Contribution of Streptococcus pseudopneumoniae and Streptococcus salivarius to vocal fold mucosal integrity and function.

Structural changes to the vocal fold (VF) epithelium, namely, loosened intercellular junctions, have been reported in VF benign lesions. The potential mechanisms responsible for the disruption of cell junctions do not address the contribution of resident microbial communities to this pathological phenomenon. In this study, we focused on determining the relationship between Streptococcus pseudopneumoniae (SP), a dominant bacterial species associated with benign lesions, and Streptococcus salivarius (SS), a commensal bacterium, with human VF epithelial cells in our three-dimensional model of the human VF mucosa. This experimental system enabled direct deposition of bacteria onto constructs at the air/liquid interface, allowing for the assessment of bacterium-host interactions at the cellular, molecular and ultrastructural levels. Our findings demonstrate that SP disrupts VF epithelial integrity and initiates inflammation via the exported products HtrA1 and pneumolysin. In contrast, SS attaches to the VF epithelium, reduces inflammation and induces Mmp2-mediated apical desquamation of infected cells to mitigate the impact of pathogens. In conclusion, this study highlights the complexity of microbial involvement in VF pathology and potential VF mucosal restoration in the presence of laryngeal commensals.

Piezo1-expressing vocal fold epithelia modulate remodeling via effects on self-renewal and cytokeratin differentiation.

Mechanoreceptors are implicated as functional afferents within mucosa of the airways and the recent discovery of mechanosensitive channels Piezo1 and Piezo2 has proved essential for cells of various mechanically sensitive tissues. However, the role for Piezo1/2 in vocal fold (VF) mucosal epithelia, a cell that withstands excessive biomechanical insult, remains unknown. The purpose of this study was to test the hypothesis that Piezo1 is required for VF mucosal repair pathways of epithelial cell injury. Utilizing a sonic hedgehog (shh) Cre line for epithelial-specific ablation of Piezo1/2 mechanoreceptors, we investigated 6wk adult VF mucosa following naphthalene exposure for repair strategies at 1, 3, 7 and 14 days post-injury (dpi). PIEZO1 localized to differentiated apical epithelia and was paramount for epithelial remodeling events. Injury to wildtype epithelium was most appreciated at 3 dpi. Shhcre/+; Piezo1loxP/loxP, Piezo2 loxP/+ mutant epithelium exhibited severe cell/nuclear defects compared to injured controls. Conditional ablation of Piezo1 and/or Piezo2 to uninjured VF epithelium did not result in abnormal phenotypes across P0, P15 and 6wk postnatal stages compared to heterozygote and control tissue. Results demonstrate a role for Piezo1-expressing VF epithelia in regulating self-renewal via effects on p63 transcription and YAP subcellular translocation-altering cytokeratin differentiation.

Exposure to e-cigarette vapor extract induces vocal fold epithelial injury and triggers intense mucosal remodeling.

Vaping has been reported to cause acute epiglottitis, a life-threatening airway obstruction induced by direct epithelial injury and subsequent inflammatory reaction. Here, we show that we were able to recapitulate this phenomenon in vitro. Exposure of human engineered vocal fold (VF) mucosae to 0.5% and 5% electronic cigarette (e-cigarette) vapor extract (ECVE) for 1 week induced cellular damage of luminal cells, disrupting homeostasis and innate immune responses. Epithelial erosion was likely caused by accumulation of solvents and lipid particles in the cytosol and intercellular spaces, which altered lipid metabolism and plasma membrane properties. Next, we investigated how the mucosal cells responded to the epithelial damage. We withdrew the ECVE from the experimental system and allowed VF mucosae to regenerate for 1, 3 and 7 days, which triggered intense epithelial remodeling. The epithelial changes included expansion of P63 (TP63)-positive basal cells and cytokeratin 14 (KRT14) and laminin subunit α-5 (LAMA5) deposition, which might lead to local basal cell hyperplasia, hyperkeratinization and basement membrane thickening. In summary, vaping presents a threat to VF mucosal health and airway protection, thereby raising further concerns over the safety of e-cigarette use. This article has an associated First Person interview with the first author of the paper.

