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Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38266052
202304680
10.1073/pnas.2304680121
datasetDatasetresearch-articleResearch ArticleneuroNeuroscience424
Biological Sciences
Neuroscience
Expression of Atoh1, Gfi1, and Pou4f3 in the mature cochlea reprograms nonsensory cells into hair cells
McGovern Melissa M. a 1 https://orcid.org/0000-0003-2254-8782

Hosamani Ishwar V. b 1 https://orcid.org/0000-0003-2187-8843

Niu Yichi b https://orcid.org/0000-0002-4376-7792

Nguyen Ken Y. a https://orcid.org/0009-0000-3384-0848

Zong Chenghang b https://orcid.org/0000-0002-8337-8038

Groves Andrew K. akgroves@bcm.edu
a b 2 https://orcid.org/0000-0002-0784-7998

aDepartment of Neuroscience, Baylor College of Medicine, Houston, TX 77030
bDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030
2To whom correspondence may be addressed. Email: akgroves@bcm.edu.
Edited by Marianne Bronner, California Institute of Technology, Pasadena, CA; received March 22, 2023; accepted December 8, 2023

1M.M.M. and I.V.H. contributed equally to this work.

24 1 2024
30 1 2024
24 7 2024
121 5 e230468012122 3 2023
08 12 2023
Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

The mammalian cochlea contains hair cells that detect and transduce sound to the brain. Hair cells do not naturally regenerate in mammals, so hair cell death leads to permanent hearing loss. Here, we show that nonsensory cells can be reprogrammed into hair cell–like cells by the expression of three transcription factors specific to hair cells: Gfi1, Atoh1, and Pou4f3. We suggest that nonsensory cells of the ear are a potential target for hearing restoration.

Mechanosensory hair cells of the mature mammalian organ of Corti do not regenerate; consequently, loss of hair cells leads to permanent hearing loss. Although nonmammalian vertebrates can regenerate hair cells from neighboring supporting cells, many humans with severe hearing loss lack both hair cells and supporting cells, with the organ of Corti being replaced by a flat epithelium of nonsensory cells. To determine whether the mature cochlea can produce hair cells in vivo, we reprogrammed nonsensory cells adjacent to the organ of Corti with three hair cell transcription factors: Gfi1, Atoh1, and Pou4f3. We generated numerous hair cell–like cells in nonsensory regions of the cochlea and new hair cells continued to be added over a period of 9 wk. Significantly, cells adjacent to reprogrammed hair cells expressed markers of supporting cells, suggesting that transcription factor reprogramming of nonsensory cochlear cells in adult animals can generate mosaics of sensory cells like those seen in the organ of Corti. Generating such sensory mosaics by reprogramming may represent a potential strategy for hearing restoration in humans.

inner ear
reprogramming
hair cells
cochlea
HHS | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD) 100000055 DC014832 Melissa M. McGovernAndrew K Groves HHS | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD) 100000055 DC019022 Melissa M. McGovernAndrew K Groves Cancer Prevention and Research Institute of Texas (CPRIT) 100004917 RP180672 Melissa M. McGovernIshwar V HosamaniYichi NiuKen Y NguyenChenghang ZongAndrew K Groves HHS | NIH | National Cancer Institute (NCI) 100000054 CA125123 Melissa M. McGovernIshwar V HosamaniYichi NiuKen Y NguyenChenghang ZongAndrew K Groves HHS | NIH | National Center for Research Resources (NCRR) 100000097 RR024574 Melissa M. McGovernIshwar V HosamaniYichi NiuKen Y NguyenChenghang ZongAndrew K Groves
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pmcCochlear hair cells are responsible for converting sound into electrochemical impulses that are relayed to the brain. Hair cells can be killed by loud noises, ototoxic drugs such as chemotherapeutics and aminoglycoside antibiotics, as well as the natural aging process (1, 2). Once lost, hair cells do not naturally regenerate in the mature mammalian cochlea, leading to permanent hearing loss. The failure of the organ of Corti to regenerate hair cells following damage has prompted attempts to reprogram neighboring nonsensory cells into hair cells. Hair cell reprogramming makes use of known hair cell transcription factors, such as Atoh1, to convert nonsensory cells into hair cells. Although expressing Atoh1 in neonatal nonsensory cells induces their conversion to hair cell–like cells, expression of Atoh1 alone in the mature cochlea is unable to reproducibly generate significant numbers of new hair cells (3–7). Reprogramming with multiple transcription factors has been shown to produce small numbers of new hair cells in adult animals (3, 8), and finding an optimal combination of reprogramming factors is therefore necessary to develop potential treatments for hearing loss.

Atoh1, Gfi1, and Pou4f3 are transcription factors that are required for the formation of hair cells during cochlear development (9–11). Recently, the expression of these factors in embryonic fibroblasts induced a hair cell–like phenotype (12). Expression of these three factors in the greater epithelial ridge of the neonatal cochlea robustly reprogrammed nonsensory cells into hair cell–like cells (13, 14). However, it is still unknown whether these factors are sufficient to convert mature nonsensory cells in the cochlea into hair cell–like cells.

Attempts to reprogram cells in the mature cochlea typically target the supporting cells of the organ of Corti (3, 4, 8, 15, 16). While these cells are an ideal target based on their proximity to hair cells, other cell types in the mature cochlea are promising targets for hearing restoration in patients with severe cochlear damage. In such patients, the entire organ of Corti is lost in some regions of the cochlea and is replaced by a flat cuboidal epithelium (8). Cells of the inner and outer sulci are believed to contribute to this flat epithelium, and we have targeted these cell populations in the present study. We previously showed that the Fbxo2CreERT2 mouse line recombines in cells of the inner and outer sulcus (OS), as well as the spiral limbus (SL) of the mature cochlea (17). Here, we used this mouse line to target nonsensory cells of the mature cochlea in combination with Cre-inducible mice that express either Atoh1 alone or a combination of Atoh1+Gfi1+Pou4f3 (13). We find that expression of Atoh1 alone reprograms only a small number of nonsensory cells into hair cell–like cells, but that expression of Atoh1+Gfi1+Pou4f3 in nonsensory cells of the cochlea generates significant numbers of cells that express many hair cell genes. These reprogrammed hair cells continued to increase in number and mature over the course of 9 wk and developed immature stereocilia bundles. We also observed cells with the features of supporting cells in our reprogrammed populations, suggesting that transcription factor reprogramming can generate mosaics of hair cells and supporting cells. The creation of hair cells and supporting cells is a necessary first step in regenerating functional auditory epithelium in the severely deafened ear.

