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Published in final edited form as: Hear Res. 2024 Jan 23;443:108962. doi: 10.1016/j.heares.2024.108962

Nestin-Expressing Cells are Mitotically Active in the Mammalian Inner Ear

Olivia Kalmanson 1, Hiroki Takeda 2, Sean R Anderson 3, Anna Dondzillo 1,*, Samuel Gubbels 1,*
PMCID: PMC10922748  NIHMSID: NIHMS1965362  PMID: 38295585

Abstract

Nestin expression is associated with pluripotency. Growing evidence suggests nestin is involved in hair cell development. The objective of this study was to investigate the morphology and role of nestin-expressing cells residing in the early postnatal murine inner ear. A lineage-tracing nestin reporter mouse line was used to further characterize these cells. Their cochleae and vestibular organs were immunostained and whole-mounted for cell counting. We found Nestin-expressing cells present in low numbers throughout the inner ear. Three morphotypes were observed: bipolar, unipolar, and globular. Mitotic activity was noted in nestin-expressing cells in the cochlea, utricle, saccule, and crista. Nestin-expressing cell characteristics were then observed after hair cell ablation in two mouse models. First, a reporter model demonstrated nestin expression in a significantly higher proportion of hair cells after hair cell ablation than in control cochleae. However, in a lineage tracing nestin reporter mouse, none of the new hair cells which repopulated the organ of Corti after hair cell ablation expressed nestin, nor did the nestin-expressing cells change in morphotype. In conclusion, Nestin-expressing cells were identified in the cochlea and vestibular organs. After hair cell ablation, nestin-expressing cells did not react to the insult. However, a small number of nestin-expressing cells in all inner ear tissues exhibited mitotic activity, supporting progenitor cell potential, though perhaps not involved in hair cell regeneration.

Keywords: Nestin, Hair Cell Regeneration, Organ of Corti, Utricle, Saccule, Crista

1. Introduction

Little is known about the stem cell niche of the mammalian inner ear, as limited regeneration occurs in the mammalian cochlea and vestibular organs. However, there have been some promising progenitor cells, such as Lgr5-expressing cells. Lgr-5 expressing cells appear to include a variety of supporting cell types in the greater epithelial ridge, medial to the inner hair cells (Chai et al, 2011; Shi et al, 2012; Cox et al, 2014), and have been shown to differentiate into new hair cells after insult (Cox et al, 2014; Golub et al, 2012; Bramhall et al, 2014). There is also evidence that other progenitor cell populations exist independent from Lgr5-expressing cells which can contribute to regeneration in specific contexts (Cox et al, 2014; Mellado et al, 2014; Bernal and Arranz, 2018; Xu et al, 2017; McGovern et al, 2019).

Nestin is an intermediate filament within the central and peripheral nervous systems widely viewed as a pluripotent cell marker in neural tissues including the cerebral cortex, retina, and olfactory bulb. In the central nervous system, nestin expression has been seen in glial cells, astrocytes, neurogenic cells, neural stem cells, and neural progenitor cells (Bernal and Arranz, 2018). Neurospheres, cultured isolated neural stem cells, have been shown to be rich in nestin-expressing cells (Mignone et al, 2004). The mechanism of nestin’s pluripotent ability is not fully elucidated, though its long C-terminal arm is hypothesized to link with microtubules and microfilaments to facilitate structural changes and nucleus positioning required for pluripotency (Herrmann and Aebi, 2000; Michalczyk and Ziman, 2015).

A growing body of evidence suggests that nestin may be a progenitor cell marker in the organ of Corti (Oshima et al, 2007; Lang et al, 2008; Watanabe et al, 2012). Using a Nestin-GFP mouse model, Chow and colleagues demonstrated nestin expression in cochlear and vestibular hair cells during embryonic development and subsequent downregulation as postnatal development progressed. The inner border cells located medial to inner hair cells and Deiters’ cells located under the outer hair cells, however, showed persistent nestin expression well into adulthood (Chow et al, 2016). Studying potential multipotent cells in the adult mammalian inner ear is particularly relevant to human health, as hearing and vestibular dysfunction due to hair cell loss are clinically detected after birth in the mature inner ear. Should nestin expressing cells behave as multipotent cells in the inner ear as they do in other tissues, their therapeutic potential would be substantial.

The objective of this study was to investigate nestin expression in cells residing in the mammalian inner ear. Further, we sought to determine if nestin-expressing cells could be induced to proliferate and differentiate toward hair- or supporting cell fates, thus clarifying their role as a progenitor cell in the mammalian inner ear. We hypothesized that nestin-expressing cells have proliferative capacity and would respond to hair cell ablation in the early postnatal period via transdifferentiation into primitive forms of hair- or supporting cells. To investigate our hypotheses, we employed both constitutively-expressing and lineage-tracing mouse models with and without targeted hair cell lesioning. We identified three different morphotypes of nestin-expressing cells in cochlear and vestibular tissues. A subset of Nestin-expressing cells were mitotically active in all inner ear tissues examined, but did not transdifferentiate in response to hair cell ablation, opposing our hypothesis that they played a role in hair cell regeneration.