Derivation of Three-Dimensional Human Induced Pluripotent Stem Cell-Derived Vocal Fold Mucosa for Clinical and Pharmacological Applications.

Healthy vocal fold mucosa is composed of two major cell types, non-keratinized stratified squamous epithelium and vocal fold fibroblasts. Although dysfunction of the epithelium may play a significant pathogenic role in vocal fold diseases, studies at the genetic and molecular level using primary epithelial cells or models of human vocal fold mucosa have been significantly limited by the availability of relevant tissue types, poor growth, and heterogeneity of primary vocal fold epithelial cells. Here, we describe in vitro developmental differentiation of human induced pluripotent stem cells into vocal fold basal epithelial progenitors that were reseeded on collagen-fibroblast constructs to induce stratification and generate a three-dimensional model of human vocal fold mucosa. The engineered vocal fold mucosa represents physiologically relevant and clinically useful model that can be used as a tool for disease modeling and testing of therapeutic approaches for the treatment of laryngeal and VF inflammation.

Inactivation of Lats1 and Lats2 highlights the role of hippo pathway effector YAP in larynx and vocal fold epithelium morphogenesis.

Proliferation and differentiation of vocal fold epithelial cells during embryonic development is poorly understood. We examined the role of Hippo signaling, a vital pathway known for regulating organ size, in murine laryngeal development. Conditional inactivation of the Hippo kinase genes Lats1 and Lats2, specifically in vocal fold epithelial cells, resulted in severe morphogenetic defects. Deletion of Lats1 and Lats2 caused abnormalities in epithelial differentiation, epithelial lamina separation, cellular adhesion, basement membrane organization with secondary failed cartilage, and laryngeal muscle development. Further, Lats1 and Lats2 inactivation led to failure in differentiation of p63+ basal progenitors. Our results reveal novel roles of Hippo-Lats-YAP signaling in proper regulation of VF epithelial fate and larynx morphogenesis.

Novel immortalized human vocal fold epithelial cell line: In vitro tool for mucosal biology.

Study of vocal fold (VF) mucosal biology requires essential human vocal fold epithelial cell (hVFE) lines for use in appropriate model systems. We steadily transfected a retroviral construct containing human telomerase reverse transcriptase (hTERT) into primary normal hVFE to establish a continuously replicating hVFE cell line. Immortalized hVFE across passages have cobblestone morphology, express epithelial markers cytokeratin 4, 13 and 14, induced hTERT gene and protein expression, have similar RNAseq profiling, and can continuously grow for more than 8 months. DNA fingerprinting and karyotype analysis demonstrated that immortalized hVFE were consistent with the presence of a single cell line. Validation of the hVFE, in a three-dimensional in vitro VF mucosal construct revealed a multilayered epithelial structure with VF epithelial cell markers. Wound scratch assay revealed higher migration capability of the immortalized hVFE on the surface of collagen-fibronectin and collagen gel containing human vocal fold fibroblasts (hVFF). Collectively, our report demonstrates the first immortalized hVFE from true VFs providing a novel and invaluable tool for the study of epithelial cell-fibroblast interactions that dictate disease and health of this specialized tissue.

Mechanisms of larynx and vocal fold development and pathogenesis.

The larynx and vocal folds sit at the crossroad between digestive and respiratory tracts and fulfill multiple functions related to breathing, protection and phonation. They develop at the head and trunk interface through a sequence of morphogenetic events that require precise temporo-spatial coordination. We are beginning to understand some of the molecular and cellular mechanisms that underlie critical processes such as specification of the laryngeal field, epithelial lamina formation and recanalization as well as the development and differentiation of mesenchymal cell populations. Nevertheless, many gaps remain in our knowledge, the filling of which is essential for understanding congenital laryngeal disorders and the evaluation and treatment approaches in human patients. This review highlights recent advances in our understanding of the laryngeal embryogenesis. Proposed genes and signaling pathways that are critical for the laryngeal development have a potential to be harnessed in the field of regenerative medicine.