Results

Nonsensory Cells of the Inner and Outer Sulci Can Be Reprogrammed into Hair Cell–Like Cells in the Mature Cochlea.

We first sought to test whether Atoh1 alone was sufficient to reprogram nonsensory cells into hair cell–like cells. We induced the expression of Atoh1 in nonsensory cells of the mature cochlea by breeding the Fbxo2CreERT2 mouse line with the Rosa26loxp-stop-loxp-Atoh1 mouse line (Rosa26-A; 13). We also incorporated the Rosa26loxp-stop-loxp-tdTomato (Rosa26-tdTomato; 18) reporter so that targeted cells would be permanently labeled by tdTomato (Fig. 1 A and B). As previously described, Fbxo2CreERT2 targets nonsensory cells both in the organ of Corti as well as in the surrounding tissue medial and lateral to the organ of Corti. Unless noted otherwise, we use “nonsensory cells” to describe cells in the epithelial tissue surrounding the organ of Corti and “supporting cells” to describe nonsensory cells adjacent to hair cells within the organ of Corti (i.e., inner phalangeal, border, pillar, Deiters’, and Hensen’s cells). Expression of Atoh1 and tdTomato was induced by injecting tamoxifen (TMX) on two consecutive days at 3 wk of age (Fig. 1A). Cochleas were collected and analyzed 2 wk later (Fig. 1A). In control cochleas, Myosin VIIa–positive hair cells were only observed in the organ of Corti (n = 3). Following the induction of Atoh1, very few reprogrammed cells (3.7 ± 3.8 per 200 µm compared to 0 ± 0 in the control cochlea, n = 3) were observed throughout the nonsensory regions of the cochlea suggesting that Atoh1 alone was insufficient to produce a significant number of reprogrammed hair cells (Fig. 1C). This result agrees with other studies that saw only limited regeneration in the adult cochlea by mis-expressing Atoh1 (3, 8).

Fig. 1. Nonsensory cells of the mature cochlea are reprogrammed into hair cell–like cells by expression of Atoh1, Gfi1, and Pou4f3. (A) Timeline for the activation of reprogramming factors and reporter expression. TMX was injected on two consecutive days at 3 wk of age. Cochleas were collected and analyzed at 5 wk of age. (B) The reprogramming factors and/or tdTomato reporter were expressed in nonsensory cells of the IS and OS. Hair cells are detected by Myosin VIIa staining (green) in the Fbxo2CreERT2::Rosa26tdTomato control cochlea. Hair cells are observed only in the organ of Corti, while tdTomato (red) was detected medial and lateral to the organ of Corti (n = 4). (C) Atoh1 activation alone (Fbxo2CreERT2::RosatdTomato::RosaA) is insufficient to reprogram nonsensory cells into hair cell–like cells (n = 8); however (D and D’) following expression of Atoh1+Gfi1+Pou4f3 (Fbxo2CreERT2:: RosaotdTomato:: RosaGAP), hair cells (green) are observed in the SL and OS (n = 19). (E) Quantification of ectopic hair cells in the IS and SL of Fbxo2CreERT2:: RosatdTomato:: RosaA (Atoh1) and Fbxo2CreERT2:: RosatdTomato:: RosaGAP (Gfi1+Atoh1+Pou4f3) cochleas (n = 3). (F) The hair cell protein, Parvalbumin (magenta), is limited to endogenous hair cells (green) in the organ of Corti in the control cochlea (Fbxo2CreERT2:: RosatdTomato, n = 4) but is expressed in reprogrammed hair cells in the SL following Atoh1+Gfi1+Pou4f3 expression (Fbxo2CreERT2:: RosatdTomato:: RosaGAP, n = 4). (G) Hair cells (green) were limited to the organ of Corti in the control cochlea in all cochlear turns. Following reprogramming with Atoh1, Gfi1, and Pou4f3, Myosin VIIa–positive cells were observed in nonsensory regions throughout the cochlear length. More hair cells were reprogrammed in the apex compared to the middle or basal turns of the cochlea. n = 3, *P < 0.05, **P < 0.01; (scale bar in A–D’, F, and G = 50 µm), OS = outer sulcus, OoC = organ of Corti, IS = inner sulcus, SL = spiral limbus.

We next tested whether the combined expression of three hair cell transcription factors, Atoh1+Gfi1+Pou4f3, could reprogram nonsensory cells into hair cell–like cells. We induced expression by crossing the Fbxo2CreERT2 mouse line with the Rosa26loxp-stop-loxp-Gfi1-Atoh1-Pou4f3 mouse line (Rosa26-GAP; 13). We again incorporated the Rosa26-tdTomato reporter allele to permanently label cells expressing the three transcription factors. After 2 wk of reprogramming, we sectioned temporal bones and investigated the expression of the hair cell marker Myosin VIIa. We observed robust hair cell generation in nonsensory cells outside the organ of Corti (the OS, SL, and the lateral wall) as well as a small number of reprogrammed supporting cells in the organ of Corti compared to controls (n = 19; Fig. 1 D and D’ arrowheads). We then counted the number of reprogrammed cells that express Myosin VIIa by dissecting the entire cochlea out of the temporal bone. Because this preparation removes variable amounts of the OS and lateral wall, we focused our quantification on the reprogrammed cells in the medial compartment of the cochlea (inner sulcus and SL). Combined expression of Atoh1, Gfi1, and Pou4f3 generated significantly more new hair cells in the mature cochlea compared to Atoh1 alone (29.2 ± 6.2 compared to 3.6 ± 3.8, n = 3, P = 0.0037, Student’s t test, Fig. 1E). We investigated other hair cell genes and found that Parvalbumin was expressed in nonsensory regions of the cochlea following reprogramming, while it was limited to cells of the organ of Corti in controls (n = 4; Fig. 1F).

