Makiko Arakaki

Role of Epithelial-Stem Cell Interactions during Dental Cell Differentiation

Background: The role of dental epithelium in stem cell differentiation has not been clearly elucidated. Results: SP cells differentiated into odontoblasts by epithelial BMP4, whereas iPS cells differentiated into ameloblasts when cultured with dental epithelium. Conclusion: Stem cells can be induced to odontogenic cell fates when co-cultured with dental epithelium. Significance: This is the first report to show induction of ameloblasts from iPS cells. Epithelial-mesenchymal interactions regulate the growth and morphogenesis of ectodermal organs such as teeth. Dental pulp stem cells (DPSCs) are a part of dental mesenchyme, derived from the cranial neural crest, and differentiate into dentin forming odontoblasts. However, the interactions between DPSCs and epithelium have not been clearly elucidated. In this study, we established a mouse dental pulp stem cell line (SP) comprised of enriched side population cells that displayed a multipotent capacity to differentiate into odontogenic, osteogenic, adipogenic, and neurogenic cells. We also analyzed the interactions between SP cells and cells from the rat dental epithelial SF2 line. When cultured with SF2 cells, SP cells differentiated into odontoblasts that expressed dentin sialophosphoprotein. This differentiation was regulated by BMP2 and BMP4, and inhibited by the BMP antagonist Noggin. We also found that mouse iPS cells cultured with mitomycin C-treated SF2-24 cells displayed an epithelial cell-like morphology. Those cells expressed the epithelial cell markers p63 and cytokeratin-14, and the ameloblast markers ameloblastin and enamelin, whereas they did not express the endodermal cell marker Gata6 or mesodermal cell marker brachyury. This is the first report of differentiation of iPS cells into ameloblasts via interactions with dental epithelium. Co-culturing with dental epithelial cells appears to induce stem cell differentiation that favors an odontogenic cell fate, which may be a useful approach for tooth bioengineering strategies.

Critical role of heparin binding domains of ameloblastin for dental epithelium cell adhesion and ameloblastoma proliferation

AMBN (ameloblastin) is an enamel matrix protein that regulates cell adhesion, proliferation, and differentiation of ameloblasts. In AMBN-deficient mice, ameloblasts are detached from the enamel matrix, continue to proliferate, and form a multiple cell layer; often, odontogenic tumors develop in the maxilla with age. However, the mechanism of AMBN functions in these biological processes remains unclear. By using recombinant AMBN proteins, we found that AMBN had heparin binding domains at the C-terminal half and that these domains were critical for AMBN binding to dental epithelial cells. Overexpression of full-length AMBN protein inhibited proliferation of human ameloblastoma AM-1 cells, but overexpression of heparin binding domain-deficient AMBN protein had no inhibitory effect. In full-length AMBN-overexpressing AM-1 cells, the expression of Msx2, which is involved in the dental epithelial progenitor phenotype, was decreased, whereas the expression of cell proliferation inhibitors p21 and p27 was increased. We also found that the expression of enamelin, a marker of differentiated ameloblasts, was induced, suggesting that AMBN promotes odontogenic tumor differentiation. Thus, our results suggest that AMBN promotes cell binding through the heparin binding sites and plays an important role in preventing odontogenic tumor development by suppressing cell proliferation and maintaining differentiation phenotype through Msx2, p21, and p27.

Platelet-derived Growth Factor Receptor Regulates Salivary Gland Morphogenesis via Fibroblast Growth Factor Expression

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is β-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor α is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRα, and PDGFRβ were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme.

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43−/− salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43−/− samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43−/− phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.

Two-year clinical evaluation of flowable composite resin containing pre-reacted glass-ionomer

Flowable resin restoration is a useful technique for children with caries. However, when composite resin restoration is performed by an inexperienced clinician, improper placement technique can lead to such problems as poor adaptation, voids and secondary caries formation. In this study, we examined fluoride release from surface of pre-reacted glass-ionomer (S-PRG) fillers containing flowable resin, termed flowable giomer. Beautiful Flow F02 showed a higher amount of fluoride released during the experimental period as compared with the other flowable resins tested. We also used that flowable giomer for Class I, II, and III restoration procedures in 95 primary teeth and 85 permanent ones, and evaluated the results using USPHS/Ryge criteria. Beautiful Flow F02 showed good clinical properties equal to those of conventional resin restorations previously reported. Our results indicate that a flowable giomer is useful for primary and permanent teeth esthetic restoration, which is important for the prevention of secondary caries and adhesion of bacterial flora on resin surfaces.

