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.

Establishment of Immortalized Laryngeal Epithelial Cells Transfected with Bmi1.

Primary laryngeal epithelial cells are essential to exploring the mechanisms of laryngeal and voice disorders; however, they are difficult to study and apply because of their limited life span. The purpose of this study was to develop a stable and reliable in vitro model for the comprehensive study of the pathogenesis of laryngeal and voice diseases. The pLVTHM-Bmi1 plasmid was constructed and used to immortalize primary laryngeal epithelial cells by lentiviral infection. The expressions of Bmi1, human telomerase reverse transcriptase (hTERT), p53, and pRB pathway proteins were detected by western blotting. Functional characteristics of the immortalized cell lines were verified by cell senescence ß-galactosidase staining, 5-ethynyl-2'-deoxyuridine cell proliferation test, and flow cytometry. We successfully introduced Bmi into human subglottic (hSG) cells and human ventricle (hV) cells. Both the human immortalized subglottic Bmi1 (hSG-Bmi1) cell line and the human immortalized ventricle Bmi1 (hV-Bmi1) cell line maintained normal epithelial morphology and divided successfully after more than 20 culture passages. As Bmi1 was overexpressed in these cells, the expression of human telomerase reverse transcriptase (hTERT) and phosphorylated Rb increased while p16 and p21 decreased. Following Bmi1-mediated immortalization, cell senescence decreased significantly, and cell proliferation was accelerated. Tumor formation was not observed for hSG, hV, or hSG-Bmi1, and hV-Bmi1 cells in nude mice. hSG-Bmi1 cells dominated by stratified squamous epithelium and hV-Bmi1 cells dominated by columnar cells were established. The new cell lines lay a foundation for the study of the pathogenic mechanisms of laryngeal and voice diseases.

Heterogeneity of Stem Cells in the Human Vocal Fold Mucosa.

1. There is growing evidence to suggest that the cells in the maculae flavae are tissue stem cells of the human vocal fold and maculae flavae are a candidate for a stem cell niche. 2. The latest research shows that the cells in the human maculae flavae are involved in the metabolism of extracellular matrices that are essential for viscoelasticity in the human vocal fold mucosa as a vibrating tissue and are considered to be important cells in the growth, development, and aging of the human vocal fold mucosa. 3. Recent evidence has indicated that the cells including vocal fold stellate cells in the maculae flavae of the human vocal fold mucosa are a functionally heterogenous population. 4. The cells in the human maculae flavae possess proteins of all three germ layers, indicating that they are undifferentiated and have the ability of multipotency. 5. The cell division in the human adult maculae flavae is reflective of asymmetric self-renewal, and cultured cells form a colony-forming unit. Therefore, the phenomenon gives rise to the strong possibility that the cells in the human maculae flavae are putative stem cells. 6. Recent research has suggested that the cells in the human maculae flavae arise from the differentiation of bone marrow cells via peripheral circulation. 7. Cultured cell populations in the human maculae flavae are roughly divided into three groups by morphological features: cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells. However, at the present state of our investigation, it is difficult to clarify the stem cell system and hierarchy of stem cells in the human maculae flavae. 8. Subpopulations of cells in the maculae flavae proliferate extremely slowly and retain stem cell properties. 9. Tension caused by phonation seems to regulate the behavior and heterogeneity of the cells (mechanical regulation) in the maculae flavae of the human vocal fold. 10. The putative stem cells in the maculae flavae appear to differentiate into other kind of cells in the surrounding tissue.

Methodology for the establishment of primary porcine vocal fold epithelial cell cultures.

