Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells

Mammalian cochlear outer hair cells (OHCs) are essential for hearing. Severe hearing impairment follows OHC degeneration. Previous attempts at regenerating new OHCs from cochlear supporting cells (SCs) have been unsuccessful, notably lacking expression of the key OHC motor protein, Prestin. Thus, regeneration of Prestin+ OHCs represents a barrier to restore auditory function in vivo. Here, we reported the successful in vivo conversion of adult mouse cochlear SCs into Prestin+ OHC-like cells through the concurrent induction of two key transcriptional factors known to be necessary for OHC development: Atoh1 and Ikzf2. Single-cell RNA sequencing revealed the upregulation of 729 OHC genes and downregulation of 331 SC genes in OHC-like cells. The resulting differentiation status of these OHC-like cells was much more advanced than previously achieved. This study thus established an efficient approach to induce the regeneration of Prestin+ OHCs, paving the way for in vivo cochlear repair via SC transdifferentiation.

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Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells

10.1101/2021.01.21.427665 ◽

2021 ◽

Author(s):

Suhong Sun ◽

Shuting Li ◽

Zhengnan Luo ◽

Minhui Ren ◽

Shunji He ◽

...

Keyword(s):

Single Cell ◽

Hearing Impairment ◽

Hair Cells ◽

Outer Hair Cells ◽

Supporting Cells ◽

Differentiation Status ◽

Cochlear Supporting Cells ◽

Simultaneous Expression ◽

Dual Expression

ABSTRACTMammalian cochlear outer hair cells (OHCs) are essential for hearing. OHC degeneration causes severe hearing impairment. Previous attempts of regenerating new OHCs from cochlear supporting cells (SCs) had yielded cells lacking Prestin, a key motor protein for OHC function. Thus, regeneration of Prestin+ OHCs remains a challenge for repairing OHC damage in vivo. Here, we reported that successful in vivo conversion of adult cochlear SCs into Prestin+ OHC-like cells could be achieved by simultaneous expression of Atoh1 and Ikzf2, two key transcriptional factors necessary for OHC development. New OHC-like cells exhibited upregulation of hundreds of OHC genes and downregulation of SC genes. Single cell transcriptomic analysis demonstrated that the differentiation status of these OHC-like cells was much more advanced than previously achieved. Thus, we have established an efficient approach to promote regeneration of Prestin+ OHCs and paved the way for repairing damaged cochlea in vivo via transdifferentiation of SCs.

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In vivo optogenetics reveals homeostatic control of cochlear sensitivity by supporting cells

10.21203/rs.3.rs-92461/v1 ◽

2020 ◽

Author(s):

Victoria Lukashkina ◽

Snezana Levic ◽

Patricio Simões ◽

Zhenhang Xu ◽

Joseph DiGuiseppi ◽

...

Keyword(s):

Hair Cells ◽

Dynamic Range ◽

Organ Of Corti ◽

Feedback System ◽

Supporting Cell ◽

Outer Hair Cells ◽

Supporting Cells ◽

Cochlear Supporting Cells

Abstract We used optogenetics to investigate the control of auditory sensitivity by cochlear supporting cells that scaffold outer hair cells, which transduce and amplify cochlear responses to sound. In vivo and in vitro measurements of sound-induced cochlear mechanical and electrical responses were made from mice that conditionally expressed nonselective cationic channelrhodopsins in Deiters’ and outer pillar supporting cells in the organ of Corti. We demonstrated that cochlear light-stimulation and subsequent activation of channelrhodopsins depolarized the supporting cells, changed their extracellular electrical environment, and sensitized insensitive and desensitized sensitive cochlear responses to sound. We concluded that outer hair cells, Deiters’ cells and outer pillar cells interact through feedback which regulates their immediate ionic and electrical environment and controls energy flow in the mammalian cochlea to optimize its performance over its entire dynamic range. Activation of the supporting cell channelrhodopsins shunts this feedback system and restores cochlear sensitivity to a set level.

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Hair Cell Generation in Cochlear Culture Models Mediated by Novel γ-Secretase Inhibitors

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.710159 ◽

2021 ◽

Vol 9 ◽

Author(s):

Silvia T. Erni ◽

John C. Gill ◽

Carlotta Palaferri ◽

Gabriella Fernandes ◽

Michelle Buri ◽

...

