Generation of four induced pluripotent stem cell lines (KEIUi004-A, KEIUi005-A, KEIUi006-A, and KEIUi007-A) from patients with sensorineural hearing loss with mutation in EYA4 gene.

Disease-related cells differentiated from patient-derived iPSCs are useful for elucidating the pathophysiological mechanisms underlying these diseases. In this study, four iPSC lines were established from independent patients with sensorineural hearing loss and a mutation in EYA4. These iPSCs showed pluripotency, the capacity to differentiate into three germ layers, and normal karyotypes, suggesting that these lines are useful for the pathological study of sensorineural hearing loss and drug screening for ear disorders.

Phase I/II Study of a Vascular Endothelial Growth Factor Receptor Vaccine in Patients With NF2-Related Schwannomatosis.

PURPOSE: The humanized antivascular endothelial growth factor (VEGF) antibody bevacizumab (Bev) is efficacious for the treatment of NF2-related schwannomatosis (NF2), previously known as neurofibromatosis type 2. This study evaluated the safety and efficacy of a VEGF receptor (VEGFR) vaccine containing VEGFR1 and VEGFR2 peptides in patients with NF2 with progressive schwannomas (jRCTs031180184). MATERIALS AND METHODS: VEGFR1 and VEGFR2 peptides were injected subcutaneously into infra-axillary and inguinal regions, once a week for 4 weeks and then once a month for 4 months. The primary end point was safety. Secondary end points included tolerability, hearing response, imaging response, and immunologic response. RESULTS: Sixteen patients with NF2 with progressive schwannomas completed treatment and were assessed. No severe vaccine-related adverse events occurred. Among the 13 patients with assessable hearing, word recognition score improved in five patients at 6 months and two at 12 months. Progression of average hearing level of pure tone was 0.168 dB/mo during the year of treatment period, whereas long-term progression was 0.364 dB/mo. Among all 16 patients, a partial response was observed in more than one schwannoma in four (including one in which Bev had not been effective), minor response in 5, and stable disease in 4. Both VEGFR1-specific and VEGFR2-specific cytotoxic T lymphocytes (CTLs) were induced in 11 patients. Two years after vaccination, a radiologic response was achieved in nine of 20 assessable schwannomas. CONCLUSION: This study demonstrated the safety and preliminary efficacy of VEGFR peptide vaccination in patients with NF2. Memory-induced CTLs after VEGFR vaccination may persistently suppress tumor progression.

Generation of an induced pluripotent stem cell line (KEIUi008-A) from a hearing loss patient with an A1555G mutation in mitochondrial DNA.

We report the establishment of a human induced pluripotent stem cell (iPSC) line from a 54-year-old male patient with an A1555G mutation in the mitochondrial 12S ribosomal RNA gene (MTRNR1), associated with sensorineural hearing loss. The established iPSC line expressed stemness markers or undifferentiated state markers. We also demonstrated the capacity of the cells to differentiate into the three germ layers, suggesting its pluripotency and utility in the pathological study of sensorineural hearing loss and drug screening for ear disorders.

Adeno-Associated Virus-Encapsulated Alginate Microspheres Loaded in Collagen Gel Carriers for Localized Gene Transfer.

This work reports localized in vivo gene transfer by biodegradation of the adeno-associated virus-encapsulating alginate microspheres (AAV-AMs) loaded in collagen gel carriers. AAV-AMs are centrifugally synthesized by ejecting a mixed pre-gel solution of alginate and AAV to CaCl2 solution to form an ionically cross-linked hydrogel microsphere immediately. The AAV-AMs are able to preserve the AAV without diffusing out even after spreading them on the cells, and the AAV is released and transfected by the degradation of the alginate microsphere. In addition, AAV-AMs can be stored by cryopreservation until use. By implanting this highly convenient AAV-encapsulated hydrogel, AAV-AMs can be loaded into collagen gel carriers to fix the position of the implanted AAV-AMs and achieve localized gene transfer in vivo. In vivo experiments show that the AAV-AMs loaded in collagen gel carriers are demonstrated to release the encapsulated AAV for gene transfer in the buttocks muscles of mice. While conventional injections caused gene transfer to the entire surrounding tissue, the biodegradation of AAV-AMs shows that gene transfer is achieved locally to the muscles. This means that the proposed AAV-loaded system is shown to be a superior method for selective gene transfer.

