Hair Cell Regeneration Research Articles

Long-term survival of LGR5 expressing supporting cells after severe ototoxic trauma in the adult mouse cochlea.

The leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a tissue resident stem cell marker, which it is expressed in supporting cells (SCs) in the organ of Corti in the mammalian inner ear. These LGR5+ SCs can be used as an endogenous source of progenitor cells for regeneration of hair cells (HCs) to treat hearing loss and deafness. We have recently reported that LGR5+ SCs survive 1 week after ototoxic trauma. Here, we evaluated Lgr5 expression in the adult cochlea and long-term survival of LGR5+ SCs following severe hearing loss. Lgr5GFP transgenic mice and wild type mice aged postnatal day 30 (P30) and P200 were used. P30 animals were deafened with a single dose of furosemide and kanamycin. Seven and 28 days after deafening, auditory brainstem responses (ABRs) were recorded. Cochleas were harvested to characterize mature HCs and LGR5+ SCs by immunofluorescence microscopy and quantitative reverse transcription PCR (q-RT-PCR). There were no significant age-related changes in Lgr5 expression when comparing normal-hearing (NH) mice aged P200 with P30. Seven and 28 days after ototoxic trauma, there was severe outer HC loss and LGR5 was expressed in the third row of Deiters' cells and in inner pillar cells. Seven days after induction of ototoxic trauma there was an up-regulation of the mRNA expression of Lgr5 compared to the NH condition; 28 days after ototoxic trauma Lgr5 expression was similar to NH levels. The presence of LGR5+ SCs in the adult mouse cochlea, which persists after severe HC loss, suggests potential regenerative capacity of endogenous cochlear progenitor cells in adulthood. To our knowledge, this is the first study showing not only long-term survival of LGR5+ SCs in the normal and ototoxically damaged cochlea, but also increased Lgr5 expression in the adult mouse cochlea after deafening, suggesting long-term availability of potential target cells for future regenerative therapies.

Stem Cell-Based Therapies for Auditory Hair Cell Regeneration in the Treatment of Hearing Loss.

The incidence and prevalence of hearing loss is increasing globally at an accelerated pace. Hair cells represent the sensory receptors of auditory and vestibular systems. Hair cell absence, loss or degeneration due to congenital diseases, trauma, toxicity, infection or advancing age, results in disabling hearing loss. Regenerative medicine approaches consisting in stem cell-based hair cell rescue or regeneration, gene therapy, as well as cell and tissue engineering are expected to dramatically improve the therapeutic arsenal available for addressing hearing loss. Current strategies that are using different stem cell types to rescue or to induce hair cell proliferation and regeneration are presented. Gene and cell therapy methods that modulates transdifferentiation of surrounding cell types into hair cells are presented, together with their specific advantages and limitations. Several modalities for improving therapeutic targeting to the inner ear such as nanoparticle-mediated cell and gene delivery are introduced. Further steps in building more relevant high-throughput models for testing novel drugs and advanced therapies are proposed as a modality to accelerate translation to clinical settings.

DNA methylation in the mouse cochlea promotes maturation of supporting cells and contributes to the failure of hair cell regeneration

Mammalian hair cells do not functionally regenerate in adulthood but can regenerate at embryonic and neonatal stages in mice by direct transdifferentiation of neighboring supporting cells into new hair cells. Previous work showed loss of transdifferentiation potential of supporting cells is in part due to H3K4me1 enhancer decommissioning of the hair cell gene regulatory network during the first postnatal week. However, inhibiting this decommissioning only partially preserves transdifferentiation potential. Therefore, we explored other repressive epigenetic modifications that may be responsible for this loss of plasticity. We find supporting cells progressively accumulate DNA methylation at promoters of developmentally regulated hair cell genes. Specifically, DNA methylation overlaps with binding sites of Atoh1, a key transcription factor for hair cell fate. We further show that DNA hypermethylation replaces H3K27me3-mediated repression of hair cell genes in mature supporting cells, and is accompanied by progressive loss of chromatin accessibility, suggestive of facultative heterochromatin formation. Another subset of hair cell loci is hypermethylated in supporting cells, but not in hair cells. Ten-eleven translocation (TET) enzyme-mediated demethylation of these hypermethylated sites is necessary for neonatal supporting cells to transdifferentiate into hair cells. We also observe changes in chromatin accessibility of supporting cell subtypes at the single-cell level with increasing age: Gene programs promoting sensory epithelium development loses chromatin accessibility, in favor of gene programs that promote physiological maturation and function of the cochlea. We also find chromatin accessibility is partially recovered in a chronically deafened mouse model, which holds promise for future translational efforts in hearing restoration.

