Publications by authors named "Masato Fujioka"

54 Publications

Neuronal development in the cochlea of a nonhuman primate model, the common marmoset.

Dev Neurobiol 2021 Sep 21. Epub 2021 Sep 21.

Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo, 160-8582, Japan.

Precise cochlear neuronal development is vital to hearing ability. Understanding the developmental process of the spiral ganglion is useful for studying hearing loss aimed at aging or regenerative therapy. Although inter-species differences have been reported between rodents and humans, to date, most of our knowledge about the development of cochlear neuronal development has been obtained from rodent models because of the difficulty in using human fetal samples in this field. In this study, we investigated cochlear neuronal development in a small New World monkey species, the common marmoset (Callithrix jacchus). We examined more than 25 genes involved in the neuronal development of the cochlea and described the critical developmental steps of these neurons. We also revealed similarities and differences between previously reported rodent models and this primate animal model. Our results clarified that this animal model of cochlear neuronal development is more similar to humans than rodents and is suitable as an alternative for the analysis of human cochlear development. The time course established in this report will be a useful tool for studying primate-specific neuronal biology of the inner ear, which could eventually lead to new treatment strategies for human hearing loss. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/dneu.22850DOI Listing
September 2021

Dynamic Spatiotemporal Expression Changes in Connexins of the Developing Primate's Cochlea.

Genes (Basel) 2021 Jul 16;12(7). Epub 2021 Jul 16.

Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan.

Connexins are gap junction components that are essential for acquiring normal hearing ability. Up to 50% of congenital, autosomal-recessive, non-syndromic deafness can be attributed to variants in , the gene that encodes connexin 26. Gene therapies modifying the expression of connexins are a feasible treatment option for some patients with genetic hearing losses. However, the expression patterns of these proteins in the human fetus are not fully understood due to ethical concerns. Recently, the common marmoset was used as a primate animal model for the human fetus. In this study, we examined the expression patterns of connexin 26 and connexin 30 in the developing cochlea of this primate. Primate-specific spatiotemporal expression changes were revealed, which suggest the existence of primate-specific control of connexin expression patterns and specific functions of these gap junction proteins. Moreover, our results indicate that treatments for connexin-related hearing loss established in rodent models may not be appropriate for human patients, underscoring the importance of testing these treatments in primate models before applying them in human clinical trials.
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http://dx.doi.org/10.3390/genes12071082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8307058PMC
July 2021

Investigation of the hearing levels of siblings affected by a single GJB2 variant: Possibility of genetic modifiers.

Int J Pediatr Otorhinolaryngol 2021 Oct 12;149:110840. Epub 2021 Jul 12.

Division of Hearing and Balance Research, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro-ku, Tokyo, 152-8902, Japan; Medical Genetics Center, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro-ku, Tokyo, 152-8902, Japan. Electronic address:

Objective: Variants in GJB2 can cause autosomal recessive deafness (DFNB1). There is evidence for genotype-phenotype correlations of GJB2 variants; however, several genotypes can cause varying levels of hearing loss likely attributable to differences in genetic or environmental background. As siblings share approximately 50% of their genetic background and usually have a common environmental background, analysis of phenotypes of siblings with a specific GJB2 variant may reveal factors relevant to phenotypic variation. There have been no previous analyses of differences in hearing among siblings carrying a single GJB2 genotype. Here, we investigated hearing differences between siblings with a single GJB2 variant, which can cause various levels of hearing loss.

Methods: We examined hearing levels in 16 pairs of siblings homozygous for the c.235delC variant of GJB2. Differences in hearing acuity between sibling pairs were detected by auditory evaluation.

Results: Average differences in acoustic threshold >30 dB were observed between five pairs of siblings, whereas the remaining 11 pairs had average threshold values within approximately 10 dB of one another. Hearing loss varied from moderate to profound.

Conclusion: Our results indicate that auditory acuity associated with homozygosity for GJB2 c.235delC can vary in degree; however, in approximately 70% of younger siblings, it was approximately the same as that in the first child, despite a diverse spectrum of hearing loss among different families. These results suggest that differences in genetic background may modify the phenotype associated with homozygous GJB2 c.235delC.
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http://dx.doi.org/10.1016/j.ijporl.2021.110840DOI Listing
October 2021

Three cases of otitis media caused by Mycobacterium abscessus subsp. abscessus: Importance of medical treatment and efficacy of surgery.

J Infect Chemother 2021 Aug 29;27(8):1251-1257. Epub 2021 Apr 29.