Brain-Specific Angiogenesis Inhibitor 3 Is Expressed in the Cochlea and Is Necessary for Hearing Function in Mice.

Mammalian auditory hair cells transduce sound-evoked traveling waves in the cochlea into nerve stimuli, which are essential for hearing function. Pillar cells located between the inner and outer hair cells are involved in the formation of the tunnel of Corti, which incorporates outer-hair-cell-driven fluid oscillation and basilar membrane movement, leading to the fine-tuned frequency-specific perception of sounds by the inner hair cells. However, the detailed molecular mechanism underlying the development and maintenance of pillar cells remains to be elucidated. In this study, we examined the expression and function of brain-specific angiogenesis inhibitor 3 (Bai3), an adhesion G-protein-coupled receptor, in the cochlea. We found that Bai3 was expressed in hair cells in neonatal mice and pillar cells in adult mice, and, interestingly, Bai3 knockout mice revealed the abnormal formation of pillar cells, with the elevation of the hearing threshold in a frequency-dependent manner. Furthermore, old Bai3 knockout mice showed the degeneration of hair cells and spiral ganglion neurons in the basal turn. The results suggest that Bai3 plays a crucial role in the development and/or maintenance of pillar cells, which, in turn, are necessary for normal hearing function. Our results may contribute to understanding the mechanisms of hearing loss in human patients.

Mapping of Notch signaling in the developing organ of Corti in common marmosets.

Introduction: The well-regulated development of the sensory epithelium is essential for hearing. This process involves the specification of a pro-sensory epithelium containing common progenitors that differentiate into hair and supporting cells. Notch signaling is one of the most critical pathways during these processes, and its modification is thought to be a feasible approach for treating hearing loss. Despite interspecies differences between rodents and primates or humans, most of our current knowledge regarding cochlear development has been obtained from rodent models. Methods: We therefore examined and mapped the expression patterns of Notch signal components in the developing cochlea of the common marmoset (Callithrix jacchus), a small monkey species native to the New World, a primate model animal. Results: In contrast to the preserved expression patterns of the Notch signaling components in the hair cell differentiation between primates and rodents, we unveiled relatively large interspecies differences during the maturation of supporting cells. Discussion: This improved knowledge of Notch signaling during primate cochlear development will facilitate the development of future regenerative therapies.

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound.

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l-lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.

Correlation between genotype and phenotype with special attention to hearing in 14 Japanese cases of NF2-related schwannomatosis.

NF2-related schwannomatosis (NF2) is an autosomal dominant genetic disorder caused by variants in the NF2 gene. Approximately 50% of NF2 patients inherit pathogenic variants, and the remainder acquire de novo variants. NF2 is characterized by development of bilateral vestibular schwannomas. The genetic background of Japanese NF2 cases has not been fully investigated, and the present report performed a genetic analysis of 14 Japanese NF2 cases and examined genotype-phenotype correlations. DNA samples collected from peripheral blood were analyzed by next-generation sequencing, multiplex ligation-dependent probe amplification analysis, and in vitro electrophoresis. Ten cases had pathogenic or likely pathogenic variants in the NF2 gene, with seven truncating variants and three non-truncating variants. The age of onset in all seven cases with truncating variants was < 20 years. The age of onset significantly differed among cases with truncating NF2 variants, non-truncating NF2 variants, and no NF2 variants. However, the clinical course of tumor growth and hearing deterioration were not predicted only by germline pathogenic NF2 variants. The rate of truncating variants was higher in the present study than that of previous reports. Genotype-phenotype correlations in the age of onset were present in the analyzed Japanese NF2 cases.

Apoptosis of type I spiral ganglion neuron cells in Otof-mutant mice.

