Expression of FOXI1 and POU2F3 varies among different salivary gland neoplasms and is higher in Warthin tumor.

PURPOSE: Salivary gland tumors are histologically diverse. Ionocytes and tuft cells, rare epithelial cells found in normal salivary glands, might be associated with salivary tumors. Here, we explored the expression of FOXI1 and POU2F3, master regulators of ionocytes and tuft cells, respectively, for common salivary neoplasms using immunohistochemistry. METHODS: We analyzed normal salivary tissues and nine salivary gland tumors; Warthin tumors (WT), pleomorphic adenomas (PA), basal cell adenomas, and oncocytomas were benign, whereas mucoepidermoid, adenoid cystic, acinic cell, salivary duct carcinomas, and polymorphous adenocarcinomas were malignant. RESULTS: Normal salivary glands contained a few FOXI1- and POU2F3-positive cells in the ducts instead of the acini, consistent with ionocytes and tuft cells, respectively. Among the benign tumors, only WTs and PAs consistently expressed FOXI1 (10/10 and 9/10, respectively). The median H-score of WTs was significantly higher than that of PAs (17.5 vs. 4, P = 0.01). While WTs and PAs harbored POU2F3-positive cells (10/10 and 9/10, respectively), the median H-score was higher in WTs than in PAs (10.5 vs 4, respectively). Furthermore, WTs exhibited a unique staining pattern of FOXI1- and POU2F3-positive cells, which were present in luminal and abluminal locations, respectively. Whereas none of the malignant tumors expressed FOXI1, only adenoid cystic carcinoma consistently expressed POU2F3 (5/5), with a median H-score of 4. CONCLUSION: The expression patterns of the characteristic transcription factors found in ionocytes and tuft cells vary among salivary gland tumor types and are higher in WT, which might be relevant for understanding and diagnosing salivary gland neoplasms.

Changes in the Proportion of Each Cell Type After hiPSC-Derived Airway Epithelia Transplantation.

No radical treatment is available for the regeneration of dysfunction and defects in airway epithelia. Artificial tracheae made of polypropylene and collagen sponge were used in clinical studies to reconstitute tracheae after resection. For early epithelialization of the luminal surface of the artificial trachea, a model was established, that is, an artificial trachea covered with human-induced pluripotent stem cell-derived airway epithelial cells (hiPSC-AECs) was transplanted into a tracheal defect in an immunodeficient rat. Unlike the cell types of hiPSC-derived cells that are currently used in clinical studies, AECs maintain tissues by proliferation and differentiation of basal cells into various cell types that constitute AECs constantly. Therefore, post-transplantation, the proportion of each cell type, such as ciliated and goblet cells, may change; however, no studies have examined this possibility. In this study, using our hiPSC-AEC-transplanted rat model, we investigated changes in the proportion of each cell type in hiPSC-AECs pre-transplantation and post-transplantation. As a result, the proportion of each cell type changed post-transplantation. The proportion of ciliated, basal, and club cells increased, and the proportion of goblet cells decreased post-transplantation. In addition, the proportion of each cell type in engrafted hiPSC-AECs is more similar to the proportion of each cell type in normal proximal airway tissue than the proportion of each cell type pre-transplantation. The results of this study are useful for the development of therapeutic techniques using hiPSC-AEC transplantation.

Comparative Study of Immunodeficient Rat Strains in Engraftment of Human-Induced Pluripotent Stem Cell-Derived Airway Epithelia.