OBJECTIVE: A current lack of methods for epithelial cell culture significantly hinders our understanding of the role of the epithelial and mucus barriers in vocal fold health and disease. Our first objective was to establish reproducible techniques for the isolation and culture of primary porcine vocal fold epithelial cells. Our second objective was to evaluate the functional significance of cell cultures using an in vitro exposure to an inflammatory cytokine. METHODS: Epithelial cells were isolated from porcine vocal folds and expanded in culture. Characterization of cultures was completed by immunostaining with markers for pan-cytokeratin (epithelial cells), vimentin (stromal cells), von Willebrand factor (endothelial cell), and MUC1 and MUC4 (mucin) glycoproteins. Established epithelial cell cultures were then exposed to the inflammatory cytokine tumor necrosis factor alpha (TNF-α) for 24-hours, and transcript expression of MUC1 and MUC4 was evaluated. RESULTS: Reproducible, porcine vocal fold epithelial cell cultures, demonstrating cobblestone appearance characteristic of the typical morphology of epithelial cell cultures were created. Cells showed positive staining for pan-cytokeratin with limited expression of vimentin and von Willebrand factor. Epithelial cells also expressed MUC1 and MUC4. TNF-α significantly increased transcript expression of MUC4. CONCLUSION: Here, we present the first report of successful culture of primary porcine vocal fold epithelial cells. Cultures will provide researchers with a valuable new in vitro tool to investigate vocal fold epithelium and mucus as well as the effects of common challenges, including inflammatory cytokines, on these barriers. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E355-E364, 2019.

[Influence of TNF-α on the immunomodulatory property of laryngeal mucosa mesenchymal stromal cells'].

Objective: To investigate the effect of tumor necrosis factor-alpha(TNF-α)on the immunoregulatory capacity of laryngeal mucosal mesenchymal stromal cells (LM-MSCs) and its potential molecular mechanism, and provide a theoretical basis for the study of chronic laryngitis. Methods: LM-MSCs were separated from epiglottal mucosa. The LM-MSCs cells were directly co-cultured with T cells in vitro to detect the immunomodulatory property of LM-MSCs. After long-term stimulation with inflammatory factors TNF-α in vitro, the differences were compared in the immunomodulatory ability of LM-MSCs between normal LM-MSCs and TNF-α stimulated LM-MSCs. The expression of general control non-repressed protein5(GCN5), FAS, FASL in normal LM-MSCs and TNF-α stimulated LM-MSCs was detected by Western blot and quantitative real-time RT-PCR(RT-qPCR). Results: After chronic stimulation of TNF-α, the RNA relative expression of GCN5 was 0.31±0.03 (3 days) and 0.53±0.06 (7 days) compared with control group, showing significant difference (F=13.45, P<0.05). The percentage of LM-MSC-induced T cell apoptosis was 6.27%±0.81% (3 days) and 4.99%±0.52% (7 days) in chronic stimulation group compared with control group 10.02%±1.02%. There is a significant difference among these groups (F=11.13, P<0.05). Moreover, the ability of LM-MSCs to induce T cell apoptosis is regulated by GCN5. Conclusion: With the chronic stimulation of TNF-α, the expression of GCN5 in LM-MSCs is decreased, thus impairing its immunoregulatory capacity.

Morphology of GNAT3-immunoreactive chemosensory cells in the rat larynx.

The upper airways play important roles in respiratory defensive reflexes. Although solitary chemosensory cells and chemosensory cell clusters have been reported in the laryngeal mucosa of mammalian species, the distribution and cellular morphology of chemosensory cells remain unclear. In the present study, the distribution and morphology of solitary chemosensory cells and chemosensory cell clusters were examined by immunofluorescence for GNAT3 on whole-mount preparations of the rat laryngeal mucosa. Electrophysiological experiments were performed to analyze the respiratory reflexes evoked by bitter stimuli to the laryngeal cavity. In the whole area of the laryngeal mucosa, the numbers of GNAT3-immunoreactive solitary chemosensory cells and chemosensory clusters were 421.0 ± 20.3 and 62.7 ± 6.9, respectively. GNAT3-immunoreactive solitary chemosensory cells were mainly distributed in the mucosa overlying epiglottic and arytenoid cartilage, and chemosensory clusters were mainly distributed on the edge of the epiglottis and aryepiglottic fold. GNAT3-immunoreactive solitary chemosensory cells were slender with elongated processes or had a flask-like/columnar shape. The number of GNAT3-immunoreactive cells in chemosensory clusters was 6.1 ± 0.4, ranging between 2 and 14 cells. GNAT3-immunoreactive cells in the cluster were variform and the tips of apical processes gathered at one point at the surface of the epithelium. The tips of apical cytoplasmic processes in solitary chemosensory cells and cells in the cluster were immunoreactive for espin, and faced the laryngeal cavity. Physiological experiments showed that the application of 10 mm quinine hydrochloride to the laryngeal cavity decreased respiratory frequency. The present results revealed the chemosensory field of the larynx and the morphological characteristics of the laryngeal chemosensory system for respiratory depression.

