Tissue engineering and gene therapy strategies offer new ways to repair permanent damage to mechanosensory hair cells (MHCs) by differentiating human Wharton's Jelly cells (HWJCs). Conventionally, these strategies require the classification of each cell as differentiated or undifferentiated. Automated classification tools, however, may serve as a novel method to rapidly classify these cells. In this paper, images from previous work, where HWJCs were differentiated into MHC-like cells, were examined. Various cell features were extracted from these images, and those which were pertinent to classification were identified. Different machine learning models were then developed, some using all extracted data and some using only certain features. To evaluate model performance, the area under the curve (AUC) of the receiver operating characteristic curve was primarily used. This paper found that limiting algorithms to certain features consistently improved performance. The top performing model, a voting classifier model consisting of two logistic regressions, a support vector machine, and a random forest classifier, obtained an AUC of 0.9638. Ultimately, this paper illustrates the viability of a novel machine learning pipeline to automate the classification of undifferentiated and differentiated cells. In the future, this research could aid in automated strategies that determine the viability of MHC-like cells after differentiation.

Reduced Cl- conductance causes inhibited muscle relaxation after forceful voluntary contraction due to muscle membrane hyperexcitability. This represents the pathomechanism of myotonia congenita. Due to the prevailing data suggesting that an increased potassium level is a main contributor, we studied the effect of a modulator of a big conductance Ca2+- and voltage-activated K+ channels (BK) modulator on contraction and relaxation of slow- and high-twitch muscle specimen before and after the pharmacological induction of myotonia. Human and murine muscle specimens (wild-type and BK-/-) were exposed to anthracene-9-carboxylic acid (9-AC) to inhibit CLC-1 chloride channels and to induce myotonia in-vitro. Functional effects of BK-channel activation and blockade were investigated by exposing slow-twitch (soleus) and fast-twitch (extensor digitorum longus) murine muscle specimens or human musculus vastus lateralis to an activator (NS1608) and a blocker (Paxilline), respectively. Muscle-twitch force and relaxation times (T90/10) were monitored. Compared to wild type, fast-twitch muscle specimen of BK-/- mice resulted in a significantly decreased T90/10 in presence of 9-AC. Paxilline significantly shortened T90/10 of murine slow- and fast-twitch muscles as well as human vastus lateralis muscle. Moreover, twitch force was significantly reduced after application of Paxilline in myotonic muscle. NS1608 had opposite effects to Paxilline and aggravated the onset of myotonic activity by prolongation of T90/10. The currently used standard therapy for myotonia is, in some individuals, not very effective. This in vitro study demonstrated that a BK channel blocker lowers myotonic stiffness and thus highlights its potential therapeutic option in myotonia congenital (MC).

Despite a vast amount of data generated by proteomic analysis on cochlear fluid, novel clinically applicable biomarkers of inner ear diseases have not been identified hitherto. The aim of the present study was to analyze the proteome of human perilymph from cochlear implant patients, thereby identifying putative changes of the composition of the cochlear fluid perilymph due to specific diseases. Sampling of human perilymph was performed during cochlear implantation from patients with clinically or radiologically defined inner ear diseases like enlarged vestibular aqueduct (EVA; n = 14), otosclerosis (n = 10), and Ménière's disease (n = 12). Individual proteins were identified by a shotgun proteomics approach and data-dependent acquisition, thereby revealing 895 different proteins in all samples. Based on quantification values, a disease-specific protein distribution in the perilymph was demonstrated. The proteins short-chain dehydrogenase/reductase family 9C member 7 and esterase D were detected in nearly all samples of Ménière's disease patients, but not in samples of patients suffering from EVA and otosclerosis. The presence of both proteins in the inner ear tissue of adult mice and neonatal rats was validated by immunohistochemistry. Whether these proteins have the potential for a biomarker in the perilymph of Ménière's disease patients remains to be elucidated.