TRIM71 reactivation enhances the mitotic and hair cell-forming potential of cochlear supporting cells.

Cochlear hair cell loss is a leading cause of deafness in humans. Neighboring supporting cells have some capacity to regenerate hair cells. However, their regenerative potential sharply declines as supporting cells undergo maturation (postnatal day 5 in mice). We recently reported that reactivation of the RNA-binding protein LIN28B restores the hair cell-regenerative potential of P5 cochlear supporting cells. Here, we identify the LIN28B target Trim71 as a novel and equally potent enhancer of supporting cell plasticity. TRIM71 is a critical regulator of stem cell behavior and cell reprogramming; however, its role in cell regeneration is poorly understood. Employing an organoid-based assay, we show that TRIM71 re-expression increases the mitotic and hair cell-forming potential of P5 cochlear supporting cells by facilitating their de-differentiation into progenitor-like cells. Our mechanistic work indicates that TRIM71's RNA-binding activity is essential for such ability, and our transcriptomic analysis identifies gene modules that are linked to TRIM71 and LIN28B-mediated supporting cell reprogramming. Furthermore, our study uncovers that the TRIM71-LIN28B target Hmga2 is essential for supporting cell self-renewal and hair cell formation.

Cross-species analysis and comparison of the inner ear between chickens and mice.

The inner ear of mammals includes the cochlea and vestibule, which house specialized hair cells that are responsible for hearing and balance, respectively. While cochlear hair cells fail to regenerate following damage, those of the utricle, which is part of the vestibular apparatus, show partial regeneration. In birds, the macula lagena, a unique ear structure in this clade, has the ability to regenerate hair cells similarly to the utricle. Many studies have sought to explain regeneration in terms of evolution and species differences. However, it remains unclear what the cellular and molecular basis is behind the differences in inner ear structures and between avians and mammals. In the present study, we first investigated the anatomical structures of the inner ear of both chickens and rodents. We then performed RNA sequencing (RNA-Seq) and made cross-species analyses of the expression of homologous genes obtained from the inner ear tissue from both chickens and mice. Finally, we focused on the lagena, the basilar papilla, and the utricle in chickens and identified differentially expressed genes between tissues and determined the expression patterns of genes involved in inner ear structure formation by single-cell RNA sequencing and bulk RNA-Seq. We concluded that the cellular and molecular composition of the lagena is more similar to that of the utricle than the cochlea. Taken together, our study provides a valuable resource for the study of inner ear evolution and development.

Defining developmental trajectories of prosensory cells in human inner ear organoids at single-cell resolution.

The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human embryonic stem cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analyses with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that vestibular type II hair cells arise primarily from supporting cells, rather than bi-fated prosensory cells in organoids. Moreover, ion channel- and ion-transporter-related gene sets were enriched in supporting cells vs. prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells vs. supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss or balance disorders.

The role of epigenetic modifications in sensory hair cell development, survival, and regulation.

The cochlea is the sensory organ in the periphery, and hair cells are its main sensory cells. The development and survival of hair cells are highly controlled processes. When cells face intracellular and environmental stimuli, epigenetic regulation controls the structure and function of the genome in response to different cell fates. During sensory hair cell development, different histone modifications can induce normal numbers of functional hair cells to generate. When individuals are exposed to environmental-related hair cell damage, epigenetic modification also plays a significant role in the regulation of hair cell fate. Since mammalian hair cells cannot regenerate, their loss can cause permanent sensorineural hearing loss. Many breakthroughs have been achieved in recent years in understanding the signaling pathways that determine hair cell regeneration, and it is fascinating to note that epigenetic regulation plays a significant role in hair cell regeneration. In this review, we discuss the role of epigenetics in inner ear cell development, survival and regeneration and the significant impact on hearing protection.

The ndrg2 Gene Regulates Hair Cell Morphogenesis and Auditory Function during Zebrafish Development.