Pericytes in the Human Vocal Fold Mucosa.

1. The human vocal fold is a vibrating tissue and vascular structures in organs which have the capacity to vibrate require a specific structure suitable for vibration. 2. The structure of the blood vessels is unique at the vocal fold edge as a vibrating tissue, where only small vessels, including arterioles, venules, and capillaries, are present. The capillaries are distributed in the superficial layer of the lamina propria (Reinke's space). 3. The blood vessels enter the vocal fold edge from the anterior or posterior end of the membranous vocal fold and run essentially parallel to the vocal fold edge. 4. Many pericytes can be seen around the capillaries in the human vocal fold mucosa. The cell bodies of the pericytes attach to capillary endothelial cells, and the branching processes encircle the capillaries and attach to the capillary endothelial cells at the tips. The processes of pericytes are in close contact with endothelial cells, sharing a common basement membrane with them. The tips of the processes form intercellular tight junctions with endothelial cells. 5. The pericytes in the vocal fold mucosa appear to provide mechanical support and protection to the capillary walls, particularly during phonation. The pericytes also appear to regulate the diameter of the capillary during and after phonation. Pericytes are also thought to be critical cells in vascular biology and angiogenesis, especially in revascularization following vocal fold tissue injury. 6. At birth, pericytes have already encircled the capillaries in the newborn vocal fold mucosa. The pericytes appear ready to provide support and protection of the blood vessels just after birth. 7. Vascular structures and their permeability are related to the specific structures and specific diseases of the human vocal fold mucosa as a vibrating tissue.

Long-term control of laryngeal plasma cell mucositis with systemic immunosuppression.

Plasma cell mucositis (PCM) is a rare non-neoplastic plasma cell proliferative disorder of the mucous membranes, which typically presents as soft tissue lesions involving oral, upper airway or genital mucosa. Laryngeal involvement resulting in stridor has been reported in four other cases previously, with three requiring tracheostomy. We present a case of supraglottic stenosis in a 53-year-old woman presenting with dysphonia and stridor, requiring surgical resection on three occasions accompanied by tracheostomy on two occasions; biopsy was consistent with PCM. Due to relapsing disease activity, high-dose prednisolone and mycophenolate mofetil were commenced with prednisolone eventually being ceased. After 2 years of mycophenolate mofetil therapy, the patient's disease has been controlled without need for further surgical intervention. This is the first reported case of prolonged symptomatic improvement with the use of systemic immunosuppressive therapy with mycophenolate mofetil in PCM.

Activation of the Wnt/ß-Catenin Pathway by an Inflammatory Microenvironment Affects the Myogenic Differentiation Capacity of Human Laryngeal Mucosa Mesenchymal Stromal Cells.

Various microenvironments influence the multiple differentiation potential of mesenchymal stromal cells. For example, inflammatory microenvironment can suppress the myogenic differentiation capability of laryngeal mucosa mesenchymal stromal cells (LM-MSCs). The present study therefore sought to identify the underlying molecular mechanisms regulating these processes. We isolated a novel population of MSCs, LM-MSCs, from the laryngeal mucosa tissues. The cells were cultured in osteogenic, adipogenic, and myogenic differentiation media in the presence or absence of interleukin-1ß and tumor necrosis factor α (to simulate inflammatory microenvironment). The expression of active ß-catenin, p-GSK3ß, and GSK3ß were detected by western blot and real-time polymerase chain reaction. The myogenic differentiation of LM-MSCs in inflammatory microenvironment and the regulation by Dickkopf-1 (DKK1) were tested both in vivo and in vitro. Inflammatory microenvironment could suppress the osteogenesis, adipogenesis, and myogenesis of LM-MSCs. The Wnt/ß-catenin signaling pathway was activated during myogenesis in inflammatory microenvironment. The suppressed myogenic differentiation capability of LM-MSCs in inflammatory microenvironment was reversed by DKK1. By regulating the Wnt/ß-catenin signaling pathway, DKK1 can improve the myogenic differentiation of LM-MSCs in inflammatory microenvironment. Thus, the results of this study may help improve the efficacy of LM-MSCs injection therapy for vocal fold regeneration.

