Abstract

   Mesenchymal stromal cells (MSCs) are an adult derived stem cell-like
   population that has been shown to mediate repair in a wide range of
   degenerative disorders. The protective effects of MSCs are mainly
   mediated by the release of growth factors and cytokines thereby
   modulating the diseased environment and the immune system. Within the
   inner ear, MSCs have been shown protective against tissue damage
   induced by sound and a variety of ototoxins. To better understand the
   mechanism of action of MSCs in the inner ear, mice were exposed to
   narrow band noise. After exposure, MSCs derived from human umbilical
   cord Wharton’s jelly were injected into the perilymph. Controls
   consisted of mice exposed to sound trauma only. Forty-eight hours
   post-cell delivery, total RNA was extracted from the cochlea and RNAseq
   performed to evaluate the gene expression induced by the cell therapy.
   Changes in gene expression were grouped together based on gene ontology
   classification. A separate cohort of animals was treated in a similar
   fashion and allowed to survive for 2 weeks post-cell therapy and
   hearing outcomes determined. Treatment with MSCs after severe sound
   trauma induced a moderate hearing protective effect. MSC treatment
   resulted in an up-regulation of genes related to immune modulation,
   hypoxia response, mitochondrial function and regulation of apoptosis.
   There was a down-regulation of genes related to synaptic remodeling,
   calcium homeostasis and the extracellular matrix. Application of MSCs
   may provide a novel approach to treating sound trauma induced hearing
   loss and may aid in the identification of novel strategies to protect
   hearing.

   Keywords: noise trauma, hearing loss, mesenchymal stroma cells,
   Wharton’s jelly, cochlear transcriptome, hearing protection

Introduction

   Sound trauma can lead to hearing loss with protean manifestations. With
   exposures ranging from acute blast injury to chronic noise exposure, a
   wide range of pathological effects are induced in the cochlea. The
   degree of injury that occurs is related to the energy and frequency of
   the input sound combined with the time of exposure ([35]Le et al.,
   2017). At higher levels of sound exposure, damage to stereocilia and
   loss of hair cells in a basal to apical gradient occurs resulting in a
   permanent threshold shift. Damage to the spiral ganglion can occur
   through excitotoxicity, inflammation and loss of trophic support
   ([36]Chen et al., 1997; [37]Puel et al., 1997; [38]Lin et al., 2011).
   Additionally, severe sound trauma results in degradation of the blood
   labyrinthine barrier. Sound trauma therefore causes a range of cellular
   damage that includes both hair cells and spiral ganglion neurons as
   well as changes in the cochlear blood supply. The molecular mechanisms
   involved in these processes have been extensively investigated. Initial
   sound overexposure results in oxidative stress with overproduction of
   reactive oxygen and nitrogen species leading to hair cell lipid
   membranes peroxidation and disruption of mitochondrial homeostasis
   ([39]Yamane et al., 1995). The mitochondrion has also been implicated
   as a primary generator of free radicals and appears to be intimately
   involved in the generation sound trauma related pathophysiology
   ([40]Kurabi et al., 2017). Multiple studies have shown that antioxidant
   defenses and the ability to buffer free calcium of the cells can
   prevent noise trauma ([41]Huang et al., 2000; [42]Gonzalez-Gonzalez,
   2017). Ultimately, there is an activation of a variety of
   mitogen-activated protein (MAP) kinase stress pathways including c-Jun
   N-terminal (JNK) kinase leading to programmed cell death/apoptosis.
   Apoptosis can also be activated through tumor necrosis factor alpha
   (TNFα), which is produced during sound trauma ([43]Kurabi et al.,
   2017). Other pro-inflammatory cytokines are produced in a delayed
   fashion via an nuclear factor kappa-light-chain-enhancer of activated
   B-cells (NFκB) signaling cascade ([44]Kurabi et al., 2017). More recent
   studies have focused on erythroid two related factor 2 (Nrf2) being a
   key signal mediator at the center of multiple pathways ([45]Fetoni et
   al., 2019). This molecule acts as a reactive oxygen species (ROS)
   sensor and normally exists in a bound state in the cytoplasm. It
   controls a variety of stress responses and events such as oxidative
   stress and inflammation can cause it to enter the nucleus and activate
   a variety of signaling cascades ([46]Fetoni et al., 2019). Evaluation
   of gene expression networks after sound trauma has implicated several
   key pathways in sound trauma mainly relating to inflammation, heat
   shock response and detoxification of ROS ([47]Clifford et al., 2016;
   [48]Yang et al., 2016). Changes in regulation of genes related to
   cytokine signaling and pathways related to innate immunity appear to
   play an important role in the early response to sound trauma ([49]Patel
   et al., 2013; [50]Vethanayagam et al., 2016; [51]Yang et al., 2016).
   Single cell RNAseq analysis after sound trauma demonstrates that a mix
   of adaptive and innate immune cells infiltrate the cochlea after sound
   trauma highlighting the complexity of the damage response ([52]Rai et
   al., 2020). Antioxidants and anti-inflammatory drugs clearly can
   protect against noise induced hearing loss although, given the
   complexity of the induced reactome, it is unlikely that a single
   pharmaceutical agent would adequately protect against all aspects of
   damage ([53]Rajguru, 2013; [54]Maeda et al., 2017).

   Many of the transcriptome changes seen in sound trauma are also seen in
   traumatic brain injury (TBI) and stroke, suggesting common mechanisms
   and potentially common approaches to attenuating these injuries
   ([55]Meng et al., 2017; [56]Li et al., 2020). An alternate approach to
   cochlear protection is treatment of inner ear injury with neuronal or
   mesenchymal stem cells/mesenchymal stromal cells (MSCs). MSCs have been
   demonstrated to have a wide range of immunomodulatory and protective
   effects after tissue injury and have been tested in diverse disorders
   such as stroke, acute kidney injury and myocardial infarction
   ([57]Squillaro et al., 2016; [58]Fu et al., 2017; [59]Chang et al.,
   2018; [60]Cunningham et al., 2018; [61]Harrell et al., 2019;
   [62]Tsintou et al., 2020). Fat derived MSCs have been shown to express
   growth factors in sound exposed inner ears ([63]Fetoni et al., 2014).
   Both peripheral and local delivery of MSCs have been shown to have
   protective effects in a variety of hearing loss models but the
   molecular consequences of MSC delivery after sound trauma have not been
   evaluated [Reviewed in [64]Warnecke et al. (2017); [65]Kanzaki et al.
   (2020)].