Keyword(s):

Hair Cell ◽

Notch Signaling ◽

Hair Cells ◽

Round Window ◽

Notch Pathway ◽

Outer Hair Cells ◽

Round Window Membrane ◽

Sensory Cells ◽

Supporting Cells

Sensorineural hearing loss is prevalent within society affecting the quality of life of 460 million worldwide. In the majority of cases, this is due to insult or degeneration of mechanosensory hair cells in the cochlea. In adult mammals, hair cell loss is irreversible as sensory cells are not replaced spontaneously. Genetic inhibition of Notch signaling had been shown to induce hair cell formation by transdifferentiation of supporting cells in young postnatal rodents and provided an impetus for targeting Notch pathway with small molecule inhibitors for hearing restoration. Here, the oto-regenerative potential of different γ-secretase inhibitors (GSIs) was evaluated in complementary assay models, including cell lines, organotypic cultures of the organ of Corti and cochlear organoids to characterize two novel GSIs (CPD3 and CPD8). GSI-treatment induced hair cell gene expression in all these models and was effective in increasing hair cell numbers, in particular outer hair cells, both in baseline conditions and in response to ototoxic damage. Hair cells were generated from transdifferentiation of supporting cells. Similar findings were obtained in cochlear organoid cultures, used for the first time to probe regeneration following sisomicin-induced damage. Finally, effective absorption of a novel GSI through the round window membrane and hair cell induction was attained in a whole cochlea culture model and in vivo pharmacokinetic comparisons of transtympanic delivery of GSIs and different vehicle formulations were successfully conducted in guinea pigs. This preclinical evaluation of targeting Notch signaling with novel GSIs illustrates methods of characterization for hearing restoration molecules, enabling translation to more complex animal studies and clinical research.

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Characterizing Adult cochlear supporting cell transcriptional diversity using single-cell RNA-Seq: Validation in the adult mouse and translational implications for the adult human cochlea

10.1101/742270 ◽

2019 ◽

Author(s):

Michael Hoa ◽

Rafal Olszewski ◽

Xiaoyi Li ◽

Ian Taukulis ◽

Alvin DeTorres ◽

...

Keyword(s):

Single Cell ◽

Hair Cells ◽

Adult Mouse ◽

Significant Proportion ◽

Supporting Cell ◽

Rna Seq ◽

Supporting Cells ◽

Human Cochlea ◽

Cell Transcriptome ◽

Cochlear Supporting Cells

AbstractHearing loss is a problem that impacts a significant proportion of the adult population. Cochlear hair cell loss due to loud noise, chemotherapy and aging is the major underlying cause. A significant proportion of these individuals are dissatisfied with available treatment options which include hearing aids and cochlear implants. An alternative approach to restore hearing would be to regenerate hair cells. Such therapy would require recapitulation of the complex architecture of the organ of Corti, necessitating regeneration of both mature hair cells and supporting cells. Transcriptional profiles of the mature cell types in the cochlea are necessary to can provide a metric for eventual regeneration therapies. To assist in this effort, we sought to provide the first single-cell characterization of the adult cochlear supporting cell transcriptome. We performed single-cell RNA-Seq on FACS-purified adult cochlear supporting cells from the LfngEGFP adult mouse, in which supporting cells express GFP. We demonstrate that adult cochlear supporting cells are transcriptionally distinct from their perinatal counterparts. We establish cell type-specific adult cochlear supporting cell transcriptome profiles, and we validate these expression profiles through a combination of both fluorescent immunohistochemistry and in situ hybridization co-localization and qPCR of adult cochlear supporting cells. Furthermore, we demonstrate the relevance of these profiles to the adult human cochlea through immunofluorescent human temporal bone histopathology. Finally, we demonstrate cell cycle regulator expression in adult supporting cells and perform pathway analyses to identify potential mechanisms for facilitating mitotic regeneration (cell proliferation, differentiation, and eventually regeneration) in the adult mammalian cochlea. Our findings demonstrate the importance of characterizing mature as opposed to perinatal supporting cells.

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Optimization of spectral-domain optical coherence tomography with a supercontinuum source for in vivo motion detection of low reflective outer hair cells in guinea pig cochleae

Optical Review ◽

10.1007/s10043-021-00654-8 ◽

2021 ◽

Author(s):

Fumiaki Nin ◽

Samuel Choi ◽

Takeru Ota ◽

Zhang Qi ◽

Hiroshi Hibino

Keyword(s):