The airway epithelia (AE) play a role in the clearance of foreign substances through ciliary motility and mucus secreted. We developed an artificial trachea that is made of collagen sponges and polypropylene mesh for the regeneration of the tracheal defect, and it was used for a clinical study. Then, a model in which the luminal surface of an artificial trachea was covered with a human-induced pluripotent stem cell-derived AE (hiPSC-AE) was transplanted into the tracheal defect of nude rats to promote epithelialization. In the future, this model was expected to be applied to research on infectious diseases and drug discovery as a trachea-humanized rat model. However, at present, sufficient engraftment has not been achieved to evaluate functional recovery in transplanted cells. Therefore, this study focused on immunosuppression in recipient rats. Nude rats lack T cell function and are widely used for transplantation experiments; however, more severe immunosuppressed recipients are preferred for xenotransplantation. Several strains of immunodeficient rats were created as rats that exhibit more severe immunodeficiency until now. In this study, to establish a trachea-humanized rat model in which human AE function can be analyzed to improve engraftment efficiency, engraftment efficiency in nude rats and X-linked severe combined immunodeficiency (X-SCID) rats following hiPSC-AE transplantation was compared. In the analysis of the proportion of engrafted cells in total cells at the graft site, the engraftment efficiency of epithelial cells tended to be high in X-SCID rats, although no statistical difference was found between the two groups, whereas the engraftment efficiency of mesenchymal cells was higher in X-SCID rats. Furthermore, the number of immune cells that accumulated in the grafts showed that a pan T cell marker, that is, CD3-positive cells, did not differ between the two strains; however, CD45-positive cells and major histocompatibility complex (MHC) class II-positive cells significantly decreased in X-SCID rats. These results indicate that X-SCID rats are more useful for the transplantation of hiPSC-AE into the tracheae to generate trachea-humanized rat models.

Laryngeal Cartilage Regeneration of Nude Rats by Transplantation of Mesenchymal Stem Cells Derived from Human-Induced Pluripotent Stem Cells.

Previous studies transplanted human-induced pluripotent stem cells (hiPSCs)-derived mesenchymal stem cells (iMSCs) into thyroid cartilage defect of X-liked severe combined immunodeficiency (X-SCID) rats and confirmed transplanted cell survival and cartilage regeneration. Thus, this study aimed to investigate the contribution of iMSC transplantation to thyroid cartilage regeneration of nude rats. iMSCs were induced from hiPSCs via a neural crest cell lineage. Then, clumps formed from an iMSC/extracellular matrix complex were transplanted into thyroid cartilage defects in nude rats. The larynx was removed and histological and immunohistochemical analyses were performed 4 or 8 weeks after the transplantation. Human nuclear antigen (HNA)-positive cells were observed in 11 of 12 (91.7%) rats, which indicated that transplanted iMSCs survived in thyroid cartilage defects in nude rats. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells in 8 of 12 rats (66.7%), which indicated cartilage-like regeneration. Cartilage-like regeneration in nude rats in this study was comparable to the previous report on X-SCID rats (HNA-positive cells were observed in all 14 rats and cartilage-like regeneration was observed in 10 of 14 rats). This result suggests that nude rats could be an alternative to X-SCID rats in thyroid cartilage regeneration experiments using iMSCs, and this nude rat cartilage transplantation model may develop cartilage regeneration research concerning fewer problems such as infection due to immunosuppression.

Peroxisome Proliferator-Activated Receptor-γ Agonist Attenuates Vocal Fold Fibrosis in Rats via Regulation of Macrophage Activation.

Macrophages aid in wound healing by changing their phenotype and can be a key driver of fibrosis. However, the contribution of macrophage phenotype to fibrosis following vocal fold injury remains unclear. Peroxisome proliferator-activated receptor-γ (PPARγ) is expressed mainly by macrophages during early wound healing and regulates the macrophage phenotype. This study aimed to evaluate the effects of pioglitazone (PIO), a PPARγ agonist, on the macrophage phenotype and fibrosis following vocal fold injury in rats. PIO was injected into the rat vocal folds on days 1, 3, 5, and 7 after injury, and the vocal fold lamina propria was evaluated on days 4 and 56 after injury. Moreover, THP-1-derived macrophages were treated with PIO, and the expression of proinflammatory cytokines under lipopolysaccharide/interferon-γ stimulation was analyzed. PIO reduced the expression of Ccl2 both in vivo and in vitro. Furthermore, PIO decreased the density of inducible nitric oxide synthase+ CD68+ macrophages and inhibited the expression of fibrosis-related factors on day 4 after injury. On day 56 after injury, PIO inhibited fibrosis, tissue contracture, and hyaluronic acid loss in a PPARγ-dependent manner. These results indicate that PPARγ activation could inhibit accumulation of inflammatory macrophages and improve tissue repair. Taken together, these findings imply that inflammatory macrophages play a key role in vocal fold fibrosis.