   Mesenchymal stromal cells have been postulated to work through two
   distinct mechanisms. When delivered into the peripheral circulation,
   MSCs can home into areas of damaged tissue and through a range of
   distinct signaling pathways including TGFβ, CCL2, IL6, IDO, VEGF, and
   WNT modulate inflammation and induce tissue protection and repair. It
   has been shown that in this delivery approach, the majority of injected
   MSCs are trapped in in the pulmonary circulation and are phagocytosed
   by pulmonary macrophages. This process of efferocytosis results in the
   release of factors inducing immune tolerance such as IL-10 and TGFβ,
   which may provide additional beneficial effects ([66]Galipeau and
   Sensébé, 2018). Local delivery of MSCs also induces immunomodulatory
   and protective effects through direct delivery of growth factors and
   extracellular vesicles in their secretome. Some studies suggest that
   interaction between the MSCs and the damaged tissue is needed for the
   most complete protective effects ([67]Wakabayashi et al., 2010;
   [68]Drago et al., 2013; [69]Hsieh et al., 2013). Local delivery of MSCs
   to the brain after TBI has been shown to partially normalize pathologic
   gene expression induced by trauma, resulting in a return to normal
   immune signaling, receptor mediated cell signaling, neuronal plasticity
   and glycolysis ([70]Darkazalli et al., 2017). This suggests that cell
   therapy with MSCs has the capacity to provide multi-mechanistic
   protection and repair. To evaluate the mechanisms underlying the
   effects of MSCs on the inner ear, we treated adult mice exposed to an
   acute narrow band sound trauma with Wharton’s jelly-derived MSCs (WJCs)
   directly delivered into the inner ear. Umbilical cord-derived cells
   like WJCs, were chosen since they have been shown to have a secretome
   that supports neuronal repair ([71]Hsieh et al., 2013). After
   treatment, changes in the cochlear transcriptome and in hearing
   threshold were evaluated. With this investigative approach, we
   identified up and down regulated genes in mice exposed to noise and
   treated with WJC when compared to mice exposed to noise trauma only.

Materials and Methods

   All animal procedures were approved by the Institutional Animal Care
   and Use Board. A total of 25 4 week old female C57BL/6 mice (Jackson
   Laboratories) weighing between 18 and 23 grams were utilized for the
   experiments described below.

Procurement and Expansion of Mesenchymal Stromal Cells

   Human MSCs were isolated from Wharton’s jelly of umbilical cords
   according to the protocols approved by the University of Kansas Human
   Subjects Committee (KU-Lawrence IRB approval #15402) and Stormont-Vail
   (SMV) Hospital (SMV IRB Approval conferred by IRB Chair, Jo-Ann S.
   Harris, MD). Informed consent was obtained from the patients before
   collection of the umbilical cords. Three umbilical cords (n = 3) were
   obtained from Stormont-Vail (Topeka, KS, United States). All cords were
   from males that were born at full term and delivered under normal
   delivery conditions. Human MSCs were isolated and cultured according to
   previously published protocols ([72]Mellott et al., 2016). Cells were
   cultured in medium consisting of 1% penicillin-streptomycin (Life
   Technologies), 10% fetal bovine serum (FBS) mesenchymal stem cell
   qualified (MSCq) (Life Technologies), and fibroblast basal medium
   (Lonza Group Ltd., Basel, Switzerland), and expanded to passage 5 for
   all experiments. For delivery, cells were harvested, washed in
   phosphate buffered saline (PBS) and resuspended to a concentration of 1
   × 10^6 cells/ml in sterile PBS.

Experimental Design

   Three groups were evaluated with five animals in each experimental
   condition. Group one consisted of animals (n = 5) that underwent
   assessment of baseline hearing by auditory brainstem response (ABR)
   followed by MSC delivery into the left ear. The same animals underwent
   re-evaluation of hearing with ABR 7 days post-MSC delivery. Group two
   animals were exposed unilaterally to sound followed by treatment with
   MSC (n = 5) or artificial perilymph (n = 5) at 48 h post-sound trauma.
   These animals were sacrificed 72 h post-MSC delivery for extraction of
   RNA from the treated (left-sided) cochlea. Group 3 animals were
   unilaterally exposed to sound followed by MSC (n = 5) or artificial
   perilymph (n = 5) at 48 h post-sound trauma. These animals underwent
   hearing testing with ABR at 14 days post-cell or perilymph therapy to
   evaluate rescue of hearing and thereafter sacrificed for histological
   analysis. Group 4 animals were exposed unilaterally to sound followed
   by treatment with MSC (n = 8) or artificial perilymph (n = 8) at 48 h
   post-sound trauma. These animals were sacrificed 72 h post-MSC delivery
   for extraction of RNA from the treated (left-sided) cochlea (n = 5) or
   perfused for immunohistochemistry (n = 3). An additional set of age
   matched untreated control animals underwent extraction of RNA from the
   cochlea (n = 5) or were perfused for immunohistochemistry (n = 3).

Auditory Brainstem Response Measurements

   For ABR testing, the mice were anesthetized intraperitoneally using a
   mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). Mice were then
   placed inside a double-walled, sound attenuated chamber. Body
   temperature was maintained at 37°C using a MediHeat V500Vstat Heated
   Operating Table Digital Thermostat (PECO Services). ABR measurements
   were acquired using the Smart EP program from Intelligent Hearing
   Systems. Needle electrodes were placed on the vertex (+), behind the
   left ear (−) and behind the opposite ear (ground). Tone bursts were
   presented at 4, 8, 16, and 32 kHz, with duration of 500 μs using a high
   frequency transducer. Recording was carried out using a total gain
   equal to 100 K and using 100 Hz and 15 kHz settings for the high and
   low-pass filters. A minimum of 128 sweeps was presented at 90 dB sound
   pressure level (SPL). The SPL was decreased in 10 dB steps. Near the
   threshold level, 5 dB SPL steps using up to 1024 presentations were
   carried out at each frequency. Threshold was defined as the SPL at
   which at least one of the waves could be identified in 2 or more
   repetitions of the recording.

Sound Exposure

   For the noise trauma (sound exposure), the mice were anesthetized as
   described above. Mice were then exposed to a 16 kHz pure tone presented
   at 118 dB SPL in the left ear for 4 h. Sound was delivered through a
   speaker equipped with a ribbon tweeter (Radio Shack 40-1310 Horn Super
   Tweeter). The speaker was coupled to the left ear via a short plastic
   tube, 12 mm in inner diameter and 45 mm in length. Prior to exposure,
   the sound output was calibrated using a Quest Electronics Precision
   Integrating Sound Level Meter (model 1800). The Sound Level Meter was
   calibrated using a 1000 Hz Bruel & Kjaer 4230 Sound Level Calibrator.
   After sound exposure, animals were allowed to recover for 48 h before
   initiating treatment.

Delivery of Mesenchymal Stromal Cells

   Mice were anesthetized as described above. A dorsal post-auricular
   incision was made, and the left posterior semicircular canal exposed.
   Using a microdrill, a canalostomy was created, exposing the
   perilymphatic space. Subsequently 1 μl of MSCs (1 × 10^6 cells/ml in
   sterile PBS) were injected using a Hamilton microsyringe with 0.1 μl
   graduations and a 36-gauge needle). Control animals received 1 μl
   injection of artificial perilymph into the canal. The canalostomy was
   sealed with bone wax. The mice showed no signs of vestibular
   dysfunction or head tilt post-operatively.