Previous studies highlighted a gradient of maturity of the cochlea along the apical–basal axis in neonatal animals (19, 20); however, less is known about how the mature cochlea might respond to damage and reprogramming along this axis. We therefore quantified the number of reprogrammed hair cells in the apex, middle, and basal turns of the cochlea to understand how these regions respond to activation of Atoh1, Gfi1, and Pou4f3 expression. Significantly more ectopic hair cells were observed in the apical turn of the cochlea (53.0 ± 12.5) compared to the middle (25.0 ± 6.9, P = 0.0144) and basal (25.0 ± 6.9, P = 0.0017 as determined by a one-way ANOVA with a Tukey’s post hoc test, n = 3, Fig. 1G) turns of the cochlea. This suggests that the apical region of the cochlea responds better to reprogramming factors than the middle and basal turns.

Single-Cell RNA-seq Reveals that Reprogrammed Cells Express Many Hair Cell Genes.

To further understand the reprogramming of nonsensory cells in the mature cochlea following Atoh1+Gfi1+Pou4f3 expression, we performed high-depth full-length single-cell RNA sequencing using a modified MATQ-sequencing protocol (21). Rosa26-GAP and Rosa26tdTomato expression was induced by Fbxo2CreERT2by injection of TMX on two consecutive days at 3 wk of age (Fig. 1A). Cochlear cells were stained with the epithelial marker EpCAM/CD326, which specifically labeled cochlear epithelial cells. Cells were sorted by flow cytometry based on colabeling of induced tdTomato fluorescence and EpCAM (Fig. 2 A and B). 200 cells from Fbxo2CreERT2:: Rosa26tdTomato animals and 200 cells from Fbxo2CreERT2:: Rosa26tdTomato:: Rosa26-GAP animals were sequenced at ~450,000 reads/cell. Unbiased clustering using Seurat V4.1 revealed a clear separation between cells from control and experimental samples (Fig. 2C).

Fig. 2. Single-cell RNA sequencing reveals that nonsensory cells activate hair cell networks following expression of Atoh1, Gfi1, and Pou4f3. (A) Representative image illustrating the expression pattern of Rosa26tdTomato (Red) when induced by Fbxo2CreERT2 and the epithelial marker EpCAM (Blue) at 5 wk. OS = outer sulcus, OoC = organ of Corti, IS = inner sulcus, SL = Spiral limbus. Fbxo2CreERT2::Rosa26-tdTomato (control) and Fbxo2CreERT2::Rosa26tdTomato::Rosa26-GAP cochleas were dissociated into single cells, and (B) tdTomato+EpCAM double positive cells were sorted by flow cytometry and collected for single-cell RNA sequencing. (C) Unbiased Seurat analysis revealed that control cells (blue) and reprogrammed cells (gold) largely clustered based on condition and 9 unique clusters were identified. RNA-velocity predicts that cells are transitioning from control cells into reprogrammed cells. (D) Known hair cell genes were activated at a higher level in one cluster of reprogrammed cells (Rprg HCs) than in two other reprogrammed clusters (Rprg Orig SCs and Rprg New SCs). (E) The heatmap showing the top 100 hair cell–specific genes, found previously at P1 from ref. 22, in reprogrammed cells. (F) SCENIC analysis detected the activation of genes associated with Atoh1 and Pou4f3 regulons in reprogrammed hair cells (Rprg HCs). (Scale bar in A, 50 µm.)

We first attempted to identify the four clusters of control cells. While neonatal cochlear cell types have been extensively characterized (22, 23), less is known about unique markers for mature cochlear nonsensory cells. Recent work has investigated the expression profiles of cells in the mature cochlea outside of the organ of Corti (24). We therefore compared the genes enriched in our control cell populations with the cell type–specific markers for cells within the cochlear epithelium from this published dataset (24). One cluster (Ctrl 1) expressed genes associated with interdental cells, basal stria cells, Claudius cells, IS and OS cells, Deiters’ cells, Pillar cells, and cells of Reissner’s membrane (SI Appendix, Figs. S1–S6). A second cluster (Ctrl 2) expressed genes associated with Claudius cells and IS and OS cells (SI Appendix, Fig. S3). A third cluster (Ctrl 3) expressed genes associated with interdental cells, basal stria cells, Claudius cells, ISand OS cells, Deiters’ cells, pillar cells, and cells of the Reissner’s membrane (SI Appendix, Figs. S1–S6). Based on genes that were enriched in Ctrl 1-3, we attempted to further explore the identity of these clusters through histology. We investigated 15 genes through immunolabeling; however, only one of these provided clear insight into cluster identity. Lgals1 labels cells of the IS and OS (SI Appendix, Fig. S7A) and is also enriched in cluster Ctrl 2. Therefore, this cluster is likely IS and OS cells. The fourth cluster, [Ctrl SCs (control supporting cells)] expressed genes associated with interdental cells, Deiters’ cells, and pillar cells (SI Appendix, Figs. S1, S4, and S5). This fourth cluster is likely inner phalangeal and border cells based on the expression of the supporting cell genes Sox2, Jag1, SLC1A3, and Lfng (SI Appendix, Fig. S15; Ctrl SCs), which are all known markers of this cell population (25–27). Taken together, these data suggest that control cells from our dataset are cochlear nonsensory and supporting cells.

We next investigated whether hair cell gene networks were activated in nonsensory cells following reprogramming. Expression of Atoh1+Gfi1+Pou4f3 was limited to cells from reprogrammed samples (SI Appendix, Fig. S7B). The hair cell markers Myosin VIIa and Parvalbumin were observed in reprogrammed cells by immunostaining (Fig. 1 D–G). We therefore investigated the activation of other known hair cell genes in these reprogrammed clusters. Myosin VIIa and Parvalbumin were expressed in two clusters of reprogrammed cells [Rprg HCs (reprogrammed hair cell–like cells), Rprg New SCs (reprogrammed new supporting cell–like cells), Fig. 2E and SI Appendix, Fig. S7C]. Of the three clusters from reprogrammed samples [Fig. 2C, Rprg HCs, Rprg New SCs, and Rprg Orig SCs (reprogrammed original supporting cells)], one cluster expressed Myosin VIIa and Parvalbumin at a higher level and in a larger percentage of the cells compared to other cells (Rprg HCs, Fig. 2E). In addition, other genes associated with hair cell identity (Otoferlin, Rasd2, Slc17a5, Insm1, and TOMT) were expressed in this cluster and absent from control clusters. We found that numerous stereocilia- and mechanotransduction-related genes (Myosin 15, Cadherin 23, Tmc1, Cib2, Lhfpl5, Ush2a, and Adgrv1) were also up-regulated in this cluster (Fig. 2E; Rprg HCs).