The Macula Flava of the Human Vocal Fold as a Stem Cell Microenvironment.

1. There is growing evidence to suggest that the cells in the maculae flavae are tissue stem cells of the human vocal fold and maculae flavae are a candidate for a stem cell niche. 2. The latest research shows that the cells in the human maculae flavae are involved in the metabolism of extracellular matrices that are essential for the viscoelasticity in the human vocal fold mucosa as a vibrating tissue, and considered to be important cells in the growth, development, and aging of the human vocal fold mucosa. 3. The cells in the human maculae flavae possess proteins of all three germ layers, indicating they are undifferentiated and have the ability of multipotency. 4. The cell division in the human adult maculae flavae is reflective of asymmetric self-renewal and cultured cells form a colony-forming unit. Therefore, the phenomenon gives rise to the strong possibility that the cells in the human maculae flavae are tissue stem cells. 5. Recent research suggests that the cells in the human maculae flavae arise from the differentiation of bone marrow cells via peripheral circulation. 6. The hyaluronan concentration in the maculae flavae is high and contains cells which possess hyaluronan receptors, indicating that the maculae flavae are hyaluronan-rich matrix, which is required for a stem cell niche. 7. A proper microenvironment in the maculae flavae of the human vocal fold mucosa is necessary to be effective as a stem cell niche maintaining the stemness of the contained tissue stem cells.

Microenvironment of macula flava in the human vocal fold as a stem cell niche.

BACKGROUND: There is growing evidence that the cells in the maculae flavae are tissue stem cells of the human vocal fold mucosa, and that the maculae flavae are a candidate for a stem cell niche. The role of microenvironment in the maculae flavae of the human vocal fold mucosa was investigated. METHOD: Anterior maculae flavae from six surgical specimens were cultured in a mesenchymal stem cell growth medium or a Dulbecco's modified Eagle's medium. RESULTS: Using mesenchymal stem cell growth medium, the subcultured cells formed a colony-forming unit, and cell division reflected asymmetric self-renewal. This indicates that these cells are mesenchymal stem cells or stromal stem cells in the bone marrow. Using Dulbecco's modified Eagle's medium, the subcultured cells showed symmetric cell division without a colony-forming unit. CONCLUSION: A proper microenvironment in the maculae flavae of the human vocal fold mucosa is necessary to be effective as a stem cell niche that maintains the stemness of the contained tissue stem cells.

Regeneration of Vocal Fold Mucosa Using Tissue-Engineered Structures with Oral Mucosal Cells.

OBJECTIVES: Scarred vocal folds result in irregular vibrations during phonation due to stiffness of the vocal fold mucosa. To date, a completely satisfactory corrective procedure has yet to be achieved. We hypothesize that a potential treatment option for this disease is to replace scarred vocal folds with organotypic mucosa. The purpose of this study is to regenerate vocal fold mucosa using a tissue-engineered structure with autologous oral mucosal cells. STUDY DESIGN: Animal experiment using eight beagles (including three controls). METHODS: A 3 mm by 3 mm specimen of canine oral mucosa was surgically excised and divided into epithelial and subepithelial tissues. Epithelial cells and fibroblasts were isolated and cultured separately. The proliferated epithelial cells were co-cultured on oriented collagen gels containing the proliferated fibroblasts for an additional two weeks. The organotypic cultured tissues were transplanted to the mucosa-deficient vocal folds. Two months after transplantation, vocal fold vibrations and morphological characteristics were observed. RESULTS: A tissue-engineered vocal fold mucosa, consisting of stratified epithelium and lamina propria, was successfully fabricated to closely resemble the normal layered vocal fold mucosa. Laryngeal stroboscopy revealed regular but slightly small mucosal waves at the transplanted site. Immunohistochemically, stratified epithelium expressed cytokeratin, and the distributed cells in the lamina propria expressed vimentin. Elastic Van Gieson staining revealed a decreased number of elastic fibers in the lamina propria of the transplanted site. CONCLUSION: The fabricated mucosa with autologous oral mucosal cells successfully restored the vocal fold mucosa. This reconstruction technique could offer substantial clinical advantages for treating intractable diseases such as scarring of the vocal folds.

