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Schematic strategy of the epithelial cell culture derived from human gingival tissue. The detailed scheme of the selective cell culture process was described in Materials and Methods.
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Dental enamel is the highly mineralized tissue covering the tooth surface and is formed by ameloblasts. Ameloblasts have been known to be impossible to detect in adult tooth because they are shed by apoptosis during enamel maturation and tooth eruption. Owing to these, little was known about appropriate cell surface markers to isolate ameloblast-li...
Citations
... 21,22 GO analysis and GSVA showed that bone remodeling-associated signatures and cell polarity-related pathways were enriched in c1 cells, indicating the interaction between c1 cells and bone remodeling (Fig. S3A, B). Cells in c2 expressed high levels of KRT6A, ODAM, LAMA3, LAMB3, and LAMC2, representing an ameloblast-like epithelial cell signature 23,24 with partial epithelial-mesenchymal transition (pEMT) characteristics 11,25 that might be involved in cell-cell adhesion and tooth mineralization and implying cytoskeletal organization and cell migration (Fig. S3A, B). Cells in c3 expressed S100A8, SPRR3, S100A9, KRT13, and S100A7, which are markers for differentiated epithelial cells involved in epithelial junctions (Fig. S3A, B). ...
Ameloblastoma is a benign tumor characterized by locally invasive phenotypes, leading to facial bone destruction and a high recurrence rate. However, the mechanisms governing tumor initiation and recurrence are poorly understood. Here, we uncovered cellular landscapes and mechanisms that underlie tumor recurrence in ameloblastoma at single-cell resolution. Our results revealed that ameloblastoma exhibits five tumor subpopulations varying with respect to immune response (IR), bone remodeling (BR), tooth development (TD), epithelial development (ED), and cell cycle (CC) signatures. Of note, we found that CC ameloblastoma cells were endowed with stemness and contributed to tumor recurrence, which was dominated by the EZH2-mediated program. Targeting EZH2 effectively eliminated CC ameloblastoma cells and inhibited tumor growth in ameloblastoma patient-derived organoids. These data described the tumor subpopulation and clarified the identity, function, and regulatory mechanism of CC ameloblastoma cells, providing a potential therapeutic target for ameloblastoma.
... Dental papilla cells further differentiate into odontoblasts and concurrently, enamel organ cells differentiate into ameloblasts at the late bell stage when dental hard tissue formation begins. However, despite the importance of this stage, molecular mechanisms, particularly for terminal cytodifferentiation and morphodifferentiation of the inner enamel epithelium in the enamel organ into functionally matured ameloblastic cells, have been largely unknown (Hyun et al. 2019). Furthermore, research has primarily focused on the cytodifferentiation of odontoblasts, which are relatively easy to obtain and necessary for dentin regeneration (Lesot 2000;Janebodin et al. 2021;Chen et al. 2022), ...
Cytodifferentiation of odontogenic cells, a late stage event in odontogenesis is based on gene regulation. However, studies on the identification of the involved genes are scarce. The present study aimed to search for molecules for the cytodifferentiation of ameloblastic cells in rats. Differential display-PCR revealed a differentially expressed gene between cap/early bell stage and hard tissue formation stage in molars. This gene was identified as N-myc Downregulated Gene 1 (Ndrg1), which is the first report in tooth development. Real time PCR and western blotting confirmed that the mRNA level of Ndrg1 was higher during enamel formation than the cap stage. Ndrg1 expression was upregulated in the early bell, crown, and root stages in a time-dependent manner. These patterns of expression were similar in Ndrg2, but Ndrg3 and Ndrg4 levels did not change during the developmental stages. Immunofluorescence revealed that strong immunoreactivity against Ndrg1 were detected in differentiated ameloblasts only, not inner enamel epithelium, odontoblasts and ameloblastic cells in defected enamel regions. Alkaline phosphatase and alizarin red s stains along with real time PCR, revealed that Ndrg1 and Ndrg2 were involved in cytodifferentiation and enamel matrix mineralization by selectively regulating amelogenin and ameloblastin genes in SF2 ameloblastic cells. These results suggest that Ndrg may play a crucial functional role in the cytodifferentiation of ameloblasts for amelogenesis.
... Similar to the respiratory and intestinal epithelium (28), our data show robust expression of IFN-λ transcripts (SI Appendix, Fig. S1A) as well as IFNL-R (Fig. 1A) toward the apical side of the tissue, largely localized within the epithelial cell adhesion molecule (EpCAM)-positive region. GECs, unlike gingival fibroblasts, specifically and strongly express EpCAM (29). To determine responsiveness of viral pathogen-associated molecular patterns (PAMPs), GECs were isolated from human gingival tissues and challenged in vitro with poly I:C, a synthetic doublestranded RNA analog and TLR3 agonist, as well a 60 base pair oligonucleotide sequence derived from HSV-1 (HSV60) recognized by cytosolic DNA sensors. ...