Expressions and functions of neurotrophic factors in tooth development

Neurotrophic factors are soluble growth factors predominantly expressed in vertebrate nervous systems and have been well-characterized for their critical roles in neural tissues. Recent studies have revealed that neurotrophin factors and their receptors are also expressed in multiple non-neural tissues, and play a role in a wide range of biological functions, such as regulation of cellular proliferation, survival, migration, and differentiation. The neurotrophic factor family is defined by its structural and functional similarities to 4 ligands; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4, also known as NT-5). They activate 2 different receptors, trk tyrosine kinase and p75, the latter of which is a member of the tumor necrosis factor receptor superfamily. During tooth development, observations of dynamic changes of specific expression patterns of neurotrophic factors and their receptors imply their important functions in odontogenic processes. In addition, our recent study demonstrated that NT-4 regulates proliferation and differentiation of dental epithelium, and promotes the production of enamel matrixes. In this review, we describe the expression patterns and functions of neurotrophic factors in the tooth germ, and discuss the relationships with tooth development.

Epithelial Cell Lines in the Field of Dental research: Review

The interaction between the epithelium and mesenchyme induces specific molecular and cellular changes that lead to organogenesis. These interac- tions are particularly crucial during the initiation of the development of ectodermal organs, such as teeth, skin, hair, and mammary and prostate glands. The oral epithe- lium provides the initial signaling for neuronal crest-derived ectomesenchyme development, and then both tissues interact during tooth formation. Various tran- scription factors, growth factors, and extracellular matrices are expressed by enamel matrix-producing ameloblasts during tooth development. Dental epithelium was lost after tooth eruption in human. To analysis of dental cell proliferation and differentiation, we should use the dental epithelial cells from tooth germ, for example third molar, supernumerary tooth or continuous erupting rodent incisor. However, primary culture of dental epithelium has a limited number of cells and passage times. Because of these reasons, cell lines from dental tissue are useful to clear the molecular mechanism of these processes. Here we introduce cell lines from dental tissues, especially dental epithelium.

Effective Differentiation of Induced Pluripotent Stem Cells Into Dental Cells: iPSC Differentiation Into Dental Cells

Background: A biotooth is defined as a complete living tooth, made in laboratory cultures from a spontaneous interplay between epithelial and mesenchymal cell‐based frontal systems. A good solution to these problems is to use induced pluripotent stem cells (iPSCs). However, no one has yet formulated culture conditions that effectively differentiate iPSCs into dental epithelial and dental mesenchymal cells phenotypes analogous to those present in tooth development. Results: Here, we tried to induce differentiation methods for dental epithelial cells (DEC) and dental mesenchymal cells from iPSCs. For the DEC differentiation, the conditional media of SF2 DEC was adjusted to embryoid body. Moreover, we now report on a new cultivation protocol, supported by transwell membrane cell culture that make it possible to differentiate iPSCs into dental epithelial and mesenchymal cells with abilities to initiate the first stages in de novo tooth formation. Conclusions: Implementation of technical modifications to the protocol that maximize the number and rate of iPSC differentiation, into mesenchymal and epithelial cell layers, will be the next step toward growing an anatomically accurate biomimetic tooth organ. Developmental Dynamics 248:129–139, 2019.

Epithelial-mesenchymal interaction reduces inhibitory effects of fluoride on proliferation and enamel matrix expression in dental epithelial cells

Abstract Aim Fluoride, well known as a specific and effective caries prophylactic agent, also affects the differentiation and function of ameloblasts. High dose sodium fluoride (NaF) induces enamel hypoplasia, also called enamel fluorosis, whereas the size and form of teeth except the enamel are not changed with its treatment. We examined the effects of fluoride on dental epithelium proliferation and differentiation using co-cultures of dental epithelial and mesenchymal cells. Methods Cultures of the dental epithelial cell line SF2 and dental mesenchymal cell line mDP were performed, as well as co-cultures. Enamel matrix expression in SF2 cells treated with NaF was analyzed by RT-PCR, while cell proliferation was examined using a trypan blue dye exclusion method and BrdU incorporation findings. The effects of NaF on NT-4-induced ERK1/2 phosphorylation were analyzed by western immunoblotting. Results Neurotrophic factor NT-4 induced enamel matrix expression, which was inhibited in the presence of NaF. Similar results were observed in regard to SF2 cell proliferation, but not with mDP cells. The levels of proliferation and ameloblastin expression in SF2-GFP cells co-cultured with mDP in the presence of NaF were lower as compared to those in SF2 cells cultured alone. Conclusion Our results indicate that dental epithelial cells co-cultured with dental mesenchymal cells are resistant to the inhibitory effects of NaF on proliferation and ameloblastin expression. They also suggest that the dental fluorosis phenotype may affect enamel, but not tooth size or shape, because of rescue of the inhibitory effects of NaF by culturing with dental mesenchymal cells.

Pannexin 3, a Gap Junction Protein, Regulates Chondrocyte Differentiation in Part Through Hemichannel Activity

Gap junctional communications play a crucial role for organogenesis including cartilage development. Pannexin 3 (Panx3) belongs to the new member of the gap junction pannexin family. However, the expression and function of Panx3 have not been cleared. Here, we demonstrate that Panx3 is expressed in cartridge, and regulates chondrocyte differentiation in vitro cell culture. Our observations indicate that Panx3 plays a crucial role for the differentiation of chondrocyte, and suggest that Panx3 is a key molecule for cartilage development.