Morphology of P2X3-immunoreactive nerve endings in the rat laryngeal mucosa.

The morphological characteristics of P2X3-immunoreactive nerve endings in the laryngeal mucosa were herein examined using immunohistochemistry with confocal laser microscopy. Ramified intraepithelial nerve endings immunoreactive to P2X3 were distributed in the epiglottis and arytenoid region. The axon terminals of P2X3-immunoreactive ramified endings were beaded or flat in shape. These endings were also immunoreactive to P2X2 and not identical to the nerve endings immunoreactive to Na(+)-K(+)-ATPase α3-subunit, substance P (SP), and calcitonin gene-related peptide (CGRP). P2X3-immunoreactive axon terminals were also immunoreactive to vGLUT1, vGLUT2, and vGLUT3. In addition to ramified endings, P2X3-immunoreactive nerve endings were associated with α-gustducin-immunoreactive solitary chemosensory cells and/or SNAP25-immunoreactive neuroendocrine cells. Furthermore, P2X3-immunoreactive nerve endings were also observed in the taste bud-like chemosensory cell clusters of the stratified squamous epithelium covering epiglottic and arytenoid cartilage. The P2X3-immunoreactive nerve endings that associated with sensory and/or endocrine cells and chemosensory cell clusters were also immunoreactive to P2X2, vGLUT1, vGLUT2, and vGLUT3, but not to SP or CGRP. In conclusion, P2X3-immunoreactive nerve endings may be classified into two types, i.e., intraepithelial ramified nerve endings and nerve endings associated with chemosensory cells and neuroendocrine cells.

Identification of distinct layers within the stratified squamous epithelium of the adult human true vocal fold.

OBJECTIVES/HYPOTHESIS: A precise molecular schema for classifying the different cell types of the normal human vocal fold epithelium is lacking. We hypothesize that the true vocal fold epithelium has a cellular architecture and organization similar to that of other stratified squamous epithelia including the skin, cornea, oral mucosa, and esophagus. In analogy to disorders of the skin and gastrointestinal tract, a molecular definition of the normal cell types within the human vocal fold epithelium and a description of their geometric relationships should serve as a foundation for characterizing cellular changes associated with metaplasia, dysplasia, and cancer. STUDY DESIGN: Qualitative study with adult human larynges. METHODS: Histologic sections of normal human laryngeal tissue were analyzed for morphology (hematoxylin and eosin) and immunohistochemical protein expression profile, including cytokeratins (CK13 and CK14), cornified envelope proteins (involucrin), basal cells (NGFR/p75), and proliferation markers (Ki67). RESULTS: We demonstrated that three distinct cell strata with unique marker profiles are present within the stratified squamous epithelium of the true vocal fold. We used these definitions to establish that cell proliferation is restricted to certain cell types and layers within the epithelium. These distinct cell types are reproducible across five normal adult larynges. CONCLUSION: We have established that three layers of cells are present within the normal adult stratified squamous epithelium of the true vocal fold. Furthermore, replicating cell populations are largely restricted to the parabasal strata within the epithelium. This delineation of distinct cell populations will facilitate future studies of vocal fold regeneration and cancer. LEVEL OF EVIDENCE: N/A.

Potential of biofluid components to modify silver nanoparticle toxicity.