To further characterize reprogrammed clusters, we asked whether hair cell network genes are differentially expressed among reprogrammed clusters. Previous work has described genes associated with hair cell identity at multiple time points including postnatal day 1 (P1), P7, P8, P12, and P20 (22, 24). We compared the expression of the top 100 differentially expressed genes in hair cells from these studies to our reprogrammed cell clusters. We found that many of the reported hair cell genes are activated in all reprogrammed clusters; however, more of these hair cell genes were present in one cluster of reprogrammed cells, which we labeled as Reprogrammed Hair Cells (Fig. 2F and SI Appendix, Figs. S8–S14, Rprg HCs). We further investigated the activation of hair cell network genes by performing transcription factor network analysis to interrogate which transcription factors and their downstream targets (regulons) were activated in the Rprg HCs using the SCENIC analysis package (28). We found that Atoh1 and Pou4f3 regulons were activated in reprogrammed hair cells (Fig. 2F and Datasets S1 and S2).

Reprogramming Activates Supporting Cell Gene Networks in Nonsensory Cells.

We observed two additional clusters of reprogrammed cells (Rprg New SCs and Rprg Orig SCs) that did not express hair cell network genes to the same level as Rprg HCs but instead expressed genes associated with supporting cell identity (Figs. 2 E and F and 3A and SI Appendix, Fig. S15A). As we have previously shown that reprogramming of nonsensory cells in the neonatal cochlea could generate mosaics of cells resembling hair cells and supporting cells (13), we further investigated the identity of these clusters to determine whether similar mosaics of hair cells and supporting cells could be generated in the adult cochlea. Both clusters of cells (Rprg New SCs and Rprg Orig SCs) expressed genes associated with supporting cell development (Sox2, Lfng, Jag1, Slc1a3, and S100a1; Fig. 3A). One cluster (Rprg New SCs) also expressed some hair cell network genes (i.e., Myosin VIIa, Rasd2, and Cib2), but did so less than reprogrammed hair cells (Fig. 2 E and F). This cluster activated Atoh1 and Pou4f3 regulons, but also weakly activated regulons associated with supporting cell identity (Sox2, Cux1, Isl1; Fig. 2G). We then interrogated our dataset with genes identified previously as being associated with supporting cell identity (22, 24) and found that Rprg New SCs expressed many supporting cells genes, while reprogrammed hair cells did not (SI Appendix, Figs. S16–S20). This suggests that these cells are activating supporting cell networks in response to reprogramming.

Fig. 3. Reprogrammed hair cells signal to their neighbors to activate supporting cell genes. (A) Supporting cell genes are expressed in original supporting cells (Ctrl SCs) and are maintained in reprogrammed original supporting cells (Rprg Orig SCs). These genes are also activated in nonsensory cells following reprogramming (Rprg New SCs). (B) Sox2 (magenta) is restricted to supporting cells of the control cochlea (n = 4). Following reprogramming, Sox2 is expressed in cells of the SL, and some Sox2+/Myosin VIIa–cells were detected (arrowheads, n = 9). More Sox2-positive/Myosin VIIa–negative cells were observed in the apex compared to the middle and basal turns. *P < 0.05. OS = outer sulcus, OoC = organ of Corti, IS = inner sulcus, SL = spiral limbus. (C) The proliferation marker Ki-67 was not observed in the control cochlea; however, a small number of Ki-67-positive cells were observed following reprogramming. (D) The Notch receptor Notch1 is expressed in both control and reprogrammed cells. The Notch ligands Jagged 2 (Jag2), Delta like ligand 3 (Dll3), and Delta like ligand 2 (Dll2) are expressed in some reprogrammed cells. The Notch effector Hes5, which is associated with the promotion of supporting cell identity, is also expressed in some reprogrammed cells. (Scale bar in B, 50 µm and 20 µm.)

We used scVelo to perform RNA-velocity analysis to investigate the probability of cells transitioning from one state to another (29). We identified a differentiation trajectory that shows the reprogrammed supporting cells from the Rprg New SC and Rrprg Orig SC clusters moving toward a hair cell fate (Fig. 2C). The dynamic model of scVelo, which recovers the latent time of underlying cellular processes and detects highly dynamic genes characterized by their high likelihoods, identified several hair cell–specific genes being actively transcribed in the reprogrammed cells (SI Appendix, Fig. S25). These data suggest that at least some of our reprogrammed hair cells may be generated by nonsensory cells of the cochlea activating elements of prosensory or supporting cells before finally adopting a hair cell fate.

While the transcription factor Sox2 is expressed in supporting cells of the mature cochlear duct (Fig. 3B), it is also expressed developmentally in prosensory cells as well as nascent hair cells (30). We therefore investigated the expression of Sox2 protein in cells following transcription factor reprogramming and detected its expression in the SL in both Myosin VIIa–positive cells as well as adjacent cells lacking Myosin VIIa expression (n = 9; Fig. 3B and SI Appendix, Fig. S15B, arrowheads), while Sox2 was not detected in the SL in control samples (n = 4). All Sox2-positive cells observed were also tdTomato-positive regardless of Myosin VIIa expression (Fig. 3B). This indicates that they also up-regulated the reprogramming factors Atoh1, Gfi1, and Pou4f3; however, only some reprogrammed cells activated Myosin VIIa expression. This suggested that reprogramming of cells in the inner sulcus by Atoh1+Gfi1+Pou4f3 expression results in the formation of nascent sensory patches consisting of both hair cells and supporting cells in the mature cochlea and that cells targeted with reprogramming factors were able to suppress hair cell identity and promote supporting cell identity.

