Vocal Fold Stellate Cells Research Articles

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

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. 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. 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. 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.

Glycolysis of Tissue Stem Cells in the Macula Flava of Newborn Vocal Fold

There is growing evidence that the cells in the maculae flavae are tissue stem cells and the maculae flavae are a stem cell niche of the human vocal fold mucosa. This study investigated the metabolic activity, especially glycolysis, of the tissue stem cells in the maculae flavae of the human newborn vocal fold. Three normal human newborn vocal folds obtained from autopsy cases were investigated using immunohistochemistry. Among the three phenotypes of cells (cobblestone-like polygonal cells, vocal fold stellate cell-like cells and fibroblast-like spindle cells) in the newborn maculae flavae, a small number of cobblestone-like polygonal cells strongly expressed glucose transporter-1. All three phenotypes of cells in the newborn maculae flavae expressed glycolytic enzymes (hexokinase II, glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase A). The cells did not express phosphofructokinase-1 (rate-limiting enzyme of regular glucose metabolism pathway) but sparsely express glucose-6-phosphate dehydrogenase (rate-limiting enzyme) indicating the cells relied more on the pentose phosphate pathway. The cells' expression of lactate dehydrogenase A suggests the maculae flavae of the newborn vocal fold is likely to be an anaerobic microenvironment where cells perform anaerobic glycolysis. The present study is consistent with the hypothesis that the tissue stem cells in the maculae flavae of the newborn vocal fold seem to rely more on anaerobic glycolysis, especially by the pentose phosphate pathway, for energy supply. Already at birth, the metabolism of the tissue stem cells in the maculae flavae of the newborn vocal fold is likely to prevent the formation of toxic reactive oxygen species and is likely favorable to maintaining the stemness and undifferentiated states of the tissue stem cells in the stem cell system.

Heterogeneity of Stem Cells in theHuman 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.

Origin of Vocal Fold Stellate Cells in the Human Macula Flava.

There is growing evidence that vocal fold stellate cells (VFSCs) in the human maculae flavae are tissue stem cells of the human vocal fold and that the maculae flavae are a stem cell niche. The origin of the cells in the human maculae flavae (CHMF) and the relationship with bone marrow-derived cells were investigated. Five human adult vocal fold mucosae were investigated. The CHMF were subcultured and morphological features were assessed. Immunoreactivity to antibodies directed to cytokeratin, desmin, GFAP, vimentin, CD34, CD45, and collagen type I was investigated. Cultured CHMF formed a colony-forming unit, indicating they are mesenchymal stem cells or stromal stem cells in the bone marrow. The CHMF expressed hematopoietic markers (CD34, CD45) and collagen type I, which are the major makers for bone marrow-derived circulating fibrocytes. The cultured CHMF expressed epithelium-associated, muscle-associated, neural-associated, and mesenchymal cell-associated proteins, indicating the CHMF are undifferentiated and express proteins of all 3 germ layers. The CHMF are undifferentiated cells derived from the differentiation of bone marrow cells. The results of this study are consistent with the hypothesis that the VFSCs are tissue stem cells or progenitor cells of the human vocal fold mucosa.

Mechanical regulation of human vocal fold stellate cells.

It is generally accepted that tensile and compressive strains have direct effects on cell morphology and structure, including changes in cytoskeletal structure and organization. Cytoskeletons play the role of mechanoreceptor of the cells. Vocal fold stellate cells (VFSCs) in the human maculae flavae (MFe) are inferred to be involved in the metabolism of extracellular matrices essential for the viscoelasticity of the vocal fold mucosa. Our previous studies have supported the hypothesis that the tension caused by phonation (vocal fold vibration) regulates the behavior of the VFSCs. The microstructure of the intermediate filaments and the expression of their proteins were investigated in VFSCs in the MFe, which had remained unphonated since birth. Three adult vocal fold mucosae that had remained unphonated since birth were investigated by immunohistochemistry and electron microscopy. The intermediate filaments of the VFSCs were fewer in number. The expression of their characteristic proteins (vimentin, desmin, and glial fibrillary acidic protein) was also reduced. Vocal fold vibration seems to affect VFSC morphology and structure, such as cytoskeletal structure and organization. This supports the hypothesis that vocal fold vibration regulates VFSC behavior in the human MFe. In addition to chemical factors, mechanical factors also appear to modulate VFSC behavior.

Cytoskeleton of newborn vocal fold stellate cells.

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. Histopathologic analysis of the human newborn vocal fold. Three newborn vocal fold mucosae were investigated by immunohistochemistry and electron microscopy. 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. 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. N/A.