Human induced pluripotent stem cell-derived vocal fold mucosa mimics development and responses to smoke exposure.

Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells' limited proliferative capacity and absence of cell lines. Here we report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa. Furthermore, we demonstrate mucosal inflammation upon exposure of these constructs to 5% cigarette smoke extract. Upregulation of pro-inflammatory genes in epithelium and fibroblasts leads to aberrant VF mucosa remodeling. Collectively, our results demonstrate that hiPSC-derived VF mucosa is a versatile tool for future investigation of genetic and molecular mechanisms underlying epithelium-fibroblasts interactions in health and disease.

Derivation of Epithelial Cells from Human Embryonic Stem Cells as an In Vitro Model of Vocal Mucosa.

Vocal fold epithelial cells are very difficult to study as the vocal fold epithelial cell lines do not exist and they cannot be removed from the healthy larynx without engendering a significant and unacceptable risk to vocal fold function. Here, we describe the procedure to create an engineered vocal fold tissue construct consisting of the scaffold composed of the collagen 1 gel seeded with human fibroblasts and simple epithelial progenitors seeded on the scaffold and cultivated at air-liquid interface for 19-21 days to derive the stratified squamous epithelium. This model of vocal fold mucosa is very similar in morphology, gene expression, and phenotypic characteristics to native vocal fold epithelial cells and the underlying lamina propria and, therefore, offers a promising approach to studying vocal fold biology and biomechanics in health and disease.

A three-dimensional study of alveologenesis in mouse lung.

Alveologenesis is the final step of lung maturation, which subdivides the alveolar region of the lung into smaller units called alveoli. Each of the nascent dividers serves as a new gas-exchange surface, and collectively they drastically increase the surface area for breathing. Disruption of alveologenesis results in simplification of alveoli, as is seen in premature infants diagnosed with bronchopulmonary dysplasia (BPD), a prevalent lung disease that is often associated with lifelong breathing deficiencies. To date, a majority of studies of alveologenesis rely on two-dimensional (2D) analysis of tissue sections. Given that an overarching theme of alveologenesis is thinning and extension of the epithelium and mesenchyme to facilitate gas exchange, often only a small portion of a cell or a cellular structure is represented in a single 2D plane. Here, we use a three-dimensional (3D) approach to examine the structural architecture and cellular composition of myofibroblasts, alveolar type 2 cells, elastin and lipid droplets in normal as well as BPD-like mouse lung. We found that 2D finger-like septal crests, commonly used to depict growing alveolar septae, are often artifacts of sectioning through fully established alveolar walls. Instead, a more accurate representation of growing septae are 3D ridges that are lined by platelet-derived growth factor receptor alpha (PDGFRA) and alpha smooth muscle actin (α-SMA)-expressing myofibroblasts, as well as the elastin fibers that they produce. Accordingly in 3D, both α-SMA and elastin were each found in connected networks underlying the 3D septal ridges rather than as isolated dots at the tip of 2D septal crests. Analysis through representative stages of alveologenesis revealed unappreciated dynamic changes in these patterns. PDGFRA-expressing cells are only α-SMA-positive during the first phase of alveologenesis, but not in the second phase, suggesting that the two phases of septae formation may be driven by distinct mechanisms. Thin elastin fibers are already present in the alveolar region prior to alveologenesis, suggesting that during alveologenesis, there is not only new elastin deposition, but also extensive remodeling to transform thin and uniformly distributed fibers into thick cables that rim the nascent septae. Analysis of several genetic as well as hyperoxia-induced models of BPD revealed that the myofibroblast organization is perturbed in all, regardless of whether the origin of defect is epithelial, mesenchymal, endothelial or environmental. Finally, analysis of relative position of PDGFRA-positive cells and alveolar type 2 cells reveal that during alveologenesis, these two cell types are not always adjacent to one another. This result suggests that the niche and progenitor relationship afforded by their close juxtaposition in the adult lung may be a later acquired property. These insights revealed by 3D reconstruction of the septae set the foundation for future investigations of the mechanisms driving normal alveologenesis, as well as causes of alveolar simplification in BPD.

