Simulation-predicted and -explained inheritance model of pathogenicity confirmed by transgenic mice models.

Variants in the gap junction beta-2 (GJB2) gene are the most common cause of hereditary hearing impairment. However, how GJB2 variants lead to local physicochemical and structural changes in the hexameric ion channels of connexin 26 (Cx26), resulting in hearing impairment, remains elusive. In this study, using molecular dynamics (MD) simulations, we showed that detached inner-wall N-terminal "plugs" aggregated to reduce the channel ion flow in a highly prevalent V37I variant in humans. To examine the predictive ability of the computational platform, an artificial mutant, V37M, of which the effect was previously unknown in hearing loss, was created. Microsecond simulations showed that homo-hexameric V37M Cx26 hemichannels had an abnormal affinity between the inner edge and N-termini to block the narrower side of the cone-shaped Cx26, while the most stable hetero-hexameric channels did not. From the perspective of the conformational energetics of WT and variant Cx26 hexamers, we propose that unaffected carriers could result from a conformational predominance of the WT and pore-shrinkage-incapable hetero-hexamers, while mice with homozygous variants can only harbor an unstable and dysfunctional N-termini-blocking V37M homo-hexamer. Consistent with these predictions, homozygous V37M transgenic mice exhibited apparent hearing loss, but not their heterozygous counterparts, indicating a recessive inheritance mode. Reduced channel conductivity was found in Gjb2V37M/V37M outer sulcus and Claudius cells but not in Gjb2WT/WT cells. We view that the current computational platform could serve as an assessment tool for the pathogenesis and inheritance of GJB2-related hearing impairments and other diseases caused by connexin dysfunction.

Generation of induced pluripotent stem cells (IBMSi027-A) from a patient with hearing loss carrying WFS1 c.2051C > T (p.Ala684Val) variant.

Pathogenic variants of the WFS1 gene can cause recessive-inherited Wolfram syndrome or dominant-inherited Wolfram-like syndrome with optic atrophy and hearing impairment. Using the Sendai virus delivery system, we generated induced pluripotent stem cells from the peripheral blood mononuclear cells of a female patient with the WFS1 pathogenic variant c.2051C > T (p.Ala684Val). The resulting induced pluripotent stem cells exhibited a normal karyotype and pluripotency, as confirmed using immunofluorescence staining, and differentiated into three germ layers in vivo. This cellular model provides a useful platform for investigating the pathogenic mechanisms of both blindness and deafness related to WFS1 variants.

Generation of induced pluripotent stem cells from a patient with hearing loss carrying OPA1 c.1468T>C (p.Cys490Arg) variant.

Pathogenic variants of OPA1 have been associated with autosomal dominant optic atrophy (DOA), leading to optic, auditory, and other sensorineural neuropathies and myopathies. Using the Sendai virus delivery system, we generated induced pluripotent stem cells from the peripheral blood mononuclear cells of a female patient with the OPA1 pathogenic variant c.1468T>C (p.Cys490Arg). The resulting induced pluripotent stem cells exhibited a normal karyotype and pluripotency, as confirmed using immunofluorescence staining, and differentiated into three germ layers in vivo. This cellular model is a useful platform for investigating the pathogenic mechanisms of both blindness and deafness related to OPA1 variants.

Insights into phenotypic differences between humans and mice with p.T721M and other C-terminal variants of the SLC26A4 gene.

Recessive variants of the SLC26A4 gene are an important cause of hereditary hearing impairment. Several transgenic mice with different Slc26a4 variants have been generated. However, none have recapitulated the auditory phenotypes in humans. Of the SLC26A4 variants identified thus far, the p.T721M variant is of interest, as it appears to confer a more severe pathogenicity than most of the other missense variants, but milder pathogenicity than non-sense and frameshift variants. Using a genotype-driven approach, we established a knock-in mouse model homozygous for p.T721M. To verify the pathogenicity of p.T721M, we generated mice with compound heterozygous variants by intercrossing Slc26a4+/T721M mice with Slc26a4919-2A>G/919-2A>G mice, which segregated the c.919-2A > G variant with abolished Slc26a4 function. We then performed serial audiological assessments, vestibular evaluations, and inner ear morphological studies. Surprisingly, both Slc26a4T721M/T721M and Slc26a4919-2A>G/T721M showed normal audiovestibular functions and inner ear morphology, indicating that p.T721M is non-pathogenic in mice and a single p.T721M allele is sufficient to maintain normal inner ear physiology. The evidence together with previous reports on mouse models with Slc26a4 p.C565Y and p.H723R variants, support our speculation that the absence of audiovestibular phenotypes in these mouse models could be attributed to different protein structures at the C-terminus of human and mouse pendrin.

