated in the labeling media for different time periods before being fixed and processed for the visualization of proliferating cells. The first cells to initiate DNA replication in response to the death of hair cells were supporting cells within the cochlear sensory epithelium. All of the labeled supporting cells were located within 200 microns of the hair-cell lesions. These cells first entered S-phase approximately 16 hr after the death of hair cells. The results indicate that supporting cells are the precursors of regenerated hair cells and suggest that regenerative proliferation of supporting cells is triggered by signals that act locally within the damaged epithelium.
Proliferation of supporting cells in the inner ear is the early major event occurring during hair cell regeneration after acoustic trauma or aminoglycoside treatment. In the present study, we examined the possible influence of 30 growth factors on the proliferation of pure rat utricular epithelial cells in culture. Utricular epithelial sheets were separated and partially dissociated from early postnatal rats via a combined enzymatic and mechanical method. The cultured utricular epithelial cells expressed exclusively epithelial cell antigens, but not fibroblast, glial, or neuronal antigens. With tritiated thymidine incorporation assays, we found that several fibroblast growth factor (FGF) family members, insulin-like growth factor-1 (IGF-1), IGF-2, transforming growth factor-alpha (TGF-alpha), and epidermal growth factor (EGF), stimulated proliferation of the utricular epithelial cells. In contrast, neurotrophins and other growth factors did not elicit any detectable mitogenic effects. Among all of the growth factors examined, FGF-2 was the most potent mitogen. When FGF-2 was added in combination with IGF-1 or TGF-alpha to the medium, combined effects were seen. These results were confirmed with BrdU immunocytochemistry. Thus, the present culture system provides a rapid and reliable assay system to screen novel growth factors involved in proliferation of mammalian inner ear supporting cells. Furthermore, immunostainings revealed that the cultured utricular epithelial cells expressed FGF and IGF-1 receptors, and utricular hair cells produced FGF-2 in vivo. The addition of neutralizing antibodies against FGF-2 or IGF-1 to the cultures significantly inhibited the utricular epithelial cell proliferation. This work suggests that FGF-2 and IGF-1 may regulate the proliferation step during hair cell development and regeneration.
The gene encoding myosin VIIA is responsible for the mouse shaker-1 phenotype, which consists of deafness and balance deficiency related to cochlear and vestibular neuroepithelial defects. In humans, a defective myosin VIIA gene is responsible for Usher syndrome type IB, which associates congenital deafness, vestibular dysfunction and retinitis pigmentosa. In an attempt to progress in the understanding of the function(s) of myosin VIIA, we studied the expression of the myosin VIIA gene during mouse embryonic development. Embryos from day 9 (E9) to E18 were analyzed by in situ hybridization and immunohistofluorescence. The myosin VIIA mRNA and protein were consistently detected in the same embryonic tissues throughout development. Myosin VIIA was first observed in the otic vesicle at E9, and later in a variety of tissues. The olfactory epithelium and the liver express it as early as E10. In the retinal pigment epithelium, choroid plexus, adrenal gland and tongue, expression begins at E12 and in the testis and the adenohypophysis at E13. In the small intestine, kidney and hair follicles of the vibrissae, expression of myosin VIIA starts only at E15. Myosin VIIA expression was observed only in epithelial cell types, most of which possess microvilli or cilia. Interestingly, myosin VIIA expression seems to be concomitant with the appearance of these structures in the epithelial cells, suggesting a role for this myosin in their morphogenesis. The cellular location of myosin VIIA within sensory hair cells and olfactory receptor neurons also argues for a role of this protein in the synaptic vesicle trafficking.
Despite increased interest in inner ear hair cell regeneration, it is still unclear what exact mechanisms underlie hair cell regeneration in mammals because of our limited understanding of hair cell development and the lack of specific hair cell markers. In this report, we studied hair cell development using immunohistochemistry on sections prepared from embryonic day (E) 13 to postnatal day 7 rat inner ear tissues. Of many epithelial, neuronal, and glial markers, we found that calcium-binding protein antibodies recognizing calretinin, calmodulin, or parvalbumin labeled immature hair cells in rat vestibular end organs. In particular, calretinin antiserum labeled the initial differentiating hair cells at E15, a stage immediately after the terminal mitosis of hair cell progenitors. The selective immunoreactivity of postmitotic presumptive hair cells, but not supporting cells or peripheral epithelial cells, was confirmed in utricular epithelial sheet cultures. Double labeling with calretinin and bromodeoxyuridine antibodies in long-term cultures showed that only a few mitotic utricular supporting cells became calretinin positive. Thus, although proliferation-mediated regeneration of new hair cells might directly contribute to hair cell regeneration in rat utricles after injury, it is very limited. In addition, double labeling with calretinin and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) revealed that differentiated hair cells underwent apoptosis during normal development at late embryonic and early postnatal stages in vivo and in vitro. Therefore, these experiments lay the groundwork for the time course of differentiation, regeneration, and apoptosis of mammalian vestibular hair cells. This work also suggests that calcium-binding proteins are useful markers for studies on inner ear hair cell differentiation and regeneration.