Localization of c-MYB in differentiated cells during postnatal molar and alveolar bone development.

The MYB family of transcription activators has been associated with a high proliferation rate and an undifferentiated state of cells in a number of tissues. Recently emerging data suggest that these molecules may also play a role in differentiation. In this study, the pattern of expression of c-MYB was followed during postnatal stages of mouse molar odontogenesis using immunohistochemistry on serial sections. Along with an abundance of the c-MYB protein in proliferating zones, we confirmed the presence of this protein in differentiated ameloblasts, odontoblasts, and osteoblasts. In addition, c-MYB was also found in cementoblasts and alveolar fibroblasts. These findings suggest integration of c-MYB into regulatory networks during hard-tissue differentiation and mineralization.

Morphogenesis and bone integration of the mouse mandibular third molar.

The mouse third molar (M3) develops postnatally and is thus a unique model for studying the integration of a non-mineralized tooth with mineralized bone. This study assessed the morphogenesis of the mouse M3, related to the alveolar bone, comparing M3 development with that of the first molar (M1), the most common model in odontogenesis. The mandibular M3 was evaluated from initiation to eruption by morphology and by assessing patterns of proliferation, apoptosis, osteoclast distribution, and gene expression. Three-dimensional reconstruction and explant cultures were also used. Initiation of M3 occurred perinatally, as an extension of the second molar (M2) which grew into a region of soft mesenchymal tissue above the M2, still far away from the alveolar bone. The bone-free M3 bud gradually became encapsulated by bone at the cap stage at postnatal day 3. Osteoclasts were first visible at postnatal day 4 when the M3 came into close contact with the bone. The number of osteoclasts increased from postnatal day 8 to postnatal day 12 to form a space for the growing tooth. The M3 had erupted by postnatal day 26. The M3, although smaller than the M1, passed through the same developmental stages over a similar time span but showed differences in initiation and in the timing of bone encapsulation.

Expression and characterization of c-Myb in prenatal odontogenesis.

The transcription factor c-Myb is involved in the control of cell proliferation, survival and differentiation. As these processes accompany the morphogenesis of developing teeth, this work investigates the possible role of c-Myb during odontogenesis. Analysis of the expression of c-Myb in the monophyodont mouse was followed by similar analysis in a diphyodont species, the pig, which has a dentition more closely resembling that of the human. The distribution of c-Myb was correlated with the pattern of proliferation and apoptosis and the tooth phenotype of c-Myb mutant mice was also assessed. In the mouse, c-Myb expression was detected throughout prenatal development of the first molar tooth. Negative temporospatial correlation was found between c-Myb expression and apoptosis, while c-Myb expression positively correlated with proliferation. c-Myb-positive cells, however, were more abundant than the proliferating cell nuclear antigen positive cells, suggesting other roles of c-Myb in odontogenesis. In the minipig, in contrast to the mouse, there was an asymmetrical arrangement of c-Myb positive cells, with a higher presence on the labial side of the tooth germ and dental lamina. A cluster of negative cells was situated in the mesenchyme close to the tooth bud. At later stages, the number of positive cells decreased and these cells were situated in the upper part of the dental papilla in the areas of future cusp formation. The expression of c-Myb in both species was strong in the odontoblasts and ameloblasts at the stage of dentin and enamel production suggesting a possible novel role of c-Myb during tooth mineralization.