Vocal Fold Stem Cells and Their Niche in the Human Vocal Fold

Vocal fold stellate cells (VFSCs) in the maculae flavae have many morphological differences from conventional fibroblasts in the human vocal fold mucosa. It is uncertain whether the VFSCs are derived from the same embryonic source as conventional fibroblasts. The purpose of this study was to investigate the stemness of the VFSCs and whether the pericellular matrices in the maculae flavae are a hyaluronan-rich matrix, which is required for a stem cell niche. Paraffin-embedded specimens were stained with Alcian blue (pH 2.5) for a hyaluronidase digestion study. Immunoreactivity to antibodies directed to CD44, CD133, Oct-4, Ki67, and telomerase was investigated in 5 human adult vocal fold mucosae. The VFSCs were resting cells (G0-phase) and expressed a mesenchymal stem cell marker. The VFSCs did not express hematopoietic or embryonic stem cell markers. Telomerase resided in the VFSCs. The hyaluronan concentration in the maculae flavae was high and the VFSCs expressed hyaluronan receptors, indicating that maculae flavae are characterized by a certain criterion of hyaluronan-rich matrix. There is growing evidence that the VFSCs in the human maculae flavae are somatic (mesenchymal) stem cells of the vocal fold, and that the maculae flavae may be a candidate for a stem cell niche that is a microenvironment nurturing a pool of VFSCs.

Vocal Fold Stellate Cells in the Human Macula Flava and the Diffuse Stellate Cell System

Hepatic stellate cells (HSCs) are desmin-positive cells with perinuclear vitamin A droplets that play important roles in liver fibrogenesis. Morphologically similar cells have been found at many extrahepatic sites. Consequently, the concept of a diffuse stellate cell system has been proposed. Vocal fold stellate cells (VFSCs) in the human maculae flavae (MFs) are starlike in shape and possess lipid droplets and store vitamin A. In this study, the relationship between the VFSCs in the human MFs and the diffuse stellate cell system was investigated. Light and electron microscopic investigations and immunohistochemical studies were performed in 5 samples of human adult vocal fold mucosa. The VFSCs showed the morphological features of the HSCs (ie, they were desmin-positive cells with perinuclear vitamin A droplets). Glial fibrillary acidic protein and vimentin were identified in the VFSCs in the MFs. The VFSCs in the human adult MFs express the neural and muscle-associated proteins seen in HSCs. Our present and previous investigations suggest that the VFSCs in the human MFs are a member of the diffuse stellate cell system. The VFSCs are considered a new category of cells in the human vocal fold. The MFs are proposed to be special microenvironments, known as niches, that nurture a pool of VFSCs.

Histopathologic study of human vocal fold mucosa unphonated over a decade

Conclusion: Mechanotransduction caused by vocal fold vibration could possibly be an important factor in the maintenance of extracellular matrices and layered structure of the human adult vocal fold mucosa as a vibrating tissue after the layered structure has been completed. Objectives: Vocal fold stellate cells (VFSCs) in the human maculae flavae of the vocal fold mucosa are inferred to be involved in the metabolism of extracellular matrices of the vocal fold mucosa. Maculae flavae are also considered to be an important structure in the growth and development of the human vocal fold mucosa. Tension caused by phonation (vocal fold vibration) is hypothesized to stimulate the VFSCs to accelerate production of extracellular matrices. A human adult vocal fold mucosa unphonated over a decade was investigated histopathologically. Methods: Vocal fold mucosa unphonated for 11 years and 2 months of a 64-year-old male with cerebral hemorrhage was investigated by light and electron microscopy. Results: The vocal fold mucosae (including maculae flavae) were atrophic. The vocal fold mucosa did not have a vocal ligament, Reinke's space or a layered structure. The lamina propria appeared as a uniform structure. Morphologically, the VFSCs synthesized fewer extracellular matrices, such as fibrous protein and glycosaminoglycan. Consequently, VFSCs appeared to decrease their level of activity.