RNA Extraction and Analysis

   Group 2 animals (noise trauma plus MSC or perilymph treatment) were
   anesthetized with Beuthanasia 75 mg/kg (Schering-Plough Animal Health
   Corp., Union, NJ, Canada). The treated (left) cochlea was removed and
   immediately placed in RNAlater (Qiagen, cat #76104). Total RNA was
   extracted with Trizol reagent (Thermofisher, cat #15596018) and
   purified by centrifuging with phase lock heavy gel (Tiagen, cat #
   WMS-2302830). Concentration and quality of RNA were assessed using an
   Agilent Bioanalyzer 2100. Analysis of cochlear RNAs was carried out
   using the NuGen Universal Plus Assay on a NovaSeq sequencer as per
   manufacturer instructions. Trimming, quality control and alignment was
   carried out using the A.I.R. software package^[73]1 with noise damage
   only specimens as a reference and noise damage with MSC treatment as
   the analyzed data package. There were five replicates for each
   experimental condition. The C57Bl/6 mouse genome (C57Bl6NJvi/ENSEMBL
   release) was used as a reference genome. Statistical analysis was
   carried out by the NOISeq method. Up regulated and down regulated genes
   were identified and organized according to their gene ontology (GO)
   classification. A combination of the enrichment score and the false
   discovery rate (FDR) was then used to develop a gene ontology
   enrichment analysis (GOEA). Analysis of potential biological
   significance was carried out using Ingenuity Pathway Analysis (IPA)
   software (Qiagen Bioinformatics, Redwood City, CA, United States) and
   the Reactome Knowledgebase^[74]2 ([75]Jassal et al., 2019). Using the
   IPA software package, results were organized into the most common
   pathways that were altered in response to MSC treatment and which keys
   genes were up or down regulated. The most up and down regulated genes
   were additionally evaluated using Pharos software^[76]3 for
   identification of molecules in clinical use that can down or
   up-regulate the identified genes.

   Expression of bcl2 and tnf was quantified in group 4 animals. cDNA from
   the control, noise exposed and noise + MSC samples was synthesized
   using the miScript II reverse transcription kit (Qiagen, cat#218161).
   Semi quantitative PCR for bcl-2 and tnf (Origene cat MP201255;
   NM013693) was then performed on the Bio-Rad CFX using the Quantitect
   SYBR Green PCR kit (Qiagen, cat# 204141). Tranthyretin expression was
   measured using the Bio-Rad qMMuCID0006861 primer pairs.

   All reactions were performed in triplicate and the Cq value was
   determined using the threshold calculated by the Bio-Rad software. Fold
   change was calculated using the 2^–ΔΔCT method.

Histological Analysis

   Group 3 animals were anesthetized with Beuthanasia (Merck Animal
   Health) 14 days after treatment with MSCs or artificial perilymph and
   fixed via intracardiac perfusion with 4% paraformaldehyde in PBS. The
   cochleae were removed and postfixed in 2.5% glutaraldehyde, 1.5%
   paraformaldehyde in 0.1 M phosphate buffer overnight at 4°C. Cochleae
   were decalcified in 1% glutaraldehyde in 0.12 M ethylene diamine
   tetraacetic acid (ETDA) in 0.1 M phosphate buffer for 4 days at 4°C.
   The specimen were then stained with osmium 1% for 1 h. After washing in
   distilled water specimens were dehydrated in a graded series of
   ethanol. After 30 min in propylene oxide (obtained from Electron
   Microscopy Sciences, Hatfield, PA, United States #20401), cochleae were
   transferred to araldite/P mix (obtained from Electron Microscopy
   Sciences, Hatfield, PA, United States #10900) and kept on a rotating
   shaker overnight. Specimen were then embedded in araldite in a vacuum
   oven. The blocks were baked in a 60°C oven. Serial sections at 30 μm
   were performed and stained with toluidine blue. After air drying,
   sections were mounted on slides with Permount for microscopic analysis.

   Fog group 4 animals, the temporal bones were removed, the stapes
   extracted, and the round window was opened. The temporal bones were
   postfixed overnight in 4% paraformaldehyde in PBS at 4°C. After rinsing
   in PBS three times for 30 min, the temporal bones were decalcified in
   10% ETDA for 48 h. The temporal bones were rinsed in PBS, dehydrated,
   and embedded in paraffin. Ten μm sections were cut in parallel to the
   modiolus, mounted on Fisherbrand^® Superfrost^®/Plus Microscope Slides
   (Fisher Scientific, Pittsburgh, PA, United States) and dried overnight.
   Samples were deparaffinized and rehydrated in PBS two times for 5 min,
   then three times in 0.2% Triton X-100 in PBS for 5 min and finally in
   blocking solution 0.2% Triton X-100 in PBS with 10% FBS for 30 min at
   room temperature. Sections underwent antigen retrieval in a microwave
   using 1:10 Dako^® Target Retrieval Solution (DAKO Corporation,
   Carpinteria, CA, United States) for 30 s. Immunohistochemistry was
   carried out with anti-Bcl-2 rabbit polyclonal antibody diluted 1:200
   (BD Biosciences, Inc., San Jose, CA, United States). The tissue was
   incubated overnight at 4°C in a humid chamber. After three rinses in
   PBS, immunohistochemical detection was carried out with an anti-Rabbit
   IgG (1:50; Vectastain Elite ABC Kit of Vector Laboratories, Inc.,
   Burlingame, CA, United States). The secondary antibody incubated for 3
   h at room temperature in a humid chamber and reactions carried out per
   manufacturer instructions. For TNFα immunohistochemistry the tissue was
   incubated for 48 h at 4°C in a humid chamber in TNFα polyclonal
   antibody (1:500, Thermo Fisher, PA5-19810). After three rinses in 0.2%
   Triton X-100 in PBS, immunohistochemical detection was carried out with
   an Alexa Fluor Donkey 555 anti-Rabbit (1:1000; Abcam). The secondary
   antibody was incubated for 6 h at room temperature in a humid chamber.
   The slides were rinsed in 0.2% Triton X-100 in PBS three times for 5
   min and finally cover slipped with ProLong^® Gold antifade reagent
   (Invitrogen^TM Molecular Probes, Eugene, OR, United States). For
   detection of Bcl-2 expression, sections underwent antigen retrieval in
   a microwave using 1:10 Dako^® Target Retrieval Solution (DAKO
   Corporation, Carpinteria, CA, United States) for 30 s.
   Immunohistochemistry was carried out with anti-human Bcl-2 rabbit
   polyclonal antibody diluted 1:200 (BD Biosciences, Inc., San Jose, CA,
   United States). The tissue was incubated overnight at 4°C in a humid
   chamber. After three rinses in PBS, immunohistochemical detection was
   carried out with an anti-Rabbit IgG (1:50; Vectastain Elite ABC Kit of
   Vector Laboratories, Inc., Burlingame, CA, United States). The
   secondary antibody incubated for 3 h at room temperature in a humid
   chamber.

Statistical Analysis

   Statistical analysis of hearing outcomes was performed with ANOVA for
   repeated measures. RT PCR results were analyzed using ANOVA (Prism V8).

Results

Delivery of Mesenchymal Stromal Cells to the Mouse Cochlea Does Not Induce
Hearing Loss

   After documentation of baseline hearing by ABR, 1 month old C57Bl/6
   mice (n = 5) underwent delivery of 1 μl of human Wharton’s
   Jelly-derived MSCs into their left posterior semi-circular canal. One
   week post-delivery of cells, their hearing was re-evaluated. ABR
   recording prior to treatment at 4, 8, 16, and 32 kHz showed an average
   hearing threshold of 28.3 ± 7, 35 ± 5, 30 ± 5, and 30 ± 17,
   respectively. The average hearing threshold for each frequency did not
   change significantly after cell therapy (30 ± 0, 35 ± 5, 30 ± 0, and 40
   ± 17 for 4, 8, 16, and 32 kHz, respectively) ([77]Figure 1).

FIGURE 1.

