Multicenter Study of Congenital Middle Ear Anomalies. Report on 246 Ears.

OBJECTIVES/HYPOTHESIS: Congenital middle ear anomalies represent a relatively rare condition. This study aimed to describe the characteristics and the surgical outcomes for patients with middle ear anomalies. METHODS: A multicenter study was conducted of consecutive patients with congenital middle ear anomalies who underwent primary surgical treatment between January 2008 and December 2017. Demographics, surgical procedures, and audiometric data were registered into the institutional database. Hearing changes and postoperative air-bone gap (ABG) were evaluated 1 year after surgery. RESULTS: A total of 246 patients (246 ears) (median age: 14 years, range: 4-75 years old) were included in this study. Anomalies were subdivided using the Teunissen and Cremers classification: 53 ears (22%) were categorized as class I, comprising only stapes ankylosis; 35 ears (14%) as class II, having ossicular chain anomalies with stapes ankylosis; 139 ears (57%) as class III, having ossicular chain anomalies with a mobile stapes-footplate; and 19 ears (8%) as class IV, with aplasia of the oval window. Evaluation of hearing outcomes for 198 ears with more than 1 year of follow-up revealed that good postoperative ABG (≤20 dB) was achieved in 82% of class I, 68% of class II, 74% of class III, and 23% of class IV anomalies. The postoperative ABG in class IV was significantly worse than in class I (P < .001) or class III (P < .01). CONCLUSIONS: This study demonstrated that class III anomalies comprised the majority of middle ear anomalies and surgical outcomes for class IV anomalies are unfavorable. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:E2323-E2328, 2021.

Prevalence and clinical features of hearing loss caused by EYA4 variants.

Variants in the EYA4 gene are known to lead to autosomal dominant non-syndromic hereditary hearing loss, DFNA10. To date, 30 variants have been shown to be responsible for hearing loss in a diverse set of nationalities. To better understand the clinical characteristics and prevalence of DFNA10, we performed genetic screening for EYA4 mutations in a large cohort of Japanese hearing loss patients. We selected 1,336 autosomal dominant hearing loss patients among 7,408 unrelated Japanese hearing loss probands and performed targeted genome enrichment and massively parallel sequencing of 68 target genes for all patients. Clinical information of cases with mutations in EYA4 was gathered and analyzed from medical charts. Eleven novel EYA4 variants (three frameshift variants, three missense variants, two nonsense variants, one splicing variant, and two single-copy number losses) and two previously reported variants were found in 12 probands (0.90%) among the 1,336 autosomal dominant hearing loss families. The audiometric configuration of truncating variants tends to deteriorate for all frequencies, whereas that of non-truncating variants tends to show high-frequency hearing loss, suggesting a new correlation between genotype and phenotype in DFNA10. The rate of hearing loss progression caused by EYA4 variants was considered to be 0.63 dB/year, as found in this study and previous reports.

The Prevalence and Clinical Characteristics of TECTA-Associated Autosomal Dominant Hearing Loss.

TECTA is well known as a causative gene for autosomal dominant mid-frequency hearing loss observed in various populations. In this study, we performed next-generation sequencing analysis of a large Japanese hearing loss cohort, including eight hundred and twelve (812) subjects from unrelated autosomal dominant hearing loss families, to estimate the prevalence and phenotype-genotype correlations in patients with TECTA mutations. The prevalence of TECTA mutations in Japanese autosomal dominant sensorineural hearing loss families was found to be 3.2%. With regard to the type of hearing loss, the patients with mutations in the nidogen-like domain or ZA domain of TECTA showed varied audiograms. However, most of the patients with mutations in the ZP domain showed mid-frequency hearing loss. The rate of hearing deterioration in TECTA-associated hearing loss patients and in the normal hearing Japanese control population were the same and regression lines for each group were parallel. We carried out haplotype analysis for four families which had one recurring missense variant, c.5597C>T (p.Thr1866Met). Our results revealed four different haplotypes, suggesting that this mutation occurred independently in each family. In conclusion, TECTA variants represent the second largest cause of autosomal dominant sensorineural hearing loss in Japan. The hearing loss progression observed in the patients with TECTA mutations might reflect presbycusis. The c.5597C>T mutation occurred in a mutational hot spot and is observed in many ethnic populations.