Transplantation of Human Induced Pluripotent Stem Cell-Derived Airway Cells on Vitrigel Membrane into Rat Nasal Cavity.

The nasal mucosa functions as a frontline biological defense against various foreign substances and pathogens. Maintaining homeostasis of the nasal epithelium is necessary to promote good health. Nasal epithelia are constantly replaced under normal conditions. However, hereditary diseases, including primary ciliary dyskinesia and cystic fibrosis, can result in intractable dysfunction of the nasal mucosa. Since there is no treatment for this underlying condition, extrinsic manipulation is necessary to recover and maintain nasal epithelia in cases of hereditary diseases. In this study, we explored the use of airway epithelial cells (AECs), including multiciliated airway cells, derived from human induced pluripotent stem cells (iPSCs) on porcine atelocollagen vitrigel membranes, as a candidate of a therapeutic method for irreversible nasal epithelial disorders. To confirm the regenerative capacity of iPSC-derived AECs, we transplanted them into nasal cavities of nude rats. Although the transplanted cells were found within cysts isolated from the recipient nasal respiratory epithelia, they survived in some rats. Furthermore, the surviving cells were composed of multiple cell types similar to the human airway epithelia. The results could contribute to the development of novel transplantation-related technologies for the treatment of severe irreversible nasal epithelial disorders. Impact Statement Nasal respiratory epithelia are important for the functions of nasal cavity, including humidifying the air and filtering various toxic substances. However, hereditary diseases, including primary ciliary dyskinesia and cystic fibrosis, can result in intractable dysfunction of the nasal mucosa. Our novel method to transplant airway epithelial cells derived from human induced pluripotent stem cells will be a candidate method to replace malfunctioned nasal respiratory epithelia in such a situation. To secure our method's safety, we used porcine atelocollagen vitrigel membranes, which prevent the immune response and bovine spongiform encephalopathy, as a scaffold.

In vivo regeneration of rat laryngeal cartilage with mesenchymal stem cells derived from human induced pluripotent stem cells via neural crest cells.

The laryngotracheal cartilage is a cardinal framework for the maintenance of the airway for breathing, which occasionally requires reconstruction. Because hyaline cartilage has a poor intrinsic regenerative ability, various regenerative approaches have been attempted to regenerate laryngotracheal cartilage. The use of autologous mesenchymal stem cells (MSCs) for cartilage regeneration has been widely investigated. However, long-term culture may limit proliferative capacity. Human-induced pluripotent stem cell-derived MSCs (iMSCs) can circumvent this problem due to their unlimited proliferative capacity. This study aimed to investigate the efficacy of iMSCs in the regeneration of thyroid cartilage in immunodeficient rats. Herein, we induced iMSCs through neural crest cell intermediates. For the relevance to prospective future clinical application, induction was conducted under xeno-free/serum-free conditions. Then, clumps fabricated from an iMSC/extracellular matrix complex (C-iMSC) were transplanted into thyroid cartilage defects in immunodeficient rats. Histological examinations revealed cartilage-like regenerated tissue and human nuclear antigen (HNA)-positive surviving transplanted cells in the regenerated lesion. HNA-positive cells co-expressed SOX9, and type II collagen was identified around HNA-positive cells. These results indicated that the transplanted C-iMSCs promoted thyroid cartilage regeneration and some of the iMSCs differentiated into chondrogenic lineage cells. Induced MSCs may be a promising candidate cell therapy for human laryngotracheal reconstruction.

A novel method for live imaging of human airway cilia using wheat germ agglutinin.