   FIGURE 1
   [78]Open in a new tab

   Delivery of MSCs did not have any effect on baseline hearing. Hearing
   thresholds measured by ABR at four different frequencies prior to
   (black bars) and 7 days after transplantation of MSC to the inner ear
   via the posterior semicircular canal (gray bars) are depicted. There is
   no significant difference between the pre- and post-transplantation
   thresholds.

Delivery of Mesenchymal Stromal Cells to the Mouse Inner Ear Provides
Significant Hearing Rescue After Severe Noise Trauma

   Pre-treatment ABR measurements showed normal hearing threshold in both
   groups ([79]Figure 2; control ore and MSC treated pre). After noise
   exposure, animals of the control group (treated with artificial
   perilymph) demonstrated a severe to profound hearing loss across all
   frequencies at 14 days post-sound exposure ([80]Figure 2; control
   post). There was a significant rescue effect seen in the MSC treated
   animals at 4 and 8 kHz (p < 0.01) and at 32 kHz (p < 0.05) ([81]Figure
   2; MSC treated post) when compared to the thresholds of the control
   group after noise exposure and application of artificial perilymph
   ([82]Figure 2; control post). At 16 kHz, treated animals showed better
   hearing thresholds than sound trauma and artificial perilymph treated
   animals, but this was not statistically significant. Histological
   analysis of the organ of Corti performed separately for the basal and
   mid turn showed loss of hair cells both inner and outer hair cells for
   both groups after sound trauma (MSCs and artificial perilymph treated)
   in the basal turn ([83]Figures 3A,B). When compared to the artificial
   perilymph treated animals ([84]Figure 3C), the MSC-treated animals did
   show preservation in the mid turn of the cochlea ([85]Figure 3D).
   DPOAEs were not measurable after sound trauma and were not restored by
   MSC therapy (data not shown).

FIGURE 2.

   FIGURE 2
   [86]Open in a new tab

   Delivery of MSCs rescue hearing after noise exposure. Exposure to sound
   for 4 h results in a profound hearing loss across all frequencies
   (medium gray bars; Control Post). Delivery of MSCs into the posterior
   semicircular canal of animals after sound exposure resulted in
   significant rescue of hearing at 4 and 8 kHz (p < 0.01**) as well as at
   32 kHz (p < 0.05*) (light gray bars; MSC treated Post) when compared to
   the control animals treated with artificial perilymph (Control Post).

FIGURE 3.

   FIGURE 3
   [87]Open in a new tab

   Histological evaluation of organ of Corti integrity in sound trauma
   (A,C) and sound trauma and MSC (B,D) treated cochlea. Loss of inner
   hair cells is consistently seen in the basal turns of both groups
   (A,B). The MSC treated group showed occasional survival of inner hair
   cells at the midturn (D).

Delivery of Mesenchymal Stromal Cell Alters Cochlear Gene Expression

   For control cochleae, an average of 155 ng/μl of RNA and for MSC
   treated cochleae 182 ng/μl of RNA were extracted. An average of
   13,789,205 and 13,472,844 input reads for control and MSC treated
   cochleae were performed yielding 15,671 mapped genes. Delivery of MSCs
   caused a greater down-regulatory than up-regulatory effect on gene
   expression in sound traumatized animals treated with MSCs. A total of
   2742 genes were down-regulated, and 1138 genes were found to be
   up-regulated. Significantly changed genes were considered to have an
   FDR of <0.05 and two fold change (up or down). Key down-regulated genes
   are shown in [88]Supplementary Table 1 and up-regulated genes are shown
   in [89]Supplementary Table 2.

Analysis of Gene Ontologies and Functional Networks

   The effects of MSC delivery after sound trauma were evaluated using GO
   expression analysis using IPA “Core” and “Bioprofiler” analysis. The GO
   classification for genes that are regulated by MSC delivery were broken
   down into the biological processes, molecular functions and cellular
   components and weighted according to their enrichment (FPKM) and FDR
   score. These were also visualized as a Voronoi tree plots to facilitate
   identification of key pathways. Down-regulated genes were enriched for
   processes related to neuronal function and repair with ion transport,
   nervous system transport, positive regulation of synapse assembly,
   modulation of synaptic transmission, regulation of membrane potential,
   synaptic transmission, regulation of post-synaptic membrane potential
   and long-term synaptic potentiation GO classifications dominating
   ([90]Figure 4). The GO molecular function classification showed
   significant changes in genes related to protein binding although these
   were not highly enriched. Genes related to transmitter-gated ion
   channel activity, voltage gated ion activity, syntaxin-1 binding,
   sodium channel activity and calcium channel activity were found.
   Interestingly, processes relating to dynein light and intermediate
   chain binding were also seen. The GO terms relating to cellular
   components concentrated on the excitatory synapse, components of the
   axon, dendrites, and myelin sheath as well as mitochondrion related
   annotations ([91]Figure 5). Voronoi tree-maps that improved
   hierarchical representation of the GOEA data were additionally
   evaluated and groups of genes that appear significantly represented
   were identified ([92]Bernhardt et al., 2009).

FIGURE 4.

   [93]FIGURE 4
   [94]Open in a new tab

   Down-regulated biological processes classified by gene ontology. The
   size of the category corresponds to a lower FDR with the more deeply
   red groups representing a higher enrichment score. The graphic
   representation of gene expression demonstrates that a key element in
   MSC treatment is modulation of synaptic assembly which is represented
   in multiple groups of down-regulated genes.

FIGURE 5.

   [95]FIGURE 5
   [96]Open in a new tab

   Up-regulated biological processes classified by gene ontology. The size
   of the category corresponds to a lower FDR with the more deeply red
   groups representing a higher enrichment score. Up-regulation of genes
   related to immune function are a key component of MSC treatment.

   Down-regulated genes included miRNAs such as miR-9, miR-124a,
   miR-124-2, and miR-133a. Regulation of heat shock proteins has been
   implicated in sound trauma and in this data set, delivery of MSCs
   resulted in down-regulation of Hspa1b, Hsp1l, Hsph1, Ahsa1, Hsp9, and
   Hsp90ab1 ([97]Gratton et al., 2011). Key effectors of cell death were
   also down-regulated whereas protective genes such as bcl-2 were
   up-regulated.

