Dynamic expression of Mage-D1 in rat dental germs and potential role in mineralization of ectomesenchymal stem cells.

Mage-D1 (MAGE family member D1) is involved in a variety of cell biological effects. Recent studies have shown that Mage-D1 is closely related to tooth development, but its specific regulatory mechanism is unclear. The purpose of this study was to investigate the expression pattern of Mage-D1 in rat dental germ development and its differential mineralization ability to ectomesenchymal stem cells (EMSCs), and to explore its potential mechanism. Results showed that the expression of Mage-D1 during rat dental germ development was temporally and spatially specific. Mage-D1 promotes the proliferation ability of EMSCs but inhibits their migration ability. Under induction by mineralized culture medium, Mage-D1 promotes osteogenesis and tooth-forming ability. Furthermore, the expression pattern of Mage-D1 at E19.5 d rat dental germ is similar to p75 neurotrophin receptor (p75NTR), distal-less homeobox 1 (Dlx1) and msh homeobox 1 (Msx1). In addition, Mage-D1 is binding to p75NTR, Dlx1, and Msx1 in vitro. These findings indicate that Mage-D1 is play an important regulatory role in normal mineralization of teeth. p75NTR, Dlx1, and Msx1 seem to be closely related to the underlying mechanism of Mage-D1 action.

Células madre en odontología: nuevas perspectivas / Stem cells in dentistry: new perspectives

En el campo de la odontología, prevalecen actualmente alternativas terapéuticas con una filosofía conservadora. Sin embargo, con el advenimiento de los tratamientos con células madre (CM), se amplían las posibilidades terapéuticas, que buscan la combinación y el equilibrio entre la intervención tradicional y las posibilidades de reposición de estructuras anatómicas dañadas, a través de la regeneración de tejidos utilizando células madre o sus derivados (AU)

In the dentistry field, therapeutic alternatives with a conservative philosophy currently prevail. However, with the advent of stem cell (SC) treatments, therapeutic possibilities are expanding, seeking a combination and balance between traditional intervention and the pos- sibility of replacing damaged anatomical structures through tissue regeneration, using stem cells or their derivatives (AU)

KMT2D deficiency disturbs the proliferation and cell cycle activity of dental epithelial cell line (LS8) partially via Wnt signaling.

Lysine methyltransferase 2D (KMT2D), as one of the key histone methyltransferases responsible for histone 3 lysine 4 methylation (H3K4me), has been proved to be the main pathogenic gene of Kabuki syndrome disease. Kabuki patients with KMT2D mutation frequently present various dental abnormalities, including abnormal tooth number and crown morphology. However, the exact function of KMT2D in tooth development remains unclear. In this report, we systematically elucidate the expression pattern of KMT2D in early tooth development and outline the molecular mechanism of KMT2D in dental epithelial cell line. KMT2D and H3K4me mainly expressed in enamel organ and Kmt2d knockdown led to the reduction in cell proliferation activity and cell cycling activity in dental epithelial cell line (LS8). RNA-sequencing (RNA-seq) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis screened out several important pathways affected by Kmt2d knockdown including Wnt signaling. Consistently, Top/Fop assay confirmed the reduction in Wnt signaling activity in Kmt2d knockdown cells. Nuclear translocation of ß-catenin was significantly reduced by Kmt2d knockdown, while lithium chloride (LiCl) partially reversed this phenomenon. Moreover, LiCl partially reversed the decrease in cell proliferation activity and G1 arrest, and the down-regulation of Wnt-related genes in Kmt2d knockdown cells. In summary, the present study uncovered a pivotal role of histone methyltransferase KMT2D in dental epithelium proliferation and cell cycle homeostasis partially through regulating Wnt/ß-catenin signaling. The findings are important for understanding the role of KMT2D and histone methylation in tooth development.

Isolation of dental pulp and periodontal cells from human teeth for single-cell RNA sequencing.

Teeth and the surrounding periodontal tissues are affected by many pathologies that compromise their integrity and significantly affect life quality. The study of the main dental tissues, the dental pulp and periodontium, is made arduous by their close association with highly mineralized tissues (dentin, cementum, and alveolar bone). Here we describe a protocol to isolate all cells composing human dental pulp and periodontium for single-cell RNA sequencing analysis. For complete details on the use and execution of this protocol, please refer to Pagella et al. (2021).

Conditioned medium derived from 3D tooth germs: A novel cocktail for stem cell priming and early in vivo pulp regeneration.

OBJECTIVES: Conditioned medium (CM) from 2D cell culture can mitigate the weakened regenerative capacity of the implanted stem cells. However, the capacity of 3D CM to prime dental pulp stem cells (DPSCs) for pulp regeneration and its protein profile are still elusive. We aim to investigate the protein profile of CM derived from 3D tooth germs, and to unveil its potential for DPSCs-based pulp regeneration. MATERIALS AND METHODS: We prepared CM of 3D ex vivo cultured tooth germ organs (3D TGO-CM) and CM of 2D cultured tooth germ cells (2D TGC-CM) and applied them to prime DPSCs. Influences on cell behaviours and protein profiles of CMs were compared. In vivo pulp regeneration of CMs-primed DPSCs was explored using a tooth root fragment model on nude mice. RESULTS: TGO-CM enhanced DPSCs proliferation, migration, in vitro mineralization, odontogenic differentiation, and angiogenesis performances. The TGO-CM group generated superior pulp structures, more odontogenic cells attachment, and enhanced vasculature at 4 weeks post-surgery, compared with the TGC-CM group. Secretome analysis revealed that TGO-CM contained more odontogenic and angiogenic growth factors and fewer pro-inflammatory cytokines. Mechanisms leading to the differential CM profiles may be attributed to the cytokine-cytokine receptor interaction and PI3K-Akt signalling pathway. CONCLUSIONS: The unique secretome profile of 3D TGO-CM made it a successful priming cocktail to enhance DPSCs-based early pulp regeneration.