Here, we show that Porphyromonas gingivalis ( Pg ), an endogenous oral pathogen, dampens all aspects of interferon (IFN) signaling in a manner that is strikingly similar to IFN suppression employed by multiple viral pathogens. Pg suppressed IFN production by down-regulating several IFN regulatory factors (IRFs 1, 3, 7, and 9), proteolytically degrading STAT1 and suppressing the nuclear translocation of the ISGF3 complex, resulting in profound and systemic repression of multiple interferon-stimulated genes. Pg -induced IFN paralysis was not limited to murine models but was also observed in the oral tissues of human periodontal disease patients, where overabundance of Pg correlated with suppressed IFN generation. Mechanistically, multiple virulence factors and secreted proteases produced by Pg transcriptionally suppressed IFN promoters and also cleaved IFN receptors, making cells refractory to exogenous IFN and inducing a state of broad IFN paralysis. Thus, our data show a bacterial pathogen with equivalence to viruses in the down-regulation of host IFN signaling.
... 13,54 Reduced enamel epithelium is usually lost after tooth eruption, either by its transformation in the junctional epithelium or apoptosis. 55 If the tooth does not erupt, the reduced enamel epithelium remains attached to the tooth surface and the connective tissue of the "dental sac" (here referred to as "dental follicle"). 56 The maintenance of such tissue in association to the tooth crown was described by Consolaro and Cardoso as "pericoronal tissue maintenance would protect the enamel from clastic cells activity, avoiding bone formation directly on enamel surface; acting as a barrier to the internal environment of the body during tooth eruption; and affording protection to this internal compartment from the highly contaminated environment of oral cavity." ...
Dental follicles are involved in odontogenesis, periodontogenesis, and tooth eruption. Dental follicles are unique structures, considering that their remnants can persist within the jawbones after odontogenesis throughout life if the tooth does not erupt. Pathological changes may occur in these tissues as individuals age. The changes range from benign to life threatening. Thus, the assessment of age‐related changes in dental follicles associated with unerupted teeth is of paramount importance. In this review, we summarize the physiological roles and changes in dental follicles in odontogenesis, tooth eruption, and aging, in addition to the pathological changes associated with these structures. We encourage investigators to consider this peculiar tissue as a unique model and explore its potential to clarify its importance from the viewpoints of developmental biology, tissue physiology, and pathology.
... In evaluating the relationship between IGFBP5-7 and ameloblastic differentiation of SF2 rat dental epithelial cells, IGFBP5-7 expression levels were upregulated as shown in Fig. 5. During the induced mineralization, Amelx (a representative gene for amelogenesis), OPN (a highly expressed gene in ameloblast-like cells) (Hyun et al. 2019), and ALP levels were upregulated. In contrast, IGFBP5 was unexpectedly unchanged. ...
Much information is currently available for molecules in early odontogenesis, but there is limited knowledge regarding terminal cytodifferentiation of ameloblasts and odontoblasts for the determination of normal crown morphology. The present differential display PCR (DD-PCR) revealed that insulin-like growth factor-binding protein 5 (IGFBP5) was differentially expressed in molar tooth germs between the cap (before crown mineralization) and root formation (after crown mineralization) stages. Real-time PCR confirmed that the expression levels of IGFBP1–4 were not significantly changed but those of IGFBP5–7 were upregulated in a time-dependent manner. Immunoreactivities for IGFBP5–7 were hardly seen in molar germs at the cap/early bell stage and protective-stage ameloblasts at the root formation stage. However, the reactivity was strong in odontoblasts and maturation-stage ameloblasts, which are morphologically and functionally characterized by wide intercellular space and active enamel matrix mineralization. The localization of each IGFBP was temporospatial. IGFBP5 was localized in the nuclei of fully differentiated odontoblasts and ameloblasts, while IGFBP6 was localized in the apical cytoplasm of ameloblasts and odontoblasts with dentinal tubules, and IGFBP7 was mainly found in the whole cytoplasm of odontoblasts and the intercellular space of ameloblasts. IGFBP silencing using specific siRNAs upregulated representative genes for dentinogenesis and amelogenesis, such as DMP1 and amelogenin, respectively, and augmented the differentiation media-induced mineralization, which was confirmed by alizarin red s and alkaline phosphatase staining. These results suggest that IGFBP5–7 may play independent and redundant regulatory roles in late-stage odontogenesis by modulating the functional differentiation of ameloblasts and odontoblasts.
