scholarly journals 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

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.

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

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.

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

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Suhong Sun ◽  
Shuting Li ◽  
Zhengnan Luo ◽  
Minhui Ren ◽  
Shunji He ◽  
...  
Keyword(s):  
Hair Cells ◽  
Adult Mouse ◽  
Outer Hair Cells ◽  
Auditory Function ◽  
Supporting Cells ◽  
Single Cell Rna Sequencing ◽  
Differentiation Status ◽  
Cochlear Supporting Cells ◽  
Dual Expression

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.

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

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.

Cis-Diamminedichloroplatinum (II) Ototoxicity in the Guinea Pig

1981 ◽  
Vol 89 (4) ◽  
pp. 638-645 ◽  
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.

scholarly journals Partial Hearing Restoration after ERBB2 Induction in Deafened Adult Mice

2019 ◽  
Author(s):  
Jingyuan Zhang ◽  
Daxiang Na ◽  
Miriam Dilts ◽  
Kenneth S. Henry ◽  
Patricia M. White
Keyword(s):  
Cell Proliferation ◽  
Hearing Loss ◽  
Noise Exposure ◽  
Supporting Cell ◽  
Supporting Cells ◽  
Adult Mice ◽  
Hearing Thresholds ◽  
Brainstem Response ◽  
Noise Damage ◽  
Cochlear Supporting Cells

AbstractNoise induced hearing loss (NIHL) affects over ten million adults in the United States, and there is no biological treatment to restore endogenous function after damage. We hypothesized that activation of signaling from ERBB2 receptors in cochlear supporting cells could mitigate NIHL damage. We used the Tet-On genetic expression system to drive a constitutively active variant of ERBB2 (CA-ERBB2) in cochlear supporting cells three days after permanent noise damage in young adult mice. Hearing thresholds were assessed with auditory brainstem response tests prior to noise damage, and hearing recovery was assessed over a three month period. We evaluated supporting cell proliferation, inner and outer hair cell (IHC and OHC) survival, synaptic preservation, and IHC cytoskeletal alterations with histological techniques. Mice harboring CA-ERBB2 capability had similar hearing thresholds to control littermates prior to and immediately after noise exposure, and incurred similar levels of permanent hearing loss. Two and three months after noise exposure, CA-ERBB2+ mice demonstrated a partial but significant reversal of NIHL threshold shifts at the lowest frequency tested, out of five frequencies (n=19 total mice, p=0.0015, ANOVA). We also observed improved IHC and OHC survival (n=7 total cochleae, p=5 × 10−5, Kruskal-Wallis rank sum test). There was no evidence for sustained supporting cell proliferation. Some mortality was associated with doxycycline and furosemide treatments to induce the Tet-ON system. These data suggest that ERBB2 signaling in supporting cells promotes HC repair and some functional recovery. Funded by NIH R01 DC014261, and grants from the Schmitt Foundation and UR Ventures.

Differential RA responsiveness among subsets of mouse late progenitor spermatogonia

Reproduction ◽  
2021 ◽  
Author(s):  
Shinnosuke Suzuki ◽  
John R. McCarrey ◽  
Brian P Hermann
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Single Cell ◽  
Adult Mouse ◽  
Retinoic Acid Receptor ◽  
Mouse Testis ◽  
Rna Seq ◽  
Reporter Expression ◽  
Acid Receptor ◽  
Whole Mount