Implantation of Induced Pluripotent Stem Cell-Derived Tracheal Epithelial Cells.

OBJECTIVES: Compared with using autologous tissue, the use of artificial materials in the regeneration of tracheal defects is minimally invasive. However, this technique requires early epithelialization on the inner side of the artificial trachea. After differentiation from induced pluripotent stem cells (iPSCs), tracheal epithelial tissues may be used to produce artificial tracheas. Herein, we aimed to demonstrate that after differentiation from fluorescent protein-labeled iPSCs, tracheal epithelial tissues survived in nude rats with tracheal defects. METHODS: Red fluorescent tdTomato protein was electroporated into mouse iPSCs to produce tdTomato-labeled iPSCs. Embryoid bodies derived from these iPSCs were then cultured in differentiation medium supplemented with growth factors, followed by culture on air-liquid interfaces for further differentiation into tracheal epithelium. The cells were implanted with artificial tracheas into nude rats with tracheal defects on day 26 of cultivation. On day 7 after implantation, the tracheas were exposed and examined histologically. RESULTS: Tracheal epithelial tissue derived from tdTomato-labeled iPSCs survived in the tracheal defects. Moreover, immunochemical analyses showed that differentiated tissues had epithelial structures similar to those of proximal tracheal tissues. CONCLUSIONS: After differentiation from iPSCs, tracheal epithelial tissues survived in rat bodies, warranting the use of iPSCs for epithelial regeneration in tracheal defects.

Regeneration of tracheal epithelium using mouse induced pluripotent stem cells.

Conclusion The findings demonstrated the potential use of induced pluripotent stem cells for regeneration of tracheal epithelium. Objective Autologous tissue implantation techniques using skin or cartilage are often applied in cases of tracheal defects with laryngeal inflammatory lesions and malignant tumor invasion. However, these techniques are invasive with an unstable clinical outcome. The purpose of this study was to investigate regeneration in a tracheal defect site of nude rats after implantation of ciliated epithelium that was differentiated from induced pluripotent stem cells. Method Embryoid bodies were formed from mouse induced pluripotent stem cells. They were cultured with growth factors for 5 days, and then cultured at the air-liquid interface. The degree of differentiation achieved prior to implantation was determined by histological findings and the results of real-time polymerase chain reaction. Embryoid bodies including ciliated epithelium were embedded into collagen gel that served as an artificial scaffold, and then implanted into nude rats, creating an 'air-liquid interface model'. Histological evaluation was performed 7 days after implantation. Results The ciliated epithelial structure survived on the lumen side of regenerated tissue. It was demonstrated histologically that the structure was composed of ciliated epithelial cells.

Potential of laryngeal muscle regeneration using induced pluripotent stem cell-derived skeletal muscle cells.

Conclusion Induced pluripotent stem (iPS) cells may be a new potential cell source for laryngeal muscle regeneration in the treatment of vocal fold atrophy after recurrent laryngeal nerve paralysis. Objectives Unilateral vocal fold paralysis can lead to degeneration, atrophy, and loss of force of the thyroarytenoid muscle. At present, there are some treatments such as thyroplasty, arytenoid adduction, and vocal fold injection. However, such treatments cannot restore reduced mass of the thyroarytenoid muscle. iPS cells have been recognized as supplying a potential resource for cell transplantation. The aim of this study was to assess the effectiveness of the use of iPS cells for the regeneration of laryngeal muscle through the evaluation of both in vitro and in vivo experiments. Methods Skeletal muscle cells were generated from tdTomato-labeled iPS cells using embryoid body formation. Differentiation into skeletal muscle cells was analyzed by gene expression and immunocytochemistry. The tdTomato-labeled iPS cell-derived skeletal muscle cells were transplanted into the left atrophied thyroarytenoid muscle. To evaluate the engraftment of these cells after transplantation, immunohistochemistry was performed. Results The tdTomato-labeled iPS cells were successfully differentiated into skeletal muscle cells through an in vitro experiment. These cells survived in the atrophied thyroarytenoid muscle after transplantation.

