Glycolytic Metabolism of the Tissue Stem Cells in the Maculae Flavae of the Human Vocal Fold.

OBJECTIVES: Metabolic programs in the stem cells are essential for maintaining homeostasis and protecting against stem cell aging. There is growing evidence that the tissue stem cells reside in the anterior and posterior maculae flavae of the human vocal fold mucosa. Our previous studies observed that the glycolysis of the cell in the human maculae flavae seems to rely more on anaerobic glycolysis for energy supply in comparison with oxidative phosphorylation. However, previous studies showed only the metabolic enzymes of glycolysis and functional morphology of the mitochondria, therefore, it has not yet been determined whether anaerobic glycolysis actually took place. The purpose of this study is to investigate the glycolytic metabolites of the cells in the maculae flavae of the human vocal fold in vitro. METHODS: Four normal human vocal folds were used. After extraction of the anterior maculae flavae, cells in the maculae flavae were cultured and proliferated. Glucose transporter-1 was assessed using immunocytochemistry and metabolites of glycolysis (lactate and NADPH) were measured. RESULTS: The cells in the maculae flavae expressed glucose transporter-1 in the cytoplasm and the cell membranes. In addition, the cultured cells produced lactate (metabolites of anaerobic glycolysis) and NADPH (metabolites of the pentose phosphate pathway). CONCLUSIONS: The cells in the maculae flavae of the human vocal folds were found to undergo anaerobic glycolysis via the pentose phosphate pathway. This suggests that the cells in the maculae flavae of the human vocal fold have a metabolism that favors the maintenance of stemness and undifferentiated states.

Three-dimensional fine structures of the maculae flavae of the human vocal fold using correlative light and electron microscopy.

Objectives: To analyze various aspects of complex tissue, there is increasing demand to study each sample at different length scales in biology. Correlative light and electron microscopy (CLEM) is the latest technique to correlate two different types of information on the exact same histological area of interest: histology (from light microscopy) and ultrastructure (from electron microscopy). The three-dimensional fine structures of the maculae flavae (MFe) of the human vocal fold were investigated using CLEM. Methods: Five normal human adult vocal folds as specimens embedded in paraffin, sectioned, and mounted on glass slides with/without a chemical digestion method (modified sodium hydroxide maceration method) were investigated. Observations using CLEM were performed. Results: The fine structures of cells and extracellular matrices in the MFe and their peripheral regions were able to be observed on the exact same histological area of interest with the light microscope and field emission-scanning electron microscope. Cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells were intermingled in the MFe of the human vocal fold. The extracellular matrices surrounding each three types of cell in the MFe differed, suggesting the cells were different in functional property. Conclusion: CLEM is a useful technique to observe the three-dimensional fine structures of the human vocal fold mucosa. The results of the present study are consistent with the hypothesis that the cells in the MFe of the human vocal fold have heterogeneity and each three types of cell have different properties.

Histoanatomical structures of laryngeal atresia: Functional considerations.

OBJECTIVE: To study the histoanatomical structure of laryngeal atresia with a focus on the laryngeal functional components in order to evaluate the functional prognosis of laryngeal atresia repair. METHODS: Twenty-one consecutives cases of laryngeal atresia were diagnosed at our institution between 2009 and 2016. Morphological analysis by macroscopic exam during autopsy was performed in 19 cases. Histological study of the larynx included hematoxylin and eosin staining and protein S100 immunostaining. Our analysis focused on the vocal folds, structures of the lamina propria, cricoarytenoid joints, muscles, and innervation. For each case, associated malformations were classified into two groups: severe and moderate. RESULTS: Antenatal diagnosis was suspected because of congenital high airway obstruction syndrome in nine cases (37%). Associated malformations were present in 19 cases (90%), including severe malformations in 12 cases (57%). Atresia involved the cricoid in all cases, with a residual lumen in only one case and the glottis in 18 cases. Separation between the cricoid and arytenoid cartilages was observed in all cases. Fusion of the vocal process of the arytenoids in the midline was present in 13 cases. According to the gestational age, posterior maculae flavae (MF) were present in 17 of 19 cases, with abnormal structure and median fusion in 13 cases. Anterior MF were present in nine of 18 cases, with fusion on the midline in five cases. Intrinsic abductors and adductors muscles were identifiable in all cases, with fusion of thyroarytenoids muscles in the midline in 18 cases. Both recurrent laryngeal nerves were observed in all cases. CONCLUSION: Laryngeal atresia is generally associated with other malformations, with a high risk of fatal outcomes. We observed that the functional structures of the glottic plane were present in most cases, with the exception of MF, which were frequently abnormal. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:252-256, 2020.

Heterogeneity and hierarchy of the tissue stem cells in the human newborn vocal fold mucosa.

OBJECTIVES: There is growing evidence that the cells in the maculae flavae (MFe) are tissue stem cells and the MFe are a stem cell niche of the human vocal fold mucosa. Heterogeneity and hierarchy of tissue stem cells in the MFe of newborn vocal fold were investigated in vivo. STUDY DESIGN: Histologic analysis of the human vocal folds. METHODS: Five normal human newborn vocal folds were investigated under transmission electron microscopy and light microscopy. RESULTS: Cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells were intermingled in the newborn MFe in vivo, indicating that the cells in the MFe had heterogeneity. However, cobblestone-like polygonal cells were predominant. Free ribosomes were well developed in the cytoplasm. The cells in some cases formed gap junctions with each other. The cells in some cases were attached to other cells and formed cell junctions with each other. These findings indicated cells in the newborn maculae flavae possessed features of mesenchymal cells (cells in mesenchyme). Colony-forming-unit-like cell aggregate was observed, indicating the cells in the newborn MFe had stemness. The cobblestone-like polygonal cells expressed SSEA-3 (a human pluripotent stem cell marker), indicating they were at the top of a cellular hierarchy in the stem cell system. CONCLUSIONS: The cells in the MFe of the human newborn vocal fold mucosa had heterogeneity and hierarchy in the stem cell system in vivo. At birth, newborn maculae flavae are ready to start the growth of the vocal fold mucosa as a vibrating tissue.