2. Materials & Methods

2.1. Mouse Models

Animal work was approved by the Institutional Animal Care and Use Committee of the University of Colorado and within the American Physiological Society guidelines. Male and female mice were used from all mouse models. Two nestin reporter mouse models were used in the described experiments. First, a Nestin-GFP mouse containing multiple copies of a plasmid encoding green fluorescent protein (GFP) linked to the nestin promoter on a C57BL/6 background was used (Yamaguchi et al, 2000). Nestin-GFP mice were kindly provided by Dr. Xinyu Zhao from the University of Wisconsin. It was used in experiments examining early postnatal nestin expression both alone and when bred with the heterozygous Pou4f3DTR mouse (Jackson Labs strain #028673). The Pou4f3DTR mouse is a knockin strain with one copy of human diphtheria toxin receptor (DTR) inserted to the Pou4f3 locus, replacing the coding sequence and rendering cells which are dependent on expression of Pou4f3 (i.e. hair cells) to be sensitive to diphtheria toxin (DTx) (Golub et al, 2012). This crossed Nestin-GFP model is referred to hereafter as Nestin-GFP::Pou4f3DTR.

The second nestin reporter mouse is a lineage-tracing model of nestin-expressing cells. This was achieved through a mouse model with the following two genes: 1) Nestin-Cre/ERT2 (Jackson Labs strain # 016261), a transgene that encodes an estrogen-receptor-controlled Cre recombinase linked to the nestin promoter. 2) AiRosa14 (Jackson Labs strain # 007914), a knock-in gene resulting in constitutive expression of red fluorescent protein (TdTomato) if Cre recombinase is present. This lineage-tracing mouse model is referred to here as Nestin-CreER::RosaTdTomato.

A more complex mouse model was used to lineage-trace early postnatal nestin-expressing cells after targeted hair cell ablation. The lineage-tracing Nestin-CreER::RosaTdTomato mouse described above was crossed with the Pou4f3DTR mouse described earlier. This mouse model is referred to hereafter as Pou4f3DTR::Nestin-CreER::RosaTdTomato.

The breeding schemes are depicted in supplemental figure 1. The final breeding pairs used for all lineage-tracing experiments were made up of Nestin-CreER::RosaTdTomato crossed with a heterozygous Pou4f3DTR mouse. To minimize early intervention and ensure optimal survivability, the early postnatal pups were not tagged prior the experimental treatment. Rather, all pups in each litter underwent the injection protocols described below, and genotyping was performed at the time of euthanasia with tail snips.

Genotyping was performed by Transnetyx (Cordova, TN) using quantitative or real-time PCR. The primers used were CRE(+/−), DTR Tg (+/−), eGFP (+/−), Pou4f3-1 WT (+/−), ROSA WT (+/−), and Rosa-Ai14 KO (+/−).

2.2. Injection Protocol

The mice were injected with three different drugs. 1) Tamoxifen: A solution of 15mg/mL tamoxifen (Sigma-Aldrich, cat# T5648) in sunflower oil (Sigma-Aldrich, cat# S5007) with 10% ethanol was gently heated at 38°C with periodic vortexing to facilitate dissolution. It was stored at room temperature for up to one week. 2) Diphtheria Toxin: DTx (List Labs, cat# 150) was suspended in filter sterilized phosphate buffered saline (PBS) to create stock aliquots of 2mg/mL solution, stored at −80°C. One stock aliquot was used to generate final concentration aliquots of 0.4ug/mL, which were also stored at −80°C until being thawed for use. A thawed final concentration aliquot was stored at 4°C for up to one week. Importantly, thawing and refreezing the stock solution multiple times to make dilutions resulted in a nonfunctional toxin. 3) EdU: The EdU solution was made by suspending lyophilized EdU (Invitrogen, cat# A10044) in filter-sterilized PBS to a concentration of 5mg/mL. Aliquots were stored at −20°C until needed. After thawing, an aliquot was stored at 4°C for up to one week.

To characterize the early postnatal nestin-expressing cells, three Nestin-CreER::RosaTdTomato mice received tamoxifen on P2 as an intraperitoneal injection of 180ug per gram body weight, initiating lineage-tracing of nestin-expressing cells. On P3, 4ng per gram body weight of DTx was injected intramuscularly. On P5, P6, and P7, 50ug per gram body weight of EdU was injected intraperitoneally. These mice were euthanized and genotyped on P7 at least 2 hours after the last EdU injection. Their labyrinths were immediately harvested, fixed, and stored while awaiting genotyping results. Labyrinths from mice with the Nestin-CreER::RosaTdTomato genotype were used for immunohistochemistry.

To compare nestin-expressing cells in control cochleae versus cochleae after hair cell ablation in the constitutively expressing nestin reporter mice, the Nestin-GFP::Pou4f3DTR mice underwent intramuscular injections of 4ng per gram body weight DTx on postnatal day 0–1 (P0–1), initiating hair cell ablation. On P3–4 or P7, the mice were euthanized, tails were snipped for genotyping, and the labyrinths harvested for immunohistochemistry. Labyrinths from animals with the appropriate genotypes (expressed Nestin-GFP transgene and were heterozygous for Pou4f3DTR) underwent further processing with immunohistochemistry.