The high complexity of the cellular architecture of the human inner ear and the inaccessibility for tissue biopsy hampers cellular and molecular analysis of inner ear disease. Sampling and analysis of perilymph may present an opportunity for improved diagnostics and understanding of human inner ear pathology. Analysis of the perilymph proteome from patients undergoing cochlear implantation was carried out revealing a multitude of proteins and patterns of protein composition that may enable characterisation of patients into subgroups. Based on existing data and databases, single proteins that are not present in the blood circulation were related to cells within the cochlea to allow prediction of which cells contribute to the individual perilymph proteome of the patients. Based on the results, we propose a human atlas of the cochlea. Finally, druggable targets within the perilymph proteome were identified. Understanding and modulating the human perilymph proteome will enable novel avenues to improve diagnosis and treatment of inner ear diseases.

SK2 (KCa2.2) channels are voltage-independent Ca2+-activated K+ channels that regulate neuronal excitability in brain regions important for memory formation. In this study, we investigated the distribution and expression of SK2 channels in human brain by Western blot analysis and immunohistochemistry. Immunoblot analysis of human brain indicated expression of four distinct SK2 channel isoforms: the standard, the long and two short isoforms. Immunohistochemistry in paraffin-embedded post-mortem brain sections was performed in the hippocampal formation, amygdala and neocortex. In hippocampus, SK2-like immunoreactivity could be detected in strata oriens and radiatum of area CA1-CA2 and in the molecular layer. In the amygdala, SK2-like immunoreactivity was highest in the basolateral nuclei, while in neocortex, staining was mainly found enriched in layer V. Activation of SK2 channels is thought to regulate neuronal excitability in brain by contributing to the medium afterhyperpolarization. However, SK2 channels are blocked by apamin with a sensitivity that suggests heteromeric channels. The herein first shown expression of SK2 human isoform b in brain could explain the variability of electrophysiological findings observed with SK2 channels.

Background: Survival and integrity of the spiral ganglion is vital for hearing in background noise and for optimal functioning of cochlear implants. Numerous studies have demonstrated that supplementation of supraphysiologic levels of the neurotrophins BDNF and NT-3 by pumps or gene therapy strategies supports spiral ganglion survival. The endogenous physiological levels of growth factors within the inner ear, although difficult to determine, are likely extremely low within the normal inner ear. Thus, novel approaches for the long-term low-level delivery of neurotrophins may be advantageous. Objectives: This study aimed to evaluate the long-term effects of gene therapy-based low-level neurotrophin supplementation on spiral ganglion survival. Using an adenovirus serotype 28-derived adenovector delivery system, the herpes latency promoter, a weak, long expressing promoter system, has been used to deliver the BDNF or NTF3 genes to the inner ear after neomycin-induced ototoxic injury in mice. Results: Treatment of the adult mouse inner ear with neomycin resulted in acute and chronic changes in endogenous neurotrophic factor gene expression and led to a degeneration of spiral ganglion cells. Increased survival of spiral ganglion cells after adenoviral delivery of BDNF or NTF3 to the inner ear was observed. Expression of BDNF and NT-3 could be demonstrated in the damaged organ of Corti after gene delivery. Hearing loss due to overexpression of neurotrophins in the normal hearing ear was avoided when using this novel vector-promoter combination. Conclusion: Combining supporting cell-specific gene delivery via the adenovirus serotype 28 vector with a low-strength long expressing promoter potentially can provide long-term neurotrophin delivery to the damaged inner ear.

Use of decellularized tissues has become popular in tissue engineering applications as the natural extracellular matrix can provide necessary physical cues that help induce the restoration and development of functional tissues. In relation to cochlear tissue engineering, the question of whether decellularized cochlear tissue can act as a scaffold and support the incorporation of exogenous cells has not been addressed. Investigators have explored the composition of the cochlear extracellular matrix and developed multiple strategies for decellularizing a variety of different tissues; however, no one has investigated whether decellularized cochlear tissue can support implantation of exogenous cells.