Generation of airway epithelial cells with native characteristics from mouse induced pluripotent stem cells.

Airway epithelial cells derived from induced pluripotent stem (iPS) cells are expected to be a useful source for the regeneration of airway epithelium. Our preliminary study of embryoid body (EB) formation and the air-liquid interface (ALI) method suggested that mouse iPS cells can differentiate into airway epithelial cells. However, whether the cells generated from mouse iPS cells had the character and phenotype of native airway epithelial cells remained uninvestigated. In this study, we generated airway epithelial cells from EBs by culturing them under serum-free conditions supplemented with Activin and bFGF and by the ALI method and characterized the iPS cell-derived airway epithelial cells in terms of their gene expression, immunoreactivity, morphology, and function. Analysis by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) revealed that the expression of the undifferentiated cell marker Nanog decreased time-dependently after the induction of differentiation, whereas definitive endoderm markers Foxa2 and Cxcr4 were transiently up-regulated. Thereafter, the expression of airway epithelium markers such as Tubb4a, Muc5ac, and Krt5 was detected by RT-PCR and immunostaining. The formation of tight junctions was also confirmed by immunostaining and permeability assay. Analysis by hematoxylin and eosin staining and scanning electron microscopy indicated that the cells generated from mouse iPS cells formed airway-epithelium-like tissue and had cilia, the movement of which was visualized and observed to be synchronized. These results demonstrate that the airway epithelial cells generated by our method have native characteristics and open new perspectives for the regeneration of injured airway epithelium.

Visualization of mouse induced pluripotent stem cells for evaluation of tracheal regeneration.

CONCLUSION: Visualization of mouse induced pluripotent stem (iPS) cells with the use of a fluorescent protein was successfully achieved for evaluation of tracheal regeneration. OBJECTIVES: Tracheal epithelial cells derived from iPS cells are expected to be a useful cell source for tracheal regeneration. Our previous study demonstrated that mouse iPS cells differentiated into tracheal epithelial cells. However, when they are implanted into tracheal defects in experimental animals, it is difficult to determine whether the regenerated tracheal epithelium is in fact derived from iPS cells. The purpose of this study was to develop a visualization technique for iPS cells for evaluation of tracheal regeneration. METHODS: Fluorescent marker tdTomato was transfected into iPS cells. Tracheal epithelial cells were generated from tdTomato-labeled iPS cells using embryoid body formation to detect the expression of tdTomato. The artificial material with tdTomato-labeled iPS cells was implanted into tracheal defects in nude rats. The survival and distribution of tdTomato-labeled iPS cell-derived cells were examined using the IVIS Imaging System and immunostaining. RESULTS: The expression of tdTomato was detected in both undifferentiated cells and tracheal epithelial cells in vitro. tdTomato-labeled iPS cell-derived cells were successfully detected in the tracheal defects by IVIS Imaging System and immunostaining.

Effective embryoid body formation from induced pluripotent stem cells for regeneration of respiratory epithelium.

OBJECTIVES/HYPOTHESIS: We have previously demonstrated the potential use of induced pluripotent stem (iPS) cells for regeneration of respiratory epithelium by culturing embryoid bodies (EBs). The aim of the present study was to determine the most effective conditions for EB formation from iPS cells for regeneration of respiratory epithelium. STUDY DESIGN: Experimental study. METHODS: iPS cells cultured on a gelatin-coated dish were seeded on low-attachment plates for generating EBs. Under several conditions including the air-liquid interface (ALI) method, with varying cell numbers and suspension times, EBs were transferred to a gelatin-coated dish supplemented with growth factors. The shape, size, aggregation, and adhesion of EBs for iPS cell differentiation were evaluated, and the cultured tissue was histologically examined. RESULTS: EBs appropriate for differentiation were observed using 1,000 cells after 5 days of suspension culture. Respiratory epithelium-like tissue was histologically observed. The ciliary epithelium was confirmed immunohistologically. CONCLUSIONS: Based on the varying suspension times and cell numbers with the ALI method, this study presented effective conditions for EB formation from iPS cells for regeneration of respiratory epithelium.