Department of Otolaryngology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan. Electronic address:

This study aimed to assess the clinical presentation, antibiotic therapy, surgery, and outcomes in patients with otitis media caused by Mycobacterium abscessus subsp. abscessus and discuss the efficacy of surgery. This is a retrospective case review of three patients diagnosed with otomastoiditis caused by M. abscessus subsp. abscessus. All patients had refractory otorrhea. One patient had granulation tissue in the tympanic membrane. They received medical treatment and underwent surgery. Otorrhea was resolved several months after the initiation of long-term multiantibiotic therapy in all cases. The timing of surgery varied among patients. Before initiating antibiotic therapy, mastoidectomy was performed to achieve definitive diagnosis in two patients, and wound dehiscence developed in these patients. Two patients underwent debridement after the initiation of multiantibiotic therapy. After antibiotic administration, tympanoplasty was performed to improve hearing in one patient. All patients achieved culture negativity after treatment, and no recurrences have been noted. From three cases, it is suggested that the mainstay of treatment for M. abscessus subsp. abscessus is long-term multiantibiotic therapy, and surgery itself may have little effect on achieving ear dryness. Thus, in most patients, drug therapy should be prioritized. Considering postoperative complications, surgery before achieving ear dryness should be avoided, except in emergency cases. In addition, if the diagnosis is not confirmed by repeated bacteriological tests, mastoidectomy should be performed to collect specimens. Tympanoplasty for hearing loss or eardrum perforation is recommended after discontinuation of medications.
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http://dx.doi.org/10.1016/j.jiac.2021.04.012DOI Listing
August 2021

Multicenter phase III trial of regenerative treatment for chronic tympanic membrane perforation.

Auris Nasus Larynx 2021 Dec 26;48(6):1054-1060. Epub 2021 Mar 26.

Translational Research Center for Medical Innovation, Foundation for Biomedical Research and Innovation at Kobe, 1-5-4 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.

Objective: To evaluate the efficacy and safety of regenerative treatment for tympanic membrane perforation (TMP) using gelatin sponge, basic fibroblast growth factor (bFGF), and fibrin glue.

Methods: This was a multicenter, non-randomized, single-arm study conducted at tertiary referral centers. Twenty patients with chronic TMP (age 23-78 years, 6 males, 14 females) were registered from three institutions. All treated patients were included in the safety analysis population. The edges of the TMP were disrupted mechanically by myringotomy and several pieces of gelatin sponge immersed in bFGF were placed and fixed with fibrin glue to cover the perforation. The TMP was examined 4 ± 1 weeks later. The protocol was repeated up to four times until closure was complete. The main outcome measures were closure or a decrease in size of the TMP, hearing improvement, and air-bone gap evaluated 16 weeks after the final regenerative procedure (FRP). Adverse events (AEs) were monitored throughout the study.

Results: Total closure of the TMP at 16 weeks was achieved in 15 out of 20 patients (75.0%, 95% confidence interval [CI]: 50.9%-91.3%) and the mean decrease in size was 92.2% (95%CI: 82.9%-100.0%). The ratio of hearing improvement and the air-bone gap at 16 weeks after FRP were 100% (20/20; 95%CI: 83.2%-100%) and 5.3 ± 4.2 dB (p <0.0001), respectively. Thirteen out of 20 patients (65.0%) experienced at least one AE, but no serious AEs occurred.

Conclusion: The results indicate that the current regenerative treatment for TMP using gelatin sponge, bFGF, and fibrin glue is safe and effective.
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http://dx.doi.org/10.1016/j.anl.2021.02.007DOI Listing
December 2021

Hydroxyapatite Prostheses in Endoscopic Transcanal Stapes Surgery for Otosclerosis Cases.

Ear Nose Throat J 2021 Feb 2:145561321989143. Epub 2021 Feb 2.

Department of Otolaryngology, Head and Neck Surgery, Keio University School of Medicine, Tokyo, Japan.

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.
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http://dx.doi.org/10.1177/0145561321989143DOI Listing
February 2021

Deficiency of large tumor suppressor kinase 1 causes congenital hearing loss associated with cochlear abnormalities in mice.

Biochem Biophys Res Commun 2021 01 5;534:921-926. Epub 2020 Nov 5.

Department of Otolaryngology-Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan. Electronic address:

Mammalian auditory hair cells are not spontaneously replaced. Their number and coordinated polarization are fairly well-maintained and both these factors might be essential for the cochlear amplifier. Cell cycle regulation has critical roles in regulating appropriate cell size and cell number. However, little is known about the physiological roles of the Hippo pathway, which is one of the most important signaling cascades that regulates cell growth, differentiation, and regenerative capacity in the cochlear sensory epithelium. Herein, we investigated the in vivo role of the large tumor suppressor 1 (LATS1), an essential kinase in the Hippo/yes-associated protein pathway, in the cochlea using the LATS1 knockout mice. LATS1 was expressed in hair cells and supporting cells. It was strongly expressed on the surface of the cuticular plate of the organ of Corti. We found that LATS1 knockout caused congenital hearing loss due to the irregular orientation and slightly reduced number of hair cells, whereas the number of supporting cells remained unchanged. On the surface of the hair cells, the kinocilium and stereocilia were dispersed during and after morphogenesis. However, the expression of the receptor-independent polarity regulators, such as Par3 or Gαi, was not affected. We concluded that LATS1 has an indispensable role in the maturation of mammalian auditory hair cells, but not in the development of the supporting cells, and thus, has a role in the hearing acquisition.
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http://dx.doi.org/10.1016/j.bbrc.2020.10.073DOI Listing
January 2021

Low-dose rapamycin-induced autophagy in cochlear outer sulcus cells.

Laryngoscope Investig Otolaryngol 2020 Jun 29;5(3):520-528. Epub 2020 May 29.

Department of Otorhinolaryngology, Head and Neck Surgery Keio University School of Medicine Tokyo Japan.