The application of neurotrophins such as brain-derived neurotrophic factor (BDNF) is a promising pharmacological approach in cochlear implant research. Several in vitro and in vivo studies demonstrated that treatment with neurotrophins support the spiral ganglion neuron (SGN) survival and the synapses. Of the more than 40 companies that are working in the field of inner ear therapeutics, only one company is currently advancing BDNF towards clinical translation. Thus, there are no approved clinical therapies with neurotrophins, their precursors or neurotrophin-like substances. For a better understanding of the mechanisms of BDNF in the inner ear, we analysed the expression of mature BDNF (mBDNF), its pro-form proBDNF and their respective receptors the low affinity p75 neurotrophin receptor (p75NTR) and the neurotrophic receptor tyrosine kinase 2 (NTRK2). In the adult murine inner ear, mBDNF is expressed in the inner and outer hair cells (IHC and OHC) of the organ of Corti and in the spiral ganglion of the Rosenthal's canal, whereas proBDNF is only detected in the supporting cells below the OHC. The corresponding receptors NTRK2 and p75NTR are expressed in the spiral ganglion whereof p75NTR is stronger expressed. For more insights in the effects of mBDNF and proBDNF on inner ear specific cells, we treated primary dissociated SGN with different concentrations of mBDNF and proBDNF alone and in combination. Interestingly, treatment with proBDNF is not toxic for SGN but simultaneously not protective. However, combined treatment of mBDNF and proBDNF maintained and perhaps slightly increased the protective effect of mBDNF. Thus, the mixture of mBDNF and proBDNF could be the new direction for the development of BDNF-based therapeutics in cochlear implantation and could represent more precisely the natural environment.

Recent studies have demonstrated that delivery of genes to the inner ear can achieve a variety of effects ranging from support of auditory neuron survival to protection and restoration of hair cells, demonstrating the utility of vector based gene delivery. Translation of these findings to useful experimental systems or even clinical applications requires a detailed understanding of the pharmacokinetics of gene delivery in the inner ear. Ideal gene delivery systems will employ a well tolerated vector which efficiently transduces the appropriate target cells within a tissue, but spare non-target structures. Adenovectors based on serotype 5 (Ad 5) are commonly used vectors, are easy to construct and have a long track record of efficacious gene transfer in the inner ear. In this study we demonstrate that distribution of Ad5 vector occurs in a basal to apical gradient with rapid distribution of vector to the vestibule after delivery via a round window cochleostomy. Transduction of the vector and expression of the delivered transgene occurs by 10 min post vector delivery. At 24 h post delivery only 16% of vector that was initially detectable within the inner ear by quantitative PCR remained. Perilymph sampling was used to determine that vector concentrations in perilymph peaked at 30 min post delivery and then declined rapidly. Understanding these basic distribution patterns and parameters for delivery are important for the design of gene delivery vectors and vital for modeling dose responses to achieve safe efficacious delivery of a therapeutic agent.

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.

The outcome of cochlear implantation depends on multiple variables including the underlying health of the cochlea. Brain derived neurotrophic factor (BDNF) has been shown to support spiral ganglion neurons and to improve implant function in animal models. Whether endogenous BDNF or BDNF-regulated proteins can be used as biomarkers to predict cochlear health and implant outcome has not been investigated yet. Gene expression of BDNF and downstream signaling molecules were identified in tissue of human cochleae obtained from normal hearing patients (n = 3) during skull base surgeries. Based on the gene expression data, bioinformatic analysis was utilized to predict the regulation of proteins by BDNF. The presence of proteins corresponding to these genes was investigated in perilymph (n = 41) obtained from hearing-impaired patients (n = 38) during cochlear implantation or skull base surgery for removal of vestibular schwannoma by nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). Analyzed by mass spectrometry were 41 perilymph samples despite three patients undergoing bilateral cochlear implantation. These particular BDNF regulated proteins were not detectable in any of the perilymph samples. Subsequently, targeted analysis of the perilymph proteome data with Ingenuity Pathway Analysis (IPA) identified further proteins in human perilymph that could be regulated by BDNF. These BDNF regulated proteins were correlated to the presence of residual hearing (RH) prior to implantation and to the performance data with the cochlear implant after 1 year. There was overall a decreased level of expression of BDNF-regulated proteins in profoundly hearing-impaired patients compared to patients with some RH. Phospholipid transfer protein was positively correlated to the preoperative hearing level of the patients. Our data show that combination of gene expression arrays and bioinformatic analysis can aid in the prediction of downstream signaling proteins related to the BDNF pathway. Proteomic analysis of perilymph may help to identify the presence or absence of these molecules in the diseased organ. The impact of such prediction algorithms on diagnosis and treatment needs to be established in further studies.

