This study evaluated the current practices of oral cytology in Japan, focusing on the adoption and clinical impact of liquid-based cytology (LBC) and associated challenges. An anonymous nationwide questionnaire was conducted by the Medical Practice Committee of the Japanese Society of Clinical Oral Pathology. Responses from 28 institutions provided insights into cytology implementation, tools used, Specimen preparation method, and genomic testing. LBC was adopted by 78.6 % of facilities, citing benefits such as improved sample evaluation, cleaner background, and genomic applicability. Seven institutions increased usage post-LBC introduction. However, issues like limited samples, costs, and lack of insurance coverage hinder broader use. Genetic testing was performed in 15 institutions, particularly for salivary gland tumors, though financial and procedural barriers remain. Findings highlight the need for improved reimbursement, national standardization, and education. LBC offers clear diagnostic value and should be integrated into standard oral pathology practices.

Embryonic oral epithelium is a multipotent, ectodermal tissue that gives rise to various organs, including salivary glands, tooth germs, taste buds, and anterior pituitary glands. Although oral epithelium is contiguous posteriorly with endodermal epithelium and anteriorly with surface ectoderm, it exhibits distinct gene expression profiles during development. However, the molecular mechanisms that govern fate specification between oral epithelium and surface ectoderm remain poorly understood. Here, we present a highly efficient protocol for the PITX2-positive oral epithelium induction from human induced pluripotent stem cells (hiPSCs) using a reporter system. Sonic Hedgehog (SHH) signaling activation is essential for the efficient PITX2-positive epithelial cell generation. The induced cells exhibited gene expression profiles resembling those of embryonic oral epithelium and formed epithelial spheres that replicated features of human oral epithelium. This study established a robust platform for investigating human oral epithelial development and provided a valuable foundation for organoid-based research on oral organs.

OBJECTIVES:The junctional epithelium (JE) is a specialized barrier that maintains periodontal tissue homeostasis. Although age-related changes have been reported in other epithelial tissues, the mechanisms underlying age-associated dysfunction in the JE remain largely unknown. In this study, the aims were to determine whether there is cellular senescence in the JE during natural aging, and to elucidate the underlying molecular mechanisms. METHODS:JE was harvested from the maxillae of young (eight-week-old) and aged (72-week-old) male mice, and tissue sections were prepared. To evaluate age-related changes, histological analyses, including hematoxylin-eosin (HE) staining, immunofluorescence staining, and TUNEL assays, as well as bulk RNA sequencing (RNA-seq) for gene expression profiling, were performed. Differentially expressed genes (DEGs) were identified from the RNA-seq data, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. RESULTS:Histological examination revealed no overt morphological differences in the JE between young and aged mice. However, the JE from aged mice had significantly fewer Ki67-positive cells and more TUNEL-positive apoptotic cells. RNA-seq identified DEGs in the JE of aged mice. GO and KEGG analyses indicated significant enrichment of inflammation- and aging-related pathways. RT-qPCR confirmed the increased expression of p16, Lcn2, and Defb3 in the JE of aged mice. Immunostaining also revealed increased numbers of γ-H2AX, p-STAT3, 8-OHdG, and 4-HNE positive cells in the JE of aged mice. CONCLUSIONS:The JE of aged mice had molecular hallmarks of cellular senescence. These findings highlight the role of senescence in the aging of periodontal barrier tissue.