Advances in hair cell regeneration are progressing at a rapid rate. This review will highlight and critique recent attempts to understand some of the cellular and molecular mechanisms underlying hair cell regeneration in non-mammalian vertebrates and efforts to induce regeneration in the mammalian inner ear sensory epithelium.
Hair cells, the sensory receptors of the mammalian inner ear, have long been thought to be produced only during embryogenesis, and postnatal hair cell loss is considered to be irreversible and is associated with permanent hearing and balance deficits. Little is known about the factors that regulate hair cell genesis and differentiation. The mitogenic effects of insulin and transforming growth factor alpha (TGFalpha) were assayed in vivo in normal and drug-damaged rat inner ear. Tritiated thymidine and autoradiographic techniques were used to identify cells synthesizing DNA. Simultaneous infusion of TGFalpha and insulin directly into the inner ear of adult rats stimulated DNA synthesis in the vestibular sensory receptor epithelium. New supporting cells and putative new hair cells were produced. Infusion of insulin alone or TGFalpha alone failed to stimulate significant DNA synthesis. These results suggest that exogenous growth factors may have utility for therapeutic treatment of hearing and balance disorders in vivo.
Sensory organs of the vertebrate inner ear contain two major cell types: hair cells (HCs) and supporting cells (SCs). To study the lineage relationships between these two populations, replication-defective retroviral vectors encoding marker genes were delivered to the otic vesicle of the chicken embryo. The resulting labeled clones were analyzed in the hearing organ of the chicken, called the basilar papilla (BP), after cellular differentiation. BPs were allowed to develop for 2 weeks after delivery of the retrovirus, were removed, and were processed histochemically as whole mounts to identify clones of cells. Clusters of labeled cells were evident in the sensory epithelium, the nonsensory epithelium, and in adjacent tissues. Labeled cell types included HCs, two morphologically distinct types of SCs, homogene cells, border cells, hyaline cells, ganglion cells, and connective tissue cells. Each clone was sectioned and cell-type identification was performed on sensory clones expressing retrovirally transduced beta-galactosidase. Cell composition was determined for 41 sensory clones, most of which contained both HCs and SCs. Clones containing one HC and one SC were observed, suggesting that a common progenitor exists that can remain bipotential up to its final mitotic division. The possibility that these two cell types may also arise from a mitotic precursor during HC regeneration in the mature basilar papilla is consistent with their developmental history.
Hair cell regeneration is well documented in the inner ear sensory epithelia of lower vertebrates and birds and may occur in the vestibular organs of mammals. By contrast, hair cell loss in the mature mammalian cochlea is considered irreversible. However, recent reports have suggested that an attempt at hair cell regeneration could occur in vivo in aminoglycoside-lesioned cochleas from neonatal rats. After amikacin treatment, atypical cells with apical specialization reminiscent of early differentiating stereocilia are transiently present at the apex of the intoxicated cochleas but fail to differentiate as hair cells in later stages. In the present study, we used electronic microscopy, histochemistry, and confocal microscopy to investigate the cellular rearrangements in the amikacin-lesioned organ of Corti of rat pups. In addition, we used 5-bromo-2'-deoxyuridine immunocytochemistry to determine whether mitotic processes are involved in the formation of the atypical cells. The morphologic and molecular data suggest that atypical cells are not recovering hair cells, but share characteristics of immature hair cells and supporting cells. Proliferative cells were absent from the region occupied by atypical cells, suggesting that the latter did not arise through mitotic processes. Altogether, the present results support the hypothesis that atypical cells arise through direct transformation of some of the supporting cells that reorganize during hair cell degeneration.