Histopathologic Investigations of the Unphonated Human Child Vocal Fold Mucosa

Vocal fold stellate cells (VFSCs) in the maculae flavae (MFe) located at both ends of the vocal fold mucosa are inferred to be involved in the metabolism of extracellular matrices. MFe are also considered to be an important structure in the growth and development of the human vocal fold mucosa. Tension caused by phonation (vocal fold vibration) is hypothesized to stimulate VFSCs to accelerate production of extracellular matrices. Human child vocal fold mucosae unphonated since birth were investigated histologically. Histologic analysis of human child vocal fold mucosa. Vocal fold mucosae, which have remained unphonated since birth, of two children (7 and 12 years old) with cerebral palsy were investigated by light and electron microscopy and compared with normal subjects. Vocal fold mucosae and MFe were hypoplastic and rudimentary and did not have a vocal ligament, Reinke's space, or the layered structure. The lamina propria appeared as a uniform structure. Some VFSCs in the MFe showed degeneration and not many vesicles were present at the periphery of the cytoplasm. The VFSCs synthesized fewer extracellular matrices, such as fibrous protein and glycosaminoglycan. The VFSCs appeared to have decreased activity. Vocal fold vibration (phonation) after birth is an important factor in the growth and development of the human vocal fold mucosa.

Expression and Distribution of Hyaluronic Acid and CD44 in Unphonated Human Vocal Fold Mucosa

Objectives: The tension caused by phonation (vocal fold vibration) is hypothesized to stimulate vocal fold stellate cells (VFSCs) in the maculae flavae (MFe) to accelerate production of extracellular matrices. The distribution of hyaluronic acid (HA) and expression of CD44 (a cell surface receptor for HA) were examined in human vocal fold mucosae (VFMe) that had remained unphonated since birth. Methods: Five specimens of VFMe (3 adults, 2 children) that had remained unphonated since birth were investigated with Alcian blue staining, hyaluronidase digestion, and immunohistochemistry for CD44. Results: The VFMe containing MFe were hypoplastic and rudimentary. The VFMe did not have a vocal ligament, Reinke's space, or a layered structure, and the lamina propria appeared as a uniform structure. In the children, HA was distributed in the VFMe containing MFe. In the adults, HA had decreased in the VFMe containing MFe. In both groups, the VFSCs in the MFe and the fibroblasts in the lamina propria expressed little CD44. Conclusions: This study supports the hypothesis that the tensions caused by vocal fold vibration stimulate the VFSCs in the MFe to accelerate production of extracellular matrices and form the layered structure. Phonation after birth is one of the important factors in the growth and development of the human VFMe.

Functional Histology of the Macula Flava in the Human Vocal Fold – Part 2: Its Role in the Growth and Development of the Vocal Fold

Objective: This study aims to clarify the role of the maculae flavae (MFe) during growth and development of the human vocal fold mucosa (VFM). Methods: Our current results concerning the MFe in the human newborn, infant, and child VFM are summarized. Results: Newborns already had immature MFe at the same sites as adults. They were composed of dense masses of vocal fold stellate cells (VFSCs), whereas extracellular matrix components were sparse. VFSCs in the newborn MFe had already started synthesizing extracellular matrices (EM). During infancy, the EM synthesized in the MFe appeared in the VFM to initiate the formation of the three-dimensional extracellular matrix structure of the human VFM. During childhood, MFe including VFSCs continued to synthesize EM such as collagenous, reticular, and elastic fibers, and hyaluronic acid (glycosaminoglycan), which are essential for the human VFM as a vibrating tissue. The MFe in newborns, infants and children were related to the growth and development of the human VFM. Conclusion: Human MFe including VFSCs were inferred to be involved in the metabolism of EM, essential for the viscoelasticity of the human VFM, and are considered to be an important structure in the growth and development of the human VFM.

Functional Histology of the Macula Flava in the Human Vocal Fold – Part 1: Its Role in the Adult Vocal Fold

Objective: This study aims to clarify the role of the maculae flavae (MFe) in the human adult vocal fold mucosa (VFM). Methods: Our current results concerning MFe in the human adult VFM are summarized. Results: MFe were found to be composed of dense masses of vocal fold stellate cells (VFSCs) and extracellular matrices (EM), such as fibrous proteins and glycosaminoglycans, which are essential for the EM in the human VFM. VFSCs in the MFe demonstrated marked morphologic differences from conventional fibroblasts. They were irregular and stellate in shape and possessed slender cytoplasmic processes. They had well-developed intracellular organelles. A number of vesicles were present at the periphery of the cytoplasm. They constantly synthesized EM. The VFSCs possessed lipid droplets and stored vitamin A. VFSCs formed an independent cell category of cells in the human VFM. The VFSCs in aged adult MFe decreased their activity, and had abnormal metabolism. Conclusion: Human MFe including VFSCs seem to be involved in the metabolism of EM which are essential for the viscoelasticity of the lamina propria of the VFM, and to be responsible for maintaining the characteristic layered structure of the human VFM. Age-related changes in VFSCs were found to influence the metabolism of EM in the VFM.