Objectives The junctional epithelium (JE) plays an important role in maintaining the protective integrity of periodontal tissues by forming an epithelial barrier that impedes bacterial invasion. This study examined the effects of Porphyromonas gingivalis lipopolysaccharide (PG-LPS) on JE-1 cells, a mouse-derived junctional epithelial cell line. Methods JE-1 cells were exposed to PG-LPS, and the effects on cell viability, migration, gene expression, protein expression, and barrier function at various time points were evaluated according to the experimental type. Results PG-LPS (1 μg/mL) did not affect JE-1 cell viability but significantly inhibited migration. Treatment with PG-LPS upregulated inflammatory genes (Il-6, Tlr2, Tlr4, Traf6) and altered the expression of chemokines (increased Cxcl2 and decreased Cxcl10) and protective factors (increased Nfe2l2 and Slpi). Notably, 1 μg/mL of PG-LPS increased permeability and decreased adhesion molecule expression (Cdh1 and Itgb4), whereas 10 μg/mL showed a non-linear response (apparent permeability [cm/s]: 0.868, 0.915, 1.416, 1.728, 1.224, and 1.176 with 0, 0.1, 0.5, 1, 5, and 10 μg/mL respectively), indicating compensatory mechanisms. Conclusions PG-LPS disrupts JE barrier function and alters its immunomodulatory properties, potentially contributing to periodontitis progression. These findings enhance our understanding of the underlying causes of periodontitis and inform targeted treatments to maintain JE defense against periodontopathogenic bacteria.

Functional integration of transplanted cells with host tissue remains a major challenge in cell-based therapies for tissue damage in organs with complex structures, such as exocrine glands. In this study, we investigated whether salivary gland organoids derived from human induced pluripotent stem cells (hiPSCs) could be integrated into injured salivary glands using cell sheet engineering. Cell sheet engineering has demonstrated therapeutic potential in a range of organs, including the heart, retina, and lungs. We found that hiPSC-derived salivary gland organoids contain long-term maintainable progenitor cells, and the resulting cell sheets exhibited heterogeneity, including acinar, ductal, and myoepithelial cells. Furthermore, transplantation of the organoid-derived salivary gland cell sheets into immunodeficient mice resulted in partial integration with the host salivary ducts, leading to the formation of structures that included xenogeneic chimeric ducts. These findings suggest that salivary gland cell sheet transplantation represents a promising strategy for functional salivary gland regeneration.

Sjögren's syndrome (SS) is an autoimmune disorder characterized by oral dryness that is primarily attributed to tumor necrosis factor alpha (TNF-α)-mediated reduction in saliva production. In traditional Chinese medicine, goji berries are recognized for their hydrating effect and are considered suitable to address oral dryness associated with Yin deficiency. In the present study, we used goji berry juice (GBJ) to investigate the potential preventive effect of goji berries on oral dryness caused by SS. Pretreatment of human salivary gland cells with GBJ effectively prevented the decrease in aquaporin-5 (AQP-5) mRNA and protein levels induced by TNF-α. GBJ also inhibited histone H4 deacetylation and suppressed the generation of intracellular reactive oxygen species (ROS). Furthermore, GBJ pretreatment reserved mitochondrial membrane potential and suppressed the upregulation of Bax and caspase-3, indicating that GBJ exerted an antiapoptotic effect. These findings suggest that GBJ provides protection against TNF-α in human salivary gland cells and prevents the reduction of AQP-5 expression on the cell membrane. Altogether, these results highlight the potential role of GBJ in preventing oral dryness caused by SS.

In the animal kingdom, evolutionarily conserved mechanisms known as cell competition eliminate unfit cells during development. Interestingly, cell competition also leads to apoptosis of donor cells upon direct contact with host cells from a different species during interspecies chimera formation. The mechanisms underlying how host animal cells recognize and transmit cell death signals to adjacent xenogeneic human cells remain incompletely understood. In this study, we developed an interspecies cell contact reporter system to dissect the mechanisms underlying competitive interactions between mouse and human pluripotent stem cells (PSCs). Through single-cell RNA-seq analyses, we discovered that Ephrin A ligands in mouse cells play a crucial role in signaling cell death to adjacent human cells that express EPHA receptors during interspecies PSC co-culture. We also demonstrated that blocking the Ephrin A-EPHA receptor interaction pharmacologically, and inhibiting Ephrin forward signaling genetically in the mouse cells, enhances the survival of human PSCs and promotes chimera formation both in vitro and in vivo . Our findings elucidate key mechanisms of interspecies PSC competition during early embryogenesis and open new avenues for generating humanized tissues or organs in animals, potentially revolutionizing regenerative medicine.