To compare nestin-expressing cells in control cochleae versus cochleae after hair cell ablation in the lineage tracing nestin reporter mice, three Nestin-CreER::RosaTdTomato and three Pou4f3DTR::Nestin-CreER::RosaTdTomato mice underwent tamoxifen injections on P2 and DTx injections on P3, as above. In the Pou4f3DTR::Nestin-CreER::RosaTdTomato mice, hair cell ablation was induced with the P3 DTx injection, whereas the Nestin-CreER::RosaTdTomato mice would be unaffected. On P10 they were euthanized and genotyped, and the labyrinths were harvested. Labyrinths from mice with the appropriate genotypes (Nestin-CreER::RosaTdTomato for controls and Pou4f3DTR::Nestin-CreER::RosaTdTomato for hair cell-ablated) were further processed for immunohistochemistry.

a. Immunohistochemistry

After euthanasia and decapitation, the labyrinths were immediately harvested from the temporal bones in PBS under a stereoscope and perfused with 4% paraformaldehyde (PFA) through the round and oval windows, and the whole labyrinths were post-fixed in PFA for an additional 10min before being stored in PBS at 4°C while awaiting the genotyping results (Dondzillo et al, 2021). The PFA incubation time was chosen to ensure proper fixation of the entire tissue and to avoid over-fixation, which can decrease antibody penetration in later steps. Each labyrinth from mice of the appropriate genotypes were then dissected into three cochlear turns, one utricle, one saccule, and one crista. Mice in the hair cell ablation experiments only underwent cochlear dissection (Supplemental table 1). All harvested tissues were immunostained and whole mounted onto glass slides.

Primary antibodies used in this experiment include rabbit-anti-MyosinVIIa (Proteus biosciences, RRID AB_10015251, 1:200), and conjugated rabbit anti-Sox2-647 (Santa Cruz Biotechnology, RRID AB_10842165, 1:100). Secondary antibodies were the following Alexa Fluor products: goat-anti-rabbit-405 IgG H+L (Invitrogen, 1:1500), goat-anti-rabbit-488 IgG H+L (Invitrogen, 1:1500), and goat-anti-mouse-647 IgG2a (Invitrogen, 1:1500). EdU labeling was performed with Click-iT EdU Alexa Fluor imaging kits for both 488 and 594 (Invitrogen).

The cochlear turns and vestibular organs were imaged on a Zeiss LSM 780 confocal microscope equipped with a 100x oil objective, numerical aperture (N.A.) 1.4, and the digital zoom set to 0.7 (voxel size x,y = 0.119 μm2, z = 0.4 μm) to obtain stacks of images. Each cochlear turn was imaged in one or two locations in areas of high concentration of nestin-expressing cells. The utricles and saccules were imaged in one central location, which represented on average 45% of the total macular area. The cristae were imaged adjacent to the eminentia cruciatum. The laser and photomultiplier settings were adjusted to capture the entire length of the cellular projections of the nestin-expressing cells, which at times resulted in saturation of the brighter cell body.

Supplemental Table 1 outlines the number of mice and scans included in the data analysis. Three mice were selected for the sample size of each group. A large volume of data was collected from each mouse through dense sampling of bilateral cochlear and vestibular organs.

2.4. Data Analysis

Cells were manually counted from the z-stack confocal images and statistically analyzed in version 4.2.1 of R software (R Core Team, 2020). Density of nestin-expressing cells and their morphotypes were calculated as the number of nestin-expressing cells in a defined region. In the cochlea, this was calculated per 100um length of the organ of Corti, measured as a straight line along the arc of inner hair cells within the sensory epithelium. In the utricle and saccule, this was calculated per 100um2 of macular organ, typically equating to the scanned area. In the cristae, this was calculated per 100um length of the crista sensory epithelium, measured as a straight line along the supporting cell layer. These cell density counts are expressed in the results section as mean ± standard deviation.

In addition to nestin-expressing cell density, the breakdown of the morphotypes were calculated. A mixed-effects analysis of variance (ANOVA) was implemented via version 1.1–29 of the lme4 package in R (Bates et al, 2015). Paired comparisons were completed via t-tests using estimated marginal means and Tukey corrections in version 1.7.5 of the emmeans package (Length, 2022). For the ANOVA and paired comparisons, Kenward-Roger estimated degrees of freedom were used (Kenward and Roger, 1997). Finally, non-parametric (i.e., Spearman) correlation was used to compare EdU- and nestin-expression across cell types and organs. The acceptable type I error rate (alpha level) was 0.05. The assumption of normality was confirmed via the Shapiro-Wilkes test and the assumption of homogeneity of variance for paired comparisons was confirmed via Levene’s test. All assumptions were also confirmed via model diagnostic plots. For additional details on the approach to the selected statistical analysis, see the supplemental materials.

Comparisons of the proportion of hair cells expressing nestin between control cochleae and cochleae after hair cell ablation in constitutively expressing Nestin-GFP mice at P3 and P7 were evaluated with two-way ANOVA.

Graphs were generated using version 3.3.6 of the ggplot2 package in R (Wickham 2016) or version 9.5.1 of Graphpad Prism (San Diego, CA). Figures in this manuscript are cuts or projections of the xy confocal images along the z axis or orthogonal views in the xz or yz plane.

3. Results

3.1. Breeding Outcomes

The yield of Nestin-CreER::RosaTdTomato and of Pou4f3DTR::Nestin-CreER::RosaTdTomato pups was one in eight pups for each. The breeders bore small litters with a typical amount of cannibalism.

3.2. Nestin-expressing cell Morphotypes

Nestin-CreER::RosaTdTomato mice were used to characterize nestin-expressing cell morphotypes. This model results in nestin-expressing cells and all their progeny to fluoresce red, allowing observation of the resident nestin-expressing cell fate, even if they subsequently lose nestin expression such as by transdifferentiating into a terminal cell type. Tamoxifen injection at P2 activated Cre recombinase in nestin-expressing cells, resulting in constitutive TdTomato expression in that cell and all its progeny. Nestin-expressing cells were examined in the cochlea, utricle, saccule, and crista. Based on the observed differences between Nestin-expressing cells, three morphologic subtypes were identified: bipolar, unipolar, and globular.