Bioengineered trachea using autologous chondrocytes for regeneration of tracheal cartilage in a rabbit model.

OBJECTIVES/HYPOTHESIS: In this study, a bioengineered trachea composed of autologous chondrocytes was developed, and its effect on cartilaginous regeneration was evaluated by implantation into tracheal defects in rabbits. STUDY DESIGN: Prospective controlled trial in an animal model. METHODS: The tracheal prosthesis used in this study was composed of polypropylene (the frame) and collagen sponge (the scaffold). Chondrocytes were harvested from the costal cartilage of rabbits and seeded into the tracheal prosthesis. The bioengineered trachea, consisting of the tracheal prosthesis with chondrocytes, was implanted into surgically created tracheal defects of rabbits in the bioengineered group, and a tracheal prosthesis without chondrocytes was implanted in the control group. RESULTS: After implantation, the presence of regenerated cartilage was observed in the bioengineered trachea but not in the tracheal prosthesis without chondrocytes. CONCLUSIONS: It was confirmed in this study that the implanted chondrocytes proliferated in an appropriate portion of the tracheal defect and that the partially resected tracheal cartilage was repaired with regenerated cartilaginous tissue into a ring-shaped form as a whole. These results demonstrate the feasibility of cartilaginous regeneration using a bioengineered trachea with autologous chondrocytes.

Implantation site-dependent differences for tracheal regeneration with induced pluripotent stem cells (iPS cells).

CONCLUSION: The histological findings and quantitative measurements demonstrated that there were differences in teratoma formation according to the site of implantation. Elucidating the mechanisms of the teratoma formation caused by the site of implantation moves the field another step closer to clinical use of induced pluripotent stem (iPS) cells for tracheal regeneration. OBJECTIVES: Our previous study demonstrated the potential for iPS cells to be used as a new cell source for tracheal regeneration. However, teratoma formation remains a major problem. Implantation site-dependent differences in teratoma formation have been reported. In this study, the teratoma-forming propensity after implantation into tracheal defects and abdominal subcutaneous tissue was examined histologically and quantitatively. METHODS: Mouse iPS cells were cultured in artificial material under various conditions. After cultivation in vitro, artificial materials with cultured iPS cells were then implanted into cervical tissue around tracheal defects and into abdominal subcutaneous tissue in nude rats. Teratoma formation was evaluated histologically and quantitatively with measurement of maximum diameter (MD). RESULTS: Teratoma was observed in 10 of 11 rats with cervical tissue around tracheal defects and in 3 of 11 rats with abdominal subcutaneous tissue implants. The average MD was 5.36 mm in the trachea and 0.97 mm in the abdomen.

Potential for respiratory epithelium regeneration from induced pluripotent stem cells.

OBJECTIVES: In cases of laryngeal inflammatory lesions and tracheal invasion of a malignant tumor, autologous tissue implantation techniques using skin or cartilage are often applied. However, these techniques are both invasive and unstable. The purpose of this study was to evaluate the potential use of induced pluripotent stem (iPS) cells in the regeneration of respiratory epithelium. METHODS: We seeded iPS cells on low-attachment plates in serum-free media to generate embryoid bodies (EBs). After a 3-day culture, the EBs were transferred to a gelatin-coated dish supplemented with activin A alone or with basic fibroblast growth factor (induction groups). As a control, EBs were cultured without these growth factors (control group). Cultured tissues from all groups were histologically examined for 2 weeks. RESULTS: In the induction groups, the presence of respiratory epithelium-like tissue was observed with hematoxylin and eosin staining after 14 days of culture. CONCLUSIONS: This study demonstrated the potential use of iPS cells in regeneration of the respiratory epithelium.