The mitotic marker 5-bromodeoxyuridine (BrdU) was injected twice daily (60 mg/kg) into pregnant hooded rats on one of embryonic days (E) 11, 12, 13, 15, 17, or 21, or into rat pups on postnatal day (P) 10. The principal findings were the following: (1) BrdU exposure on E11 produces profound effects on body morphology, and animals must be fed a special diet because of chronic tooth abnormalities; (2) BrdU exposure at E17 or earlier produces a change in coat spotting pattern, the precise pattern varying with age; (3) BrdU exposure on E15 or earlier produces a reduction in both brain and body weight; (4) BrdU exposure on E17 or earlier reduces cortical thickness; (5) BrdU exposure on E11-E13 and at P10 reduces cerebellar size relative to cerebral size; (6) spatial learning is significantly affected after injections of BrdU at E11-E17, but the largest effect is on E17; (7) the deficit in spatial learning may be related in part to a reduction in visual acuity; and (8) skilled forelimb ability is most disrupted after BrdU exposure at E15 but is also impaired after injections on E13 or earlier. BrdU thus has teratological effects on body, brain, and behavior that vary with the developmental age of the fetus or infant.
The mammalian cochlea contains an invariant mosaic of sensory hair cells and non-sensory supporting cells reminiscent of invertebrate structures such as the compound eye in Drosophila melanogaster. The sensory epithelium in the mammalian cochlea (the organ of Corti) contains four rows of mechanosensory hair cells: a single row of inner hair cells and three rows of outer hair cells. Each hair cell is separated from the next by an interceding supporting cell, forming an invariant and alternating mosaic that extends the length of the cochlear duct. Previous results suggest that determination of cell fates in the cochlear mosaic occurs via inhibitory interactions between adjacent progenitor cells (lateral inhibition). Cells populating the cochlear epithelium appear to constitute a developmental equivalence group in which developing hair cells suppress differentiation in their immediate neighbours through lateral inhibition. These interactions may be mediated through the Notch signalling pathway, a molecular mechanism that is involved in the determination of a variety of cell fates. Here we show that genes encoding the receptor protein Notch1 and its ligand, Jagged 2, are expressed in alternating cell types in the developing sensory epithelium. In addition, genetic deletion of Jag2 results in a significant increase in sensory hair cells, presumably as a result of a decrease in Notch activation. These results provide direct evidence for Notch-mediated lateral inhibition in a mammalian system and support a role for Notch in the development of the cochlear mosaic.
The aim of this study was to test the possible regenerative potential of several molecules and growth factors such as retinoic acid (RA), insulin, epidermal growth factor (EGF) and transforming growth factors alpha (TGFalpha) and beta (TGFbeta) on the neonatal cochlea in vitro after neomycin intoxication. Our studies show that cochlear sensory epithelium behaves differently while maintained in various culture conditions, although we did not observe regeneration whatever the molecules or growth factors tested. The ototoxic action of neomycin in vitro produced a specific death of hair cells, except in the apical region. Organ of Corti of rats 3 days after birth always presented two regions that responded differently to the antibiotic: a widespread scar region extending from the basal cochlea up to the beginning of the apical turn, where most hair cells had disappeared, and a second region called the resistance region localized in the apex, and which was more or less developed depending on culture conditions. The length of the resistance region was modulated by molecules or growth factors added to the feeding solution suggesting that some of them could produce a protective action on hair cells against neomycin. Slight protection effects may be found with RA and insulin, however, the most definite protection results from the combination of insulin with TGFalpha as shown by the large increase in the length of the resistance region compared to organ of Corti treated with antibiotic alone. The tested molecules and growth factors did not promote cochlear hair cell regeneration in vitro after neomycin treatment, however some of them may offer a protective action against ototoxicity.
During embryonic development of the inner ear, the sensory primordium that gives rise to the organ of Corti from within the cochlear epithelium is patterned into a stereotyped array of inner and outer sensory hair cells separated from each other by non-sensory supporting cells. Math1, a close homolog of the Drosophila proneural gene atonal, has been found to be both necessary and sufficient for the production of hair cells in the mouse inner ear. Our results indicate that Math1 is not required to establish the postmitotic sensory primordium from which the cells of the organ of Corti arise, but instead is limited to a role in the selection and/or differentiation of sensory hair cells from within the established primordium. This is based on the observation that Math1 is only expressed after the appearance of a zone of non-proliferating cells that delineates the sensory primordium within the cochlear anlage. The expression of Math1 is limited to a subpopulation of cells within the sensory primordium that appear to differentiate exclusively into hair cells as the sensory epithelium matures and elongates through a process that probably involves radial intercalation of cells. Furthermore, mutation of Math1 does not affect the establishment of this postmitotic sensory primordium, even though the subsequent generation of hair cells is blocked in these mutants. Finally, in Math1 mutant embryos, a subpopulation of the cells within the sensory epithelium undergo apoptosis in a temporal gradient similar to the basal-to-apical gradient of hair cell differentiation that occurs in the cochlea of wild-type animals.