Initiation of spermatogonial differentiation in the mouse testis begins with the response to retinoic acid (RA) characterized by activation of KIT and STRA8 expression. In the adult, spermatogonial differentiation is spatiotemporally coordinated by a pulse of RA every 8.6 days that is localized to stages VII-VIII of the seminiferous epithelial cycle. Dogmatically, progenitor spermatogonia that express retinoic acid receptor gamma (RARG) at these stages will differentiate in response to RA, but this has yet to be tested functionally. Previous single-cell RNA-seq data identified phenotypically and functionally distinct subsets of spermatogonial stem cells (SSCs) and progenitor spermatogonia, where late progenitor spermatogonia were defined by expression of RARG and Dppa3. Here, we found late progenitor spermatogonia (RARGhigh KIT-) were further divisible into two subpopulations based on Dppa3 reporter expression (Dppa3-ECFP or Dppa3-EGFP) and were observed across all stages of the seminiferous epithelial cycle. However, nearly all Dppa3+ spermatogonia were differentiating (KIT+) late in the seminiferous epithelial cycle (stages X-XII), while Dppa3- late progenitors remained abundant, suggesting that Dppa3+ and Dppa3- late progenitors differentially responded to RA. Following acute RA treatment (2-4hr), significantly more Dppa3+ late progenitors induced KIT, including at the midpoint of the cycle (stages VI-IX), than Dppa3- late progenitors. Subsequently, single-cell analyses indicated a subset of Dppa3+ late progenitors expressed higher levels of Rxra, which we confirmed by RXRA whole-mount immunostaining. Together, these results indicate RARG alone is insufficient to initiate a spermatogonial response to RA in the adult mouse testis and suggest differential RXRA expression may discriminate responding cells.

scholarly journals Expression of the Na+/K+-ATPase Subunits in Adult Mouse Brain Analyzed by Single-Cell RNA-Seq Profiling

2019 ◽  
Vol 116 (3) ◽  
pp. 129a
Author(s):  
Song Jiao ◽  
Cristina Moreno Vadillo ◽  
Miguel Holmgren
Keyword(s):  
Single Cell ◽  
Mouse Brain ◽  
Adult Mouse ◽  
Rna Seq ◽  
Adult Mouse Brain ◽  
Atpase Subunits

scholarly journals LIN28B/let-7control the ability of neonatal murine auditory supporting cells to generate hair cells through mTOR signaling

2020 ◽  
Vol 117 (36) ◽  
pp. 22225-22236
Author(s):  
Xiao-Jun Li ◽  
Angelika Doetzlhofer
Keyword(s):  
Hair Cell ◽  
Hair Cells ◽  
Rna Binding ◽  
Mammalian Target Of Rapamycin ◽  
Cell Loss ◽  
Supporting Cell ◽  
Dependent Manner ◽  
Cell Plasticity ◽  
Supporting Cells ◽  
Cochlear Hair Cells

Mechano-sensory hair cells within the inner ear cochlea are essential for the detection of sound. In mammals, cochlear hair cells are only produced during development and their loss, due to disease or trauma, is a leading cause of deafness. In the immature cochlea, prior to the onset of hearing, hair cell loss stimulates neighboring supporting cells to act as hair cell progenitors and produce new hair cells. However, for reasons unknown, such regenerative capacity (plasticity) is lost once supporting cells undergo maturation. Here, we demonstrate that the RNA binding protein LIN28B plays an important role in the production of hair cells by supporting cells and provide evidence that the developmental drop in supporting cell plasticity in the mammalian cochlea is, at least in part, a product of declining LIN28B-mammalian target of rapamycin (mTOR) activity. Employing murine cochlear organoid and explant cultures to model mitotic and nonmitotic mechanisms of hair cell generation, we show that loss of LIN28B function, due to its conditional deletion, or due to overexpression of the antagonistic miRNAlet-7g, suppressed Akt-mTOR complex 1 (mTORC1) activity and renders young, immature supporting cells incapable of generating hair cells. Conversely, we found that LIN28B overexpression increased Akt-mTORC1 activity and allowed supporting cells that were undergoing maturation to de-differentiate into progenitor-like cells and to produce hair cells via mitotic and nonmitotic mechanisms. Finally, using the mTORC1 inhibitor rapamycin, we demonstrate that LIN28B promotes supporting cell plasticity in an mTORC1-dependent manner.

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