Expression and Distribution of Hyaluronic Acid and CD44 in Unphonated Human Vocal Fold Mucosa

The tension caused by phonation (vocal fold vibration) is hypothesized to stimulate vocal fold stellate cells (VFSCs) in the maculae flavae (MFe) to accelerate production of extracellular matrices. The distribution of hyaluronic acid (HA) and expression of CD44 (a cell surface receptor for HA) were examined in human vocal fold mucosae (VFMe) that had remained unphonated since birth. Five specimens of VFMe (3 adults, 2 children) that had remained unphonated since birth were investigated with Alcian blue staining, hyaluronidase digestion, and immunohistochemistry for CD44. The VFMe containing MFe were hypoplastic and rudimentary. The VFMe did not have a vocal ligament, Reinke's space, or a layered structure, and the lamina propria appeared as a uniform structure. In the children, HA was distributed in the VFMe containing MFe. In the adults, HA had decreased in the VFMe containing MFe. In both groups, the VFSCs in the MFe and the fibroblasts in the lamina propria expressed little CD44. This study supports the hypothesis that the tensions caused by vocal fold vibration stimulate the VFSCs in the MFe to accelerate production of extracellular matrices and form the layered structure. Phonation after birth is one of the important factors in the growth and development of the human VFMe.

Stellate cells in the human child vocal fold macula flava

Vitamin A-storing vocal fold stellate cells (SCs) in the macula flava (MF) are hypothesized to be involved in the metabolism of extracellular matrices (EM) of the human vocal fold mucosa (VFM). The SCs in the child VFM were examined. Histologic analysis of cadaveric human child VFM. Light and electron microscopic investigations of SCs in the human child VFM were carried out on three excised human child larynges. The SCs were stellate and dense in the child MF. They had cytoplasmic processes, possessed lipid droplets, and stored vitamin A in their cytoplasm. The SCs had a small nucleus-cytoplasm ratio and rough endoplasmic reticula, suggesting constant protein synthesis in these cells. There were vesicles along the periphery of the cytoplasm, and newly released amorphous materials were seen on the cell surface. There were dense reticular, collagenous, and elastic fibers around SCs. The SCs exhibited strong cytoplasm staining with periodic acid-Schiff stain and type III collagen. Most of the VFSCs showed CD44 (a cell surface receptor for hyaluronic acid) expression, and a large amount of hyaluronic acid was present immediately adjacent to SCs. This study revealed several morphologic differences between the SCs in the MF and the conventional fibroblasts in the VFM. The SCs in the child MF constantly synthesized EM, essential for viscoelastic properties of the lamina propria of the human VFM, in the stage of vocal fold growth and development. The SCs in the MF were inferred to be involved in the growth and development of human VFM.

Roles of Vitamin a and Macula Flava in Maintaining Vocal Folds

Vitamin A plays important roles in development, growth, and regeneration. Vitamin A-storing stellate cells have been identified in several organs. The functional roles of vitamin A in the vocal folds are still unknown, although vitamin A-storing vocal fold stellate cells have been observed in the macula flava of human and rat vocal folds. The purpose of this study was to investigate the roles of vitamin A in vocal folds. Vitamin A-deficient rats were generated, and the vocal folds were examined histologically. Messenger RNA was extracted from the vocal folds and analyzed by real-time polymerase chain reaction. Immunohistochemical analysis of normal vocal folds revealed expression of retinoic acid receptor a in vocal fold stellate cells. The cells in the macula flava of vitamin A-deficient rats showed a larger nucleus/cytoplasm ratio than did those of vitamin A-sufficient rats, but messenger RNA expression of major extracellular matrix components in the macula flava of vitamin A-deficient rats did not present a remarkable change except for procollagen type I. Expression of hyaluronic acid, collagen types I and III, and elastin did not show a significant change in vitamin A-deficient rat vocal folds. These results indicate that vitamin A is not essential to maintaining the extracellular matrix of normal adult vocal folds, although vocal fold stellate cells participate in vitamin A storage.