Objectives: Autophagy is an intracellular housekeeping process that degrades cytoplasmic organelles, damaged molecules, and abnormal proteins or pathogens and is essential for normal hearing. Recent studies revealed the essential roles of autophagy in hearing and balance. The aim of this study was to evaluate the activation state of rapamycin-induced autophagy in cochlear outer sulcus cells (OSCs).

Methods: We used autophagy reporter transgenic mice expressing the green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) fusion protein and counted GFP-LC3 puncta in cochlear OSCs to evaluate the activation state of autophagy after oral administration of rapamycin.

Results: We observed basal level GFP-LC3 expression and an increase in the number of GFP-LC3 puncta in cochlear OSCs by oral administration of rapamycin. This increase was detected when the daily rapamycin intake was as low as 0.025 mg/kg, and it was dose dependent. The increased number of puncta was more at the basal turn than the apical turn.

Conclusion: Oral intake of low-dose rapamycin activates autophagy in cochlear OSCs.

Level Of Evidence: NA.
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http://dx.doi.org/10.1002/lio2.392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314457PMC
June 2020

Differences in hearing levels between siblings with hearing loss caused by GJB2 mutations.

Auris Nasus Larynx 2020 Dec 15;47(6):938-942. Epub 2020 Jun 15.

Division of Hearing and Balance Research, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro-ku, Tokyo 152-8902, Japan; Medical Genetics Center, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro, Tokyo 152-8902, Japan. Electronic address:

Objective: Hearing loss caused by GJB2 mutations is inherited in an autosomal recessive manner (DFNB1); thus siblings of an affected child have a 25% chance of also being affected. Hearing loss among subsequent siblings carrying the same GJB2 mutation is a concern for parents and a frequent topic of enquiry during genetic counseling. Evidence exists for genotype-phenotype correlations of GJB2 mutations; however, no analysis of differences in hearing among siblings, in whom the common genetic background may decrease variation, has been reported. The purpose of the present study was to investigate hearing differences between siblings with identical GJB2 mutations.

Methods: We examined the hearing levels of 12 pairs of siblings; each pair had the same pathogenic GJB2 mutations. Differences in hearing acuity between sibling pairs detected by auditory evaluation.

Results: No significant correlation was detected between the average hearing levels of first and second affected siblings. Average differences in acoustic threshold >30 dB were observed between four pairs of siblings, whereas the remaining eight pairs had average threshold values within 20 dB of one another.

Conclusion: Our results indicate that auditory acuity would be expected to approximate that found in the first child in approximately 70% of subsequent children with GJB2-mediated hearing loss, whereas 30% of subsequent siblings would have average differences of >30 dB.
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http://dx.doi.org/10.1016/j.anl.2020.05.008DOI Listing
December 2020

A phase I/IIa double blind single institute trial of low dose sirolimus for Pendred syndrome/DFNB4.

Medicine (Baltimore) 2020 May;99(19):e19763

Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, Tokyo, Japan.

Introduction: Pendred syndrome (PDS)/DFNB 4 is a disorder with fluctuating and progressive hearing loss, vertigo, and thyroid goiter. We identified pathophysiology of a neurodegenerative disorder in PDS patient derived cochlear cells that were induced via induced pluripotent stem cells and found sirolimus, an mTOR inhibitor, as an inhibitor of cell death with the minimum effective concentration less than 1/10 of the approved dose for other diseases. Given that there is no rational standard therapy for PDS, we planned a study to examine effects of low dose oral administration of sirolimus for the fluctuating and progressive hearing loss, and the balance disorder of PDS by daily monitor of their audio-vestibular symptoms.

Methods And Analysis: This is a phase I/IIa double blind parallel-group single institute trial in patient with PDS/DFNB4. Sixteen of outpatients with fluctuating hearing diagnosed as PDS in SLC26A4 genetic testing aged in between 7 and 50 years old at the time of consent are given either placebo or sirolimus tablet (NPC-12T). In NPC-12T placebo arm, placebo will be given for 36 weeks; in active substance arm, placebo will be given for 12 weeks and the NPC-12T for 24 weeks. Primary endpoints are safety and tolerability. The number of occurrences and types of adverse events and of side effects will be sorted by clinical symptoms and by abnormal change of clinical test results. A 2-sided 95% confidence interval of the incidence rate by respective dosing arms will be calculated using the Clopper-Pearson method. Clinical effects on audio-vestibular tests performed daily and precise physiological test at each visit will also be examined as secondary and expiratory endpoints.

Trial Registration Number: JMA-IIA00361; Pre-results.
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http://dx.doi.org/10.1097/MD.0000000000019763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7220392PMC
May 2020

The common marmoset as suitable nonhuman alternative for the analysis of primate cochlear development.

FEBS J 2021 01 15;288(1):325-353. Epub 2020 May 15.

Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, Tokyo, Japan.