In addition, we investigated whether the apical–basal gradient of hair cell reprogramming was also reflected in the presence of Sox2-positive/Myosin VIIa–negative cells. We counted these cells per 200 µm in the SL in each turn of the cochlea. We found significantly more singly labeled Sox2-positive cells in the apical turn of the cochlea (31.3 ± 7.5) compared to the middle (9.7 ± 5.5, P = 0.0464) or basal turns (Fig. 3B, 9.0 ± 11.4, P = 0.0412, as determined by a one-way ANOVA with a Tukey’s post hoc test, n = 3). This again suggested that the apical turn of the cochlea is more sensitive to reprogramming factors compared to the middle or basal turns. In addition, we found Sox2-positive/Myosin VIIa–negative cells in the basal turn of the cochlea that were not in proximity to Myosin VIIa–positive cells (SI Appendix, Fig. S15B). Therefore, it is possible that Sox2 is activated in reprogrammed hair cells prior to other hair cell identifiers such as Myosin VIIa.

We next investigated how Sox2-positive/Myosin VIIa–negative cells were generated. During development, cell division increases the pool of prosensory cells that will become hair cells and supporting cells; however, cells within the cochlear duct are postmitotic during adulthood (31, 32). Therefore, we investigated whether cells targeted for reprogramming were undergoing cell division. We sectioned samples following 2 wk of reprogramming and stained them with the hair cell marker Parvalbumin and Ki-67, which marks cell division. We found a small number of Ki-67-positive cells in sections from reprogrammed samples (five cells total from three cochlear samples) and none in samples from controls (Fig. 3C). The Ki-67-positive cells did not coexpress Parvalbumin but were located adjacent to Parvalbumin-positive cells. This suggests that a small number of cells re-enter the cell cycle in the process of being reprogrammed and may become supporting cell–like cells.

During development, cochlear cells establish a mosaic of hair cells and supporting cells through Notch signaling (33). However, this signaling pathway is largely down-regulated over the first postnatal week and there is little evidence to suggest that it can regulate cell identity in the mature cochlea (34). We asked whether the new sensory patches we observed after reprogramming deployed this developmental strategy in establishing mosaics of hair cells and supporting cells. We found that the Notch1 receptor was expressed in most cells from control and reprogrammed samples (Fig. 3D). Three Notch ligands, Jagged 2 (Jag2), Delta Like Ligand 1 (Dll1), and Delta Like Ligand 3 (Dll3) were expressed in reprogrammed cells. Dll3 was restricted to reprogrammed hair cells (Fig. 3D, Rprg HCs), but Jag2 and Dll1, which are known to signal from developing hair cells and induce supporting cell fate in their neighbors (35), were expressed in hair cells (Rprg HCs) and reprogrammed new supporting cells (Rprg New SCs; Fig. 3D). Finally, the Notch effector Hes5, which promotes supporting cell identity and directly suppresses Atoh1 (36), was expressed specifically in Rprg New SCs (Fig. 3D). This suggests that reprogrammed hair cells are signaling to their neighboring cells through Notch signaling and promoting a supporting cell identity.

We observed a second cluster of supporting cell–like cells following reprogramming (Fig. 3A; Rprg Orig SCs). These cells failed to express most hair cell genes (Fig. 2 D and E and SI Appendix, Figs. S8–S14 or activate hair cell regulons (Fig. 2F). In addition, the regulons that were expressed in Ctrl SCs (Sox2, Cux1, Isl1, Klf5 extended, Pou3f2 extended, Rorb, and Nr1d2 extended) were also expressed in this cluster following reprogramming (Fig. 2F). Based on the expression of supporting cell genes (Sox2, Lfng, Jag1, Slc1a3, and S100a1, Fig. 3A) and their similarity to Ctrl SCs (Fig. 2D) we identified these as inner phalangeal and border cells. These cells also express Atoh1, Gfi1, and Pou4f3 (SI Appendix, Fig. S7B), and, therefore, they have been targeted for reprogramming; however, they weakly activate the Pou4f3 regulon while strongly activating supporting cell regulons (Fig. 2F). Few reprogrammed hair cells were observed at this time point in the inner phalangeal and border cells region of the cochlea based on histological analysis (Fig. 1 B and C). In addition, we are preparing a second manuscript that demonstrates that supporting cells of the mature cochlea are unresponsive to reprogramming without hair cells first being killed. Taken together, these data suggest that these cells are either not responsive to transcription factor reprogramming, or they require additional factors as well as time to activate hair cell networks.

Finally, we asked whether the reprogramming of nonsensory cells effectively suppressed nonsensory cell gene networks or whether both the gene networks of hair cells and nonsensory cells continued to be active in the reprogrammed cells. We identified the top 20 differentially expressed genes among the control clusters (Ctrl 1, Ctrl2, Ctrl3, Ctrl SCs). We then investigated the expression of these genes within clusters of reprogrammed cells. We found that genes associated with control clusters 1-3 are generally not expressed in reprogrammed cells suggesting that these cells are changing their identities to become hair cells and supporting cells (SI Appendix, Fig. S21). In contrast, genes associated with Ctrl SCs were expressed in Rprg Orig SCs, further suggesting that supporting cells within the organ of Corti do not completely convert into hair cells following 2 wk of reprogramming (SI Appendix, Fig. S21). Taken together, these data suggest that nonsensory cells outside the organ of Corti are more responsive to reprogramming compared to the supporting cells of the organ of Corti. This agrees with previous work in the neonatal cochlea which suggests that cells within the organ of Corti do not respond to reprogramming with Atoh1+Gfi1+Pou4f3 compared to those in the greater epithelial ridge (13, 37)

Cellular Reprogramming Continues over 9 wk Following Expression of Atoh1, Gfi1, and Pou4f3.

We next investigated whether prolonged expression of reprogramming factors would increase the number or induce the maturation of ectopic hair cells. We activated the expression of Atoh1+Gfi1+Pou4f3 on two consecutive days at 3 wk of age and collected cochleas for analysis after 2, 5 or 9 wk of reprogramming (Fig. 4A). Following 2 wk of reprogramming, Myosin VIIa was detected in nonsensory regions of the apical turn of reprogrammed cochleas (n = 19; Fig. 4B). Following 5 and 9 wk of reprogramming we detected additional Rprg HCs were added in nonsensory regions progressing into the middle and basal turns of the cochlea (n = 5; Fig. 4B and SI Appendix, Fig. S22 A and B).

