Vocal fold epithelial cells are very difficult to study as the vocal fold epithelial cell lines d... more 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.
Study of vocal fold (VF) mucosal biology requires essential human vocal fold epithelial cell (hVF... more 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.
Abstract Teeth develop as epithelial-mesenchymal organs in context with the surrounding structure... more Abstract Teeth develop as epithelial-mesenchymal organs in context with the surrounding structures, particularly the alveolar (and jaw) bone. There is a dynamic in the development of both structures; moreover, the tooth mesenchyme contributes directly to formation of the bone tissue. Additionally, the bone undergoes an active remodeling as the tooth grows, and the balance of both structures must be re-established after eruption and maintained throughout the functional life of dentition. In this chapter, the molecular signals regulating tooth initiation and morphogenesis are described to explain patterning of the odontogenic region, terminal differentiation and mineralization of the dental structures, and prevention of mineralization of the soft parts as the dental pulp. The basic principles of bone remodeling are introduced, and the interactions between the tooth and bone summarized. The mouse dentition, as the most common model in odontogenesis research, is used to explain the events accompanying odontogenesis, along with osteogenesis and bone remodeling, and to outline the major disorders found in humans.
Mechanoreceptors are implicated as functional afferents within mucosa of the airways and the rece... more 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 ...
Vaping has been reported to cause acute epiglottitis, a life-threatening airway obstruction induc... more Vaping has been reported to cause acute epiglottitis, a life-threatening airway obstruction induced by the direct epithelial injury and subsequent inflammatory reaction. Here we show that we were able to recapitulate this phenomenon in vitro. Exposure of human engineered VF mucosae to 0.5% and 5% e-cig vapor extract (ECVE) for one 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 let VF mucosae to regenerate for 1, 3 and 7 days which triggered intense epithelial remodeling. The epithelial changes included P63+ basal cell expansion and Cytokeratin 14 and Laminin alpha 5 deposition that may lead to local basal cell hype...
The larynx, trachea, and esophagus share origin and proximity during embryonic development. Clini... more The larynx, trachea, and esophagus share origin and proximity during embryonic development. Clinical and experimental evidence support the existence of neurophysiological, structural, and functional interdependencies before birth. This investigation provides the first comprehensive transcriptional profile of all three organs during embryonic organogenesis, where differential gene expression gradually assembles the identity and complexity of these proximal organs from a shared origin in the anterior foregut. By applying bulk RNA sequencing and gene network analysis of differentially expressed genes (DEGs) within and across developing embryonic mouse larynx, esophagus, and trachea, we identified co-expressed modules of genes enriched for key biological processes. Organ-specific temporal patterns of gene activity corresponding to gene modules within and across shared tissues during embryonic development (E10.5-E18.5) are described, and the laryngeal transcriptome during vocal fold deve...
Vocal fold epithelial cells are very difficult to study as the vocal fold epithelial cell lines d... more 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.
Study of vocal fold (VF) mucosal biology requires essential human vocal fold epithelial cell (hVF... more 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.
Abstract Teeth develop as epithelial-mesenchymal organs in context with the surrounding structure... more Abstract Teeth develop as epithelial-mesenchymal organs in context with the surrounding structures, particularly the alveolar (and jaw) bone. There is a dynamic in the development of both structures; moreover, the tooth mesenchyme contributes directly to formation of the bone tissue. Additionally, the bone undergoes an active remodeling as the tooth grows, and the balance of both structures must be re-established after eruption and maintained throughout the functional life of dentition. In this chapter, the molecular signals regulating tooth initiation and morphogenesis are described to explain patterning of the odontogenic region, terminal differentiation and mineralization of the dental structures, and prevention of mineralization of the soft parts as the dental pulp. The basic principles of bone remodeling are introduced, and the interactions between the tooth and bone summarized. The mouse dentition, as the most common model in odontogenesis research, is used to explain the events accompanying odontogenesis, along with osteogenesis and bone remodeling, and to outline the major disorders found in humans.
Mechanoreceptors are implicated as functional afferents within mucosa of the airways and the rece... more 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 ...
Vaping has been reported to cause acute epiglottitis, a life-threatening airway obstruction induc... more Vaping has been reported to cause acute epiglottitis, a life-threatening airway obstruction induced by the direct epithelial injury and subsequent inflammatory reaction. Here we show that we were able to recapitulate this phenomenon in vitro. Exposure of human engineered VF mucosae to 0.5% and 5% e-cig vapor extract (ECVE) for one 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 let VF mucosae to regenerate for 1, 3 and 7 days which triggered intense epithelial remodeling. The epithelial changes included P63+ basal cell expansion and Cytokeratin 14 and Laminin alpha 5 deposition that may lead to local basal cell hype...
The larynx, trachea, and esophagus share origin and proximity during embryonic development. Clini... more The larynx, trachea, and esophagus share origin and proximity during embryonic development. Clinical and experimental evidence support the existence of neurophysiological, structural, and functional interdependencies before birth. This investigation provides the first comprehensive transcriptional profile of all three organs during embryonic organogenesis, where differential gene expression gradually assembles the identity and complexity of these proximal organs from a shared origin in the anterior foregut. By applying bulk RNA sequencing and gene network analysis of differentially expressed genes (DEGs) within and across developing embryonic mouse larynx, esophagus, and trachea, we identified co-expressed modules of genes enriched for key biological processes. Organ-specific temporal patterns of gene activity corresponding to gene modules within and across shared tissues during embryonic development (E10.5-E18.5) are described, and the laryngeal transcriptome during vocal fold deve...
Uploads