An important requirement for gene therapy in the inner ear is to achieve efficient gene delivery without damaging residual inner ear function. This can be achieved by delivering a high concentration of vector in a minimal volume. Adenovectors are well suited to meet these requirements since high quality concentrated vector with a high capacity for a gene payload can be produced. To reduce the number of vector particles and volume of delivery to the inner ear, we tested vectors with enhancements in cell binding and cell entry properties. We compared delivery of a marker gene to the inner ear using two different advanced generation serotype 5 adenovector designs. The first adenovector tested, AdRGD, has a restricted tropism of entry into cells. AdRGD is an Ad5 capsid vector with an arg-gly-asp (RGD) motif built into the adenovector fiber that has also been modified to abolish the fiber-CAR and penton-integrin interactions that provide the normal well characterized two-step entry pathway for adenovirus. The AdRGD vector has enhanced binding to alphanu integrins. The second vector, AdF2K, contains 7 lysine residues within the fiber knob and has been shown to have expanded tropism for cells in vitro and in vivo. AdF2K maintains its normal CAR and integrin receptors interactions and has an additional mechanism of entry via its ability to interact with heparan sulfate. Both vectors demonstrated effective delivery to the inner ear and more uniform labeling of the inner ear sensory epithelia than native capsid vector, when tested in vivo. Analysis of expression efficiency using quantitative PCR was tested in vitro on cultured macular organs and demonstrated that vector delivery with the AdF2K vector design yielded optimal delivery. The present study demonstrates that retargeting strategies can improve delivery to the inner ear.

Sound damage induced hearing loss has been shown to elicit changes in auditory and non-auditory brain regions. A protein critical for neuronal migration and brain development, doublecortin (DCX), has been used as a marker of central nervous system (CNS) neuroplasticity. DCX is expressed in unipolar brush cells (UBCs) of the dorsal cochlear nucleus (DCN), cerebellar parafloccular lobe (PFL) and neuronal precursor cells in the sub-granular zone of the hippocampal dentate gyrus (DG). Sound damage induced hearing loss has been shown to differentially impact DCX expression months later. To identify earlier alterations in DCX expression, we utilized immunohistochemistry to detect DCX protein in three brain regions (DCN, PFL, DG) approximately one month following unilateral sound damage. Auditory brainstem response was used to measure hearing loss. Unilateral hearing loss was evident in all sound damaged animals. Hearing loss related decreases in DCX expression were evident bilaterally in the DG while hearing loss related increases in DCX expression were evident bilaterally in the PFL. No changes to DCX expression were evident in the auditory DCN. Gap detection was used to assess whether this sound damage paradigm induced tinnitus-like behavior. However, results obtained from this behavioral test as used here were inconclusive and are presented here only as a guide to others wishing to design similar studies.

Hypothesis: Menière's disease microRNA (miRNA) profiles are unique and are reflected in the perilymph and serum of patients. Background: Development of effective biomarkers for Menière's disease are needed. miRNAs are small RNA sequences that downregulate mRNA translation and play a significant role in a variety of disease states, ultimately making them a promising biomarker. miRNAs can be readily isolated from human inner ear perilymph and serum, and may exhibit disease-specific profiles. Methods: Perilymph sampling was performed in 10 patients undergoing surgery; 5 patients with Meniere's disease and 5 patients with otosclerosis serving as controls. miRNAs were isolated from the serum of 5 patients with bilateral Menière's disease and compared to 5 healthy age-matched controls. For evaluation of miRNAs an Agilent miRNA gene chip was used. Analysis of miRNA expression was carried out using Qlucore and Ingenuitey Pathway Analysis software. Promising miRNAs biomarkers were validated using qPCR. Results: In the perilymph of patients with Menière's disease, we identified 16 differentially expressed miRNAs that are predicted to regulate over 220 different cochlear genes. Six miRNAs are postulated to regulate aquaporin expression and twelve miRNAs are postulated to regulate a variety of inflammatory and autoimmune pathways. When comparing perilymph with serum samples, miRNA-1299 and-1270 were differentially expressed in both the perilymph and serum of Ménière's patients compared to controls. Further analysis using qPCR confirmed miRNA-1299 is downregulated over 3-fold in Meniere's disease serum samples compared to controls. Conclusions: Patients with Ménière's disease exhibit distinct miRNA expression profiles within both the perilymph and serum. The altered perilymph miRNAs identified can be linked to postulated Ménière's disease pathways and may serve as biomarkers. miRNA-1299 was validated to be downregulated in both the serum and perilymph of Menière's patients.