Fig. 4. Hair cell–like cells are added over the course of 9 wk following the expression of Atoh1, Gfi1, and Pou4f3. (A) Reprogramming was activated at 3 wk of age in nonsensory cells of the inner ear and cochlea were analyzed at 5, 8, or 12 wk of age. (B) In the control cochlea, the normal complement of inner and outer hair cells (green) was observed n = 4. At 5 wk, Myosin VIIa+ hair cells were observed in the SL and OS in the apical turn and the density of these cells declined toward the basal turn. At 8 wk, the density of hair cells had increased in the SL and OS throughout the length of the cochlea. At 12 wk, cells throughout the length of the cochlea, in the SL, the IS, and the OS are robustly converted into hair cell–like cells. (C) Quantification of hair cells per 200 µm region of the IS and SL of Atoh1 expressing cochlea at 5 and 8 wk and Atoh1+Gfi1+Pou4f3 expressing cochlea at 5, 8, and 12 wk of age. (D) Quantification cells in the apical turn comparing the medial compartment and lateral compartment of control or Atoh1+Gfi1+Pou4f3 expressing cochlea at 12 wk of age. (E) Higher magnification images of Atoh1+Gfi1+Pou4f3 expressing cochlea at 8 and 12 wk of age. Quantification of reprogrammed hair cells per 200 µm reveals that there is an increase in the number of reprogrammed cells between 5 and 8 wk of age in the middle and base, while there is an increase in the apex between 8 and 12 wk of age. *P < 0.05, **P < 0.0, ***P < 0.001, and ****P < 0.0001.

We quantified the number of reprogrammed hair cells per 200 µm in the medial compartment of cochleas and found that the number of ectopic hair cells increased following the expression of Atoh1, Gfi1, and Pou4f3 between 5 wk of age (29.2 ± 6.2, n = 3) and 8 wk (95.4 ± 15.7, n = 3, P < 0.0001), as well as between 8 and 12 wk (Fig. 4 B and C, 139.3 ± 5.8, n = 3, P = 0.0009, as determined by a one-way ANOVA with a Tukey’s post hoc test). Because there was a significant increase in the number of ectopic hair cells with additional reprogramming time, we asked whether Atoh1 alone could also reprogram significantly given additional time. There was no significant increase in the number of ectopic hair cells following Atoh1 expression between 5 wk of age (3.7 ± 3.8, n = 3) and 8 wk of age (24.1 ± 8.0, n = 3, P = 0.1045, as determined by a one-way ANOVA with a Tukey’s post hoc test, Fig. 4C and SI Appendix, Fig. S23). At both 5 and 8 wk of age, significantly more ectopic hair cells were observed with combined Atoh1, Gfi1, and Pou4f3 expression compared to Atoh1 alone (Figs. 4C and 5 wk P = 0.0351, 8 wk P < 0.0001, as determined by a one-way ANOVA with a Tukey’s post hoc test). This indicates that over the course of 9 wk, the three-factor combined reprogramming continues to generate new hair cells; however, a single-factor reprogramming is still insufficient to generate significant numbers of hair cells.

Fig. 5. Reprogrammed cells express multiple mature hair cell genes. (A) Reprogramming was activated at 3 wk of age in nonsensory cells of the inner ear and cochlea were analyzed at 5, 8, or 12 wk of age. (B) Parvalbumin (magenta) continues to be expressed in Rprg HCs in the SL and inner sulcus. Quantification of Parvalbumin-positive cells reveals that there is a significant increase in positive cells in the medial compartment (medial to the tunnel of Corti) but not in the Organ of Corti or floor of the cochlear duct or lateral wall (n = 3). (C) Prestin (magenta) is expressed in outer hair cells in the control cochlea and is expressed in some Rprg HCs (determined by the colabeling of Myosin VIIa in green and tdTomato in red) adjacent to the organ of Corti. The organ of Corti is disorganized following reprogramming, and the arrow indicates an endogenous hair cell and arrowheads indicate Rprg HCs. Significantly more Prestin-positive/tdTomato-positive cells were observed following reprogramming (n = 3). (D) Phalloidin labeling (white) is limited to the organ of Corti in the control cochlea (n = 6); however, numerous phalloidin+ cells are observed in the SL, IS, and OS following reprogramming (n = 2). Higher magnification analysis reveals distinct stereocilia atop Rprg HCs. (E) Sox2 expression (magenta) is limited to the organ of Corti in the control cochlea but is detected in the SL, Is, and OS following reprogramming (n = 2). Several cells were observed to be Sox2-positive and Myosin VIIa–negative (green; arrowheads). There was not a significant increase in the number of Sox2-positive/Myosin VIIa–negative cells with increased duration of reprogramming. Inner hair cells are indicated by asterisks, and outer hair cells are indicated with brackets in A and C–E. TMX = tamoxifen, OHCs = outer hair cells, IHCs = inner hair cells. (Scale bar in B, 200 µm; (C and D) low mag = 20 µm; E low mag = 50 µm; E med mag = 10 µm, high mag = 2 µm.) OS = outer sulcus, OoC = organ of Corti, IS = inner sulcus, SL = spiral limbus. *P < 0.05 and ***P < 0.001.

We next sought to better understand the efficiency of reprogramming of different regions of the cochlea along both the medial–lateral axis as well as the apical–basal axis. We sectioned the cochlea at 80 µm and divided sections into medial and lateral compartments at the tunnel of Corti. We quantified the number of Myosin VIIa–positive cells in the medial region and the lateral region of the apical turn and found that both regions have significantly more hair cells following reprogramming than control sections (Fig. 4D, medial 11.0 ± 3.6 vs. 75.0 ± 13.5, P < 0.0001, lateral 19.3 ± 4.0 vs. 51.0 ± 1.0, P = 0.0014, n = 3, as determined by a two-way ANOVA with Sidak’s post hoc test). In addition, we found that the medial compartment had significantly more reprogrammed hair cells than the lateral compartment following reprogramming (Fig. 4D, 75.0 ± 13.5 vs. 51.0 ± 1.0, P = 0.0076, n = 3, as determined by a two-way ANOVA with Sidak’s post hoc test).