Strict control of cellular proliferation is required to shape the complex structures of the developing embryo. The organ of Corti, the auditory neuroepithelium of the inner ear in mammals, consists of two types of terminally differentiated mechanosensory hair cells and at least four types of supporting cells arrayed precisely along the length of the spiral cochlea. In mice, the progenitors of greater than 80% of both hair cells and supporting cells undergo their terminal division between embryonic day 13 (E13) and E14. As in humans, these cells persist in a non-proliferative state throughout the adult life of the animal. Here we report that the correct timing of cell cycle withdrawal in the developing organ of Corti requires p27(Kip1), a cyclin-dependent kinase inhibitor that functions as an inhibitor of cell cycle progression. p27(Kip1) expression is induced in the primordial organ of Corti between E12 and E14, correlating with the cessation of cell division of the progenitors of the hair cells and supporting cells. In wild-type animals, p27(Kip1) expression is downregulated during subsequent hair cell differentiation, but it persists at high levels in differentiated supporting cells of the mature organ of Corti. In mice with a targeted deletion of the p27(Kip1) gene, proliferation of the sensory cell progenitors continues after E14, leading to the appearance of supernumerary hair cells and supporting cells. In the absence of p27(Kip1), mitotically active cells are still observed in the organ of Corti of postnatal day 6 animals, suggesting that the persistence of p27(Kip1) expression in mature supporting cells may contribute to the maintenance of quiescence in this tissue and, possibly, to its inability to regenerate. Homozygous mutant mice are severely hearing impaired. Thus, p27(Kip1) provides a link between developmental control of cell proliferation and the morphological development of the inner ear.
Many studies have documented the decline in auditory function with age. We broaden that data base in this the first of a series of reports emanating from the auditory testing of the Framingham cohort during biennial exam 18. The results of the auditory questionnaire, hearing sensitivity, acoustic compliance measures, and word recognition tests obtained from 1662 men and women in their 60th through 90th decades are presented. Pure-tone thresholds increased with age but the rate of change with age did not differ by gender even though men had poorer threshold sensitivity. Maximum word recognition ability declined with age more rapidly in men than in women and was poorer in men than in women at all ages. Acoustic compliance and middle ear pressure did not vary with gender or age. Acoustic reflex thresholds to a contralat-era1 stimulus at 1 kHz increased slightly with age, more in women than in men; ipsilateral acoustic reflex thresholds did not vary with age or gender. Hearing aids were being used in only 10% of subjects likely to benefit from amplification.
The 12th–12.5th gestational day inner ear otocyst from the CBA/ CBA mouse was explanted to organ culture with and without surrounding mesenchyme. One group of otocysts from which the mesenchyme had been removed was cultured in conditioned medium (i.e., medium in which mesenchyme alone had been cultured but had been removed prior to explantation of the stripped otocyst, without adjacent mesenchyme). Morphogenesis was good in organ cultures with preserved mesenchyme, acceptable in stripped otocysts cultured in conditioned medium, but very poor (or even lacking) in specimens deprived of mesenchyme and cultured in normal medium. In the two latter groups, only a small number of hair cells were identified. Although morphogenesis can be induced in specimens initially deprived of adjacent mesenchyme, a normal tissue relationship seems essential for cytodifferentiation of a normal number of hair cells.
Inductive tissue interactions were studied in the otocyst of the CBA/CBA mouse. Otocysts with surrounding mesenchyme explanted at the 12.5–13th gestational day and cultured in vitro for 4 days underwent morphogenesis with formation of semicircular canals, vestibular organs, and some cochlear coiling. Without their surrounding mesenchyme only little, if any, development was seen. However, otocysts without mesenchyme but grown in a medium precultured with mesenchyme did develop normally. A soluble and diffusible induction factor is apparently produced by mesenchyme and also by other fetal organs. Cell-cell contact may not be needed for induction.