Damages of sensory hair cells (HCs) are mainly responsible for sensorineural hearing loss, however, its pathological mechanism is not yet fully understood due to the fact that many potential deafness genes remain unidentified. N-myc downstream-regulated gene 2 (ndrg2) is commonly regarded as a tumor suppressor and a cell stress-responsive gene extensively involved in cell proliferation, differentiation, apoptosis and invasion, while its roles in zebrafish HC morphogenesis and hearing remains unclear. Results of this study suggested that ndrg2 was highly expressed in the HCs of the otic vesicle and neuromasts via in situ hybridization and single-cell RNA sequencing. Ndrg2 loss-of-function larvae showed decreased crista HCs, shortened cilia, and reduced neuromasts and functional HCs, which could be rescued by the microinjection of ndrg2 mRNA. Moreover, ndrg2 deficiency induced attenuated startle response behaviors to sound vibration stimuli. Mechanistically, there were no detectable HC apoptosis and supporting cell changes in the ndrg2 mutants, and HCs were capable of recovering by blocking the Notch signaling pathway, suggesting that ndrg2 was implicated in HC differentiation mediated by Notch. Overall, our study demonstrates that ndrg2 plays crucial roles in HC development and auditory sensory function utilizing the zebrafish model, which provides new insights into the identification of potential deafness genes and regulation mechanism of HC development.

Progress of research on the role of Atoh1 gene in the regeneration of mammalian auditory hair cells

Atoh1 gene encodes a helix-loop-helix transcription factor which is involved in the generation and differentiation of mammalian auditory hair cells and supporting cells, and regulation of the proliferation of cochlear cells, therefore plays an important role in the pathogenesis and recovery of sensorineural deafness. This study reviews the progress of the Atoh1 gene in hair cell regeneration, with the aim of providing a reference for the study of hair cell regeneration gene therapy for sensorineural deafness.

The dark kinase STK32A regulates hair cell planar polarity opposite of EMX2 in the developing mouse inner ear.

The vestibular maculae of the inner ear contain sensory receptor hair cells that detect linear acceleration and contribute to equilibrioception to coordinate posture and ambulatory movements. These hair cells are divided between two groups, separated by a line of polarity reversal (LPR), with oppositely oriented planar-polarized stereociliary bundles that detect motion in opposite directions. The transcription factor EMX2 is known to establish this planar polarized organization in mouse by regulating the distribution of the transmembrane receptor GPR156 at hair cell boundaries in one group of cells. However, the genes regulated by EMX2 in this context were previously not known. Using mouse as a model, we have identified the serine threonine kinase STK32A as a downstream effector negatively regulated by EMX2. Stk32a is expressed in hair cells on one side of the LPR in a pattern complementary to Emx2 expression in hair cells on the opposite side. Stk32a is necessary to align the intrinsic polarity of the bundle with the core planar cell polarity (PCP) proteins in EMX2-negative regions, and is sufficient to reorient bundles when ectopically expressed in neighboring EMX2-positive regions. We demonstrate that STK32A reinforces LPR formation by regulating the apical localization of GPR156. These observations support a model in which bundle orientation is determined through separate mechanisms in hair cells on opposite sides of the maculae, with EMX2-mediated repression of Stk32a determining the final position of the LPR.

Gene therapy: an emerging therapy for hair cells regeneration in the cochlea.

Sensorineural hearing loss is typically caused by damage to the cochlear hair cells (HCs) due to external stimuli or because of one's genetic factors and the inability to convert sound mechanical energy into nerve impulses. Adult mammalian cochlear HCs cannot regenerate spontaneously; therefore, this type of deafness is usually considered irreversible. Studies on the developmental mechanisms of HC differentiation have revealed that nonsensory cells in the cochlea acquire the ability to differentiate into HCs after the overexpression of specific genes, such as Atoh1, which makes HC regeneration possible. Gene therapy, through in vitro selection and editing of target genes, transforms exogenous gene fragments into target cells and alters the expression of genes in target cells to activate the corresponding differentiation developmental program in target cells. This review summarizes the genes that have been associated with the growth and development of cochlear HCs in recent years and provides an overview of gene therapy approaches in the field of HC regeneration. It concludes with a discussion of the limitations of the current therapeutic approaches to facilitate the early implementation of this therapy in a clinical setting.