Bipolar cells have a cell body flanked by two elongated ends oriented radially (Fig. 1A). This morphotype was located in the cochlear spiral lamina and among the neurites exiting the vestibular organs. Unipolar cells have one or two elongations on a single side of the cell body reaching toward the hair cells (Fig. 1B). They were located outside the sensory epithelium, immediately below and medial to the supporting cells under the cochlear inner hair cells and below the supporting cell layer in the vestibular organs. Globular cells lacked any polarity. Some were globular in shape without any projections (Fig. 1C), while others were astrocytic with many short projections irregularly oriented around the cell body. These were located among the cochlear supporting cells under the inner hair cells and occasionally within the vestibular organs.

Figure 1. Nestin-expressing cell morphotypes and mitotic activity.

Figure 1.

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NestinCreER::RosaTdTomato mice lacking the Pou4f3DTR gene, and therefore remained unlesioned, were used in this figure. Panels A-C demonstrate examples of the different morphotypes of nestin-expressing cells, all taken from the cochlea. Nestin-TdTomato is magenta, MyoVIIa is yellow, and Sox2 is white. Some nestin-expressing cells exhibited two elongated ends extending radially along the spiral lamina, deemed a “Bipolar” morphotype (A). Others exhibited single-sided extensions reaching up among the supporting cells immediately under the inner hair cells, deemed a “Unipolar” morphotype (B). Few nestin-expressing cells lacked any directionality, ranging from globular-appearing to astrocytic, deemed a “Globular” morphotype (C). These three morphotypes were identified in cochlear and vestibular tissue. Panels D-G demonstrate the proliferative capacity of nestin-expressing cells. Nestin-TdTomato is again magenta, Sox2 is white, and MyoVIIa is yellow to highlight hair cells. Orthogonal views of a cochlea (D), utricle (E), saccule (F), and crista (G) demonstrating colabeling (white arrows) of TdTomato (magenta) with 5-ethynyl-2´-deoxyuridine (EdU, blue). Violin plots in (H) show all nestin-expressing cells (upper) and those that colabel with EdU (lower). Unipolar cells were the most likely morphotype to label with EdU across all organs. There was no statistically significant difference in EdU colabeling between organs (cochlea versus vestibular).

In the cochlea, the nestin-expressing cell density was an average of 8.8±4.3 cells per 100um. The percentage breakdown of cochlear morphotypes was calculated by dividing the density of a morphotype by the density of all nestin-expressing cells in that organ. These varied greatly, with an average of 40.4±13.4% bipolar cells, 53.3±12.2% unipolar cells, and 6.0±8.2% globular cells.

In the vestibular organs, the unipolar subtype was consistently dominant. The utricular nestin-expressing cell density was an average of 32.9±18.1 cells per 100um2. The percent breakdown of utricular morphotypes was 15.0±10.4% bipolar cells, 82.6±7.2% unipolar cells, and 2.4±3.7% globular cells. In the saccule, the nestin-expressing cell density was an average of 29.0±9.6 cells per 100um2. The breakdown of saccular morphotypes was 9.9±6.0% bipolar cells, 88.1±7.4% unipolar cells, and 2.0±3.3% globular cells. In the crista, the nestin-expressing cell density was an average of 10.6±9.9 cells per 100um. The breakdown of crista morphotypes was 17.2±19.1% bipolar cells, 80.0±18.6% unipolar cells, and 2.8±4.6% globular cells.

To assess the nestin expression within the cochlea and vestibular system, a mixed-effects ANOVA was used, containing fixed-effects of cell type (unipolar, bipolar, and globular) and organ (cochlea or vestibular system) and dependent variable the cube-root of nestin-expressing cells within 100 μm. The results indicated a significant effect of cell type [F(2,102)=60.579, p<0.0001], but no significant effect of organ [F(1,102)=1.720, p=0.193]. Paired comparisons revealed that there were significantly more unipolar cells than bipolar cells [t(102)=5.253, p<0.0001] and globular cells [t(102)=11.003, p<0.0001]. There were also more bipolar cells than globular cells [t(102)=5.751, p<0.0001]. Including a random effect of animal and cube-root transformed data allowed for use of the raw data rather than percentages in the model. No additional variables were assessed because when they were included in the model, they resulted in substantial deviation from the assumption of linearity and showed bimodal residuals, which resulted because of floor effects associated with cell/morphotype counts of zero. Thus, co-varying factors like ear, cochlear turn, or vestibular organ were treated as replicate observations. To see the breakdown of nestin-expression by cochlear turn and vestibular organ, see Fig. 1H.

3.3. Mitotic Activity

NestinCreER::RosaTdTomato mice were used to evaluate mitotic activity of nestin-expressing cells in cochlear and vestibular epithelia. EdU injections were performed, which labels the nuclei of mitotically active cells with a nucleic acid analog, allowing for subsequent fluorescent labeling. Mitotically active nestin-expressing cells were identified in all tissue types examined: basal, middle, and apical cochlear turns, utricle, saccule, and crista (Fig. 1D–G). Bipolar and unipolar subtypes were seen to incorporate EdU.