Cochlear development is a complex process with precise spatiotemporal patterns. A detailed understanding of this process is important for studies of congenital hearing loss and regenerative medicine. However, much of our understanding of cochlear development is based on rodent models. Animal models that bridge the gap between humans and rodents are needed. In this study, we investigated the development of hearing organs in a small New World monkey species, the common marmoset (Callithrix jacchus). We describe the general stages of cochlear development in comparison with those of humans and mice. Moreover, we examined more than 25 proteins involved in cochlear development and found that expression patterns were generally conserved between rodents and primates. However, several proteins involved in supporting cell processes and neuronal development exhibited interspecific expression differences. Human fetal samples for studies of primate-specific cochlear development are extremely rare, especially for late developmental stages. Our results support the use of the common marmoset as an effective alternative for analyses of primate cochlear development.
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http://dx.doi.org/10.1111/febs.15341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818239PMC
January 2021

Author Correction: A VEGF receptor vaccine demonstrates preliminary efficacy in neurofibromatosis type 2.

Nat Commun 2020 04 21;11(1):2028. Epub 2020 Apr 21.

Department of Neurosurgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-16007-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7174327PMC
April 2020

A VEGF receptor vaccine demonstrates preliminary efficacy in neurofibromatosis type 2.

Nat Commun 2019 12 17;10(1):5758. Epub 2019 Dec 17.

Department of Neurosurgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan.

The anti-VEGF antibody bevacizumab has shown efficacy for the treatment of neurofibromatosis type 2 (NF2). Theoretically, vascular endothelial growth factor receptors (VEGFRs)-specific cytotoxic T lymphocytes (CTLs) can kill both tumor vessel cells and tumor cells expressing VEGFRs. Here we show an exploratory clinical study of VEGFRs peptide vaccine in seven patients with progressive NF2-derived schwannomas. Hearing improves in 2/5 assessable patients (40%) as determined by international guidelines, with increases in word recognition scores. Tumor volume reductions of ≥20% are observed in two patients, including one in which bevacizumab had not been effective. There are no severe adverse events related to the vaccine. Both VEGFR1-specific and VEGFR2-specific CTLs are induced in six patients. Surgery is performed after vaccination in two patients, and significant reductions in the expression of VEGFRs in schwannomas are observed. Therefore, this clinical immunotherapy study demonstrates the safety and preliminary efficacy of VEGFRs peptide vaccination in patients with NF2.
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http://dx.doi.org/10.1038/s41467-019-13640-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917794PMC
December 2019

Distribution of tight junctions in the primate cochlear lateral wall.

Neurosci Lett 2020 01 12;717:134686. Epub 2019 Dec 12.

Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, Tokyo, Japan.

The blood-labyrinth barrier (BLB) is a major structure that separates the inner ear from the systemic blood circulation. Many drugs cannot access the inner ear because of their inability to cross the BLB. In the cochlea, the BLB is mainly distributed in the lateral wall. However, the ultrastructure of the cochlear lateral wall, including the distribution of tight junctions (TJs), which are its main component, has not been thoroughly examined in primates. This study investigated the distribution of TJs in the cochlear lateral wall of the common marmoset by performing immunohistochemistry for TJ markers and transmission electron microscopy. As previously reported in rodents, TJs were distributed at the lumenal side of stria marginal cells and basal cells. In outer sulcus cells, which are more developed in primates than in rodents, TJs were distributed at the side with the endolymph but not at the side with the spiral ligament, where many capillaries were located. These findings indicate that drugs and small compounds circulating systemically in the blood can easily access outer sulcus cells, but have a limited ability to enter endolymph. No structural differences were detected between species, indicating that the in vivo distribution of drugs in cochlear lateral wall cells, including outer sulcus cells, in primates can be predicted by performing rodent experiments.
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http://dx.doi.org/10.1016/j.neulet.2019.134686DOI Listing
January 2020

Comprehensive analysis of syndromic hearing loss patients in Japan.

Sci Rep 2019 08 19;9(1):11976. Epub 2019 Aug 19.

Department of Otorhinolaryngology - Head and Neck Surgery, Kyoto Prefectural University of Medicine, Kyoto, Japan.

More than 400 syndromes associated with hearing loss and other symptoms have been described, corresponding to 30% of cases of hereditary hearing loss. In this study we aimed to clarify the mutation spectrum of syndromic hearing loss patients in Japan by using next-generation sequencing analysis with a multiple syndromic targeted resequencing panel (36 target genes). We analyzed single nucleotide variants, small insertions, deletions and copy number variations in the target genes. We enrolled 140 patients with any of 14 syndromes (BOR syndrome, Waardenburg syndrome, osteogenesis imperfecta, spondyloepiphyseal dysplasia congenita, Stickler syndrome, CHARGE syndrome, Jervell and Lange-Nielsen syndrome, Pendred syndrome, Klippel-Feil syndrome, Alport syndrome, Norrie disease, Treacher-Collins syndrome, Perrault syndrome and auditory neuropathy with optic atrophy) and identified the causative variants in 56% of the patients. This analysis could identify the causative variants in syndromic hearing loss patients in a short time with a high diagnostic rate. In addition, it was useful for the analysis of the cases who only partially fulfilled the diagnostic criteria.
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http://dx.doi.org/10.1038/s41598-019-47141-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700179PMC
August 2019

Generation of a human iPS cell line (CGMH.SLC26A4919-2) from a Pendred syndrome patient carrying SLC26A4 c.919-2A>G splice-site mutation.

Stem Cell Res 2019 10 31;40:101524. Epub 2019 Jul 31.