Objectives Details about salivary gland tumor histogenesis remain unknown. Here, we established a newly generated murine salivary gland tumor model that could overexpress pleomorphic adenoma gene 1 (PLAG1) and attempted to clarify the events that occur during the early phase of salivary gland tumor histogenesis. Methods Salivary gland tumors were generated using murine models (Sox9IRES-CreERT2; ROSA26-PLAG1). Lineage tracing of Sox9-expressing cells was performed using Sox9IRES-CreERT2; ROSA26-tdTomato mice, which were generated by crossing Sox9CreERT2/- and ROSA26-tdTomato mice (expressing the tdTomato fluorescent protein). Organ-cultured embryonic salivary glands from the murine model were morphologically analyzed, and mRNA sequencing was conducted two days after tumor induction for gene enrichment and functional annotation analysis. Results Salivary gland tumors exhibited epithelial features with acinar-like structures because of gene rearrangements in the luminal cells. Structural disturbances in the duct-acinar unit of the salivary gland were observed and cancer-related pathways were enriched among the differentially upregulated genes in the early phase of tumor induction in an organ-cultured embryonic salivary gland tumor model. Conclusions The newly generated murine salivary gland tumor model may show that the tumorization of luminal stem/progenitor cells can result in the development of salivary gland tumors comprising only luminal cells.

Mesothelial cells, in the outermost layer of internal organs, are essential for both organ development and homeostasis. Although the parietal mesothelial cell is the primary origin of mesothelioma that may highjack developmental signaling, the signaling pathways that orchestrate developing parietal mesothelial progenitor cell (MPC) behaviors, such as MPC pool expansion, maturation, and differentiation, are poorly understood. To address it, we established a robust protocol for culturing WT1+ MPCs isolated from developing pig and mouse parietal thorax. Quantitative qPCR and immunostaining analyses revealed that BMP4 facilitated MPC differentiation into smooth muscle cells (SMCs). In contrast, FGF2 significantly promoted MPC progenitor pool expansion but blocked the SMC differentiation. BMP4 and FGF2 counterbalanced these effects, but FGF2 had the dominant impact in the long-term culture. A Wnt activator, CHIR99021, was pivotal in MPC maturation to CALB2+ mesothelial cells, while BMP4 or FGF2 was limited. Our results demonstrated central pathways critical for mesothelial cell behaviors.

Objectives This study aimed to investigate the regulatory mechanisms governing dental mesenchymal cell commitment during tooth development, focusing on odontoblast differentiation and the role of epigenetic regulation in this process. Methods We performed single-cell RNA sequencing (scRNA-seq) of dental cells from embryonic day 14.5 (E14.5) mice to understand the heterogeneity of developing tooth germ cells. Computational analyses including gene regulatory network (GRN) assessment were conducted.We validated our findings using immunohistochemistry (IHC) and in vitro loss-of-function analyses using the DNA methyltransferase 1 (DNMT1) inhibitor Gsk-3484862 in primary dental mesenchymal cells (DMCs) isolated from E14.5 mouse tooth germs. Bulk RNA-seq of Gsk-3484862-treated DMCs was performed to identify potential downstream targets of DNMT1. Results scRNA-seq analysis revealed diverse cell populations within the tooth germs, including epithelial, mesenchymal, immune, and muscle cells. Using single-cell regulatory network inference and clustering (SCENIC), we identified Dnmt1 as a key regulator of early odontoblast development. IHC analysis showed the ubiquitous expression of DNMT1 in the dental papilla and epithelium. Bulk RNA-seq of cultured DMCs showed that Gsk-3484862 treatment upregulated odontoblast-related genes, whereas genes associated with cell division and the cell cycle were downregulated. Integrated analysis of bulk RNA-seq data with scRNA-seq SCENIC profiles was used to identify the potential Dnmt1 target genes. Conclusions Dnmt1 may negatively affect odontoblast commitment and differentiation during tooth development. These findings contribute to a better understanding of the molecular mechanisms underlying tooth development and future development of hard-tissue regenerative therapies.