Toward the Pathogenicity of the SLC26A4 p.C565Y Variant Using a Genetically Driven Mouse Model.

Recessive variants of the SLC26A4 gene are globally a common cause of hearing impairment. In the past, cell lines and transgenic mice were widely used to investigate the pathogenicity associated with SLC26A4 variants. However, discrepancies in pathogenicity between humans and cell lines or transgenic mice were documented for some SLC26A4 variants. For instance, the p.C565Y variant, which was reported to be pathogenic in humans, did not exhibit functional pathogenic consequences in cell lines. To address the pathogenicity of p.C565Y, we used a genotype-based approach in which we generated knock-in mice that were heterozygous (Slc26a4+/C565Y), homozygous (Slc26a4C565Y/C565Y), and compound heterozygous (Slc26a4919-2A>G/C565Y) for this variant. Subsequent phenotypic characterization revealed that mice with these genotypes demonstrated normal auditory and vestibular functions, and normal inner-ear morphology and pendrin expression. These findings indicate that the p.C565Y variant is nonpathogenic for mice, and that a single p.C565Y allele is sufficient to maintain normal inner-ear physiology in mice. Our results highlight the differences in pathogenicity associated with certain SLC26A4 variants between transgenic mice and humans, which should be considered when interpreting the results of animal studies for SLC26A4-related deafness.

Low intensity ultrasound enhances cisplatin uptake in vitro by cochlear hair cells.

Drug delivery to the inner ear has been challenging due to the blood-labyrinth barrier. Intracochlear drug delivery is an invasive alternative with less pharmacokinetic variables. In this study, the effect of low intensity ultrasound on drug uptake by hair cells is investigated. Cochlear explants harvested from newborn mice were cultured in a medium containing cisplatin to emulate drug delivered to the endolymph. The results demonstrated the exposure to ultrasound stimulation effectively enhanced cisplatin uptake by hair cells. The uptake started from the apical side of the hair cells and progressed inward as the exposure time increased.

Establishment of an induced pluripotent stem cell (iPSC) line from a 7-year-old male patient with profound hearing loss carrying c.235delC in GJB2 gene.

Gap junction protein beta 2 gene (GJB2) mutations are the most frequent cause of hereditary hearing impairment. The recessive c.235delC mutation in the GJB2 gene is the most common mutation causing severe to profound sensorineural hearing loss in the Asian population. The induced pluripotent stem cell (iPSC) line was generated using the integration-free Sendai virus method from peripheral blood mononuclear cells (PBMCs) of a hearing-impaired patient with homozygous GJB2 c.235delC mutation. This cell line may serve as a cellular model for studying the pathogenic mechanisms of deafness caused by GJB2 mutations.

Consumption of betel quid contributes to sensorineural hearing impairment through arecoline-induced oxidative stress.

Betel quid is one of the most widely used psychoactive substances, and is consumed by approximately 10% of the world's population. In addition to its carcinogenicity, betel quid has also been reported to affect many organs, including the brain, heart, lungs, gastrointestinal tract, and reproductive organs. As betel quid contains several neurotoxic ingredients, we hypothesize that it also possesses ototoxicity and may lead to sensorineural hearing impairment (SNHI). In this study, we investigated the contribution of betel quid consumption to SNHI in a large clinical cohort, and validated the pathogenetic mechanisms in ex vivo tissue explants. We enrolled a total of 2364 volunteers, and determined their audiologic results based on Z-scores converted from their original frequency-specific hearing thresholds. Using generalized linear regression, we identified a positive correlation between betel quid consumption and the Z-scores across different frequencies. Subsequently, we explored the toxicity of arecoline, the main neuroactive component of betel quid, on tissue explants from murine cochleae. Arecoline reduced cell activity in the explant cultures and induced apoptosis in the hair cells, probably through the effects of oxidative stress. These findings have expanded the potential hazards of betel quid to common neurological disorders, and provide insights into preventive strategies against SNHI caused by neurotoxic substances.

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

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.

Unique spectra of deafness-associated mutations in Mongolians provide insights into the genetic relationships among Eurasian populations.