Department of Otolaryngology, National Taiwan University Hospital, Taipei, Taiwan. Electronic address:

SLC26A4 is the second most frequent gene implicated in congenital hearing loss after GJB2 mutations. Here, we report the generation of induced pluripotent stem cells (iPSCs), from a patient who was carrying a homozygous c.919-2A>G variant in the SLC26A4 gene. This is the most common variant of SLC26A4 gene in the Chinese population and the second most prevalent one in other Asian countries. The established patient-derived iPSC displayed all the features of pluripotent stem cell markers and had the ability to differentiate into all of the three germ layers and possessed a normal karyotype.
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http://dx.doi.org/10.1016/j.scr.2019.101524DOI Listing
October 2019

Anatomical and Surgical Evaluation of the Common Marmoset as an Animal Model in Hearing Research.

Front Neuroanat 2019 6;13:60. Epub 2019 Jun 6.

Division of Regenerative Medicine, The Jikei University School of Medicine, Tokyo, Japan.

Recent studies have indicated that direct administration of viral vectors or small compounds to the inner ear may aid in the treatment of Sensorineural hearing loss (SNHL). However, due to species differences between humans and rodents, translating experimental results into clinical applications remains challenging. The common marmoset (), a New World monkey, is considered a pre-clinical animal model. In the present study, we describe morphometric data acquired from the temporal bone of the common marmoset in order to define the routes of topical drug administration to the inner ear. Dissection and diffusion tensor tractography (DTT) were performed on the fixed cadaverous heads of 13 common marmosets. To investigate potential routes for drug administration to the inner ear, we explored the anatomy of the round window, oval window (OW), semicircular canal, and endolymphatic sac (ES). Among these, the approach the round window with posterior tympanotomy appeared feasible for delivering drugs to the inner ear without manipulating the tympanic membrane, minimizing the chances of conductive hearing loss. The courses of four critical nerves [including the facial nerve (FN)] were visualized using three-dimensional (3D) DTT, which may help to avoid nerve damage during surgery. Finally, to investigate the feasibility of actual drug administration, we measured the volume of the round window niche (RWN), which was approximately 0.9 μL. The present findings may help to establish experimental standards for evaluating new therapies in this primate model.
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http://dx.doi.org/10.3389/fnana.2019.00060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563828PMC
June 2019

Estimating the concentration of therapeutic range using disease-specific iPS cells: Low-dose rapamycin therapy for Pendred syndrome.

Regen Ther 2019 Jun 17;10:54-63. Epub 2018 Dec 17.

Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi Shinjuku-ku, Tokyo 160-8582, Japan.

Introduction: Pendred syndrome is an autosomal-recessive disease characterized by congenital hearing loss and thyroid goiter. Previously, cell stress susceptibilities were shown to increase in patient-derived cells with intracellular aggregation using an acute cochlear cell model derived from patient-specific pluripotent stem (iPS) cells. Moreover, we showed that rapamycin can relieve cell death. However, studies regarding long-term cell survival without cell stressors that mimic the natural course of disease or the rational minimum concentration of rapamycin that prevents cell death are missing.

Methods: In this report, we first investigated the rational minimum concentration of rapamycin using patient-specific iPS cells derived-cochlear cells with three different conditions of acute stress. We next confirmed the effects of rapamycin in long-term cell survival and phenotypes by using cochlear cells derived from three different patient-derived iPS cells.

Results: We found that inner ear cells derived from Pendred syndrome patients are more vulnerable than those from healthy individuals during long-term culturing; however, this susceptibility was relieved via treatment with low-dose rapamycin. The slow progression of hearing loss in patients may be explained, in part, by the vulnerability observed in patient cells during long-term culturing. We successfully evaluated the rational minimum concentration of rapamycin for treatment of Pendred syndrome.

Conclusion: Our results suggest that low-dose rapamycin not only decreases acute symptoms but may prevent progression of hearing loss in Pendred syndrome patients.
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http://dx.doi.org/10.1016/j.reth.2018.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299162PMC
June 2019

Visualization of nerve fibers around the carotid bifurcation with use of a 9.4 Tesla microscopic magnetic resonance diffusion tensor imaging with tractography.

Head Neck 2018 10 26;40(10):2228-2234. Epub 2018 Jun 26.

Department of Otolaryngology - Head and Neck Surgery, Keio University School of Medicine, Tokyo, Japan.

Background: Precise imaging of nerves have been challenging in the head and neck region, mainly due to low spatial resolution. Here, we investigated how nerves in the head and neck region could be visualized using an ultra-high magnetic field MR system.

Methods: We used formol-carbol-fixed human cadaveric necks and obtained MR diffusion tensor images (DTIs) using a 9.4 Tesla (T) ultra-high magnetic field MR system. Afterward, we prepared tissue sections and checked the anatomic relationships between the neurons and the carotid artery in order to confirm that the visualized fibers are indeed neuron fibers.

Results: We were able to identify nerves, including the vagus nerve, the hypoglossal nerve, and the spinal-accessory nerve. Hematoxylin-eosin stained histological sections confirmed neuron fibers in the same anatomic position.

Conclusion: This technique has the feasibility to be applied for a more accurate anatomic understanding, maybe even close to a histological level.
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http://dx.doi.org/10.1002/hed.25318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220873PMC
October 2018

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus).

J Vis Exp 2018 02 27(132). Epub 2018 Feb 27.