... The generation of ameloblasts is still an obstacle. Although several mouse ameloblast-like cell lines, such as ALC and LS8, have been established, they do not generate enamel [98,99] . Human gingival epithelial cells have been a source of ameloblast-like cells induced by BMPs and TGF-β. ...
Tooth enamel, a highly mineralized tissue covering the outermost area of teeth, is always damaged by dental caries or trauma. Tooth enamel rarely repairs or renews itself, due to the loss of ameloblasts and dental epithelial stem cells (DESCs) once the tooth erupts. Unlike human teeth, mouse incisors grow continuously due to the presence of DESCs that generate enamel-producing ameloblasts and other supporting dental epithelial lineages. The ready accessibility of mouse DESCs and wide availability of related transgenic mouse lines make mouse incisors an excellent model to examine the identity and heterogeneity of dental epithelial stem/progenitor cells; explore the regulatory mechanisms underlying enamel formation; and help answer the open question regarding the therapeutic development of enamel engineering. In the present review, we update the current understanding about the identification of DESCs in mouse incisors and summarize the regulatory mechanisms of enamel formation driven by DESCs. The roles of DESCs during homeostasis and repair are also discussed, which should improve our knowledge regarding enamel tissue engineering.
... After preparation, separated tissue was minced using surgical blades. In order to isolate fibroblasts, digestion in a collagenase solution was used [23,24]. Therefore, fragments of tissue (approximately 0.5 mm 3 ) were incubated with 0.05% collagenase I (Sigma Aldrich, Madison, WI, USA) for 40 min at 37 • C in a shaking water bath. ...
The mechanisms of wound healing and vascularization are crucial steps of the complex morphological process of tissue reconstruction. In addition to epithelial cells, fibroblasts play an important role in this process. They are characterized by dynamic proliferation and they form the stroma for epithelial cells. In this study, we have used primary cultures of oral fibroblasts, obtained from porcine buccal mucosa. Cells were maintained long-term in in vitro conditions, in order to investigate the expression profile of the molecular markers involved in wound healing and vascularization. Based on the Affymetrix assays, we have observed three ontological groups of markers as wound healing group, response to wounding group and vascularization group, represented by different genes characterized by their expression profile during long-term primary in vitro culture (IVC) of porcine oral fibroblasts. Following the analysis of gene expression in three previously identified groups of genes, we have identified that transforming growth factor beta 1 (TGFB1), ITGB3, PDPN, and ETS1 are involved in all three processes, suggesting that these genes could be recognized as markers of repair specific for oral fibroblasts within the porcine mucosal tissue.
... However, regeneration of tooth enamel has been a major challenge, even its reshaping or endogenous regeneration is not feasible, as it is formed before tooth eruption (Huang, 2011). However, mechanisms have been seeked to obtain it at a stage after the dentinogenic process, using human fetuses and rat models (Morotomi et al., 2005;Yan et al., 2006) and it has been reported that the only producing sources would be those derived of the same embryonic origin as Hertwing's epithelial sheath and Malassez's epithelial cells and from alternative sources such as bone marrow, embryonic cells, induced pluripotent stem cells (IPSCs) and skin epithelial, which require differentiation by stimuli with growth factors, cytokines (TGF, FGF, Wnts and BMP) and extracellular matrix compounds generated by the epithelium and the mesenchyme (Hyun et al., 2019). In the present work and using tissue engineering techniques, the pluripotential capacity of hDPSCs to express tooth enamel proteins that participate in amelogenesis was demonstrated (Campos-Navarro et al.) as amelogenin, ameloblastine, tuftelin, enamelisin and parvalbumin by induction generated by natural scaffolds made from pigskin glue and rat tail tendon (Maurer et al., 2018;Davison-Kotler et al., 2019), which according to the results reported by Yang & Shu, its composition is 90% to 95% of type I collagen. ...
Mesenchymal stem cells are present in adult tissues such as the human dental pulp. They are pluripotent and can differentiate into various specialized cell types in vitro through appropriate stimuli. Ameloblasts produce human tooth enamel only during embryonic development before tooth eruption, so endogenous regeneration is not possible. Various efforts have been aimed at generating natural or artificial substitutes for dental enamel with properties similar to the specific components of said tissue. The purpose of this study was to induce human dental pulp stem cells to produce enamel proteins using extracellular matrix derived from the rat tail tendon and pigskin. Primary cultures of human dental pulp stem cells were established and characterized by RT-PCR and immunofluorescence, using mesenchymal cell markers such as CD14, CD40, CD44, CD105, and STRO-1. The cells were then incubated with the extracellular matrix for fourteen days and labeled with specific antibodies to detect the expression of dental enamel proteins such as amelogenin, ameloblastin, enamelisin, tuftelin, and parvalbumin, characteristics of the phenotype of ameloblasts. This work demonstrated a positive effect of the extracellular matrix to induce the expression of enamel proteins in the stem cells of the human dental pulp.