   Up-regulated genes were enriched for processes involved in adaptive and
   innate immune responses, extracellular matrix organization, cell
   adhesion and a wide range of cell repair and developmental processes
   ([98]Table 1). Additional processes included transcription factor
   function, wound healing, regulation of apoptotic process and response
   to hypoxia. Molecular function annotations centered on a range of
   protein receptor signaling processes and processes related to matrix
   binding. Signaling processes related to insulin like growth factor
   binding, TGFβ, fibroblast growth factor and Wnt binding were
   represented. Annotations showed that these genes were related to the
   cell surface, the extracellular matrix and the Golgi apparatus.
   Processes involving membrane repair and transmembrane tyrosine kinase
   signaling were also represented. Also represented were processes such
   as negative regulation of neuronal apoptosis, protein stabilization and
   sensory response to sound, which we would expect to see in a repair
   process involving the inner ear. Up-regulated genes related to response
   to sound are specific to the ear, associated with development of the
   inner ear, associated with syndromes that include hearing loss or when
   mutated are associated with sensorineural hearing loss ([99]Shearer et
   al., 1993). We identified 14 up-regulated genes including Myo1A
   ([100]Kwon et al., 2014), Ripor2 ([101]Diaz-Horta et al., 2018), Nipbl,
   Ccdc50 ([102]Modamio-Høybjør et al., 2007), Lhfpl5 ([103]Ge et al.,
   2018), Fgfr1 ([104]Pirvola et al., 2002), Diaph1 ([105]Neuhaus et al.,
   2017), Pou4f3 ([106]Walters et al., 2017), P2rx2 ([107]Mittal et al.,
   2016), Otog ([108]Schraders et al., 2012), Otoa ([109]Lukashkin et al.,
   2012), Cntn5, Icam1 ([110]Schmutzhard et al., 2011), and Hexa. The
   identified genes ranged from hair cell, spiral ganglion and stria
   specific to affecting tectorial membrane function, emphasizing that the
   MSC treatment affects multiple areas of the inner ear. Treatment with
   MSC resulted in both up and down regulation of genes that have been
   associated with noise induced hearing loss in other studies. Regulation
   of complement and toll receptor signaling has been shown to be
   associated with noise induced hearing loss ([111]Patel et al., 2013;
   [112]Vethanayagam et al., 2016; [113]Yang et al., 2016). Members of the
   complement cascade identified included C1qtnf4, C1ql2, and Cfd
   (down-regulated) and C1rl and C3 (up-regulated). Other members of the
   innate immune system that showed differential expression were members
   of the Toll signaling system including Tlr4, Tlr6, and Tiarp, which
   were up-regulated. To more clearly identify effects of cell therapy the
   IPA software was used to compare only significantly up- and
   down-regulated genes to evaluate the relationship between expression
   data and potential pathways involved in injury and protection. Key
   canonical pathways identified were calcium signaling, synaptogenesis
   signaling, endocaniboid neuronal synapse pathway, GABA receptor
   signaling and CREB signaling in neurons. Key predicted upstream
   regulators include BDNF, which has been shown to be produced by MSCs
   and has been identified as a mechanism of MSC protection of neurons in
   stroke models ([114]Gu et al., 2014; [115]Mukai et al., 2018). When
   graphically represented, TNFα can be seen as playing a central role in
   the predicted changes in biological function and signaling pathways
   ([116]Figure 6). This was further evaluated using immunohistochemistry
   and semi quantitative PCR ([117]Figures 7–[118]9). Immunofluorescent
   labeling showed that MSC therapy reduced expression of TNFα protein in
   the lateral wall ([119]Figures 7B vs. C), however expression was higher
   than controls in the inner hair cells of both sound trauma only
   ([120]Figure 7B) and sound trauma + MSC treated animals ([121]Figure
   7C). RNAseq analysis demonstrated a 0.8 fold up-regulation of TNFα mRNA
   in MSC vs. sound trauma treated animals. Evaluation using RT-PCR
   demonstrated that both sound trauma only and sound trauma + MSC treated
   animals showed significantly up-regulated mRNA levels compared to
   controls (p = 0.0021; p = 0.0002) but were not significantly different
   from each other (p = 0.2517). IPA comparison analysis ([122]Figure 6)
   also identified NeuroD1 as a significantly down-regulated transcription
   factor that is predicted to modulate multiple pathways related to
   neuronal growth and survival. Many of the survival effects are mediated
   through neurotrophin signaling. One of the downstream effects of neuroD
   signaling are alterations in the balance of bcl-2 and bax modulating
   apoptosis ([123]Wy, 2013). MSCs have also been shown to modulate bcl-2
   expression in a variety of injury models ([124]Xiang et al., 2009;
   [125]Gu et al., 2014). Our data showed that there was a 0.4 fold
   up-regulation of bcl-2 in MSC treated animals vs. sound only controls.
   There were no identifiable changes in bax expression in our data
   suggesting induction of an anti-apoptotic state. This was confirmed
   using immunohistochemistry and RT-PCR ([126]Figure 8). MSC treated
   animals showed an increase of bcl-2 immunolabeling in the organ of
   Corti, particularly in the supporting cells ([127]Figure 8C) and
   demonstrated an up-regulation of bcl-2 mRNA ([128]Figure 8D). One of
   the most down-regulated genes identified in our study was transthyretin
   (5.377 fold decrease in expression after MSC treatment). Transthyretin
   plays multiple roles in the nervous system and is a key modulator of
   metabolism, which was predicted to be altered in the IPA comparison
   analysis ([129]Figure 6). We confirmed down-regulation of transthyretin
   by semi quantitative RT-PCR ([130]Figure 9). Finally, we identified
   molecules in clinical use that could modulate most significantly
   regulated genes, identifying 46 drugs that could potentially be used to
   study some of the effects induced by MSCs ([131]Table 2). Transthyretin
   can be down-regulated using thyroxine hormone (T4). Based on the Pharos
   analysis, multiple modulators of GABA neurotransmission are available,
   raising the possibility of using small molecules to modulate this
   pathway.

TABLE 1.

   Down- and up-regulated canonical pathways.
   Down-regulated canonical pathways Up-regulated canonical pathways
   Calcium Signaling Cardiovascular Disease, Cardiovascular System
   Development and Function
   Synaptogenesis Signaling Pathway Cancer, Organismal Injury and
   Abnormalities
   GABA Receptor Signaling Cell-To-Cell Signaling and Interaction,
   Hematological System Development and Function, Inflammatory Response,
   Organismal Functions
   Endocannabinoid Neuronal Synapse Pathway Hematological System
   Development and Function, Immune Cell Trafficking, Inflammatory
   Response, Tissue Development
   Oxidative Phosphorylation Cancer, Hematological Disease, Immunological
   Disease, Organismal Injury and Abnormalities
   Opioid Signaling Pathway Connective Tissue Development and Function,
   Skeletal and Muscular System Development and Function, Tissue
   Development
   Mitochondrial Dysfunction Cancer, Gastrointestinal Disease, Organismal
   Injury and Abnormalities
   GNRH Signaling Cellular Function and Maintenance
   Synaptic Long Term Depression Cell-To-Cell Signaling and Interaction,
   Cellular Function and Maintenance, Inflammatory Response
   Corticotropin Releasing Hormone Signaling Cancer, Gastrointestinal
   Disease, Organismal Injury and Abnormalities
   [132]Open in a new tab

FIGURE 6.

   [133]FIGURE 6
   [134]Open in a new tab

   Graphical representation of IPA analysis of up- and down-regulated
   genes and processes. Using IPA core analysis of down-regulated genes
   and processes (blue) predicted up-regulation of genes and processes
   (orange). There is an overall down-regulation of pathways related to
   synaptogenesis, development of neurons and excitatory signaling. TNFα
   is a key factor that influences multiple processes and genes.

FIGURE 7.

   [135]FIGURE 7
   [136]Open in a new tab

   Expression of TNFα protein (Red A–C) and semi quantitative RT PCR of
   TNFα mRNA (D). Control cochlea showed minimal TNF expression in the
   hair cells and lateral wall (A). After sound trauma there was a clear
   increase in TFN alpha immunolabeling in hair cells, the spiral ganglion
   (arrow) and the lateral wall (B). Treatment with MSCs after sound
   trauma reduced TNFα expression in the lateral wall and outer hair cells
   but expression remained high in the inner hair cell (arrow C). Semi
   quantitative RT-PCR showed an up-regulation of TNF gene expression
   after sound trauma. This was slightly reduced after MSC treatment but
   not to control levels (D).