Morphogenesis of the cartilaginous otic capsule is directed by interactions between the epithelial anlage of the membranous labyrinth (otocyst) and its associated periotic mesenchyme. Utilizing a developmental series of high-density (micromass) cultures of periotic mesenchyme to model capsule chondrogenesis, we have shown that the early influence of otic epithelium in cultures of 10.5- or 14-gestation day (gd) periotic mesenchyme results in initiation or suppression of chondrogenesis, respectively. Furthermore, we have shown that introduction of otic epithelium at two distinct times during in vitro development to cultures of 10.5-gd mesenchyme cells results first in an initiation and then in an inhibition of their chondrogenic response. These influences of epithelial tissue on chondrogenic differentiation by periotic mesenchyme are not tissue specific but are characterized by temporal selectivity. The ability of otic epithelium to influence chondrogenesis and the competence of the periotic mesenchyme to respond to its signals are dependent upon the developmental stage of both tissues. This study provides conclusive evidence that otic epithelium acts as a developmental "switch" during otic capsule morphogenesis, signaling first the turning on and then the turning off of chondrogenic programs in the responding cephalic mesenchyme.
Many studies have documented the decline in auditory function with age. We broaden that data base in this the first of a series of reports emanating from the auditory testing of the Framingham cohort during biennial exam 18. The results of the auditory questionnaire, hearing sensitivity, acoustic compliance measures, and word recognition tests obtained from 1662 men and women in their 60th through 90th decades are presented. Pure-tone thresholds increased with age but the rate of change with age did not differ by gender even though men had poorer threshold sensitivity. Maximum word recognition ability declined with age more rapidly in men than in women and was poorer in men than in women at all ages. Acoustic compliance and middle ear pressure did not vary with gender or age. Acoustic reflex thresholds to a contralateral stimulus at 1 kHz increased slightly with age, more in women than in men; ipsilateral acoustic reflex thresholds did not vary with age or gender. Hearing aids were being used in only 10% of subjects likely to benefit from amplification.
• This study examines the temporal pattern of hair cell loss in the chick basilar papilla following ten days of gentamicin administration in hatchling chicks. Chicks were subsequently killed at ages 11, 18, 25, and 32 days. The basilar papillae were embedded in plastic and serially sectioned for light microscopic analysis. Hair cell counts were obtained at 100-μm intervals throughout the length of the papilla. Significant hair cell loss was documented basally in the 11-day-old chicks, and spread apically over time to maximal loss in the 18-day-old animals. Relative to the control chicks, there was a 36% hair cell loss in these animals. Interestingly, there appears to be a progressive partial recovery of the normal hair cell counts in the 25- and 32-day-old animals.
(Arch Otolaryngol Head Neck Surg 1987;113:1058-1062)
The developing inner ears of mice (CBA/CBA), ages ranging from gestational day 12 through postnatal day 21, were examined using scanning electron microscopy following the epoxy-embedding/freeze-fracture technique. This technique provides unique three-dimensional views of surface and fractured structures of the developing inner ear, thus allowing excellent preservation of the relationships between the developing sensory epithelium and the overlying membranes (i.e. the tectorial membrane and cupula) during their development. The tectorial membrane is formed of two distinct parts: the major (medial) and the minor (distal). The major portion is produced by the cells of the greater epithelial ridge prior to the formation of the minor part, which is produced largely by the primordial supporting cells of the lesser epithelial ridge. The developing tectorial membrane has two types of fibers: radial (found mainly in the major part) and slanted (found mainly in the minor part). The slanted fibers become the cover net, which fuses with the marginal band. The marginal zone of the developing tectorial membrane is completely sealed during development by the third row of Deiters' cells. The surfaces of cells that produce the tectorial membrane are characterized by numerous long microvilli which are largely lost when the tectorial membrane completely forms and separates from the supporting cells. The surface of developing auditory sensory cells is initially covered with numerous microvilli, some of which become future stereocilia. Stereocilia form stepped rows in the shape of a "W", with the tallest row located at the periphery of the cell. As the sensory cell matures, the short transitional stereocilia gradually disappear. Kinocilia on hair cells are still seen in the 14-day-old mouse (even though the organ of Corti is morphologically mature) but not in the 21-day-old mouse, indicating that complete maturation of the sensory cells in all turns is attained by 21 days of age. The mouse has upper radial tunnel fibers and basal tunnel fibers. Neural contacts of the upper radial tunnel fibers with the outer hair cells at the apical portions are frequent in the developing organ of Corti. The external sulcus cells undergo drastic changes during development, forming numerous pits that are often covered with mucus-like droplets or grape-like spherical structures of varying sizes. This phenomenon was observed only during postnatal days 6 and 14. The developing cupula starts as a thin amorphous membrane, which later becomes compact and fibrotic-like as the mass increases. By the 6th postnatal day well-developed cupular canals occur.(ABSTRACT TRUNCATED AT 400 WORDS)