In the cochlea, colabeling with EdU occurred in an average of 13.1±11.5% of nestin-expressing cells, made up of 45.8±39.6% bipolar morphotype and 54.2±39.6% unipolar morphotype. No globular morphotypes colabeled with EdU in the isolated cochleae.

In the utricle, EdU colabeled with an average of 3.7±4.5% of nestin-expressing cells, made up of 17.9±35.7% bipolar morphotype and 82.1±35.7% unipolar morphotype. No globular morphotypes colabeled with EdU in the isolated utricles.

In the saccule, EdU colabeled with an average of 7.9±9.2% of nestin-expressing cells, 100% of which were the unipolar morphotype. No bipolar or globular morphotypes colabeled with EdU in any isolated saccules.

In the crista, EdU colabeled with an average of 15.3±15.3% of nestin-expressing cells, made up of 17.0±2.9% bipolar morphotype and 83.1±2.9% unipolar morphotype. No globular morphotypes colabeled with EdU in any of the isolated cristae.

It was not possible to fit a model to the data for EdU-expressing cells without violating assumptions or substantially reducing statistical power because of the large number of images containing no EdU-expressing cells. However, as shown in Fig. 1H, there was a large correspondence between nestin- and EdU-expression. Thus, as a compromise, a non-parametric correlation was computed, wherein the data were ranked and the degree to which they corresponded to one another was assessed. This avoids the necessity of a linear relationship that is clearly absent given the floor effects in Fig. 1H. The results revealed a high correspondence between nestin- and EdU-expression [ρ=0.635, p<0.0001]. In grouping data from all animals together, we violate the assumption of independence associated with the Spearman rank coefficient. As an alternative approach, we also computed the non-parametric correlation for each animal. Results showed that mouse 1 [ρ=0.456, p=0.005], mouse 2 [ρ=0.725, p<0.0001], and mouse 3 [ρ=0.583, p=0.0002] all showed this relationship. No corrections were made to p-values because they were far below the threshold for significance (0.05).

3.4. Nestin-expressing cells in cochleae with and without hair cell ablation

3.4.1. Nestin-GFP reporter mouse model

Initial experiments were performed with Nestin-GFP and Nestin-GFP::Pou4f3DTR mice to assess cochlear nestin expression in the early postnatal period and in response to hair cell ablation (Fig. 2). In this mouse, cells fluoresce green at the time of nestin expression and lose fluorescence if nestin expression stops. Additionally, DTx is used in this model to selectively ablate hair cells and observe nestin-expressing (GFP+) cells. Control cochleae demonstrated some nestin-expressing hair cells at P3, as seen in a whole-mounted organ of Corti imaged through the hair cell nuclei (Fig. 2A) and in an orthogonal view of the same mouse (Fig. 2B). After hair cell ablation, there were fewer hair cells and a higher proportion of nestin-expressing hair cells present in the organ of Corti, see in the xy plane (Fig. 2C) and orthogonal plane (Fig. 2D). Statistical evaluation with 2-way ANOVA revealed a significantly higher percentage of nestin+ hair cells in cochleae after hair cell ablation compared to controls (p=0.0005 at P3, p=0.0006 at P7) (Fig. 2E). At P3, there were Nestin-expressing hair cells present in the apical turns of control cochleae and in all turns of cochleae after hair cell ablation. At P7, there were no Nestin-expressing hair cells in the control cochleae, while they were present in all turns of the cochleae after hair cell ablation, though predominantly in the apical and middle turns (Fig. 2F). Further characterization on the P7 animals revealed that 75% of nestin expression in hair cells was identified in outer hair cells, and 25% in inner hair cells. After hair cell ablation, 25% of outer hair cells and 38% of inner hair cells expressed nestin. To more thoroughly investigate the potential regenerative capacity of Nestin-expressing cells, we used the lineage-tracing mouse model.

Figure 2. Patterns of nestin-expression after hair cell ablation in constitutively expressing Nestin-GFP model.

Figure 2.

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Pups of the genotypes Nestin-GFP (control) and Nestin-GFP::Pou4f3DTR (hair cell-ablated) underwent diphtheria toxin (DTx) injection on P1 and were euthanized on P3 or P7. Control cochlea demonstrated some nestin-expressing hair cells at P3, as seen in a whole-mounted organ of Corti imaged through the hair cell nuclei (A) and in an orthogonal view of the same mouse (B). After hair cell ablation, there were fewer hair cells and a higher proportion of nestin-expressing hair cells present in the organ of Corti, see in the xy plane (C) and orthogonal plane (D). There was a significantly higher percentage of nestin-expressing hair cells after hair cell ablation than in control cochleae at both P3 and P7 (E). The nestin-expressing hair cells were predominantly located in the middle and apical turns at both P3 and P7 (F).

3.4.2. Lineage-tracing mouse model

To fully evaluate the role of nestin-expressing cells in hair cell regeneration, a lineage-tracing model was employed. Because all progeny of nestin-expressing cells are also fluorescent in this model, it enabled differentiation between transient nestin expression in hair cells during regeneration and replacement of hair cells through transdifferentiation of nestin-expressing cells. Further, because fluorescence is induced by tamoxifen administration, only cells which express nestin postnatally (on P2) were evaluated. In this experiment, the Pou4f3DTR::Nestin-CreER::RosaTdTomato mouse is the experimental animal undergoing hair cell ablation, and the control mouse was Nestin-CreER::RosaTdTomato. Both genotypes resulted from the same litters and received the same injections, but the control mouse (Nestin-CreER::RosaTdTomato) would not experience hair cell ablation in response to DTx administration due to the absence of the Pou4f3DTR gene.