FIGURE 9.

   FIGURE 9
   [137]Open in a new tab

   Expression of cochlear transthyretin by semi quantitative RT-PCR. Sound
   trauma treated animals showed a relative increase in expression of
   transthyretin mRNA. MSC treatment resulted in a significant reduction
   in expression of transthyretin mRNA after sound trauma (p = 0.001).

FIGURE 8.

   [138]FIGURE 8
   [139]Open in a new tab

   Expression of bcl-2 (brown A–C) and semi quantitative RT PCR of bcl-2
   mRNA (D). Control cochlea showed bcl-2 expression in the pillar, in the
   inferior portion of the outer hair cells (arrows) and neurites (A).
   After sound trauma there was an occasional increase in bcl-2
   immunolabeling in isolated supporting cells (arrow) (B). Treatment with
   MSCs after sound trauma increased expression of bcl-2 in supporting
   cells (arrow C). Semi quantitative RT-PCR showed reduction of bcl-2
   mRNA expression after sound trauma. Treatment with MSC after sound
   trauma showed a significant up-regulation of bcl-2 mRNA compared to
   both controls and sound trauma only treated animals (p = 0.003) (D).

TABLE 2.

   Pharos analysis of genes showing greatest up- and down-regulation that
   can be modulated by small molecules in clinical use.
   Symbol Gene name Symbol Gene name
   Cacna1a Voltage-dependent calcium channel subunit alpha-1/alpha Hrh3
   Histamine H3 receptor
   Cacna1i Voltage-dependent T-type calcium channel subunit alpha Htr2c
   5-hydroxytryptamine receptor 2C
   Car4 Carbonic anhydrase 4 Irs4 Insulin receptor substrate 4
   Cck Cholecystokinin Kcnd2 Potassium voltage-gated channel subfamily D
   member 2
   Ccl3 C-C motif chemokine 3 Kcnh6 Potassium voltage-gated channel,
   subfamily H, member 6
   Cnr1 Cannabinoid receptor 1 Kcns2 Potassium voltage-gated channel
   subfamily S member 2
   Gabra1 Gamma-aminobutyric acid receptor subunit alpha-1 Krt1 Keratin,
   type II cytoskeletal 1
   Gabra4 Gamma-aminobutyric acid receptor subunit alpha-4 Lipf Gastric
   triacylglycerol lipase
   Gabra6 Gamma-aminobutyric acid receptor subunit alpha-6 Muc1 Mucin 1,
   transmembrane
   Gabrb1 Gamma-aminobutyric acid receptor subunit beta-1 Ngf Beta-nerve
   growth factor
   Gabrb2 Gamma-aminobutyric acid receptor subunit beta-2 Pou1f1
   Pituitary-specific positive transcription factor 1
   Gabrp Gamma-aminobutyric acid receptor subunit pi Prl Prolactin
   Gal Galanin peptides Prlr Prolactin receptor
   Gh Growth hormone Rnf112 RING finger protein 112
   Gnrhr Gonadotropin releasing hormone receptor Scn2a1 Sodium channel
   protein
   Gp2 Pancreatic secretory granule membrane major glycoprotein GP2 Slc5a1
   SGLT1 protein
   Gria1 Glutamate receptor 1 Slc6a1 Sodium- and chloride-dependent GABA
   transporter 1
   Gria2 Glutamate receptor 2 Slc6a5 Sodium-dependent noradrenaline
   transporter
   Grik2 Glutamate receptor ionotropic, kainate 2 Sstr3 Somatostatin
   receptor type 3
   Grin2a Glutamate receptor ionotropic, NMDA 2A; NMDA 2B; NMDA 2D Stum
   Protein stum homolog
   Grin2b Glutamate receptor ionotropic, NMDA 2B Tnr Tenascin-R
   Grin2c Glutamate receptor, ionotropic, NMDA2C Trpm8 Transient receptor
   potential cation channel subfamily M member 8
   Grm5 Metabotropic glutamate receptor 5 Ttr Transthyretin
   [140]Open in a new tab

   Besides neurotrophins, MSCs produce a variety of immunomodulatory
   molecules and other growth factors. Using the predictive functions of
   IPA, we analyzed the potential effect of several growth factors and
   cytokines known to be delivered by MSC on our data set and found that
   the observed changes in HSP90, complement factor 3 and toll like
   receptor 4 could potentially be modulated by MSCs ([141]Figure 10).

FIGURE 10.

   FIGURE 10
   [142]Open in a new tab

   The known WJC secretome regulates multiple genes found to be altered
   after sound trauma and MSC delivery. Components of the WJC secretome
   (boxed area) were evaluated for potential interactions with genes that
   we found to undergo significant change using the IPA knowledgebase.
   This demonstrates the potential biological basis for the protective
   effects that WJCs have on noise trauma.

Discussion

   Intra-labyrinthine application of WJCs after noise trauma showed
   significant protection of hearing threshold at the lower frequencies at
   4 and 8 kHz and to a lesser degree at 32 kHz in mice. Histological
   changes related to sound trauma could be identified in the organ of
   Corti in both groups. There was preservation of some mid turn hair
   cells in the MSC treated group. The changes underlying the altered
   threshold could also be the result of damage to other regions, for
   example of the stria vascularis or the synapses. Since the focus of the
   present study was the functional restoration and RNA changes induced by
   cell therapy rather than the histological changes, synapses and the
   stria vascularis were not investigated. We identified more than 2700
   down-regulated and 1138 up-regulated genes after treatment with WJC. To
   understand the biological effects of the differentially regulated
   genes, the cellular components, molecular function, and biological
   process were analyzed by GO annotation. Using IPA, specific analysis of
   genes related to cell death and survival showed the regulation of 365
   molecules related to necrosis and 353 molecules related to apoptosis
   with a predictive effect of decreasing both death mechanisms and
   increasing cell survival. We could demonstrate that there are MSC
   induced reductions of TNFα expression in the cochlear lateral wall
   ([143]Figure 7) and MSC induced increases in bcl-2 expression in the
   organ of Corti ([144]Figure 8). TNFα up-regulation has been associated
   with sound trauma ([145]Frye et al., 2019). Our data demonstrated an
   up-regulation in TNFα gene expression which could not be confirmed by
   PCR ([146]Figure 7D). However, distribution of TNFα expression was
   reduced in the lateral wall of the cochlea ([147]Figures 7C vs. B).
   Increased expression of TNFα in the sound damaged cochlea has been
   demonstrated for days 1–3 post-sound trauma followed by a decrease and
   then a further expression increase at 28 days post-noise exposure
   ([148]Fujioka et al., 2006). Additional time points will be needed to
   evaluate the long term effects of MSC treatment. We additionally
   evaluated the effect of MSC treatment on bcl-2 expression in the sound
   damaged inner ear. Our RNAseq data demonstrated a down-regulation of
   cell death effector genes and an up-regulation of the antiapoptotic
   bcl-2 ([149]Figure 8) consistent with other MSC experiments ([150]Gu et
   al., 2014). Up-regulation of bcl-2 has been shown to be an underlying
   mechanism in sound conditioning protection from noise trauma ([151]Niu
   et al., 2003).