Whole salivary gland generation and transplantation offer potential therapies for salivary gland dysfunction. However, the specific lineage required to engineer complete salivary glands has remained elusive. In this study, we identify the Foxa2 lineage as a critical lineage for salivary gland development through conditional blastocyst complementation (CBC). Foxa2 lineage marking begins at the boundary between the endodermal and ectodermal regions of the oral epithelium before the formation of the primordial salivary gland, thereby labeling the entire gland. Ablation of Fgfr2 within the Foxa2 lineage in mice leads to salivary gland agenesis. We reversed this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts, resulting in mice that survived to adulthood with salivary glands of normal size, comparable to those of their littermate controls. These findings demonstrate that CBC-based salivary gland regeneration serves as a foundational experimental approach for future advanced cell-based therapies.

The number of neurons in the brain increases over time, even in adults. This phenomenon, known as “neurogenesis”, is associated with neural stem cells present only in the dentate gyrus of the hippocampus. However, the frequency of neurogenesis is regulated by various factorsand mastication has been suggested as one such factor. In the present study, we examined the effects of varying the input of masticatory stimulation on neural stem cells using feed with different degrees of hardness. Male C57BL/5 mice were used in a comparative study. After weaning at 3weeks old, experimental mice were fed a soft or hard diet for 4 or 11weeks. To study the dynamic behavior of neural stem cells, the expression of two markers （nestin, a neural stem/progenitor cell marker; and doublecortin, a microtubule-associated protein） was quantitatively analyzed via immunostaining, whereas gene expression was analyzed by real-time polymerase chain reaction （RT-PCR）. Immunostaining-based quantification and gene expression analysis by RT-PCR revealed the decreased expression of markers in the hippocampus in the soft diet group compared to that in the hard diet group. Based on these results, the repression of neurogenesis by a soft diet was associated with changes in neural stem cells in the hippocampus and the frequency of neurogenesis.

31 Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ 32 transplants. Identifying the crucial lineage and the program for lung organogenesis could facilitate 33 designing whole-lung bioengineering . Using lineage-tracing mice and human iPSC-derived lung- 34 directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung 35 mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung 36 mesenchyme progressively increased and occupied more than half of the mesenchyme niche, 37 including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 38 gene depletion caused the lung agenesis phenotype in mice. Importantly, wild-type donor mouse 39 iPSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective 40 niche in the lung epithelium and mesenchyme. Donor cell is shown to replace the entire lung 41 epithelial and robust mesenchymal niche during early chimeric lung development, resulting in 42 efficient complementation of the nearly entire lung niche at the late stage of lung development. 43 These results suggest that lung complementation based on the Foxa2 lineage is a unique model for 44

Objectives: Aging-related salivary gland changes, such as lymphocyte infiltration and acinar cell loss decrease saliva secretion, thereby affecting quality of life. The precise molecular mechanisms underlying these changes remain unclear. Methods: We here performed single-cell RNA sequencing to clarify gene expression changes in each cell type comprising the submandibular glands (SMGs) of adult and aged mice. Results: The proportion of acinar cells decreased in various epithelial clusters annotated with cell type-specific marker genes. Expression levels of the cellular senescence markers, Cdkn2a/p16 and Cdkn1a/p21, were increased in the basal and striated ducts of aged SMGs relative to their levels in those of adult SMGs. In contrast, senescence-associated secretory phenotype-related genes, except transforming growth factor-beta, exhibited little change in expression in aged SMGs relative to adult SMGs. Conclusions: Gene Ontology analysis revealed increased expression levels of genes encoding major histocompatibility complex (MHC) class I components in the ductal component cells of aged SMGs. MHC class I expression may thus be associated with salivary gland aging.