Regeneration of Hair Cells from Endogenous Otic Progenitors in the Adult Mammalian Cochlea: Understanding Its Origins and Future Directions.

Sensorineural hearing loss is caused by damage to sensory hair cells and/or spiral ganglion neurons. In non-mammalian species, hair cell regeneration after damage is observed, even in adulthood. Although the neonatal mammalian cochlea carries regenerative potential, the adult cochlea cannot regenerate lost hair cells. The survival of supporting cells with regenerative potential after cochlear trauma in adults is promising for promoting hair cell regeneration through therapeutic approaches. Targeting these cells by manipulating key signaling pathways that control mammalian cochlear development and non-mammalian hair cell regeneration could lead to regeneration of hair cells in the mammalian cochlea. This review discusses the pathways involved in the development of the cochlea and the impact that trauma has on the regenerative capacity of the endogenous progenitor cells. Furthermore, it discusses the effects of manipulating key signaling pathways targeting supporting cells with progenitor potential to promote hair cell regeneration and translates these findings to the human situation. To improve hearing recovery after hearing loss in adults, we propose a combined approach targeting (1) the endogenous progenitor cells by manipulating signaling pathways (Wnt, Notch, Shh, FGF and BMP/TGFβ signaling pathways), (2) by manipulating epigenetic control, and (3) by applying neurotrophic treatments to promote reinnervation.

The role of Espin in the stereocilia regeneration and protection in Atoh1-overexpressed cochlear epithelium.

Hair cells (HCs) in mammals cannot spontaneously regenerate after damage. Atoh1 overexpression can promote HC regeneration in the postnatal cochlea, but the regenerated HCs do not possess the structural and functional characteristics of HCs in situ. The stereocilia on the apical surface of HCs are the first-level structure for sound conduction, and regeneration of functional stereocilia is the key basis for the reproduction of functional HCs. Espin, as an actin bundling protein, plays an important role in the development and structural maintenance of the stereocilia. Here, we found that the upregulation of Espin by AAV-ie was able to induced the aggregation of actin fibres in Atoh1-induced HCs in both cochlear organoids and explants. In addition, we found that persistent Atoh1 overexpression resulted in impaired stereocilia in both endogenous and newly formed HCs. In contrast, the forced expression of Espin in endogenous and regenerative HCs was able to eliminate the stereocilia damage caused by persistent Atoh1 overexpression. Our study shows that the enhanced expression of Espin can optimize the developmental process of stereocilia in Atoh1-induced HCs and can attenuate the damage to native HCs induced by Atoh1 overexpression. These results suggest an effective method to induce the maturation of stereocilia in regenerative HCs and pave the way for functional HC regeneration via supporting cell transdifferentiation.

Regulation of hair cell and stomatal size by a hair cell-specific peroxidase in the grass Brachypodium distachyon

The leaf epidermis is the outermost cell layer forming the interface between plants and the atmosphere that must both provide a robust barrier against (a)biotic stressors and facilitate carbon dioxide uptake and leaf transpiration.1 To achieve these opposing requirements, the plant epidermis developed a wide range of specialized cell types such as stomata and hair cells. Although factors forming these individual cell types are known,2,3,4,5 it is poorly understood how their number and size are coordinated. Here, we identified a role for BdPRX76/BdPOX, a class III peroxidase, in regulating hair cell and stomatal size in the model grass Brachypodium distachyon. In bdpox mutants, prickle hair cells were smaller and stomata were longer. Because stomatal density remained unchanged, the negative correlation between stomatal size and density was disrupted in bdpox and resulted in higher stomatal conductance and lower intrinsic water-use efficiency. BdPOX was exclusively expressed in hair cells, suggesting that BdPOX cell-autonomously promotes hair cell size and indirectly restricts stomatal length. Cell-wall autofluorescence and lignin stainings indicated a role for BdPOX in the lignification or crosslinking of related phenolic compounds at the hair cell base. Ectopic expression of BdPOX in the stomatal lineage increased phenolic autofluorescence in guard cell (GC) walls and restricted stomatal elongation in bdpox. Together, we highlight a developmental interplay between hair cells and stomata that optimizes epidermal functionality. We propose that cell-type-specific changes disrupt this interplay and lead to compensatory developmental defects in other epidermal cell types.