This study used epidemiologic methods to examine hearing loss in the elderly. The Framingham Heart Study Cohort was the reference population. The participants were 935 men and 1358 women, aged 57 to 89 years. Using a conservative definition of hearing loss as threshold levels greater than 20 dB above audiometric zero for at least one frequency from 0.5 to 4 kHz, the prevalence was estimated to be 83%. The majority of cases displayed a sensorineural hearing loss. There were no statistically significant differences by sex at 1 kHz and below. Women had significantly better hearing than men at 2 kHz and above. A multivariate model was constructed to determine which variables had a significant impact upon hearing loss. Under the model, age, sex, illness, family history of hearing loss, Meniere's disease, and noise exposure were significant population risk factors. Age was by far the most critical risk factor.
The 12th-12.5th gestational day inner ear otocyst from the CBA/CBA mouse was explanted to organ culture with and without surrounding mesenchyme. One group of otocysts from which the mesenchyme had been removed was cultured in conditioned medium (i.e., medium in which mesenchyme alone had been cultured but had been removed prior to explantation of the "stripped" otocyst, without adjacent mesenchyme). Morphogenesis was good in organ cultures with preserved mesenchyme, acceptable in "stripped" otocysts cultured in conditioned medium, but very poor (or even lacking) in specimens deprived of mesenchyme and cultured in normal medium. In the two latter groups, only a small number of hair cells were identified. Although morphogenesis can be induced in specimens initially deprived of adjacent mesenchyme, a normal tissue relationship seems essential for cytodifferentiation of a normal number of hair cells.
The purpose of this study was to obtain information about factors controlling the interkinetic movement of neuroepithelial cells and the cortical migration of neuroblasts in the developing mouse neocortex. In the first experiment 15-day pregnant mice were injected with a single dose of fluorodeoxyuridine, a compound which inhibits the enzyme thymidylate synthetase thus blocking DNA-replication. The animals were sacrificed at various hours and days following treatment. Two hours after treatment the mitotic index of the neuroepithelial cells was severely reduced and cells with abnormal nuclei appeared in the DNA-synthesizing zone of the neuroepithelial layer. By five hours more abnormal cells appeared and many showed degenerating nuclei. By 12 hours cells with abnormal nuclei were seen in the outer half of the neuroepithelial layer and some in the migratory zone. Since they were not seen at the lumen, these observations indicate that fluorodeoxyuridine-affected nuclei do not move to the lumen and do not divide. By 24 hours abnormal nuclei were seen in the migratory zone and cortical plate suggesting cortical migration without preceding cell division. In the second experiment 15-day pregnant mice were treated with col-cemid, a compound which arrests dividing cells in metaphase. Two and four hours after injection a large number of neuroepithelial cells in metaphase accumulated at the lumen. By seven and 12 hours the number of arrested metaphases decreased rapidly, but at the same time cells with dense, darkly stained nuclei appeared in the adjacent neuroepithelium. By 24 hours these abnormal nuclei were present in the migratory zone and cortical plate, suggesting that they were migrating towards the surface of the cortex despite the failure to complete mitosis. In the third experiment 15-day pregnant mice were injected with bromodeoxyuridine, a compound incorporated into DNA and thought to interfere with differentiation. During the first 12 hours after treatment a striking increase in the number of normal mitotic figures was seen at the lumen. By 16 hours, however, abnormal mitotic figures started to appear. Since the generation time of neuroepithelial cells at this stage of development is about 13 hours, the abnormal mitotic figures must represent cells which have gone through two DNA-synthetic periods since the beginning of the treatment. The abnormal mitotic figures give rise to cells with dense pycnotic nuclei which by 24 hours are found throughout the width of the developing neocortex. Thus in each experiment cells with abnormal, presumably non-functional, nuclei were produced. Despite the failure to divide or to carry out a normal division, the nuclei migrate from the neuroepithelial layer towards the cortical plate in a fashion similar to that of normal postmitotic neuroblasts.