   Many of the identified genes and GO annotations have been found in or
   belong to a similar GO pattern seen in stroke or TBI suggesting that
   severe sound trauma and TBI ([152]Spaethling et al., 2008;
   [153]Darkazalli et al., 2017; [154]Meng et al., 2017; [155]Trivedi et
   al., 2019; [156]Li et al., 2020) involve similar molecular mechanisms.
   By evaluating the diverse pathways activated in this recovering system,
   we may be able to select new targets for the pharmacological mitigation
   of sound trauma. Significant advances have been made in the
   pharmacologic treatment of brain injury and many of the drugs that are
   in experimental or clinical trials ([157]Diaz-Arrastia et al., 2014)
   may also be applicable to the inner ear. Use of the Pharos database
   identified that 46 of the most up and down regulated genes could
   potentially be targeted by molecules that are already in use. NMDA
   receptors in particular have already been targeted in clinical trials
   for sound induced tinnitus, demonstrating that MSC induced protection
   in part functions through previously identified mechanisms ([158]Bing
   et al., 2015; [159]Staecker et al., 2017).

   In the cellular component category, the most represented term is “AMPA
   glutamate receptor complex,” followed by “post-synaptic density
   membrane” and “excitatory and inhibitory synapses” for the
   down-regulated genes. In the molecular function category, the term
   “transmitter-gated ion channel activity involved in regulation of
   post-synaptic membrane potential” and “syntaxin-1 binding was found to
   be represented. The underlying biological processes in the
   down-regulated genes were related to synaptic functions. Most striking,
   synapse-specific processes like excitatory as well as inhibitory
   synapse, AMPA glutamate receptor complex synapse assembly, regulation
   of post-synaptic membrane potential, modulation of synaptic
   transmission and long-term synaptic potentiation were significantly
   down-regulated. This suggests that neuronal repair and control of
   excitotoxicity could be key components of recovery from severe sound
   trauma. For the up-regulated genes, “trans-Golgi network transport
   vesicles” is the most over-represented term in the cellular component
   category. Looking at molecular functions, extracellular binding,
   Wnt-protein and insulin-like growth factor binding were most abundant
   alongside with cytokine receptor activity. In the biological processes
   category, the terms extracellular matrix organization, adaptive immune
   response as well as transmembrane receptor protein tyrosine kinase
   (Trk) signaling pathway were found to be strongly represented.
   Immunomodulation and activation of Trk signaling pathways are known to
   be potent enhancers of survival. For example, systemic transplantation
   of MSC in a TBI model led to a lower density of microglia, macrophages
   as well as peripheral infiltrating leukocytes at the injury site, to
   reduced levels of pro-inflammatory cytokines and increased
   anti-inflammatory cytokines ([160]Zhang et al., 2013). Even in a model
   of xenotransplantation of MSC derived from adipose tissue after ventral
   root avulsion, an increased neuronal survival and partial preservation
   of synaptophysin-positive nerve terminals as well as a reduction of
   pro-inflammatory reactions were elicited ([161]Ribeiro et al., 2015).
   It is assumed that by suppressing lymphocyte, astroglia and microglia
   effects, MSC can prevent a second delayed neuronal injury ([162]Ribeiro
   et al., 2015). Furthermore, MSCs can prevent neuronal cell death not
   only via restoration of the local microenvironment but also by the
   release of classic inhibitors of apoptosis ([163]Tran and Damaser,
   2014).

   Looking at the top down-regulated genes, mainly genes coding for
   transcription factors or for proteins and receptors related to synaptic
   function are among the most regulated. Down-regulation of oxidative
   stress pathways might be key for the mediation of protective effects.
   According to our RNA sequencing data, transthyretin (Ttr), a neuronal
   stress marker, is one of the most significantly down-regulated genes by
   the application of MSC after noise-induced trauma. It has been shown
   that Ttr level correlate with ROS or reactive nitrogen species (RNS)
   ([164]Saito et al., 2005; [165]Fong and Vieira, 2013). In addition, the
   expression of the Ttr gene is regulated by glucocorticoids, which are
   stress hormones ([166]Li et al., 2011; [167]Martinho et al., 2012). On
   a protein level, Ttr can initiate oxidative stress in the endoplasmic
   reticulum and is thereby involved in the unfolded protein response
   (UPR) ([168]Teixeira et al., 2006; [169]Genereux et al., 2015;
   [170]Chen J. et al., 2016). Thus, down-regulation of ttr might result
   in inhibition of formation of reactive oxygen and nitrogen species
   thereby reducing the extent of trauma-associated damage. Recent TBI
   studies using drop-seq have demonstrated that up-regulation of ttr is
   one of the major markers of trauma in a wide range of cell types
   ([171]Arneson et al., 2018). This study also documented functional
   recovery induced by dosing TBI animals with thyroxine (T4) which
   down-regulates transthyretin. We demonstrated an MSC induced
   normalization of ttr gene expression by RNAseq and semiquantitative PCR
   ([172]Figure 9) raising the possibility that thyroxine could be used to
   limit sound trauma induced damage. Other factors involved in the
   production of ROS belong to the kallikrein-kinin system. This system
   includes plasma or tissue related kinins and they mediate vascular
   permeability, edema formation, inflammation and the release of
   glutamate in the case of neurodegenerative disease associated with
   neuroinflammation such as TBI or Alzheimer’s disease
   ([173]Naffah-Mazzacoratti Mda et al., 2014; [174]Hopp and
   AlbertWeissenberger, 2015). Tissue-derived kallikreins belong to the
   chymotrypsin- and trypsin-like serine endopeptidases. Especially
   kallikrein-7, originally identified as an inflammation−induced
   proteolytic enzyme in the skin, is down regulated by the application of
   WJS after sound trauma ([175]Kidana et al., 2018). In addition,
   stress-related heat shock proteins that are involved in the response to
   stress and metabolic damage to the inner ear such as Hspa1b, Hsp1l,
   Hsph1, Ahsa1, Hsp9, and Hsp90ab1 are also down-regulated by MSC
   treatment ([176]Sheehan-Rooney et al., 2013).

   Moreover, several transcription factors, e.g., BarHl2, Neurod2, EN2,
   and Pax6, are down-regulated by application of MSC in noise trauma as
   demonstrated by our results. The homeobox protein BarH-like 2 (Barhl2)
   acts as transcription factor and possesses an enhancer that can be
   activated by the basic helix-loop-helix factor Atoh1 and can drive
   spinal cord-specific gene expression. Interestingly, this gene is
   significantly down-regulated when comparing noise-damaged cochlea
   treated with MSC to the sham-treated. Similar is observed for the
   neuronal differentiation factor 2, Pax6 and homeobox protein
   engrailed-2 (EN2). The later may alter the excitation/inhibition
   balance, and its down-regulation may rescue or regain homeostasis by
   MSC to reduce damage. NeuroD2 and Pax6 are important regulators of
   neuronal development in the central nervous system and their
   down-regulation after treatment with MSC seems quite surprising
   ([177]Yogarajah et al., 2016). Whether damage leads to
   dedifferentiation and induction of neurogenesis thereby making the
   inner ear more susceptible to secondary damage mediated by oxidative
   stress and therewith associated metabolites remains speculative and
   needs further investigation. In addition to neuronal development,
   NeuroD2 balances synaptic neurotransmission and intrinsic excitability
   in the adult system, thereby decreasing neuronal excitability of
   cortical pyramidal neurons both in vitro and in vivo ([178]Chen F. et
   al., 2016). After trauma, as has been shown for example in models of
   TBI, there is an excitation/inhibition imbalance as also reflected by
   the GABA/glutamate imbalance. Excitatory pathways in the brain are
   regulated by GABA and loss of GABA releasing cells after trauma further
   enhances cell injury and apoptosis ([179]Guerriero et al., 2015).
   Although glutamate is the excitatory transmitter in the brain required
   for proper function, post-traumatic burst release of glutamate leads to
   excitotoxicity and subsequent cellular injury as has been also shown
   after noise-induced damage of the inner ear. By down-regulation of
   both, glutamate and GABA receptor subunits, MSC might down regulate the
   neuronal activity thereby possibly preventing further damage and
   transforming the cochlea to a state for protection and regeneration.
   The availability of multiple small molecules modulating this pathway
   ([180]Table 2) raises the possibility of novel treatments of sound
   trauma.