We next quantified the expansion of hair cells along the apical–basal axis. There was a significant increase in the number of ectopic hair cells in the middle and basal turns between 5 wk of age (middle = 25.0 ± 6.9, base = 9.7 ± 1.5) and 8 wk (middle = 102.3 ± 24.6, P = 0.0003, base = 91.7 ± 8.5, P = 0.001) but not in the apex (53.0 ± 12.5 vs. 92.3 ± 17.0). Conversely, there was an increase in the number of hair cells in the apex between 8 and 12 wk of age (92.3 ± 17.0 vs. 170.7 ± 25.1, n = 3, P = 0.0002, as determined by a two-way ANOVA with a Tukey’s post hoc test), but not in the middle (102.3 ± 24.6 vs. 132.0 ± 4.4, n = 3) or base (91.7 ± 8.5 vs. 115.3 ± 37.3, n = 3, Fig. 4E). In addition, while there was a significant apical to basal gradient at 5 wk of age (Fig. 1G), we found no significant difference in the number of ectopic hair cells along the apical–basal gradient at 8 wk (apex = 92.3 ± 17.0, middle = 102.3 ± 24.6, base = 91.7 ± 8.5, n = 3) or 12 wk of age (apex = 170.7 ± 25.1, middle = 132.0 ± 4.4, base = 115.3 ± 37.7, n = 3). Taken together these data suggest that the apical turn of the cochlea responds to reprogramming before the middle and basal turns, but that the middle and basal turns produce a similar number of hair cells over time. In addition, all turns of the cochlea continue to add reprogrammed cells with prolonged exposure to reprogramming factors.

In addition to increasing the overall number of hair cells present, we also investigated whether prolonged reprogramming increased the maturation of ectopic hair cells. We counted the number of Parvalbumin-positive cells in three regions of the cochlear apex: the medial region from the tunnel of Corti to the where Reissner’s membrane contacts the SL, the organ of Corti and the floor of the cochlear duct, and the lateral wall above the spiral prominence. We found that there were significantly more Parvalbumin-positive cells in the medial region of the cochlea compared to controls (46.9 ± 17.9 vs. 1.0 ± 1.7, n = 3, P < 0.0001 as determined by a two-way ANOVA with Sidak’s post hoc test) but not in the rest of the cochlear duct (Fig. 5B and SI Appendix, Fig. S2E). We also examined expression of Prestin, which is a protein expressed specifically in mature outer hair cells. At 12 wk of age, we found Rprg HCs within the organ of Corti and along the lateral border that were colabeled with tdTomato/Myosin VIIA and Prestin. We quantified the number of tdTomato-positive/Prestin-positive cells in control and reprogrammed samples. Significantly more dual labeled cells were observed following reprogramming with Atoh1+Gfi1+Pou4f3 compared to control samples (27.7 ± 10.2 vs. 0.0 ± 0.0, n = 3, P = 0.0423 as determined by Student’s t test; Fig. 5C; arrowheads), suggesting that these cells were expressing some features of outer hair cells. Functional cochlear hair cells also require mechanosensory hair bundles that can be visualized with fluorescently labeled phalloidin. At 12 wk of age, we observed disorganized stereocilia bundles in the IS, SL, and OS (n = 2; Fig. 5D). Innervation of the endogenous organ of Corti was visualized by labeling with Parvalbumin or Neurofilament heavy chain; however, innervation of Rprg HCs was not detected at any time (n = 2; Fig. 5B and SI Appendix, Fig. S22C).

Finally, we asked whether Sox2-positive/Myosin VIIa–negative cells were still present with additional reprogramming time. We did not see evidence of Sox2-positive cells in nonsensory regions of the cochlea at any age in control tissue; however, we observed Sox2-positive/Myosin VIIa–negative cells in the SL at 5, 8, and 12 wk of age following reprogramming. We quantified the number of Sox2-positive/Myosin VIIa–negative cells following reprogramming at these ages and found there was no difference in the number of cells at any time point (Fig. 5E, n = 2 to 3), suggesting that the number of Sox2 single-positive cells remains consistent over time.

Discussion

Here, we describe a potential target for hearing restoration: nonsensory cells medial and lateral to the organ of Corti. Although the mature cochlea is normally unable to regenerate hair cells following damage, we demonstrate that many cells on either side of the organ of Corti are responsive to cellular reprogramming with Atoh1, Gfi1, and Pou4f3. These cells readily activate hair cell gene programs, and they also activate the Notch signaling pathway, a well characterized developmental pathway that is involved in hair cell–supporting cell signaling during the development of the organ of Corti. It is possible that reprogrammed hair cells are redeploying this pathway to create a new sensory mosaic in the mature cochlea. The ability to produce both new hair cells and new supporting cells in vivo in the mature cochlea may provide a novel strategy for replacing lost hair cells in the severely damaged inner ear.

Previously, strategies to reprogram cochlear cells of the inner ear into hair cells largely focused on organ of Corti supporting cells (3, 8, 16). While these cells are an ideal target based on their proximity to hair cells, in cases of severe damage these cells are also lost and the organ of Corti is replaced by a flat cuboidal epithelium (38, 39). One hypothesis suggests that the cells of the IS and OS are the source for this flat epithelium (40) and, therefore, the ability of these cell populations to convert into hair cells and supporting cells in the mature epithelium presents a potential target for hearing restoration. Investigating the formation of the flat epithelium and whether it is responsive to transcription factor reprogramming is a crucial next step in evaluating the feasibility of this approach to hearing restoration.