WE ARE greatly indebted to a few investigators for our fine morphological understanding of the vestibular organs.1-7 Ultrastructural observations of the otolithic membranes were reported by Carlström and Engström,8 Iurato and de Petris,9 and others.10 Directional sensitivity of the lateral line organs of the fish was studied by Flock,11 and that of the vestibular sensory receptors by Wersäll et al12 with the electron microscope. The scanning electron microscopic observation of statolithic sensory organs of the Eledone (octopus family) was first attempted by Barber and Boyde.13This investigation concerns the surface topography of the sensory epithelia of the vestibular sensory organs with special emphasis on the arrangement of stereocilia and their relationship to the otolithic membrane. This part of the study was supplementary information to add more details to what the authors have already reported.14Materials and Methods
Fourteen guinea pig, five pigeon
Under conditions where muscle actin only partially polymerizes, or where it does not polymerize at all, a significant enhancement of polymerization was observed if equimolar phalloidin was also present. The increased extent of polymerization in the the presence of phalloidin can be explained by the reduced critical actin concentration of partially polymerized populations at equilibrium. Under such conditions, the rate of polymerization, as judged by the length of time to reach half the viscosity plateau, was found to be essentially independent of the phalloidin concentration. Moreover, the initial rate of polymerization of actin was also found to be independent of phalloidin concentration. However, phalloidin apparently causes a reduction in the magnitude of the reverse rates in the polymerization reaction, as was demonstrated by the lack of depolymerization of phalloidin-treated actin polymers. This effect of phalloidin is also supported by the identification of actin nuclei and short polymers in populations of G-actin incubated with phalloidin in the absence of added KCl. Our conclusion, then, is that phalloidin influences the polymerization of actin by stabilizing nuclei and polymers as they are formed.
Inductive tissue interactions were studied in the otocyst of the CBA/CBA mouse. Otocysts with surrounding mesenchyme explanted at the 12.5-13th gestational day and cultured in vitro for 4 days underwent morphogenesis with formation of semicircular canals, vestibular organs, and some cochlear coiling. Without their surrounding mesenchyme only little, if any, development was seen. However, otocysts without mesenchyme but grown in a medium precultured with mesenchyme did develop normally. A soluble and diffusible induction factor is apparently produced by mesenchyme and also by other fetal organs. Cell-cell contact may not be needed for induction.
The distribution of S-100-like immunoreactivity in mouse and hamster auditory and vestibular end organs was determined by the use of immunohistochemistry. Within the organ of Corti, the cytoplasm of cells of Deiter and Hensen were strongly immunoreactive. Inner hair cells and the peripheral processes and cell bodies of the spiral ganglion were weakly immunoreactive for S-100, whereas the supranuclear regions of outer hair cells and cells underlying the basilar membrane were unstained. Immunoreactivity was observed near the base of outer hair cells. In the lateral wall of the cochlea, cellular components of the spiral ligament and a subpopulation of epithelial cells in the stria vascularis, identified as predominantly basal cells, were immunoreactive. For the saccule, utricle, and semicircular canals, S-100 immunoreactivity was observed in vestibular hair cells, types I and II, and the nerve calyces surrounding type I hair cells as well as in nerve fibers underlying the sensory epithelium. Weak S-100-like immunoreactivity was associated with vestibular nerve fibers and cell bodies in the vestibular ganglion. The localization of S-100-like immunoreactivity to the sensory cells and nerve fibers of the peripheral auditory and vestibular end organs is consistent with a functional role for S-100 proteins at these sites.
The recent discovery of hair cell regeneration in the avian inner ear raises the possibility that hair cell regeneration might occur in the mammalian cochlea as well. The authors used 3H-thymidine labeling to detect mitotic activity in the cochleas of normal 3-week old gerbils exposed to acoustic trauma. Following an acoustic insult that caused progressively more severe damage in an apical to basal progression, 3H-thymidine was injected for 5 days. Control animals were not exposed to the acoustic insult. The gerbils' cochleas were sectioned and processed for autoradiography. In the control cochleas, there were extremely rare labeled cells in the stria, the spiral ligament, and the glial cells around the acoustic nerve fibers. In the damaged cochleas, no evidence of hair cell regeneration or of any cell division within the normal sensory epithelial structures was seen. Three labeled cells were seen in intercellular spaces within the sensory epithelium; they appeared to be macrophages. Frequent cell division was seen in numerous other regions of the damaged cochleas and among glial cells adjacent to the acoustic nerve fibers. It is concluded that there is no evidence for hair cell regeneration following acoustic trauma in the gerbil, but acoustic trauma does induce cell division in numerous other areas of the cochlea.