... The immortalized HERS-SV40 and primary HERS/ERM showed the same morphological characteristics, immunophenotypes, and gene expression profiles [9]. However, these cell lines are established from primary HERS/ERM cells, which are generally not easy to obtain from teeth extracted from adults [33]. ...
Tooth development and regeneration occur through reciprocal interactions between epithelial and ectodermal mesenchymal stem cells. However, the current studies on tooth development are limited, since epithelial stem cells are relatively difficult to obtain and maintain. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be alternative options for epithelial cell sources. To differentiate hESCs/hiPSCs into dental epithelial-like stem cells, this study investigated the hypothesis that direct interactions between pluripotent stem cells, such as hESCs or hiPSCs, and Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cell line may induce epithelial differentiation. Epithelial-like stem cells derived from hES (EPI-ES) and hiPSC (EPI-iPSC) had morphological and immunophenotypic characteristics of HERS/ERM cells, as well as similar gene expression. To overcome a rare population and insufficient expansion of primary cells, EPI-iPSC was immortalized with the SV40 large T antigen. The immortalized EPI-iPSC cell line had a normal karyotype, and a short tandem repeat (STR) analysis verified that it was derived from hiPSCs. The EPI-iPSC cell line co-cultured with dental pulp stem cells displayed increased amelogenic and odontogenic gene expression, exhibited higher dentin sialoprotein (DSPP) protein expression, and promoted mineralized nodule formation. These results indicated that the direct co-culture of hESCs/hiPSCs with HERS/ERM successfully established dental epithelial-like stem cells. Moreover, this differentiation protocol could help with understanding the functional roles of cell-to-cell communication and tissue engineering of teeth.
... Although EMT plays major roles in tumor invasion and metastasis, it also occurs during the process of odontogenesis. EMT has an important role in differentiation of enamel-forming ameloblast [23]. Studies have shown that epithelial cells differentiate into hard tissues such as cementum in the presence of TGF-β-1 during EMT [23][24][25][26]. ...
... EMT has an important role in differentiation of enamel-forming ameloblast [23]. Studies have shown that epithelial cells differentiate into hard tissues such as cementum in the presence of TGF-β-1 during EMT [23][24][25][26]. A recent study also suggested that EMT drives odontogenic epithelial stem cells to develop supernumerary teeth through interaction with various MSCs [27]. ...
Background:
The differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts (OBs) is a prerequisite for bone formation. However, little is known about the definitive surface markers for OBs during osteogenesis.
Methods:
To study the surface markers on OBs, we generated and used monoclonal antibodies (MAbs) against surface molecules on transforming growth factor-β1 (TGF-β1)-treated cancer cells. The generated MAbs were further selected toward expression changes on hMSCs cultured with TGF-β1/bone morphogenetic protein-2 (BMP-2) or osteogenic differentiation medium (ODM) by flow cytometry. Immunoprecipitation and mass spectrometry were performed to identify target antigens of selected MAbs. Expression changes of the target antigens were evaluated in hMSCs, human periodontal ligament cells (hPDLCs), and human dental pulp cells (hDPCs) during osteogenic and adipogenic differentiation by quantitative polymerase chain reaction (qPCR) and flow cytometry. hMSCs were also sorted by the MAbs using magnetic-activated cell sorting system, and osteogenic potential of sorted cells was evaluated via Alizarin Red S (ARS) staining and qPCR.
Results:
The binding reactivity of MR14-E5, one of the MAbs, was downregulated in hMSCs with ODM while the binding reactivity of ER7-A7, ER7-A8, and MR1-B1 MAbs was upregulated. Mass spectrometry and overexpression identified that MR14-E5, ER7-A7/ER7-A8, and MR1-B1 recognized integrin α2, α3, and αV, respectively. Upon osteogenic differentiation of hMSCs, the expression of integrin α2 was drastically downregulated, but the expression of integrin α3 and αV was upregulated in accordance with upregulation of osteogenic markers. Expression of integrin α3 and αV was also upregulated in hPDLCs and hDPCs during osteogenic differentiation. Cell sorting showed that integrin αV-high hMSCs have a greater osteogenic potential than integrin αV-low hMSCs upon the osteogenic differentiation of hMSCs. Cell sorting further revealed that the surface expression of integrin αV is more dramatically induced even in integrin αV-low hMSCs.
Conclusion:
These findings suggest that integrin α3 and αV induction is a good indicator of OB differentiation. These findings also shed insight into the expression dynamics of integrins upon osteogenic differentiation of hMSCs and provide the reason why different integrin ligands are required for OB differentiation of hMSCs.