Efforts toward hair cell reprogramming in the mature cochlea will likely require the modulation of a combination of multiple transcription factors in nonsensory cells and in the flat epithelium. We have found that, just as Atoh1 alone is not sufficient to reprogram supporting cells in the neonatal organ of Corti to a hair cell fate (3, 8), Atoh1 alone is also insufficient to reprogram most nonsensory cells in the mature cochlea into hair cell–like cells. Moreover, flat cochlear epithelium created by high doses of aminoglycoside antibiotics that destroy the organ of Corti is also refractory to reprogramming with Atoh1 alone (41). While Atoh1 is one of the first hair cell transcription factors to be expressed in developing hair cells, Atoh1 target genes are epigenetically silenced during the maturation of the murine cochlea and become largely inaccessible to Atoh1 within the first postnatal week (42). A second hair cell transcription factor, Pou4f3, has been recently discovered to be a pioneer factor that increases the accessibility of many Atoh1 target genes, allowing Atoh1 to bind and drive their expression (43). Moreover, the combined expression of Pou4f3 with Atoh1 increased the number of supporting cells reprogrammed into hair cell–like cells in the mature cochlea (3). Taken together, this suggests that the coexpression of Pou4f3 with Atoh1 increases the accessibility of Atoh1 target genes thus enhancing hair cell reprogramming. In primary somatic cells, the addition of a third hair cell transcription factor, Gfi1, increased the production of hair cell–like cells compared to that of combined Atoh1 and Pou4f3 expression (44, 45); however, the role of Gfi1 in reprogramming is less clear. Additional work will be required to understand the epigenetic changes that occur following combined Atoh1, Gfi1, and Pou4f3 expression compared to Atoh1 expression alone.

While we have designated reprogrammed cells as either reprogrammed hair cells or reprogrammed new supporting cells, these clusters both express markers of hair cell and supporting cell identity. We suggest two possible explanations for this phenomenon. First, as cells are being reprogrammed, they may transition through a progenitor/supporting cell period prior to becoming hair cell–like cells. This is supported by the RNA-velocity analysis which suggests that the nonsensory cells transition to a supporting cell–like state, and then onto hair cell–like cells. In addition, we found activation of Sox2, the progenitor and supporting cell marker, in cells that are not adjacent to hair cell–like cells, which suggests the presence of a transitional state resembling progenitor cells that cells pass through prior to becoming hair cell–like. This would result in the coexpression of genes associated with both hair cell and supporting cell identity within these transitional cells. It is possible that these transitional cells will decrease over time as reprogrammed cells fully commit to a hair cell or supporting cell fate. Second, the transcription factors Atoh1, Gfi1, and Pou4f3 are permanently expressed in reprogrammed cells under the control of the Rosa26 promoter in our reprogramming mice, unlike the developing cochlea where they regulate one another and affect the epigenetic state of each other’s target genes (43). It is possible that this persistent expression of hair cell transcription factors may lead to a hybrid cell state where genes regulated by Atoh1, Gfi1, and Pou4f3 may remain expressed alongside elements of the supporting cell gene network.

Of note, the Fbxo2CreERT2 mouse line we have used for this project is a knock-in mouse line that removes one copy of the Fbxo2 allele. Fbxo2 was previously identified to be involved in the maintenance of cochlear cells in mice when both copies of the allele were lost (46). Other genes, such as Sox2, have been shown to exhibit a haploinsufficient effect in the inner ear in the context of regeneration (47). Fbxo2 is a member of the ubiquitin ligase pathway and it is possible that loss of a single copy of Fbxo2 in combination with the mis-expression of Atoh1, Gfi1, and Pou4f3 could increase the responsiveness of nonsensory cells to reprogramming. Further work will be required to understand the role that Fbxo2 might play in plasticity of the inner ear.

We found that nonsensory cells along the apical–basal axis of the cochlea are differentially responsive to hair cell reprogramming factors: Cells in the apical turn of the cochlea responded much faster to reprogramming than did their middle and basal counterparts. Moreover, it takes several weeks for the reprogramming to progress from the apical turn into the basal turn. During development of the inner ear, cells exit the cell cycle in an apical to basal gradient followed a few days later by the differentiation and maturation of hair cells in a basal to apical gradient (31). Our data suggest that differences along this axis remain in the mature cochlea and investigating these differences in reprogramming responsiveness may increase our understanding of transcription factor reprogramming as a therapeutic strategy.

Methods

Fbxo2CreERT2::Rosa26tdTomato::Rosa26GAP or Fbxo2CreERT2::Rosa26tdTomato::Rosa26A mice as well as Fbxo2CreERT2::Rosa26tdTomatocontrol animals were injected with TMX (9 mg/40 g) on two consecutive days at 3 wk of age and analyzed at 5, 8, or 12 wk of age. Fluorescence histology was performed as previously described (17, 48), and samples were imaged using Zeiss, Leica, or Olympus confocal microscopy systems. Samples used for RNA sequencing were prepared following a modified protocol previously described (13). Single-cell SMART-MATQ RNA sequencing was performed and modified from the protocol previously described. All methods are described in detail in SI Appendix.

Supplementary Material

Appendix 01 (PDF)

Click here for additional data file.

Dataset S01 (XLSX)

Click here for additional data file.

Dataset S02 (XLSX)

Click here for additional data file.

Dataset S03 (XLSX)

Click here for additional data file.

Dataset S04 (XLSX)

Click here for additional data file.

We acknowledge members of the Groves lab for their help and advice. Specifically, we thank Alyssa Crowder and Hongyuan Zhang for support and assistance with animal care as well as the staff at the Cytometry and Cell Sorting Core at Baylor College of Medicine for technical assistance. This study was funded by the following grants: F32 DC019022-01 (M.M.M.), RO1 DC014832 (A.K.G.), and a Hearing Restoration Project Consortium award from the Hearing Health Foundation (A.K.G.). The project was supported by the following cores at Baylor College of Medicine: The Optical Imaging & Vital Microscopy Core with assistance from Jason Kirk; the Cytometry and Cell Sorting Core with funding from the CPRIT Core Facility Support Award (CPRIT-RP180672), and the NIH (P30 CA125123 and S10 RR024574) and assistance from Joel M. Sederstrom, Madhavi Chintalapati and Amanda White. We also acknowledge the members of the r/bioinformatics reddit and slack channels for helpful discussions and suggestions.

Author contributions

M.M.M., I.V.H., and A.K.G. designed research; M.M.M., I.V.H., Y.N., and K.Y.N. performed research; Y.N. and C.Z. contributed new reagents/analytic tools; M.M.M., I.V.H., Y.N., K.Y.N., C.Z., and A.K.G. analyzed data; and M.M.M. and A.K.G. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

Sequencing data have been deposited in GEO (GSE224627) (49). Code to analyze the sequencing data have been deposited in GitHub (50). Sequencing data can be visualized on the gEAR website (51).

Supporting Information

This article is a PNAS Direct Submission.
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