Supporting cells in the vestibular sensory epithelia from the ears of mature guinea pigs and adult humans proliferate in vitro after treatments with aminoglycoside antibiotics that cause sensory hair cells to die. After 4 weeks in culture, the epithelia contained new cells with some characteristics of immature hair cells. These findings are in contrast to expectations based on previous studies, which had suggested that hair cell loss is irreversible in mammals. The loss of hair cells is responsible for hearing and balance deficits that affect millions of people.
It has long been thought that hair cell loss from the inner ears of mammals is irreversible. This report presents scanning electron micrographs and thin sections of the utricles from the inner ears of guinea pigs that show that, after hair cell loss caused by treatment with the aminoglycoside gentamicin, hair cells reappeared. Four weeks after the end of treatment, a large number of cells with immature hair bundles in multiple stages of development could be identified in the utricle. Thin sections showed that lost type 1 hair cells were replaced by cells with a morphology similar to that of type 2 hair cells. These results indicate an unexpected capacity for hair cell regeneration in vivo in the mature mammalian inner ear.
The deafness mouse (dn/dn) is a well known model of hereditary deafness uncomplicated by behavioral and motor disturbances. The organ of Corti in this mouse develops a normal complement of sensory and supporting cell structures, yet animals homozygous for this gene never demonstrate any hearing capacity. They are profoundly deaf from birth. Soon after development, the organ of Corti rapidly degenerates, most sensory cells having vanished by 50 days of age. Published observations have suggested that apical regions of the organ of Corti may regenerate some supporting cell structures by 90 days of age. We have quantified changes in organ of Corti structure from 15 to 130 days of age using several different measures. Measures of peak height and total cross-sectional area. as well as a subjective rating scale, all demonstrate consistent degenerative changes during this time period. No evidence for regeneration of supporting or sensory cell structures is noted, although a surprising degree of variability is present in all regions of the organ of Corti which may account for previous claims.
The inner ear is a complex sensory organ that forms from a simple epithelial placode. The expression patterns of cell adhesion molecules and extracellular matrix components that have been described in the developing inner ear to date are summarized. Whilst our knowledge of the distribution of some of the known elements involved in cell-cell and cell-matrix interactions is in some instances quite limited, these studies generally suggest many potential roles for cell-cell and cell-matrix interactions in various aspects of inner ear development. However, there is a serious need for experimental studies to assess these possibilities.
Strict control of cellular proliferation is required to shape the complex structures of the developing embryo. The organ of Corti, the auditory neuroepithelium of the inner ear in mammals, consists of two types of terminally differentiated mechanosensory hair cells and at least four types of supporting cells arrayed precisely along the length of the spiral cochlea. In mice, the progenitors of greater than 80% of both hair cells and supporting cells undergo their terminal division between embryonic day 13 (E13) and E14. As in humans, these cells persist in a non-proliferative state throughout the adult life of the animal. Here we report that the correct timing of cell cycle withdrawal in the developing organ of Corti requires p27(Kip1), a cyclin-dependent kinase inhibitor that functions as an inhibitor of cell cycle progression. p27(Kip1) expression is induced in the primordial organ of Corti between E12 and E14, correlating with the cessation of cell division of the progenitors of the hair cells and supporting cells. In wild-type animals, p27(Kip1) expression is downregulated during subsequent hair cell differentiation, but it persists at high levels in differentiated supporting cells of the mature organ of Corti. In mice with a targeted deletion of the p27(Kip1) gene, proliferation of the sensory cell progenitors continues after E14, leading to the appearance of supernumerary hair cells and supporting cells. In the absence of p27(Kip1), mitotically active cells are still observed in the organ of Corti of postnatal day 6 animals, suggesting that the persistence of p27(Kip1) expression in mature supporting cells may contribute to the maintenance of quiescence in this tissue and, possibly, to its inability to regenerate. Homozygous mutant mice are severely hearing impaired. Thus, p27(Kip1) provides a link between developmental control of cell proliferation and the morphological development of the inner ear.