Optical Coherence Tomography ◽

High Resolution ◽

Guinea Pig ◽

Hair Cells ◽

Sensory Epithelium ◽

Outer Hair Cells ◽

Acquisition Time ◽

Total Acquisition Time ◽

Sd Oct

AbstractSound evokes sub-nanoscale vibration within the sensory epithelium. The epithelium contains not only immotile cells but also contractile outer hair cells (OHCs) that actively shrink and elongate synchronously with the sound. However, the in vivo motion of OHCs has remained undetermined. The aim of this work is to perform high-resolution and -accuracy vibrometry in live guinea pigs with an SC-introduced spectral-domain optical coherence tomography system (SD-OCT). In this study, to reveal the effective contribution of SC source in the recording of the low reflective materials with the short total acquisition time, we compare the performances of the SC-introduced SD-OCT (SCSD-OCT) to that of the conventional SD-OCT. As inanimate comparison objects, we record a mirror, a piezo actuator, and glass windows. For the measurements in biological materials, we use in/ex vivo guinea pig cochleae. Our study achieved the optimization of a SD-OCT system for high-resolution in vivo vibrometry in the cochlear sensory epithelium, termed the organ of Corti, in mammalian cochlea. By introducing a supercontinuum (SC) light source and reducing the total acquisition time, we improve the axial resolution and overcome the difficulty in recording the low reflective material in the presence of biological noise. The high power of the SC source enables the system to achieve a spatial resolution of 1.72 ± 0.00 μm on a mirror and reducing the total acquisition time contributes to the high spatial accuracy of sub-nanoscale vibrometry. Our findings reveal the vibrations at the apical/basal region of OHCs and the extracellular matrix, basilar membrane.

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Cis-Diamminedichloroplatinum (II) Ototoxicity in the Guinea Pig

Otolaryngology ◽

10.1177/019459988108900424 ◽

1981 ◽

Vol 89 (4) ◽

pp. 638-645 ◽

Cited By ~ 83

Author(s):

Scott A. Estrem ◽

Richard W. Babin ◽

Jai H. Ryu ◽

Kenneth C. Moore

Keyword(s):

Electron Microscopy ◽

Hair Cells ◽

Supporting Cell ◽

Cell Ultrastructure ◽

Outer Hair Cells ◽

Detectable Change ◽

Supporting Cells ◽

Apical Portion ◽

Ultrastructural Evidence ◽

Transmission Electron

Cochleas from 12 guinea pigs were evaluated using light, scanning, and transmission electron microscopy after systemic administration of cis-diamminedichloroplatinum (cis-DDP). Administration of cis-DDP resulted in loss of the Preyer reflex and degeneration of outer hair cells (OHC) with increased dose. The OHC degeneration was most pronounced in the basal turns of the cochlea with greatest severity in the inner row. Ultrastructural evidence of OHC degeneration included dilatation of the parietal membranes, softening of the cuticular plate, increased vacuolization and increased numbers of lysosome-like bodies in the apical portion of the cell. Supporting cells appeared more sensitive than OHC. Alteration of supporting cell ultrastructure preceded detectable change in OHC. Injury to the supporting cells was noted with intracellular vesiculation and increased autophagocytosis.

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Vibration of the organ of Corti within the cochlear apex in mice

Journal of Neurophysiology ◽

10.1152/jn.00306.2014 ◽

2014 ◽

Vol 112 (5) ◽

pp. 1192-1204 ◽

Cited By ~ 48

Author(s):

Simon S. Gao ◽

Rosalie Wang ◽

Patrick D. Raphael ◽

Yalda Moayedi ◽

Andrew K. Groves ◽

...

Keyword(s):

Mechanical Properties ◽

Phase Difference ◽

Basilar Membrane ◽

Organ Of Corti ◽

Lateral Compartment ◽

Outer Hair Cells ◽

Supporting Cells ◽

Tuning Curves ◽

Cochlear Amplification

The tonotopic map of the mammalian cochlea is commonly thought to be determined by the passive mechanical properties of the basilar membrane. The other tissues and cells that make up the organ of Corti also have passive mechanical properties; however, their roles are less well understood. In addition, active forces produced by outer hair cells (OHCs) enhance the vibration of the basilar membrane, termed cochlear amplification. Here, we studied how these biomechanical components interact using optical coherence tomography, which permits vibratory measurements within tissue. We measured not only classical basilar membrane tuning curves, but also vibratory responses from the rest of the organ of Corti within the mouse cochlear apex in vivo. As expected, basilar membrane tuning was sharp in live mice and broad in dead mice. Interestingly, the vibratory response of the region lateral to the OHCs, the “lateral compartment,” demonstrated frequency-dependent phase differences relative to the basilar membrane. This was sharply tuned in both live and dead mice. We then measured basilar membrane and lateral compartment vibration in transgenic mice with targeted alterations in cochlear mechanics. Prestin499/499, Prestin−/−, and TectaC1509G/C1509G mice demonstrated no cochlear amplification but maintained the lateral compartment phase difference. In contrast, SfswapTg/Tg mice maintained cochlear amplification but did not demonstrate the lateral compartment phase difference. These data indicate that the organ of Corti has complex micromechanical vibratory characteristics, with passive, yet sharply tuned, vibratory characteristics associated with the supporting cells. These characteristics may tune OHC force generation to produce the sharp frequency selectivity of mammalian hearing.