Differential cell adhesion to vocal fold extracellular matrix constituents

The human vocal folds are a complex layering of cells and extracellular matrix. Vocal fold extracellular matrix uniquely contributes to the biomechanical viscoelasticity required for human phonation. We investigated the adhesion of vocal fold stellate cells, a novel cell type first cultured by our laboratory, and fibroblasts to eight vocal fold extracellular matrix components: elastin, decorin, fibronectin, hyaluronic acid, laminin and collagen types I, III and IV. Our data demonstrate that these cells adhere differentially to said substrates at 5 to 120 min. Cells were treated with hyaluronidase and Y-27632, a p160ROCK-specific inhibitor, to test the role of pericellular hyaluronan and Rho-ROCK activation in early and mature adhesion. Reduced adhesion resulted; greater inhibition of fibroblast adhesion was observed. We modulated the fibronectin affinity exhibited by both cell types using Nimesulide, an inhibitor of fibronectin integrin receptors alpha5beta1 and alphavbeta3. Our results are important in understanding vocal fold pathologies, wound healing, scarring, and in developing an accurate organotypic model of the vocal folds.

Changes in expression of extracellular matrix genes, fibrogenic factors, and actin cytoskeletal organization in retinol treated and untreated vocal fold stellate cells

The regulation of extracellular matrix (ECM) constituency is critical in maintaining vocal cord biomechanical viscoelasticity required for phonation. Recently our laboratory successfully isolated and cultured a novel cell called a vocal fold stellate cell (VFSC), thought to play a central role in laryngeal ECM metabolism, aging, scarring and cancer. Our laboratory has shown that these cells undergo transdifferentiation that is partially reversed by exposure to all-trans retinol (ATROH). Here we make the first report on the expression of various ECM components, MMPs, TIMPs, pro-fibrogenic cytokines, and other ECM modulators in transdifferentiated and deactivated VFSCs. We show that VFSCs maintain an ECM expression pattern similar to laryngeal cancer and scars but distinct from tracheal fibroblasts. Exposure to ATROH differentially affects the VFSC expression of ECM components, matrix-regulating enzymes, and fibrogenic factors suggesting that the inhibitory effects of this synthetic cofactor should be studied further in laryngeal fibrosis and scarring. We also show that increased exposure to retinol induces sequential reorganization of the actin cytoskeleton in activated VFSCs. Our findings demonstrate that VFSCs are capable of regulating vocal fold ECM constituency important throughout normal laryngeal development. Furthermore, our results implicate VFSC activation in ECM misregulation which is a hallmark of several laryngeal pathologies.

Transdifferentiation of vocal-fold stellate cells and all-trans retinol-induced deactivation

The maculae flavae of the human vocal folds include dense extracellular matrices and compacted cells with a stellate morphology. These vocal-fold stellate cells are thought to participate in the metabolism of extracellular matrices essential in maintaining vocal-fold viscoelasticity required for phonation. We have isolated and cultured these new cells and have tested the hypothesis that they maintain a distinct cellular and biochemical phenotype. We have compared proliferation rates, changes on immunophenotype, and intracellular lipid and vitamin A storage. Vocal-fold stellate cells undergo culture-induced transdifferentiation to a myofibroblast-like phenotype with an altered phenotype resembling, but not identical to, activated hepatic and pancreatic stellate cells. Our results reveal that these cells are capable of responding to exogenous all-trans retinol in culture. Exposure to this synthetic co-factor causes deactivation characterized by decreased proliferation, loss of the activated stellate cell marker, alpha-smooth muscle actin, and restoration of intracellular lipid and vitamin A metabolite storage. These data establish a new and distinct cellular target for future investigations of the viscoelastic properties of the vocal-fold mucosa during normal phonation, aging, vocal-fold scarring, laryngeal fibrosis, and myofibroblastoma.

Vitamin A-Storing Stellate Cells in the Human Newborn Vocal Fold

Vocal fold stellate cells (VFSCs) in the newborn vocal fold were examined and compared with VFSCs in the adult vocal fold. Light and electron microscopic investigation of VFSCs was carried out on 5 human newborn larynges. The VFSCs were distributed in human newborn maculae flavae. They were stellate or oval in shape, and possessed cytoplasmic processes. The VFSCs in some cells formed a gap junction with each other. A few lipid droplets were present in the cytoplasm, but they were much fewer than those of an adult. The nucleus-cytoplasm ratio was high, and the intracellular organelles were not very well developed. Free ribosomes were well developed in the cytoplasm. The VFSCs in some cells showed strong cytoplasm staining with periodic acid-Schiff stain and type III collagen. There were vesicles along the periphery of the cytoplasm of the VFSCs, and newly released amorphous materials were seen, but fewer were observed on the cell surface. The newborn VFSCs had started to synthesize extracellular matrices such as collagenous fibers, reticular fibers, elastic fibers, and glycosaminoglycan. The VFSCs in the newborn maculae flavae were immature, but had already started the synthesis of extracellular matrices essential for the viscoelastic properties of the vocal fold mucosa.