Otof, which encodes otoferlin, knockout mice are considered model mice for auditory neuropathy spectrum disorder, which is characterized by an absent auditory brainstem response (ABR) despite preserved distortion product otoacoustic emission (DPOAE). Although otoferlin-deficient mice lack neurotransmitter release at the inner hair cell (IHC) synapse, it remains unclear how the Otof mutation affects spiral ganglions. Thus, we used Otof-mutant mice carrying the Otoftm1a(KOMP)Wtsi allele (Otoftm1a) and analyzed spiral ganglion neurons (SGNs) in Otoftm1a/tm1a mice by immunolabeling type Ⅰ SGNs (SGN-Ⅰ) and type II SGNs (SGN-II). We also examined apoptotic cells in SGNs. Four-week-old Otoftm1a/tm1a mice had an absent ABR but normal DPOAEs. The number of SGNs was significantly lower in Otoftm1a/tm1a mice on postnatal day 7 (P7), P14, and P28 compared with that of wild-type mice. Moreover, significantly more apoptotic SGNs were observed in Otoftm1a/tm1a mice than in wild-type mice on P7, P14, and P28. SGN-IIs were not significantly reduced in Otoftm1a/tm1a mice on P7, P14, and P28. No apoptotic SGN-IIs were observed under our experimental conditions. In summary, Otoftm1a/tm1a mice showed a reduction in SGNs accompanied by apoptosis of SGN-Ⅰs even before the onset of hearing. We speculate that the reduction in SGNs with apoptosis is a secondary defect caused by a lack of otoferlin in IHCs. Appropriate glutamatergic synaptic inputs may be important for the survival of SGNs.

Generation of two induced pluripotent stem cell lines from individuals without auditory disorders.

We report the establishment of two human induced pluripotent stem cell (iPSC) lines from individuals without auditory disorders. Extensive audiometry tests were performed to confirm normal hearing. The generated iPSC lines expressed pluripotency genes and showed differentiation capability into the three germ layers. The iPSC lines will be used as controls for pathological analysis and drug screening for ear disorders.

Hydroxyapatite Prostheses in Endoscopic Transcanal Stapes Surgery for Otosclerosis Cases.

OBJECTIVES: Hydroxyapatite is a commonly used material for medical applications due to its excellent biocompatibility. We use hydroxyapatite prosthesis for the reconstruction of the ossicular chain in stapes surgery. In this study, we report a case series of endoscopic ear surgery using a basket-type hydroxyapatite prosthesis. METHODS: We retrospectively examined 8 cases of endoscopic transcanal stapes surgery using hydroxyapatite prostheses. We evaluated the postoperative results and complications. RESULTS: The average postoperative air-bone gaps were within 10 dB in all cases. Postoperative sensorineural hearing loss was not observed in any case. There was an intraoperative complication with the chorda tympani in 1 patient. We were able to preserve the chorda tympani of all patients, including this case. Postoperative transient dizziness and transient taste disorder were observed in 50% of cases. No other complications, including facial nerve palsy, tympanic membrane perforation, or postoperative infection, were observed. CONCLUSIONS: The postoperative results and complications were comparable to those of surgery under a microscope. The hydroxyapatite prosthesis could be a possible alternative for the piston-type titanium or polytetrafluoroethylene prosthesis.

How Can Dupilumab Cause Eosinophilic Pneumonia?

Reports of eosinophilic pneumonia (EP) as a side effect of dupilumab administration are limited in previous studies. Herein, we report two cases in which EP developed subsequent to the administration of dupilumab for eosinophilic chronic rhinosinusitis (ECRS). Case 1: A 55-year-old woman presented with ECRS, eosinophilic otitis media, and bronchial asthma, and was treated with dupilumab for ECRS. Five weeks later, fever and dyspnea developed, and infiltration shadows were observed in her lungs. The peripheral blood eosinophil count (PBEC) was 3848/µL (26%), bronchoalveolar lavage fluid showed eosinophilic infiltration, and EP was subsequently diagnosed. Her condition improved following prednisolone treatment. Case 2: A 59-year-old man presented with fatigue and dyspnea after receiving dupilumab for ECRS. He had infiltrative shadows throughout his left lung field, and his PBEC was 4850/µL (26.5%). Prednisolone was initiated, and his condition improved. EP developed in both patients during the period of elevated PBEC after dupilumab administration, and dupilumab was suspected to be the causative agent in their EP. Hence, EP should be considered as a differential diagnosis when fever and dyspnea appear following dupilumab administration.

Potential Usefulness of Tracking Head Movement via a Wearable Device for Equilibrium Function Testing at Home.