Multiciliated epithelial cells in the airway are essential for mucociliary clearance. Their function relies on coordinated, metachronal and directional ciliary beating, appropriate mucus secretion and airway surface hydration. However, current conventional methods for observing human airway ciliary movement require ciliated cells to be detached from airway tissues. Determining the directionality of cilia is difficult. We developed a novel method to stain airway epithelial cilia to observe their movement without releasing ciliated cells. Human tracheae were obtained from patients (n = 13) who underwent laryngectomies to treat malignancies or swallowing disorders. The tracheae were treated with fluorescently labeled wheat germ agglutinin, which interacts with the acidic mucopolysaccharides present on the cilia. Epithelial surfaces were observed using an epi-fluorescence microscope equipped with a water-immersion objective lens and a high-speed camera. Ciliary movement was observable at 125 fps (13/13 samples). Ciliated cells in close proximity mostly exhibited well-coordinated ciliary beats with similar directionalities. These findings indicated that wheat germ agglutinin renders ciliary beats visible, which is valuable for observing human airway ciliary movements in situ.

Recurrent laryngeal nerve regeneration using a self-assembling peptide hydrogel.

OBJECTIVES/HYPOTHESIS: To regenerate defected recurrent laryngeal nerves (RLNs), various methods have been developed. However, no consistently effective treatments are currently available because of their insufficient functional recovery. RADA16-I, a self-assembling peptide used clinically as a hemostat, reportedly supports neurite outgrowth and functional synapse formation in vitro. The purpose of this study was to investigate the effect of RADA16-I hydrogels on transected RLNs in rats. STUDY DESIGN: Animal experiments with controls. METHODS: Fifteen adult rats were divided into the following three groups: RADA16-I (+), RADA16-I (-), and neurectomy. A 6-mm gap of the left RLN was bridged using an 8-mm silicone tube in the RADA16-I (-) and RADA16-I (+) groups. Subsequently, RADA16-I hydrogel was injected into the tube in the RADA16-I (+) group. The surgical incisions were closed without any further treatment in the neurectomy group. After 8 weeks, laryngoscopy and electrophysiological and histological examinations were performed to evaluate the effect of RADA16-I on nerve regeneration and thyroarytenoid muscle atrophy. RESULTS: Although most rats in the three groups exhibited no improvements of their vocal fold movement, partial recovery was observed in one rat in the RADA16-I (+) group. The neurofilament-positive areas and the number of myelinated nerves in the RADA16-I (+) group were significantly higher than in the RADA16-I (-) group. The area of the left thyroarytenoid muscle in the RADA16-I (+) group was significantly larger than that of the neurectomy group. CONCLUSIONS: Our results suggested that RADA16-I hydrogel was effective for RLN regeneration. LEVEL OF EVIDENCE: NA Laryngoscope, 130:2420-2427, 2020.

[Prognosis of Papillary Thyroid Carcinoma with Local Invasion].

OBJECTIVE: The aim of this study was to detect prognostic factors in patients with locally advanced papillary thyroid carcinoma. PATIENTS: The study included 72 patients (T4a/T4b 72/0, N0/N1a/N1b 25/15/32, M0/M1 68/4, mean follow-up 8.1 4.4 years) who underwent initial surgical treatment at Osaka Red Cross Hospital between April 1993 and April 2011. RESULTS: Eleven patients died of PTC, 3 patients with recurrence died of unrelated disease and 10 patients are alive with recurrence. The overall 5-year survival rate was 88.3%, and the 10-year survival rate was 73.4%. The disease-specific 5-year survival rate was 91.4%, and the 10-year survival rate was 88.6%. The 5-year local control rate was 94.1%, and the 10-year local control rate was 85.4%. Patients with distant metastasis (M1), tracheal invasion and/or multiple organs invasion showed a significantly worse disease-specific survival rate based on a univariate analysis, which also revealed that tracheal invasion, laryngeal invasion, esophageal invasion and multiple organs invasion were risk factors linked to the development of distant metastasis during follow-up (recurrence as distant metastasis). The following were found to be clinically significant risk factors, based on the multivariate analysis among tracheal invasion, laryngeal invasion, esophageal invasion and recurrent laryngeal nerve invasion : Tracheal invasion was a risk factor for disease-specific survival, and tracheal invasion and laryngeal invasion were risk factors for recurrence as distant metastasis. CONCLUSIONS: In this study, distant metastasis, multiple organs invasion, tracheal invasion and/or laryngeal invasion were shown to be higher risk factors.