Genetic factors are an important cause of idiopathic sensorineural hearing impairment (SNHI). From the epidemiological perspective, mutations of three deafness genes: GJB2, SLC26A4, and MT-RNR1, are much more prevalent than those of other genes worldwide. However, mutation spectra of common deafness genes differ remarkably across different populations. Here, we performed comprehensive genetic examination and haplotype analyses in 188 unrelated Mongolian families with idiopathic SNHI, and compared their mutation spectra and haplotypes to those of other European and Asian cohorts. We confirmed genetic diagnoses in 18 (9.6%) of the 188 families, including 13 with bi-allelic GJB2 mutations, three with bi-allelic SLC26A4 mutations, and two with homoplasmic MT-RNR1 m.1555A>G mutation. Moreover, mono-allelic mutations were identified in 17 families (9.0%), including 14 with mono-allelic GJB2 mutations and three with mono-allelic SLC26A4 mutations. Interestingly, three GJB2 mutations prevalent in other populations, including c.35delG in Caucasians, c.235delC in East Asians, and c.-23+1G>A in Southwest and South Asians, were simultaneously detected in Mongolian patients. Haplotype analyses further confirmed founder effects for each of the three mutations, indicating that each mutation derived from its ancestral origin independently. By demonstrating the unique spectra of deafness-associated mutations, our findings may have important clinical and scientific implications for refining the molecular diagnostics of SNHI in Mongolian patients, and for elucidating the genetic relationships among Eurasian populations.

Generation of induced pluripotent stem cells from a patient with hearing loss carrying GJB2 p.V37I mutation.

Recessive mutations in the GJB2 gene are the most common genetic cause of hearing loss in humans. By using the Sendai-virus delivery system, we generated induced pluripotent stem cells (iPSCs) from the peripheral blood mononuclear cells of a female patient with the p.V37I (c.109G > A) mutation, a GJB2 mutation highly prevalent in the Asian population. The resulting iPSCs had a normal karyotype. The iPSCs also showed pluripotency, as confirmed by immunofluorescence staining, and differentiated into the three germ layers in vivo. This cellular model will provide a useful platform for investigating the pathogenic mechanisms of deafness related to GJB2 mutations.

Increased mucociliary differentiation and aquaporins formation of respiratory epithelial cells on retinoic acid-loaded hyaluronan-derivative membranes.

While playing a major role in maintaining the mucociliary phenotype of respiratory epithelial cells (RECs), retinoids are critical determinants of their normal function. However, despite being a powerful biological agent, retinoic acid (RA) is generally not used in regenerative medicine due to its scarce bioavailability via conventional administration. Therefore, the ability to incorporate RA into biomaterials allows for a combination of the biological effects of RA and biomaterials in influencing cellular behavior. This study attempts to develop RA-loaded hyaluronan-derivative membrane (RA-HAm) and investigates how this membrane affects the mucociliary differentiation and aquaporins (AQP) formation of RECs. In a simulated in vitro culture condition, the RA release from membranes is maintained for 7days. On the seventh day, the cumulative release rate of RA from supportive biomaterials is ~87% under detect limitation. RECs cultured on RA-HAm reveal numerous mature ciliated cells and microvilli compared to aggregated cilia-like structures on hyaluronan-derivative membrane (HAm). Moreover, the expression levels of MUC5AC and AQP on RA-HAm are higher than those on HAm. The proposed model elucidates the release of hydrophobic RA from hyaluronan-derivative biomaterials. We believe that RA-loaded hyaluronan biomaterials are highly promising biomaterials for use in sinonasal surgery and tissue engineering of the respiratory system.

Formation of post-confluence structure in human parotid gland acinar cells on PLGA through regulation of E-cadherin.

As a potential solution for patients to retrieve their lost salivary gland functions, tissue engineering of an auto-secretory device is profoundly needed. Under serum-free environment, primary human parotid gland acinar (PGAC) cells can be obtained. After reaching confluence, PGAC cells spontaneously form three-dimension (3D) cell aggregations, termed post-confluence structure (PCS), and change their behaviors. Poly (lactic-co-glycolic acid) (PLGA) has been widely used in the field of biomedical applications because of its biodegradable properties for desired functions. Nonetheless, the role of PLGA in facilitating PGAC cells to form PCS has seldom been explored to recover epithelial characteristics. In this study, PGAC cells were found to have a greater tendency to form PCS on PLGA than on tissue culture polystyrene (TCPS). By tracing cell migration paths and modulating E-cadherin activity with specific inhibitor or antibody, we demonstrated that the static force of homophilic interaction on surfaces of individual cells, but not the dynamics of cell migration, played a more important role in PCS formation. Thus, PLGA was successfully confirmed to support PGAC cells to form more PCS through the effects on enhancing E-cadherin expression, which is associated with FAK/ILK/Snail expression in PGAC cells. This result indicates that selective appropriate biomaterials may be potentially useful in generating 3D PCS on two-dimension (2D) substrate without fabricating a complex 3D scaffold.