Reprogramming by drug-like molecules leads to regeneration of cochlear hair cell–like cells in adult mice

Strategies to overcome irreversible cochlear hair cell (HC) damage and loss in mammals are of vital importance to hearing recovery in patients with permanent hearing loss. In mature mammalian cochlea, co-activation of Myc and Notch1 reprograms supporting cells (SC) and promotes HC regeneration. Understanding of the underlying mechanisms may aid the development of a clinically relevant approach to achieve HC regeneration in the nontransgenic mature cochlea. By single-cell RNAseq, we show that MYC/NICD "rejuvenates" the adult mouse cochlea by activating multiple pathways including Wnt and cyclase activator of cyclic AMP (cAMP), whose blockade suppresses HC-like cell regeneration despite Myc/Notch activation. We screened and identified a combination (the cocktail) of drug-like molecules composing of small molecules and small interfering RNAs to activate the pathways of Myc, Notch1, Wnt and cAMP. We show that the cocktail effectively replaces Myc and Notch1 transgenes and reprograms fully mature wild-type (WT) SCs for HC-like cells regeneration invitro. Finally, we demonstrate the cocktail is capable of reprogramming adult cochlea for HC-like cells regeneration in WT mice with HC loss invivo. Our study identifies a strategy by a clinically relevant approach to reprogram mature inner ear for HC-like cells regeneration, laying the foundation for hearing restoration by HC regeneration.

Adenovirus-mediated effects of Wnt and Notch signalling pathways on hair cell regeneration in mice

Some genes are delivered to cochleae by adenoviruses to restore partial hearing function. This provides promising prospects for gene therapies for hearing loss from hair cell damage. To study the adenovirus (AD)-mediated effect of the Wnt and Notch signalling pathways on hair cell regeneration in the mouse cochlea, we constructed a β-catenin-adenovirus (β-catenin-AD) to increase the activity of the Wnt signalling pathway and a NICD (intracellular domain of Notch1)-RNAi-adenovirus to decrease the activity of the Notch signalling pathway (NICD-RNAi-AD). Our study indicated that approximately 40% of supporting cells in the cochleae damaged by gentamicin were infected with the adenoviruses. Following the β-catenin-AD-mediated increase in Wnt signalling pathway activity, mitotic regeneration was increased, while direct transdifferentiation was increased after the NICD-RNAi-AD-mediated decrease in Notch signalling pathway activity. The expected synergistic interaction on hair cell regeneration was not obtained after coinfection of β-catenin-AD and NICD-RNAi-AD into the damaged cochleae, which might be due to the low cotransfection efficiency to supporting cells. Our study indicated that it may be possible to develop AD mediated gene therapies for hearing loss that act by regulating the Wnt and Notch signalling pathways.

Rps14 upregulation promotes inner ear progenitor proliferation and hair cell regeneration in the neonatal mouse cochlea

Sensorineural hearing loss a result from hair cell damage, which is irreversible in mammals owing to the lack of hair cell regeneration, but recent researches have shown that Lgr5+ supporting cells are progenitors capable of regenerating hair cells. RPS14 (ribosomal protein S14) is a 40S ribosomal subunit component and is associated with erythrocyte differentiation, and in this study, we used a novel adeno-associated virus-inner ear system to upregulate Rps14 expression in cultured hair cell progenitors and observed an enhancement on their ability to proliferate and differentiate into hair cells. Similarly, Rps14 overexpression in the mice cochlea could promote supporting cells proliferation by activating the Wnt signalling pathway. In addition, over-expressing Rps14 induced hair cells regeneration in the organ of Corti, and lineage tracing showed that the new hair cells had transformed from Lgr5+ progenitors. In conclusion, our analysis reveals the potential role of Rps14 in driving hair cell regeneration in mammalian.

Valproic Acid Inhibits Progressive Hereditary Hearing Loss in a KCNQ4 Variant Model through HDAC1 Suppression.