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Overexpression of SK2 Channels Enhances Efferent Suppression of Cochlear Responses Without Enhancing Noise Resistance

Journal of Neurophysiology ◽

10.1152/jn.01183.2006 ◽

2007 ◽

Vol 97 (4) ◽

pp. 2930-2936 ◽

Cited By ~ 15

Author(s):

Stéphane F. Maison ◽

Lisan L. Parker ◽

Lucy Young ◽

John P. Adelman ◽

Jian Zuo ◽

...

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Hair Cells ◽

Otoacoustic Emissions ◽

Outer Hair Cell ◽

Outer Hair Cells ◽

Sk Channels ◽

Cochlear Function ◽

Cochlear Hair Cells ◽

Efferent Suppression

Cochlear hair cells express SK2, a small-conductance Ca2+-activated K+ channel thought to act in concert with Ca2+-permeable nicotinic acetylcholine receptors (nAChRs) α9 and α10 in mediating suppressive effects of the olivocochlear efferent innervation. To probe the in vivo role of SK2 channels in hearing, we examined gene expression, cochlear function, efferent suppression, and noise vulnerability in mice overexpressing SK2 channels. Cochlear thresholds, as measured by auditory brain stem responses and otoacoustic emissions, were normal in overexpressers as was overall cochlear morphology and the size, number, and distribution of efferent terminals on outer hair cells. Cochlear expression levels of SK2 channels were elevated eightfold without striking changes in other SK channels or in the α9/α10 nAChRs. Shock-evoked efferent suppression of cochlear responses was significantly enhanced in overexpresser mice as seen previously in α9 overexpresser mice; however, in contrast to α9 overexpressers, SK2 overexpressers were not protected from acoustic injury. Results suggest that efferent-mediated cochlear protection is mediated by other downstream effects of ACh-mediated Ca2+ entry different from those involving SK2-mediated hyperpolarization and the associated reduction in outer hair cell electromotility.

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Cochlear progenitor number is controlled through mesenchymal FGF receptor signaling

eLife ◽

10.7554/elife.05921 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 39

Author(s):

Sung-Ho Huh ◽

Mark E Warchol ◽

David M Ornitz

Keyword(s):

Growth Factors ◽

Hair Cells ◽

Fibroblast Growth Factors ◽

Organ Of Corti ◽

Sensory Epithelium ◽

Feedback Mechanism ◽

Outer Hair Cells ◽

Fibroblast Growth ◽

Supporting Cells ◽

Fgf Receptor

The sensory and supporting cells (SCs) of the organ of Corti are derived from a limited number of progenitors. The mechanisms that regulate the number of sensory progenitors are not known. Here, we show that Fibroblast Growth Factors (FGF) 9 and 20, which are expressed in the non-sensory (Fgf9) and sensory (Fgf20) epithelium during otic development, regulate the number of cochlear progenitors. We further demonstrate that Fgf receptor (Fgfr) 1 signaling within the developing sensory epithelium is required for the differentiation of outer hair cells and SCs, while mesenchymal FGFRs regulate the size of the sensory progenitor population and the overall cochlear length. In addition, ectopic FGFR activation in mesenchyme was sufficient to increase sensory progenitor proliferation and cochlear length. These data define a feedback mechanism, originating from epithelial FGF ligands and mediated through periotic mesenchyme that controls the number of sensory progenitors and the length of the cochlea.

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Aquaporin-6 Expression in the Cochlear Sensory Epithelium Is Downregulated by Salicylates

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/264704 ◽

2010 ◽

Vol 2010 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Paola Perin ◽

Simona Tritto ◽

Laura Botta ◽

Jacopo Maria Fontana ◽

Giulia Gastaldi ◽

...

Keyword(s):

Inner Ear ◽

Expression Pattern ◽

Hair Cells ◽

Organ Of Corti ◽

Sensory Epithelium ◽

Outer Hair Cells ◽

Rt Pcr ◽

Supporting Cells ◽

Sensory Epithelia ◽

Mechanical Compliance

We characterize the expression pattern of aquaporin-6 in the mouse inner ear by RT-PCR and immunohistochemistry. Our data show that in the inner ear aquaporin-6 is expressed, in both vestibular and acoustic sensory epithelia, by the supporting cells directly contacting hair cells. In particular, in the Organ of Corti, expression was strongest in Deiters' cells, which provide both a mechanical link between outer hair cells (OHCs) and the Organ of Corti, and an entry point for ion recycle pathways. Since aquaporin-6 is permeable to both water and anions, these results suggest its possible involvement in regulating OHC motility, directly through modulation of water and chloride flow or by changing mechanical compliance in Deiters' cells. In further support of this role, treating mice with salicylates, which impair OHC electromotility, dramatically reduced aquaporin-6 expression in the inner ear epithelia but not in control tissues, suggesting a role for this protein in modulating OHCs' responses.

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