Human salivary gland acinar cells spontaneously form three-dimensional structures and change the protein expression patterns.

Applying tissue engineering principles to design an auto-secretory device is a potential solution for patients suffering loss of salivary gland function. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of reports on the in vitro cell biology and physiology of human salivary gland cells. This study used a low-calcium culture system to selectively cultivate human parotid gland acinar (PGAC) cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α-amylase) and function-related proteins (aquaporin-3, aquaporin-5, and ZO-1). Notably, when the cells reached confluence, three-dimensional (3D) cell aggregates were observed in crowded regions. These self-formed cell spheres were termed post-confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function-related proteins compared to the two-dimensional (2D) cells. Translocation of aquoporin-3 from cytosolic to alongside the cell boundaries, and of ZO-1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form PCSs without the help of 3D scaffolds and retain certain differentiation and polarity. This phenomenon implies that it is possible to introduce 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering.

Selective culture of different types of human parotid gland cells.

BACKGROUND: Advances in salivary gland tissue engineering can benefit patients diagnosed with xerostomia. Complexity of the gland explains the urgent demand for a reliable protocol to isolate and expand various gland cells that can be used for further study. METHODS: Three cells with different morphologies were isolated from the same human parotid glands using different culture medium systems and then were identified by the expressions from mRNA to the protein level. RESULTS: Among the 34 specimens, parotid gland acinar cells, myoepithelial cells, and fibroblasts expressing specific markers that belonged to individual cell types, were successfully isolated and expanded from 30 specimens without a complex mechanical process and expensive flow technique. CONCLUSION: The proposed protocol is simple with a high success rate to culture various gland cells, making it highly promising for use in future tissue engineering studies.

Clinical and biomechanical analyses to select a suture material for uvulopalatopharyngeal surgery.

OBJECTIVE: The selection of the degradation pattern of suture materials is critical in uvulopalatopharyngeal surgery because of the complex oral environment such as the constant presence of saliva; the microbial accumulation; and the functions related to speech, mastication, and swallowing. The aim of the study was to identify appropriate suture materials for uvulopalatopharyngeal surgery by analyzing the clinical and biomechanical results obtained using four different absorbable suture materials. STUDY DESIGN: A randomized open-label trial. SETTING: Tertiary referral center. SUBJECTS AND METHODS: Eighty patients with obstructive sleep apnea syndrome who had undergone microdebrider-assisted extended uvulopalatoplasty were randomized into the four suture groups: sutures of polyglycolide (Dexon) and copolymers of polyglycolide and other degradable polymers (Polysorb, Monocryl, Maxon). Handling characteristics, surgical outcomes, Young's modulus reduction, morphologic changes by scanning electron microscope, and saliva absorption test were analyzed. RESULTS: Monocryl (poly(glycolide-co-ε-caprolactone)) had the optimal handling characteristics and exhibited two-stage degradation: sufficient tensile strength was maintained initially to allow wound healing, and then the suture degraded rapidly to avoid irritation. In contrast, the hydrolytic degradation of Dexon, Polysorb, and Maxon seemed to involve only an approximately single-stage process in the oral environment. Moreover, a large amount of debris was observed among filaments in Dexon and Polysorb, whereas only some scattered debris accumulated on Monocryl and Maxon under scanning electron microscope. CONCLUSION: Poly(glycolide-co-ε-caprolactone) has good handling characteristics and degrades in two stages. It is a suitable suture material for uvulopalatopharyngeal surgery.

Regulation of ciliary differentiation of human respiratory epithelial cells by the receptor for hyaluronan-mediated motility on hyaluronan-based biomaterials.

Selecting a scaffold that facilitates ciliary differentiation of respiratory epithelial cells (RECs) is crucial in developing tissue engineered respiratory epithelium. Hyaluronan derivative membranes, consisting of an esterified form of hyaluronan (HYAFF), have been proved to promote ciliary differentiation of RECs in the presence of retinoic acid (RA). However, the regulatory mechanism of ciliary differentiation-promoting effect of hyaluronan-based biomaterials remains unknown. In addition to investigating the ciliary differentiation of RECs on HYAFF with/without RA compared with that on collagen with/without RA, this study elucidates the role of the receptor for hyaluronan-mediated motility (RHAMM) in promoting ciliary differentiation of RECs. Analytical results of culturing RECs on collagen and HYAFF indicate that only HYAFF can increase the ciliary differentiation of RECs under RA-free conditions. The expression level of RHAMM mRNA of RECs more significantly decreases on collagen than that on HYAFF without RA. Therefore, by using lentiviral vector-based short hairpin RNA targeting RHAMM, the study further reveals that knockdown of RHAMM obviously inhibits the ciliary differentiation of RECs on collagen with RA and on HYAFF with/without RA. In addition to demonstrating that hyaluronan-based biomaterials partially "replace" RA in the ciliary differentiation of RECs, which is regulated by RHAMM, this study establishes that RHAMM regulates the ciliary differentiation-promoting effect of RA on RECs.