Division of Regenerative Medicine, Jikei University School of Medicine;

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.
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http://dx.doi.org/10.3791/56574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931374PMC
February 2018

Engraftment of Human Pluripotent Stem Cell-derived Progenitors in the Inner Ear of Prenatal Mice.

Sci Rep 2018 01 31;8(1):1941. Epub 2018 Jan 31.

Departments of Otolaryngology-Head and Neck Surgery, Kumamoto University Graduate School of Medicine, 1-1-1 Honjo, Chuoku, Kumamoto city, Japan.

There is, at present, no curative treatment for genetic hearing loss. We have previously reported that transuterine gene transfer of wild type CONNEXIN30 (CX30) genes into otocysts in CX30-deleted mice could restore hearing. Cell transplantation therapy might be another therapeutic option, although it is still unknown whether stem cell-derived progenitor cells could migrate into mouse otocysts. Here, we show successful cell transplantation of progenitors of outer sulcus cell-like cells derived from human-derived induced pluripotent stem cells into mouse otocysts on embryonic day 11.5. The delivered cells engrafted more frequently in the non-sensory region in the inner ear of CX30-deleted mice than in wild type mice and survived for up to 1 week after transplantation. Some of the engrafted cells expressed CX30 proteins in the non-sensory region. This is the first report that demonstrates successful engraftment of exogenous cells in prenatal developing otocysts in mice. Future studies using this mouse otocystic injection model in vivo will provide further clues for developing treatment modalities for congenital hearing loss in humans.
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http://dx.doi.org/10.1038/s41598-018-20277-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792596PMC
January 2018

Expression pattern of EYA4 in the common marmoset (Callithrix jacchus) cochlea.

Neurosci Lett 2018 Jan 18;662:185-188. Epub 2017 Oct 18.

Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Electronic address:

The eyes absent (EYA)-like genes are essential for the formation of sensory organs among fly (Drosophila melanogaster) and mammals. EYA4, one of the vertebrate genes of Eya family, is reported to be causative for late-onset mid-frequency sensorineural hearing loss in humans, while Eya4-deficient mice exhibited congenital profound deafness and otitis media with effusion due to the eustachian tube dysmorphology. Because of the species difference in the phenotype, the pathophysiology of EYA4 in the human cochlea has yet to be elucidated. Here, we examine the expression pattern of EYA4 in the cochlea of common marmoset (Callithrix jacchus), a non-human primate. The results indicated a distinct expression pattern of EYA4 in the adult marmoset cochleae, especially strong in all supporting cells, while in mouse their expressions were diminished. Interestingly, EYA4 expression in the hair cells, supporting cells and neurons was co-localized with sine oculis homeobox-SIX1, a transcription factor essential for the transcriptional activity of EYA4. The results revealed inter-species differences in the expression pattern of EYA4 gene in supporting cells between primates and rodents. The results also indicated a fundamental role of EYA4 in the primate auditory cells. Experiments with primate models such as marmosets or with human cochlear cells may provide cues about the unknown pathogenesis of EYA4-related hearing loss.
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http://dx.doi.org/10.1016/j.neulet.2017.10.030DOI Listing
January 2018

Prevalence of TECTA mutation in patients with mid-frequency sensorineural hearing loss.

Orphanet J Rare Dis 2017 09 25;12(1):157. Epub 2017 Sep 25.

Department of Otolaryngology, National Hospital Organization Tokyo Medical Center, 2-5-1 Higashigaoka, Meguro, Tokyo, 152-8902, Japan.

Background: To date, 102 genes have been reported as responsible for non-syndromic hearing loss, some of which are associated with specific audiogram features. Four genes have been reported as causative for mid-frequency sensorineural hearing loss (MFSNHL), among which TECTA is the most frequently reported; however, the prevalence of TECTA mutations is unknown. To elucidate the prevalence of TECTA mutation in MFSNHL and clarify genotype-phenotype correlations, we analyzed the genetic and clinical features of patients with MFSNHL.

Methods: Subjects with bilateral non-syndromic hearing loss were prescreened for GJB2 and m.1555A > G and m.3243A > G mitochondrial DNA mutations, and patients with inner ear malformations were excluded. We selected MFSNHL patients whose audiograms met the U-shaped criterion proposed by the GENDEAF study group, along with those with shallow U-shaped audiograms, for TECTA analysis. All TECTA exons were analyzed by Sanger sequencing. Novel missense variants were classified as possibly pathogenic, non-pathogenic, and variants of uncertain significance, based on genetic data. To evaluate novel possibly pathogenic variants, we predicted changes in protein structure by molecular modeling.

Results: Pathogenic and possibly pathogenic variants of TECTA were found in 4 (6.0%) of 67 patients with MFSNHL. In patients with U-shaped audiograms, none (0%) of 21 had pathogenic or possibly pathogenic variants. In patients with shallow U-shaped audiograms, four (8.7%) of 46 had pathogenic or possibly pathogenic variants. Two novel possibly pathogenic variants were identified and two previously reported mutations were considered as variant of unknown significance. The clinical features of patients with pathogenic and possibly pathogenic variants were consistent with those in previous studies. Pathogenic or possibly pathogenic variants were identified in 3 of 23 families (13.0%) which have the family histories compatible with autosomal dominant and 1 of 44 families (2.3%) which have the family histories compatible with sporadic or autosomal recessive.