Genetic or congenital hearing loss still has no definitive cure. Among genes related to genetic hearing loss, the potassium voltage-gated channel subfamily Q member 4 (KCNQ4) is known to play an essential role in maintaining ion homeostasis and regulating hair cell membrane potential. Variants of the KCNQ4 show reductions in the potassium channel activity and were responsible for non-syndromic progressive hearing loss. KCNQ4 has been known to possess a diverse variant. Among those variants, the KCNQ4 p.W276S variant produced greater hair cell loss related to an absence of potassium recycling. Valproic acid (VPA) is an important and commonly used histone deacetylase (HDAC) inhibitor for class I (HDAC1, 2, 3, and 8) and class IIa (HDAC4, 5, 7, and 9). In the current study, systemic injections of VPA attenuated hearing loss and protected the cochlear hair cells from cell death in the KCNQ4 p.W276S mouse model. VPA activated its known downstream target, the survival motor neuron gene, and increased acetylation of histone H4 in the cochlea, demonstrating that VPA treatment directly affects the cochlea. In addition, treatment with VPA increased the KCNQ4 binding with HSP90β by inhibiting HDAC1 activation in HEI-OC1 in an in vitro study. VPA is a candidate drug for inhibiting late-onset progressive hereditary hearing loss from the KCNQ4 p.W276S variant.

AAV-Net1 facilitates the trans-differentiation of supporting cells into hair cells in the murine cochlea.

Mechanosensitive hair cells (HCs) in the cochlear sensory epithelium are critical for sound detection and transduction. Mammalian HCs in the cochlea undergo cytogenesis during embryonic development, and irreversible damage to hair cells postnatally is a major cause of deafness. During the development of the organ of Corti, HCs and supporting cells (SCs) originate from the same precursors. In the neonatal cochlea, damage to HCs activates adjacent SCs to act as HC precursors and to differentiate into new HCs. However, the plasticity of SCs to produce new HCs is gradually lost with cochlear development. Here, we delineate an essential role for the guanine nucleotide exchange factor Net1 in SC trans-differentiation into HCs. Net1 overexpression mediated by AAV-ie in SCs promoted cochlear organoid formation and HC differentiation under two and three-dimensional culture conditions. Also, AAV-Net1 enhanced SC proliferation in Lgr5-EGFPCreERT2 mice and HC generation as indicated by lineage tracing of HCs in the cochleae of Lgr5-EGFPCreERT2/Rosa26-tdTomatoloxp/loxp mice. We further found that the up-regulation of Wnt/β-catenin and Notch signaling in AAV-Net1-transduced cochleae might be responsible for the SC proliferation and HC differentiation. Also, Net1 overexpression in SCs enhanced SC proliferation and HC regeneration and survival after HC damage by neomycin. Taken together, our study suggests that Net1 might serve as a potential target for HC regeneration and that AAV-mediated gene regulation may be a promising approach in stem cell-based therapy in hearing restoration.

Generation of p27icreER transgenic mice: A tool for inducible gene expression in supporting cells in the cochlea

The loss of cochlear hair cells (HCs) is an important cause of sensorineural hearing loss, and finding ways to regenerate HCs would be the ideal way forward for restoring hearing. In this research field, tamoxifen-inducible Cre recombinase (iCreER) transgenic mice and the Cre-loxp system are widely used to manipulate gene expression in supporting cells (SCs), which lie beneath the sensory HCs and are a natural source for HC regeneration. However, many iCreER transgenic lines are of limited utility because they cannot target all subtypes of SCs or they cannot be used in the adult stage. In this study, a new line of iCreER transgenic mice, the p27-P2A-iCreERT2 knock-in mouse strain, was generated by inserting the P2A-iCreERT2 cassette immediately in front of the stop codon of p27, which kept the endogenous expression and function of p27 intact. Using a reporter mouse line with tdTomato fluorescence, we showed that the p27iCreER transgenic line can target all subtypes of cochlear SCs, including Claudius cells. p27-CreER activity in SCs was observed in both the postnatal and the adult stage, suggesting that this mouse strain can be useful for research work in adult cochlear HC regeneration. We then overexpressed Gfi1, Pou4f3, and Atoh1 in p27+ SCs of P6/7 mice using this strain and successfully induced many new Myo7a/tdTomato double-positive cells, further confirming that the p27-P2A-iCreERT2 mouse strain is a new and reliable tool for cochlear HC regeneration and hearing restoration.