The Pou4f3DTR::Nestin-CreER::RosaTdTomato pups underwent cochlear hair cell ablation, and the nestin-expressing cells were observed during hair cell repopulation. A total of three control mice (Nestin-CreER::RosaTdTomato, six ears) and three mice after hair cell ablation (Pou4f3DTR::Nestin-CreER::RosaTdTomato, six ears) were used in this experiment (Supp. Table 1), in which diphtheria toxin was injected on P3.

With the lineage-tracing model, there were no cells coexpressing MyosinVIIa and nestin, unlike in the constitutively expressing Nestin-GFP model. When compared with the control organs of Corti (Fig. 3A), cochleae after hair cell ablation demonstrated round hair cells with a disorganized organ of Corti (Fig. 3B), similar to what has been captured by other investigators (Cox et al, 2014; Bramhall et al, 2014). This indicated that either hair cells in the apical turn resist cell death by DTx or are repopulating, again as shown by other investigators (Cox et al, 2014; Bramhall et al, 2014). In the basal turn, the control mice (Nestin-CreER::RosaTdTomato) had 45.19 myosinVIIa-staining hair cells per 100μm, while the mice with hair cell ablation (Pou4f3DTR::Nestin-CreER::RosaTdTomato) exhibited only 9.48 hair cells per 100μm (p=0.0014). In the middle turn, control cochleae had 43.85 hair cells per 100μm, while the cochleae after hair cell ablation had 23.80 hair cells per 100μm (p=0.0080). In the apical turn, the control cochleae had 45.35 hair cells per 100μm, while the cochleae after hair cell ablation had 38.86 hair cells per 100μm (p=0.1353) (Fig. 3C). Again, the hair cells in the apical turn after DTx administration appeared disorganized and unlike the apical hair cells of the control animals, suggesting they are new hair cells rather than hair cells which survived after DTx. None of the new hair cells expressed TdTomato, implying that the regenerated hair cells did not result from the resident nestin-expressing cells. The nestin-expressing cells were present in the same locations and densities in control cochleae and cochleae with hair cell ablation.

Figure 3. Response of nestin-expressing cells to hair cell ablation in lineage-tracing model.

Figure 3.

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Pups of the genotypes Nestin-CreER::RosaTdTomato (control) and Pou4f3DTR::Nestin-CreER::RosaTdTomato (hair cell-ablated) underwent tamoxifen injection on P2 to induce TdTomato expression in nestin-expressing cells, diphtheria toxin injection on P3 to ablate hair cells, and were euthanized on P10. In assessing control cochleae (A) versus cochleae with hair cell ablation (B), no hair cells expressed nestin-TdTomato, and present hair cells appeared disorganized. The disorganized hair cells appeared most frequently in the apical turns and least commonly in the basal turns (C). Additionally, the violin plot in (D) shown there was no difference in predominance of nestin-expressing cell morphotype (bipolar vs unipolar vs globular) or location (basal vs middle vs apical turns).

Specifically, the density of nestin-expressing cells did not change significantly between control mice and mice after hair cell ablation of the same age. The density of nestin-expressing cells in control cochleae is 9.3 ± 4.5 cells per 100um, and the density in cochleae after hair cell ablation is 4.9 ± 3.2 cells per 100um. While these density values appear to differ considerably, the difference is not significant when the litter was taken into account, as some litters exhibited more TdTomato expression and therefore more fluorescent nestin-expressing cells than others.

The breakdown of the morphotypes was also compared between control cochleae and cochleae after hair cell ablation and found not to differ (Fig. 3D). Bipolar cells made up 23.2±13.7% and 30.1±26.0% of control and hair cell-ablated cochlear nestin-expressing cell populations, respectively. Unipolar cells made up 53.6±20.6% and 47.4±28.8% and globular cells made up 23.2±13.6% and 22.5±23.8%, respectively.

To assess the nestin-expression within the cochlea in control mice and mice after hair cell ablation, a mixed-effects ANOVA was used, containing fixed-effects of treatment group (hair cell-ablated or control), cochlear turn (apical, middle, and basal), cell type (unipolar, bipolar, and globular), and their interactions with dependent variable the square-root of nestin-expressing cells within 100 μm. The results indicated only one significant effect of cell type [F(2,185)=31.499, p<0.0001]. Treatment group [F(1,4)=2.736, p=0.238] and cochlear turn [F(2,185)=2.736, p=0.067] were not significant, contrary to the hypothesis that nestin-expressing cells played a role in hair cell regeneration. Paired comparisons revealed that there were significantly more unipolar cells than bipolar cells [t(185)=6.306, p<0.0001] and globular cells [t(185)=7.328, p<0.0001]. There were was no significant difference between the number of bipolar and globular cells [t(185)=1.022, p=0.564]. Notably, the interaction between treatment group and cell type was just above the threshold for significance [F(2,185)=2.408, p=0.093]. This effect was in the opposite direction of the hypotheses, with the control group showing more unipolar nestin-expressing cells than the group with hair cell ablation. This suggests that the lack of effect was not due to the statistical procedures. Including a random effect of animal and square-root transformed data allowed for use of the raw data rather than percentages in the model. Co-varying factors like ear were treated as replicate observations.