Many studies have reported the use of wearable devices to acquire biological data for the diagnosis and treatment of various diseases. Balance dysfunction, however, is difficult to evaluate in real time because the equilibrium function is conventionally examined using a stabilometer installed on the ground. Here, we used a wearable accelerometer that measures head motion to evaluate balance and examined whether it performs comparably to a conventional stabilometer. We constructed a simplified physical head-feet model that simultaneously records "head" motion measured using an attached wearable accelerometer and center-of-gravity motion at the "feet", which is measured using an attached stabilometer. Total trajectory length (r = 0.818, p -false discovery rate [FDR] = 0.004) and outer peripheral area (r = 0.691, p -FDR = 0.026) values measured using the wearable device and stabilometer were significantly positively correlated. Root mean square area values were not significantly correlated with wearable device stabilometry but were comparable. These results indicate that wearable, widely available, non-medical devices may be used to assess balance outside the hospital setting, and new approaches for testing balance function should be considered.

MRI tractography reveals the human olfactory nerve map connecting the olfactory epithelium and olfactory bulb.

The olfactory nerve map describes the topographical neural connections between the olfactory epithelium in the nasal cavity and the olfactory bulb. Previous studies have constructed the olfactory nerve maps of rodents using histological analyses or transgenic animal models to investigate olfactory nerve pathways. However, the human olfactory nerve map remains unknown. Here, we demonstrate that high-field magnetic resonance imaging and diffusion tensor tractography can be used to visualize olfactory sensory neurons while maintaining their three-dimensional structures. This technique allowed us to evaluate the olfactory sensory neuron projections from the nasal cavities to the olfactory bulbs and visualize the olfactory nerve maps of humans, marmosets and mice. The olfactory nerve maps revealed that the dorsal-ventral and medial-lateral axes were preserved between the olfactory epithelium and olfactory bulb in all three species. Further development of this technique might allow it to be used clinically to facilitate the diagnosis of olfactory dysfunction.

Critical roles of FGF, RA, and WNT signalling in the development of the human otic placode and subsequent lineages in a dish.

Introduction: Efficient induction of the otic placode, the developmental origin of the inner ear from human pluripotent stem cells (hPSCs), provides a robust platform for otic development and sensorineural hearing loss modelling. Nevertheless, there remains a limited capacity of otic lineage specification from hPSCs by stepwise differentiation methods, since the critical factors for successful otic cell differentiation have not been thoroughly investigated. In this study, we developed a novel differentiation system involving the use of a three-dimensional (3D) floating culture with signalling factors for generating otic cell lineages via stepwise differentiation of hPSCs. Methods: We differentiated hPSCs into preplacodal cells under a two-dimensional (2D) monolayer culture. Then, we transferred the induced preplacodal cells into a 3D floating culture under the control of the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), retinoic acid (RA) and WNT signalling pathways. We evaluated the characteristics of the induced cells using immunocytochemistry, quantitative PCR (qPCR), population averaging, and single-cell RNA-seq (RNA-seq) analysis. We further investigated the methods for differentiating otic progenitors towards hair cells by overexpression of defined transcription factors. Results: We demonstrated that hPSC-derived preplacodal cells acquired the potential to differentiate into posterior placodal cells in 3D floating culture with FGF2 and RA. Subsequent activation of WNT signalling induced otic placodal cell formation. By single-cell RNA-seq (scRNA-seq) analysis, we identified multiple clusters of otic placode- and otocyst marker-positive cells in the induced spheres. Moreover, the induced otic cells showed the potential to generate hair cell-like cells by overexpression of the transcription factors ATOH1, POU4F3 and GFI1. Conclusions: We demonstrated the critical role of FGF2, RA and WNT signalling in a 3D environment for the in vitro differentiation of otic lineage cells from hPSCs. The induced otic cells had the capacity to differentiate into inner ear hair cells with stereociliary bundles and tip link-like structures. The protocol will be useful for in vitro disease modelling of sensorineural hearing loss and human inner ear development and thus contribute to drug screening and stem cell-based regenerative medicine.

Early development of the cochlea of the common marmoset, a non-human primate model.

BACKGROUND: Fine-tuned cochlear development is essential for hearing. Owing to the difficulty in using early human fetal samples, most of our knowledge regarding cochlear development has been obtained from rodents. However, several inter-species differences in cochlear development between rodents and humans have been reported. To bridge these differences, we investigated early otic development of a non-human primate model animal, the common marmoset (Callithrix jacchus). METHODS: We examined 20 genes involved in early cochlear development and described the critical developmental steps for morphogenesis, which have been reported to vary between rodents and marmosets. RESULTS: The results revealed that several critical genes involved in prosensory epithelium specifications showed higher inter-species differences, suggesting that the molecular process for hair cell lineage acquisition in primates differs considerably from that of rodents. We also observed that the tempo of cochlear development was three times slower in the primate than in rodents. CONCLUSIONS: Our data provide new insights into early cochlear development in primates and humans and imply that the procedures used for manipulating rodent cochlear sensory cells cannot be directly used for the research of primate cells due to the intrinsic inter-species differences in the cell fate determination program.