Establishing realistic exposure scenarios is critical for cytotoxic investigation of silver nanoparticles (AgNP) in the gastrointestinal tract. This study investigated the potential interaction with and effect of biofluid components, namely cholic acid, deoxycholic acid and ursodeoxycholic acid, on AgNP toxicity. Two cell lines corresponding to organs related to the biofluid components were employed. These were HepG-2 a hepatocellular carcinoma derived from liver tissue and Hep2 an epithelial cell line. Physiochemical and cytotoxic screening was performed and the ability of biofluid components to modify AgNP cytotoxicity was explored. No alteration to the physiochemical characteristics of AgNP by biofluid components was demonstrated. However, biofluid component addition resulted in alteration of AgNP toxicity. Greater reactive oxygen species induction was noted in the presence of cholic acid and deoxycholic acid. Ursodeoxycholic acid demonstrated no modification of toxicity in HepG-2 cells; however, significant modification was noted in Hep2 cells. It is concluded that biofluid components can modify AgNP toxicity but this is dependent on the biofluid component itself and the location where it acts.

Comparative cytotoxicity of dolomite nanoparticles in human larynx HEp2 and liver HepG2 cells.

Dolomite is a natural mineral of great industrial and commercial importance. With the advent of nanotechnology, natural minerals including dolomite in the form of nanoparticles (NPs) are being utilized in various applications to improve the quality of products. However, safety or toxicity information of dolomite NPs is largely lacking. This study evaluated the cytotoxicity of dolomite NPs in two widely used in vitro cell culture models: human airway epithelial (HEp2) and human liver (HepG2) cells. Concentration-dependent decreased cell viability and damaged cell membrane integrity revealed the cytotoxicity of dolomite NPs. We further observed that dolomite NPs induce oxidative stress in a concentration-dependent manner, as indicated by depletion of glutathione and induction of reactive oxygen species (ROS) and lipid peroxidation. Quantitative real-time PCR data demonstrated that the mRNA level of tumor suppressor gene p53 and apoptotic genes (bax, CASP3 and CASP9) were up-regulated whereas the anti-apoptotic gene bcl-2 was down-regulated in HEp2 and HepG2 cells exposed to dolomite NPs. Moreover, the activity of apoptotic enzymes (caspase-3 and caspase-9) was also higher in both kinds of cells treated with dolomite NPs. It is also worth mentioning that HEp2 cells seem to be marginally more susceptible to dolomite NPs exposure than HepG2 cells. Cytotoxicity induced by dolomite NPs was efficiently prevented by N-acetyl cysteine treatment, which suggests that oxidative stress is primarily responsible for the cytotoxicity of dolomite NPs in both HEp2 and HepG2 cells. Toxicity mechanisms of dolomite NPs warrant further investigations at the in vivo level.

Utility of cell viability assays for use with ex vivo vocal fold epithelial tissue.

OBJECTIVES/HYPOTHESIS: Ex vivo models are routinely used to investigate the barrier function of the vocal fold epithelium. However, there are limited reports on assays that can be used to investigate the effect of clinically relevant challenges on vocal fold epithelial tissue viability. Our objective was to determine the utility of two assays routinely used in cell culture-a cellular metabolic activity assay and a cell membrane integrity assay-to investigate the viability of ex vivo porcine vocal fold epithelium. STUDY DESIGN: Prospective, ex vivo animal study. METHODS: Porcine vocal folds were exposed to acrolein, hydrochloric acid, or hydrogen peroxide challenge. An untreated, sham challenge was included as a control. Assays including metabolic activity, cell membrane integrity, and histology were used to determine whether challenges reduced epithelial viability as compared to sham. RESULTS: Cell membrane integrity and metabolic activity assays detected reductions in viability following hydrochloric acid and hydrogen peroxide challenges but not acrolein challenge as compared to sham. No challenge produced significant changes in epithelial appearance as evidenced by light microscopy. CONCLUSIONS: Metabolic activity and cell membrane integrity assays are valuable tools that can be used to evaluate the viability of ex vivo vocal fold epithelial tissue following clinically relevant challenges. As viability is reduced, the ability of epithelial tissue to maintain its barrier function is compromised. Accurate assessment of viability may provide us clues into understanding mechanisms underlying vocal fold epithelial injury and disease. LEVEL OF EVIDENCE: NA Laryngoscope, 125:E180-E185, 2015.

[Myogenic differentiation of laryngeal mucosal mesenchymal stem cells].