4. Discussion

This series of experiments investigated nestin-expressing cells in the early postnatal inner ear tissues of mice. The constitutively-expressing nestin reporter model, Nestin-GFP mice, demonstrated nestin expression within native cochlear hair cells. The lineage-tracing nestin reporter model, Nestin-CreER::RosaTdTomato mice, did not. The lineage-tracing model was more specific to the postnatal inner ear, and was used to examine the resident nestin-expressing cells. The cochleae and vestibular organs were included in this evaluation. Three morphologic subtypes of nestin-expressing cells were identified, which varied in number between the various inner ear tissues (Fig. 1). Not all nestin-expressing cells were within the sensory epithelium; for example, bipolar cells were not. However, work from Hakuba et al demonstrated that injected nestin-expressing cells demonstrated tropism to the region below the inner hair cells via the tunnel of Corti, and may be protective against ischemic damage (Hakuba et al, 2005).

The motivation behind using the lineage-tracing mouse model occurred after seeing nestin expressed in early postnatal hair cells both in a normally developing cochlea (Fig. 2A,B) and in a cochlea recovering from hair cell ablation (Fig. 2C,D) in the Nestin-GFP::Pou4f3DTR mice. Further, the cochlea with hair cell ablation had a higher proportion of nestin-expressing hair cells than control cochleae (Fig. 2E). Prior studies have also noticed nestin expression in early postnatal hair cells that is lost in mature cochleae through both mRNA analysis (Kolla et al, 2020) and genetic reporter models (Chow et al, 2016). These results are reminiscent of findings from Mignone et al, who found that ex vivo neurospheres of nestin-expressing cells lose nestin expression as the cells differentiate (Mignone et al, 2004). Expression of nestin in developing hair cells suggests that they could be transdifferentiating from resident nestin-expressing cells, but it is also possible that hair cells transiently express nestin during transdifferentiation from other progenitor cells, or that the nestin-expressing hair cells are more likely to survive ablation with diphtheria toxin. The lineage-tracing mouse model (Nestin-CreER::RosaTdTomato) distinguished between these possibilities by permanently labeling all nestin-expressing cells and their progeny with TdTomato at P2.

In the control cochleae of Nestin-CreER::RosaTdTomato mice, there were no hair cells which expressed nestin. This suggests that the nestin-expressing hair cells seen in the Nestin-GFP model fluoresced green from persistent GFP expression after nestin-expression had ceased, sometime prior to P2. This is consistent with the findings in the Nestin-GFP model, which exhibited loss of GFP in hair cells before P7. However, an alternative hypothesis for the difference in results between the Nestin-GFP and Nestin-CreER::RosaTdTomato models could lie in the regulatory elements which may or may not have been included in each construct, causing varying expression patterns.

In the cochleae of Pou4f3DTR::Nestin-CreER::RosaTdTomato mice which underwent hair cell ablation, there was robust hair cell repopulation in the apical turns compared with other reports (Mahrt et al, 2013). However, none of the new hair cells repopulating the lesioned organ of Corti expressed TdTomato labeling. This outcome suggests that the regenerated cochlear hair cells do not arise from a reservoir of nestin-expressing cells, and that the nestin expression seen in the early postnatal Nestin-GFP mouse model is likely due to either transient expression of nestin within developing hair cells or survival of hair cells which recently expressed nestin. When the density of nestin-expressing cells was examined, no difference was noted between control cochleae and cochleae after hair cell ablation, suggesting that even if the nestin-expressing cells are multiplying in response to hair cell ablation, the process is counteracted by the cell death. This finding is in contrast to the work of Watanabe and colleagues who found an increase in the area of nestin-positive cells after noise-induced lesioning of the organ of Corti (Watanabe et al, 2012). The discrepancy between ours and Watanabe findings might originate from the differences in the quantification or the lesioning methods used. Finally, the breakdown of the morphologic subtypes was evaluated in both groups, and no change in the ratio of the subtypes was observed between control cochleae and cochleae with hair cell ablation. It appeared that hair cell ablation using diphtheria toxin does not trigger changes in the cochlear nestin-expressing cells.

The potential of inner ear nestin-expressing cells to act as progenitor cells for other cell types (non-hair cells) is not lost, however. Nestin is thought to be essential in facilitating proliferation and self-renewal by assisting with nucleus positioning and mediating interactions between intermediate filaments, microtubules, and microfilaments (Bernal and Arranz, 2018; Herrmann and Aebi, 2018; Chou et al, 2003). Our findings here of postnatal EdU colabeling with nestin across multiple inner ear tissues demonstrate the presence of mitotic activity, a key feature of nestin+ stem cells from other tissues (Bernal and Arranz, 2018). In our study, nestin-expressing cells demonstrated mitotic activity through EdU labeling in all tissue types examined: the cochlea, utricle, saccule, and crista. There was a predominance of unipolar subtypes, which were also the most mitotically active subtypes.

Nestin-expressing cells of the embryonic murine spiral ganglia were previously noted to colabel with EdU (Chow et al, 2016), suggesting further investigation into the inner ear ganglia is warranted. Scarpa’s ganglion in rodents has demonstrated considerable plasticity, which may be a major player in the functional recovery achieved in the vestibular system (Travo et al, 2012), but the etiology behind this plasticity is unknown. In the zebrafish statoacoustic ganglion, the spiral ganglia equivalent, nestin-expressing cells act as a pool of progenitor cells (Schwarzer et al, 2020). Whether Nestin-expressing cells may act as neuronal progenitor cells in the spiral and Scarpa’s ganglia could have considerable implications for the success of cochlear and vestibular implants.