Transcanal endoscopic ear surgery for management of ossicular malformation: clinical outcomes of 17 cases.

BACKGROUND: In recent years, transcanal endoscopic ear surgery (TEES) is known as an innovative and minimally invasive surgery. AIMS: To clarify the usefulness of TEES for the ossicular malformation, we performed a retrospective study. MATERIAL AND METHODS: We examined cases of ossicular malformation performed using TEES at our hospital between April 2015 and April 2020. RESULTS: The hearing level results were countable for 16 cases. Post-operative hearing levels were significantly improved. Transient nausea, tongue paralysis, and taste disorders were observed; however, no other complications were observed. In 2015-2018, some of the cases required the assistance of a surgical microscope. In contrast, all the cases were performed by TEES after 2019 using a powered device to curve the canal. CONCLUSIONS AND SIGNIFICANCE: TEES requires considerable training, and the sensation of depth is difficult to acquire. However, this less invasive method is also helpful for ossicular malformation cases.

Otic Organoids Containing Spiral Ganglion Neuron-like Cells Derived from Human-induced Pluripotent Stem Cells as a Model of Drug-induced Neuropathy.

The spiral ganglion of the cochlea is essential for hearing and contains primary bipolar neurons that relay action potentials generated by mechanosensory hair cells. Injury to spiral ganglion neurons (SGNs) causes permanent hearing loss because these cells have limited regenerative capacity. Establishment of human cell-derived inner ear tissue in vitro could facilitate the development of treatments for hearing loss. Here, we report a stepwise protocol for differentiating human-induced pluripotent stem cells (hiPSCs) into otic organoids that contain SGN-like cells and demonstrate that otic organoids have potential for use as an experimental model of drug-induced neuropathy. Otic progenitor cells (OPCs) were created by 2D culture of hiPSCs for 9 days. Otic spheroids were formed after 2D culture of OPCs for 2 days in a hypoxic environment. Otic organoids were generated by 3D culture of otic spheroids under hypoxic conditions for 5 days and normoxic conditions for a further 30 days or more. The protein expression profile, morphological characteristics, and electrophysiological properties of SGN-like cells in otic organoids were similar to those of primary SGNs. Live-cell imaging of AAV-syn-EGFP-labeled neurons demonstrated temporal changes in cell morphology and revealed the toxic effects of ouabain (which causes SGN-specific damage in animal experiments) and cisplatin (a chemotherapeutic drug with ototoxic adverse effects). Furthermore, a cyclin-dependent kinase-2 inhibitor suppressed the toxic actions of cisplatin on SGN-like cells in otic organoids. The otic organoid described here is a candidate novel drug screening system and could be used to identify drugs for the prevention of cisplatin-induced neuropathy.

Complete Removal of Stage II Congenital Cholesteatoma Destroying Temporomandibular Joints Using Transcanal Endoscopic Ear Surgery With a Mirror Technique: A Case Report.

Transcanal endoscopic ear surgery (TEES) provides wide-angle clear vision for otologic surgery. We report the utility of TEES with the mirror technique for the complete removal of a congenital cholesteatoma in a 3-year-old boy. A white mass was observed through the tympanic membrane, and a congenital cholesteatoma was suspected during the conservative treatment of otitis media with effusion. Pre-operative computed tomography (CT) revealed an irregular mass lesion in the left middle ear, with bone erosion in the hypotympanum. During surgery, an open-type cholesteatoma was observed, mainly in the middle lower tympanum. The cholesteatoma had destroyed the periphery of the temporomandibular joint, which was widely exposed. The cholesteatoma had also spread to the tympanic sinus. Careful observation with a forward-oblique viewing endoscope and a variable angle tympanic mirror enabled complete removal of the mass under endoscopic guidance. No recurrence was observed during the postoperative follow-up, although residual open-type congenital cholesteatoma may often result in recurrence. We believe that careful removal of the lesion under TEES, which allowed us to perform surgery under a secure view with illumination and magnification, may have facilitated complete removal and prevented recurrence.