Conclusions: TECTA mutations were identified in 6.0% of MFSNHL. These mutations were more frequent in patients with shallow U-shaped audiograms than those with U-shaped audiograms, and in families which have the family histories compatible with autosomal dominant than those with the family histories compatible with sporadic or autosomal recessive.
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http://dx.doi.org/10.1186/s13023-017-0708-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613382PMC
September 2017

Characterization of slow-cycling cells in the mouse cochlear lateral wall.

PLoS One 2017 20;12(6):e0179293. Epub 2017 Jun 20.

Department of Otorhinolaryngology, School of Medicine, Keio University,35 Shinanomachi, Shinjuku-ku, Tokyo, Japan.

Cochlear spiral ligament fibrocytes (SLFs) play essential roles in the physiology of hearing including ion recycling and the generation of endocochlear potential. In adult animals, SLFs can repopulate after damages, yet little is known about the characteristics of proliferating cells that support SLFs' self-renewal. Here we report in detail about the characteristics of cycling cells in the spiral ligament (SL). Fifteen P6 mice and six noise-exposed P28 mice were injected with 5-bromo-2'-deoxyuridine (BrdU) for 7 days and we chased BrdU retaining cells for as long as 60 days. Immunohistochemistry revealed that the BrdU positive IB4 (an endotherial marker) negative cells expressed an early SLF marker Pou3f4 but negative for cleaved-Caspase 3. Marker studies revealed that type 3 SLFs displayed significantly higher percentage of BrdU+ cells compared to other subtypes. Notably, the cells retained BrdU until P72, demonstrating they were dividing slowly. In the noise-damaged mice, in contrast to the loss of the other types, the number of type 3 SLFs did not altered and the BrdU incorporating- phosphorylated Histone H3 positive type 3 cells were increased from day 1 to 14 after noise exposure. Furthermore, the cells repopulating type 1 area, where the cells diminished profoundly after damage, were positive for the type 3 SLF markers. Collectively, in the latral wall of the cochlea, type 3 SLFs have the stem cell capacity and may contribute to the endogenous regeneration of lateral wall spiral ligament. Manipulating type 3 cells may be employed for potential regenerative therapies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179293PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478125PMC
September 2017

Autophagy is essential for hearing in mice.

Cell Death Dis 2017 05 11;8(5):e2780. Epub 2017 May 11.

Department of Otolaryngology, Faculty of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.

Hearing loss is the most frequent sensory disorder in humans. Auditory hair cells (HCs) are postmitotic at late-embryonic differentiation and postnatal stages, and their damage is the major cause of hearing loss. There is no measurable HC regeneration in the mammalian cochlea, and the maintenance of cell function is crucial for preservation of hearing. Here we generated mice deficient in autophagy-related 5 (Atg5), a gene essential for autophagy, in the HCs to investigate the effect of basal autophagy on hearing acuity. Deletion of Atg5 resulted in HC degeneration and profound congenital hearing loss. In autophagy-deficient HCs, polyubiquitinated proteins and p62/SQSTM1, an autophagy substrate, accumulated as inclusion bodies during the first postnatal week, and these aggregates increased in number. These findings revealed that basal autophagy has an important role in maintenance of HC morphology and hearing acuity.
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http://dx.doi.org/10.1038/cddis.2017.194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520715PMC
May 2017

Cochlear Cell Modeling Using Disease-Specific iPSCs Unveils a Degenerative Phenotype and Suggests Treatments for Congenital Progressive Hearing Loss.

Cell Rep 2017 01;18(1):68-81

Department of Physiology, Keio University School of Medicine, 35 Shinanomachi Shinjyuku-ku, Tokyo 160-8582, Japan. Electronic address:

Hearing impairments are the most common symptom of congenital defects, and they generally remain intractable to treatment. Pendred syndrome, the most frequent syndromic form of hereditary hearing loss, is associated with mutations in the anion exchanger pendrin. Loss of pendrin function as an anion exchanger is thought to be causative, but rodent models do not exhibit progressive deafness. Here, we report a degenerative phenotype exhibiting mutant pendrin aggregates and increased susceptibility to cellular stresses in cochlear epithelial cells induced from patient-derived induced pluripotent stem cells (iPSCs). These degenerative phenotypes were rescued by site-specific gene corrections. Moreover, low-dose rapamycin and metformin reduced aggregation and cell death. Our results provide an unexpected, comprehensive understanding of deafness due to "degenerative cochlear disease" and may contribute to rational therapeutic development. This iPSC-based disease model provides an approach to the study of pathogenesis and therapeutic development for hereditary hearing loss.
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http://dx.doi.org/10.1016/j.celrep.2016.12.020DOI Listing
January 2017

Distinct Expression Pattern of a Deafness Gene, KIAA1199, in a Primate Cochlea.

Biomed Res Int 2016 14;2016:1781894. Epub 2016 Jun 14.