OBJECTIVE: To investigate the myogenic differentiation of laryngeal mucosal mesenchymal stem cells (LM-MSCs) and the possibility of LM-MSCs as new alternative seed cells for laryngeal tissue engineering. METHODS: LM-MSCs were separated from normal epiglottis mucosa and the cell surface markers including CD44, CD105, CD90, CD29, CD34 and CD45 were analyzed through flow cytometry. The osteogenesis and adipogenesis differentiation of LM-MSCs were investigated by oil red staining and alizarin red S staining. Immunofluorescence staining and RT-PCR were used to detect the expressions of myogenic differentiation markers including Myod1, Myogenin and myosin heavy chain (MyHc). RESULTS: The separated LM-MSCs were in a fibrocyte-like form with long fusiform shape and grew adherent. The expression rates of cell surface markers LM-MSCs were CD44 (100.0%), CD105 (90.4%), CD90(99.9%), CD29 (93.0%), CD34 (0.4%) and CD45(1.3%) respectively. A number of beaded lipid drops and mineral deposition were observed after 14 days of adipogenesis differentiation and 21 days of osteogenesis differentiation. Myod1, Myogenin and MyHc genes appeared after 1 week and 3 weeks of myogenesis differentiation respectively. CONCLUSIONS: The LM-MSCs have the properties of mesenchymal stem cells and could be differentiated into myoblasts, providing with the possibility to repair the damaged vocal cords with LM-MSCs through tissue engineering techniques.

Cytoskeleton of newborn vocal fold stellate cells.

OBJECTIVES/HYPOTHESIS: Vocal fold stellate cells (VFSCs) in the human maculae flavae located at both ends of the vocal fold mucosa are inferred to be involved in the metabolism of extracellular matrices of the vocal fold mucosa. Tension caused by phonation (vocal fold vibration) likely regulates the behavior of the VFSCs in the human maculae flava. Tensile and compressive strains have direct effects on cell morphology and structure, including changes in cytoskeletal structure and organization. Cytoskeletons play a role as mechanoreceptors for the cells. The microstructure of the intermediate filaments and the expression of their characteristic proteins were investigated regarding the human newborn VFSCs. STUDY DESIGN: Histopathologic analysis of the human newborn vocal fold. METHODS: Three newborn vocal fold mucosae were investigated by immunohistochemistry and electron microscopy. RESULTS: The intermediate filaments in the cytoplasm of the newborn VFSCs were few in number. However, their characteristic proteins (vimentin, desmin, GFAP [Glial fibrillary acidic protein], cytokeratin) had already expressed. CONCLUSION: The function and fate of VFSCs are regulated by various microenvironmental factors. Not only chemical factors but also mechanical factors could also modulate VFSC behaviors. The cytoskeletal structure of the newborn VFSCs is under development. And the newborn VFSCs have not acquired mechanical regulation. LEVEL OF EVIDENCE: N/A.

The use of laryngeal mucosa mesenchymal stem cells for the repair the vocal fold injury.

Stem cell transplantation is a kind of attractive and new approach that complements traditional restorative or surgical techniques for the regeneration of injured or pathologically damaged laryngeal tissues. However, the best cell delivery strategy remains to be identified. The objective of this study was to establish a new strategy to the healing of injured vocal fold, using laryngeal mucosa mesenchymal stem cells differentiating into myofibroblasts or fibroblasts and improving the reconstruction microenvironment in the vocal fold injury as a new alternative as seed cells for laryngeal tissue engineering. After isolation and expansion, cells were identified as adherent mesenchymal cells with substantial proliferation potential in vitro, and were also characterized by flow cytometry. The differentiation potential of mesenchymal cells was maintained during proliferation as confirmed by culturing for adipogenesis, osteogenesis and chondrocyte. When LM-MSC was transplanted into the injured vocal fold, it has the potent differentiated into myofibroblasts and fibroblasts, which could regulate extracellular matrix, block collagen and the fibronectin rapid increased, inhibit the rapidly decrease of elastic fiber and HA, decrease the microenvironment inflammatory reaction, and prevent the formation of vocal fold scar.