Clinical translation of gene therapy has been challenging, due to limitations in current delivery vehicles such as traditional viral vectors. Herein, we report the use of gRNA:Cas9 ribonucleoprotein (RNP) complexes engineered extracellular vesicles (EVs) for in vivo gene therapy. By leveraging a novel high-throughput microfluidic droplet-based electroporation system (μDES), we achieved 10-fold enhancement of loading efficiency and more than 1000-fold increase in processing throughput on loading RNP complexes into EVs (RNP-EVs), compared with conventional bulk electroporation. The flow-through droplets serve as enormous bioreactors for offering millisecond pulsed, low-voltage electroporation in a continuous-flow and scalable manner, which minimizes the Joule heating influence and surface alteration to retain natural EV stability and integrity. In the Shaker-1 mouse model of dominant progressive hearing loss, we demonstrated the effective delivery of RNP-EVs into inner ear hair cells, with a clear reduction of Myo7ash1 mRNA expression compared to RNP-loaded lipid-like nanoparticles (RNP-LNPs), leading to significant hearing recovery measured by auditory brainstem responses (ABR).

Objective To evaluate the safety and tolerability of repeated intratympanic administration of the gel-formulated NMDA receptor antagonist AM-101 in acute patients with inner ear tinnitus. Study Design Prospective, double-blind, randomized, placebo-controlled study. Setting Sixty-nine secondary and tertiary sites in North America, Europe, and Asia. Subjects and Methods In total, 343 subjects with persistent acute tinnitus after traumatic cochlear injury or otitis media were randomized to receive 3 intratympanic doses of either AM-101 0.87 mg/mL or placebo over 3 to 5 days. They were followed for 84 days. The primary safety end point was the incidence of a clinically meaningful hearing deterioration from baseline to study day 35. Further safety assessments included tympanic membrane closure rates, analysis of adverse events, hematology, blood chemistry, and vital signs. In addition, data were collected on applied anesthetics and injection techniques. Results The treatment was well tolerated, with no intervention-related serious adverse events. The incidence of clinically meaningful hearing deterioration was low, comparable between treatment groups ( P = .82 for the primary safety end point) and not different between treated and untreated ears in unilaterally treated subjects. The rate of treatment and procedure-related adverse events was similar among treatment groups. The tympanic membrane was closed in 92% of subjects within 1 week and in all subjects by study day 84. Blood values and vital signs were inconspicuous. Conclusion Repeated intratympanic injections of AM-101 over a 3- to 5-day period appear to be safe and well tolerated, demonstrating the ability to potentially use this delivery approach over longer time periods.

Calcium-activated potassium channels have been shown to be critically involved in neuronal function, but an elucidation of their detailed roles awaits identification of the microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the large-conductance calcium-activated potassium channels (called BK, KCa1.1, or Slo1) in the somatodendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane. At distal dendritic compartments, BK channels were scattered over the plasma membrane of dendritic shafts and spines but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, whereas the other pool was clustered in plasma membrane domains overlying subsurface cisterns. The labeling density ratio of clustered to scattered channels was about 60:1, established in SDS-FRL. Subsurface cisterns, also called hypolemmal cisterns, are subcompartments of the endoplasmic reticulum likely representing calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones, and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells.

The lack of approved anti-inflammatory and neuroprotective therapies in otology has been acknowledged in the last decades and recent approaches are heralding a new era in the field. Extracellular vesicles (EVs) derived from human multipotent (mesenchymal) stromal cells (MSC) can be enriched in vesicular secretome fractions, which have been shown to exert effects (eg, neuroprotection and immunomodulation) of their parental cells. Hence, MSC-derived EVs may serve as novel drug candidates for several inner ear diseases. Here, we provide first evidence of a strong neuroprotective potential of human stromal cell-derived EVs on inner ear physiology. In vitro, MSC-EV preparations exerted immunomodulatory activity on T cells and microglial cells. Moreover, local application of MSC-EVs to the inner ear significantly attenuated hearing loss and protected auditory hair cells from noise-induced trauma in vivo. Thus, EVs derived from the vesicular secretome of human MSC may represent a next-generation biological drug that can exert protective therapeutic effects in a complex and nonregenerating organ like the inner ear.