Department of Otorhinolaryngology, Head and Neck Surgery, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

Deafness is one of the most common types of congenital impairments, and at least half of the cases are caused by hereditary mutations. Mutations of the gene KIAA1199 are associated with progressive hearing loss. Its expression is abundant in human cochlea, but interestingly the spatial expression patterns are different between mouse and rat cochleae; the pattern in humans has not been fully investigated. We performed immunohistochemical analysis of a nonhuman primate, common marmoset (Callithrix jacchus), cochlea with a KIAA1199-specific antibody. In the common marmoset cochlea, KIAA1199 protein expression was more widespread than in rodents, with all epithelial cells, including hair cells, expressing KIAA1199. Our results suggest that the primate pattern of KIAA1199 expression is wider in comparison with rodents and may play an essential role in the maintenance of cochlear epithelial cells.
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http://dx.doi.org/10.1155/2016/1781894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4923552PMC
February 2017

Expression pattern of wolframin, the WFS1 (Wolfram syndrome-1 gene) product, in common marmoset (Callithrix jacchus) cochlea.

Neuroreport 2016 08;27(11):833-6

Departments of aOtorhinolaryngology, Head and Neck Surgery bPhysiology, Keio University School of Medicine, Tokyo, Japan.

Wolfram syndrome is an autosomal recessive disorder of the neuroendocrine system, known as DIDMOAD (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy and Deafness) syndrome, and considered an endoplasmic reticulum disease. Patients show mutations in WFS1, which encodes the 890 amino acid protein wolframin. Although Wfs1 knockout mice develop diabetes, their hearing level is completely normal. In this study, we examined the expression of wolframin in the cochlea of a nonhuman primate common marmoset (Callithrix jacchus) to elucidate the discrepancy in the phenotype between species and the pathophysiology of Wolfram syndrome-associated deafness. The marmoset cochlea showed wolframin immunoreactivity not only in the spiral ligament type I fibrocytes, spiral ganglion neurons, outer hair cells, and supporting cells, but in the stria vascularis basal cells, where wolframin expression was not observed in the previous mouse study. Considering the absence of the deafness phenotype in Wfs1 knockout mice, the expression of wolframin in the basal cells of primates may play an essential role in the maintenance of hearing. Elucidating the function of wolframin protein in the basal cells of primates would be essential for understanding the pathogenesis of hearing loss in patients with Wolfram syndrome, which may lead to the discovery of new therapeutics.
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http://dx.doi.org/10.1097/WNR.0000000000000624DOI Listing
August 2016

Sustained Effect of Hyaluronic Acid in Subcutaneous Administration to the Cochlear Spiral Ganglion.

PLoS One 2016 21;11(4):e0153957. Epub 2016 Apr 21.

Department of Otolaryngology Head and Neck Surgery, Keio University, Shinjuku-ku, Tokyo, Japan.

The spatiotemporal distribution of drugs in the inner ear cannot be precisely evaluated because of its small area and complex structure. In the present study, we used hyaluronic acid (HA)-dispersed luciferin to image transgenic mice and to determine the effect of HA on controlled drug delivery to the cochlea. GFAP-luc mice, which express luciferase in cochlear spiral ganglion cells, were subcutaneously administered HA-luciferin (HA-sc) or luciferin dissolved in saline (NS-sc) or intraperitoneally administered luciferin dissolved in saline (NS-ip). The bioluminescence of luciferin was monitored in vivo in real time. The peak time and half-life of fluorescence emission were significantly increased in HA-sc-treated mice compared with those in NS-sc- and NS-ip-treated mice; however, significant differences were not observed in peak photon counts. We detected differences in the pharmacokinetics of luciferin in the inner ear, including its sustained release, in the presence of HA. The results indicate the clinical potential of using HA for controlled drug delivery to the cochlea.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0153957PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839654PMC
September 2016

Overlapping expression of anion exchangers in the cochlea of a non-human primate suggests functional compensation.

Neurosci Res 2016 Sep 19;110:1-10. Epub 2016 Apr 19.

Keio University School of Medicine, Department of Otorhinolaryngology, Head and Neck Surgery, 35 Shinanomachi, Shinjuku-ku 160-8582, Japan.

Ion homeostasis in the inner ear is essential for proper hearing. Anion exchangers are one of the transporters responsible for the maintenance of homeostasis, but their expression profile in the primate cochlea has not been fully characterized. However, species-specific overlapping expression patterns and functional compensation in other organs, such as the kidney, pancreas and small intestine, have been reported. Here, we determined the expression patterns of the anion exchangers SLC26A4, SLC26A5, SLC26A6, SLC26A7, SLC26A11, SLC4A2 and SLC4A3 in the cochlea of a non-human primate, the common marmoset (Callithrix jacchus). Although the pattern of expression of SLC26A4 and SLC26A5 was similar to that in rodents, SLC26A7, SLC4A2, SLC4A3 exhibited different distributions. Notably, five transporters, SLC26A4, SLC26A6, SLC26A11 SLC4A2 and SLC4A3, were expressed in the cells of the outer sulcus. Our results reveal a species-specific distribution pattern of anion exchangers in the cochlea, particularly in the outer sulcus cells, suggesting functional compensation among these exchangers. This "primate-specific" pattern may be related to the human-specific hearing loss phenotypes of channelopathy disorders, including the SLC26A4-related diseases Pendred syndrome/DFNB4.
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http://dx.doi.org/10.1016/j.neures.2016.04.002DOI Listing
September 2016
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