Increased mucociliary differentiation of human respiratory epithelial cells on hyaluronan-derivative membranes.

The selection of a scaffold to facilitate mucociliary differentiation of respiratory epithelial cells (RECs) is crucial in the development of tissue engineering of respiratory epithelium. However, how the differentiation of RECs is influenced by the biomaterials has never been thoroughly explored. Previously, hyaluronan derivatives were considered as unsuitable biomaterials for culture of respiratory epithelium. In contrast, this study demonstrates that the membranous scaffolds made from benzyl esters of hyaluronic acids are capable of providing a more preferential environment for human RECs than conventionally used collagen-based scaffolds. The proliferation and mucociliary differentiation of RECs were examined by MTT assays, scanning electron microscopy, immunofluorescence, immunoblotting and gene expression. The percentage of ciliated cells in cultured RECs increased from 12.4% on collagen to 20.4% on hyaluronan-derivative membranes with a pseudostratified polarized layer that closely resembled the composition of the native epithelium. The expression levels of MUC5AC and MUC5B mRNA were higher on hyaluronan-based scaffolds than those on collagen. The presence of a hyaluronan-binding domain, CD44 and the receptor for hyaluronan-mediated motility of RECs were also demonstrated. Accordingly, the mucociliary differentiation-promoting effect of hyaluronan-derivative membranes indicates that it may be applied to the tissue engineering of respiratory epithelium.

Culture of nasal epithelial cells using chitosan-based membranes.

OBJECTIVES/HYPOTHESIS: The aim of this study was to evaluate whether chitosan-based membranes can be used as scaffolds for growth and differentiation of nasal epithelial cells (NECs). Our final goal was to establish a novel methodology for enhancing the regeneration of the respiratory system. STUDY DESIGN: Prospective study. METHODS: Human NECs were cultured on three various substrates, e.g., chitosan membranes, collagen, and chitosan-collagen membranes. Morphology of NECs was examined via light and electron microscopy, the area of ciliated cells was measured by confocal microscopy, and ciliary beat frequency was also evaluated. Expression of mucin genes was investigated with reverse-transcription polymerase chain reaction. RESULTS: NECs were found to be successfully adhesive with collagen and chitosan-collagen membranes at day 3 after seeding, but not with chitosan membranes. The cilia area on collagen were 6.1% +/- 1.2%, 8.4% +/- 1.4%, and 12.5% +/- 1.9% at days 7, 14, and 21 after confluence, respectively, compared with 5.1% +/- 0.9%, 8.6% +/- 1.6%, and 12.3% +/- 2.1% in chitosan-collagen membranes, exhibited nonsignificant difference (P > .05). There were no significant differences in ciliary beat frequency between each group. The expression levels of mucin genes, namely, MUC5AC, MUC5B, and MUC2, in NECs on both collagen and chitosan-collagen membranes did not differ significantly (P > .05). CONCLUSIONS: A small amount collagen mixed with chitosan substrate may improve the biocompatibility and promote the mucociliary differentiation in NECs. It appears that chitosan-collagen membrane is a promising scaffold for culture of the nasal epithelium, which sets the stage for studying tissue regeneration in the respiratory system.

Formation of salivary acinar cell spheroids in vitro above a polyvinyl alcohol-coated surface.

Tissue engineering of salivary glands offers the potential for future use in the treatment of patients with salivary hypofunction. Biocompatible materials that promote acinar cell aggregation and function in vitro are an essential part of salivary gland tissue engineering. In this study, rat parotid acinar cells assembled into three-dimensional aggregates above the polyvinyl alcohol (PVA)-coated surface. These aggregates developed compact acinar cell spheroids resembling in vivo physiological condition, which were different from the traditional monolayered morphology in vitro. Cells remained viable and with better functional activity in response to acetylcholine in the spheroids and could form monolayered acinar cells when they were reinoculated on tissue culture polystyrene wells. To interpret the phenomenon further, we proposed that the formation of acinar cell spheroids on the PVA is mediated by a balance between two competing forces: the interactions of cell-PVA and cell-cell. This study demonstrated the formation of functional cell spheroids above a PVA-coated surface may provide an in vitro system for investigating cell behaviors for tissue engineering of artificial salivary gland.