Limitations of this study include using the qualitative appearance of disarrayed MyosinVIIa-stained hair cells in the organ of Corti after ablation as a marker for regeneration. Additionally, the mouse models used in this study required strict breeding schemes and bore small litters, resulting in slow and laborious data collection. However, our group sizes (6 cochleae/utricles/saccules/cristae) are within the range of other seminal work in this field (Cox et al, 2014; Golub et al, 2012). The statistical results suggest that, at least for within-subject variables like cell type, there was high correspondence between the results and inferences. This may be in part due to the high suitability of mixed-effects ANOVA for the unique challenges in this study (multiple levels of nested data), as well as the choice to use procedures that did not reduce statistical power. Test assumptions were checked rigorously and met despite these challenges. Results were consistent across animals within- and between-groups as evidenced in figures 1 and 3.

5. Conclusions

In conclusion, we have enhanced the understanding of inner ear nestin-expressing cells in the cochlear and vestibular epithelia using both a constitutively-expressing nestin reporter mouse model as well as a novel, complex lineage-tracing mouse model. Nestin-expressing cells were identified in the cochlea, utricle, saccule, and crista. Three morphologic subtypes were identified and further analyzed with respect to their inner ear distribution, proliferative capacity, and response to hair cell ablation. After precise hair cell ablation in the organ of Corti, new hair cells did not arise from resident nestin-expressing cells. The cochlear nestin-expressing cells did not appear to react to this specific insult. However, nestin-expressing cells in all inner ear tissues exhibited some degree of mitotic activity.

Supplementary Material

1
2

Supplemental Figure 1. Breeding Scheme. Hemizygous Nestin-CreER mice were bred with homozygous RosaTdTomato mice to obtain Nestin-CreER::RosaTdTomato pups in the F1 generation. F1 mice were bred with heterozygous Pou4f3DTR mice to obtain a mix of genotypes, including Pou4f3DTR::Nestin-CreER::RosaTdTomato (experimental mice with hair cell ablation) and Nestin-CreER::RosaTdTomato (control mice).

3

Supplemental Table 1. Description of sample sizes. For characterizing nestin-expressing cell morphotypes and proliferative capability with EdU, the labyrinths from three control (Nestin-CreER::RosaTdTomato) mice were used. Three cochlear turns (apical, “A”, middle “M”, and basal “B”), the utricle (U), saccule (S), and one crista (C) from each labyrinth were used for immunohistochemistry. To evaluate the role of nestin after hair cell ablation, apical (A), middle (M), and basal (B) turns of the cochleae from three control mice (Nestin-CreER::RosaTdTomato) and three mice with hair cells ablated (Pou4f3DTR::Nestin-CreER::RosaTdTomato) were used. Each organ was imaged in one or two locations. As a result, a total of 105 scans in 18 ears and 9 mice were performed.

Highlights.

  • Nestin is associated with pluripotency and is expressed in the inner ear

  • A lineage-tracing model tracked nestin-expressing cells in early postnatal mice

  • Nestin-expressing cells are mitotically active in cochlear and vestibular tissues

  • Nestin-expressing cells did not facilitate hair cell regeneration after ablation

  • Further research into the mitotic activity of nestin-expressing cells is warranted

Acknowledgements

Imaging was performed in the Advanced Light Microscopy Core Facility of the NeuroTechnology Center at the University of Colorado Anschutz Medical Campus, which is supported in part by Rocky Mountain Neurological Disorders Core Grant (P30 NS048154) and by Diabetes Research Center Grant (P30 DK116073).

Funding Sources

Author OK received grant funding from the NIH/NIDCD (#T32 DC-012280) and the University of Colorado School of Medicine (Women in Surgery grant), which were used to perform the research in this manuscript. SG received funding from the NIH/NIDCD (#R01 DC013912) for this work.

Footnotes

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Conflicts of Interest

Authors report no conflict of interest relevant to this manuscript.

Declaration of Interest: Sean R. Anderson is an employee of Cochlear, Ltd.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1965362-supplement-1.docx (13KB, docx)
2

Supplemental Figure 1. Breeding Scheme. Hemizygous Nestin-CreER mice were bred with homozygous RosaTdTomato mice to obtain Nestin-CreER::RosaTdTomato pups in the F1 generation. F1 mice were bred with heterozygous Pou4f3DTR mice to obtain a mix of genotypes, including Pou4f3DTR::Nestin-CreER::RosaTdTomato (experimental mice with hair cell ablation) and Nestin-CreER::RosaTdTomato (control mice).

NIHMS1965362-supplement-2.tiff (247.1KB, tiff)
3

Supplemental Table 1. Description of sample sizes. For characterizing nestin-expressing cell morphotypes and proliferative capability with EdU, the labyrinths from three control (Nestin-CreER::RosaTdTomato) mice were used. Three cochlear turns (apical, “A”, middle “M”, and basal “B”), the utricle (U), saccule (S), and one crista (C) from each labyrinth were used for immunohistochemistry. To evaluate the role of nestin after hair cell ablation, apical (A), middle (M), and basal (B) turns of the cochleae from three control mice (Nestin-CreER::RosaTdTomato) and three mice with hair cells ablated (Pou4f3DTR::Nestin-CreER::RosaTdTomato) were used. Each organ was imaged in one or two locations. As a result, a total of 105 scans in 18 ears and 9 mice were performed.

NIHMS1965362-supplement-3.doc (61.5KB, doc)

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