   Several miRNAs (miR-9, miR-124a, miR-124-2, and miR-133a) are also
   down-regulated after treatment with MSCs. These miRNAs play a variety
   of important developmental roles; miRNA-9 is the most significantly
   up-regulated miRNA in the developing brain, targeting the transcription
   factors FoxG1, Hes1, or Tlx, thereby playing an important role in the
   regulation of neuronal progenitors ([181]Coolen et al., 2013).
   MicroRNA-9 has also been shown to regulate Schwann cell migration in
   neuronal injury, and is thought to play an important role in peripheral
   neuronal repair ([182]Zhou et al., 2014). Similarly, miRNA-124a is an
   important developmental regulator and is involved in neurogenesis of
   the hippocampus and retinal cones ([183]Sanuki et al., 2011). This
   miRNA has also been shown to be up-regulated in repetitive brain injury
   ([184]Huang et al., 2018). Therefore, the down-regulation of miR-124
   observed in this study may represent a positive effect of MSC
   treatment. Both miR-9 and -124 have been identified as biomarkers of
   TBI ([185]Ji et al., 2016). Thus, MSCs regulate not only transcription
   factors involved in neurogenesis, but also miRNAs involved in the
   regulation of transcription factors. Another miRNA that we found to be
   down-regulated (miR-133) is involved in organogenesis, regulating the
   hedgehog pathway in myogenesis ([186]Mok et al., 2018; [187]Yu et al.,
   2019). This miRNA has been found to protect neurons when up-regulated
   but is also involved in the remodeling of connective tissue after
   injury ([188]Xin et al., 2012; [189]Li et al., 2018). To fully
   understand the relevance of the changes induced by MSC in the inner
   ear, investigation of further time points after damage and treatment or
   even transcriptomics at single cell resolution will be needed.

   The biological effects of MSCs have been widely studied. MSCs produce a
   wide range of growth factors, extracellular vesicles and miRNAs that
   taken as a whole have a wide range of potential effects on a tissue.
   The secretome of umbilical cord MSCs has been shown to contain Ntf3,
   Egf, Mdk, Hbegf, Cxcl5, Cxcl2, and Fgf9 ([190]Hsieh et al., 2013).
   Interestingly, many of these factors are involved in neurogenesis and
   in vitro, they up-regulate a variety of transcription factors that
   enhance local release of some of the same growth factors, thereby
   amplifying the repair process. Many of the regulated genes we also
   identified in this study. Immunomodulation is also a well characterized
   feature of MSCs ([191]Hsieh et al., 2013). We evaluated the potential
   effect of some of these reported growth factors by modeling their
   regulation of genes we saw change using IPA software. As depicted in
   [192]Figure 10, the IPA knowledge base predicts that growth factors
   produced by WJCs can regulate factors such as C3, Tlr4, bcl2, and Ttr
   that we identified. Fully characterizing the links between MSC
   signaling and our findings is difficult since interactions of MSCs with
   their target tissue may actually alter their secretome ([193]Drago et
   al., 2013). Evaluation of extracellular vesicles produced by WJCs for
   the mitigation of sound trauma has demonstrated that similar protective
   effects can be achieved using only the secretome alone ([194]Warnecke
   et al., 2020).

   Several limitations are associated with the present study. The study
   was focused on identifying the effects of MSC cell therapy on the
   cochlear transcriptome after noise exposure. Further insights could be
   gained by including analysis of the transcriptome from a group of mice
   not exposed to noise with and without treatment with MSC. Sound
   trauma-induced changes in gene expression have been evaluated in
   numerous publications ([195]Cai et al., 2012; [196]Patel et al., 2013).
   We chose a severe form of sound trauma to test the ability of WJC type
   MSCs to rescue an inner ear function. The identified pathways are
   similar to the ones seen in other MSC-based rescue of models and in
   analysis of TBI induced neuronal injury. To further characterize these
   effects, a more moderate sound trauma paradigm with multiple sample
   time points after MSC delivery and verification of the effects using
   PCR and immunohistochemistry will be needed. Despite these limitations,
   main cell signaling pathways involved in MSC-mediated inner ear
   protection after noise trauma have been identified by applying pathway
   enrichment analysis. In future studies, we could mimic the effects of
   MSCs or inhibit potential protective pathways with small molecules to
   better characterize regulatory pathways by using large-scale
   transcriptome analysis in well-defined sets of experiments ([197]Islam
   et al., 2019).

Conclusion

   Delivery of WJC type MSCs after sound trauma has a significant rescue
   effect on hearing. RNAseq analysis demonstrates regulatory changes in
   multiple genes including genes related to excitatory neurotransmission
   and the innate immune system. Further studies using different sound
   trauma paradigms and different timing of cell delivery as well as
   additional replicates are needed to better characterize the effects
   mediated by MSCs in the present study.

Data Availability Statement

   The original contributions presented in the study are publicly
   available. The data presented in the study are deposited in the NCBI
   GEO repository, accession number [198]GSE187029:
   [199]https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE187029.

Ethics Statement

   The studies involving human participants were reviewed and approved by
   University of Kansas Human Subjects Committee (KU-Lawrence IRB approval
   #15402) and Stormont-Vail (SMV) Hospital (SMV IRB Approval conferred by
   IRB Chair, Jo-Ann S. Harris, MD). The patients/participants provided
   their written informed consent to participate in this study. The animal
   study was reviewed and approved by University of Kansas Health System,
   Institutional Animal Care and Use Board Committee.

Author Contributions

   AW and HS research design, data analysis, and writing of the
   manuscript. JH and MD data analysis and writing of the manuscript. MS
   electrophysiology and manuscript approval. AM preparation and delivery
   of MSC and manuscript approval. IM bioinformatics and manuscript
   approval. All authors contributed to the article and approved the
   submitted version.

Conflict of Interest

   HS is a consultant for MedEl GmbH and Otonomy Inc. and holds shares in
   Rescue Hearing Inc. AM is a shareholder in Ronawk Inc. The remaining
   authors declare that the research was conducted in the absence of any
   commercial or financial relationships that could be construed as a
   potential conflict of interest.

Publisher’s Note

   All claims expressed in this article are solely those of the authors
   and do not necessarily represent those of their affiliated
   organizations, or those of the publisher, the editors and the
   reviewers. Any product that may be evaluated in this article, or claim
   that may be made by its manufacturer, is not guaranteed or endorsed by
   the publisher.

Acknowledgments