Research regarding the process of salivary gland development and elucidation of related mechanisms are considered essential for development of effective treatments for conditions associated with salivary disease. Various reports regarding the effects of bone morphogenetic protein (BMP)-2 on hard tissue cells have been presented, though few have examined those related to salivary gland formation. Using an organ culture system, the present study was conducted to investigate the function of BMP-2 in salivary gland formation. Salivary glands obtained from embryonic day 13.5 mice and treated with BMP-2 showed suppression of primordial cell differentiation and also gland formation in a concentration-dependent manner. Furthermore, gland formation inhibition was suppressed by concurrent treatment with dorsomorphin, an inhibitor of the Smad pathway. Expression levels of AQP5, a marker gene for acinar cells, and Prol1, an opiorphin expressed in the lacrimal gland, were decreased in salivary glands treated with BMP-2. The present findings indicate that suppression of salivary gland formation, especially acinar differentiation, is induced by BMP-2, a phenomenon considered to be related to the Smad pathway.

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Generating the whole organ in conjunction with the thymus is a significant milestone for organ transplantation because the thymus is the central organ to educate immune cells. Using lineage-tracing mice and human pluripotent stem cell (PSC)-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung and thymus agenesis phenotype in mice. Wild-type donor mouse PSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme and thymic epithelium. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during lung development, efficiently complementing the nearly entire lung niche. Importantly, those mice survived until adulthood with normal lung function. These results suggest that our Foxa2 lineage-based model is unique for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and thymus generation, which can provide critical insights into studying lung transplantation post-transplantation shortly.

Various patients suffer from dry mouth due to salivary gland dysfunction. Whole salivary gland generation and transplantation is a potential therapy to resolve this issue. However, the lineage permissible to design the entire salivary gland generation has been enigmatic. Here, we discovered Foxa2 as a lineage critical for generating a salivary gland via conditional blastocyst complementation (CBC). Foxa2 linage, but not Shh nor Pitx2, initiated to label between the boundary region of the endodermal and the ectodermal oral mucosa before primordial salivary gland formation, resulting in marking the entire salivary gland. The salivary gland was agenesis by depleting Fgfr2 under the Foxa2 lineage in the mice. We rescued this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts. Those mice survived until adulthood with normal salivary glands compatible in size compared with littermate controls. These results indicated that CBC-based salivary gland generation is promising for next-generation cell-based therapy.

Millions of people suffer from end-stage refractory diseases. The ideal treatment option for terminally ill patients is organ transplantation. However, donor organs are in absolute shortage, and sadly, most patients die while waiting for a donor organ. To date, no technology has achieved long-term sustainable patient-derived organ generation. In this regard, emerging technologies of chimeric human organ production via blastocyst complementation (BC) holds great promise. To take human organ generation via BC and transplantation to the next step, we reviewed current emerging organ generation technologies and the associated efficiency of chimera formation in human cells from the standpoint of developmental biology.

Objectives: Tissue differentiation is regulated by transcription factors. This study aimed to identify candidate transcription factors that induce periodontal ligament (PDL) cell differentiation in human pluripotent stem cells (hPSCs). Methods: Human PDL tissues were scraped from the root surfaces of extracted teeth for orthodontic treatment and cultured using the explant culture method. We used RNA-seq to generate gene expression profiles of third-passage PDL cells and compared them with those of undifferentiated human induced pluripotent stem cells (hiPSCs) and human embryonic stem cell (hESC)-derived neural crest (NC) cells (publicly available data). Results: Primary cultured PDL cells exhibited a spindle-shaped fibroblast-like appearance and the gene expression of several PDL cell-specific markers. The gene expression profiles of PDL cells were relatively similar to those of hESC-derived NC cells but not those of undifferentiated hiPSCs. Thirty-seven tran-scription factors were identified as upregulated genes in PDL cells. Pathway analysis showed that differentially expressed genes were enriched in several functional groups and pathways, including the SMAD 2/3 nuclear pathway. Conclusions: We identified 37 upregulated transcription genes in primary cultured PDL cells compared with hESC-derived NC cells. Regulating these genes and the SMAD signaling pathway may be promising ways to induce PDL cells from hPSC-derived NC cells.(c) 2023 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.