Sinking Our Teeth in Getting Dental Stem Cells to Clinics for Bone Regeneration.

Dental stem cells have been isolated from the medical waste of various dental tissues. They have been characterized by numerous markers, which are evaluated herein and differentiated into multiple cell types. They can also be used to generate cell lines and iPSCs for long-term in vitro research. Methods for utilizing these stem cells including cellular systems such as organoids or cell sheets, cell-free systems such as exosomes, and scaffold-based approaches with and without drug release concepts are reported in this review and presented with new pictures for clarification. These in vitro applications can be deployed in disease modeling and subsequent pharmaceutical research and also pave the way for tissue regeneration. The main focus herein is on the potential of dental stem cells for hard tissue regeneration, especially bone, by evaluating their potential for osteogenesis and angiogenesis, and the regulation of these two processes by growth factors and environmental stimulators. Current in vitro and in vivo publications show numerous benefits of using dental stem cells for research purposes and hard tissue regeneration. However, only a few clinical trials currently exist. The goal of this review is to pinpoint this imbalance and encourage scientists to pick up this research and proceed one step further to translation.

Strategies for differentiation of hiPSCs into dental epithelial cell lineage.

Different stem cell-based strategies, especially induced pluripotent stem cells (iPSCs), have been exploited to regenerate teeth or restore biological and physiological functions after tooth loss. Further research is needed to establish an optimized protocol to effectively differentiate human iPSCs (hiPSCs) into dental epithelial cells (DECs). In this study, various factors were precisely modulated to facilitate differentiation of hiPSCs into DECs, which are essential for the regeneration of functional teeth. Embryoid bodies (EBs) were formed from hiPSCs as embryo-like aggregates, retinoic acid (RA) was used as an early ectodermal inducer, and bone morphogenic protein 4 (BMP4) activity was manipulated. The characteristics of DECs were enhanced and preserved after culture in keratinocyte serum-free medium (K-SFM). The yielded cell population exhibited noticeable DEC characteristics, consistent with the expression of epithelial cell and ameloblast markers. DECs demonstrated odontogenic abilities by exerting an inductive effect on human dental pulp stem cells (hDPSCs) and forming a tooth-like structure with the mouse tooth mesenchyme. Overall, our differentiation protocol provides a practical approach for applying hiPSCs for tooth regeneration.

Cells: Are They (Still) Essential for Dental Regeneration?

Tissue regeneration in dentistry has demonstrated impressive progress over during the last decades compared to other medical sciences [...].

Dual roles of TGF-ß signaling in the regulation of dental epithelial cell proliferation.

The purpose of this study is to investigate the molecular mechanisms and biological function of TGF-ß-activated Smad1/5 in dental epithelium. Immunohistochemistry was used to detect the expressions of TGF-ß signaling-related gene in mice molar germ. Primary dental epithelial cells were cultured and treated with TGF-ß1 at a concentration of 0.5 or 5 ng/mL. Small molecular inhibitors, SB431542 and ML347, was used to inhibite ALK5 and ALK1/2, respectively. Small interfering RNA was used to knock down Smad1/5 or Smad2/3. The proliferation rate of cells was evaluated by EdU assay. In the basal layer of dental epithelial bud TGF-ß1 and p-Smad1/5 were highly expressed, and in the interior of the epithelial bud TGF-ß1 was lowly expressed, whereas p-Smad2/3 was highly expressed. In primary cultured dental epithelial cells, low concentration of TGF-ß1 activated Smad2/3 but not Smad1/5, while high concentration of TGF-ß1 was able to activate both Smad2/3 and Smad1/5. SB431542 but not ML347 was able to block the phosphorylation of Smad2/3 by TGF-ß1. Either SB431542 or ML347 was able to block the phosphorylation of Smad1/5 by TGF-ß1. EdU staining showed that high concentration of TGF-ß1 promoted dental epithelial cell proliferation, which was reversed by silencing Smad1/5, whereas low concentration of TGF-ß1 inhibited cell proliferation, which was reversed by silencing Smad2/3. In conclusions, TGF-ß exhibits dual roles in the regulation of dental epithelial cell proliferation through two pathways. On the one hand, TGF-ß activates canonical Smad2/3 signaling through ALK5, inhibiting the proliferation of internal dental epithelial cells. On the other hand, TGF-ß activates noncanonical Smad1/5 signaling through ALK1/2-ALK5, promoting the proliferation of basal cells in the dental epithelial bud.

[Dental stem cells: myth or hope in neuroregenerative medicine?] / Cellules souches dentaires : mythe ou espoir en médecine neurorégénératrice ?

The use of dental stem cells has raised many hopes in the development of new treatments for neurodegenerative diseases. According to current statistics, about 1 in 6 people in the world would be affected by a neurological disease. This number continues to increase as the world's population ages, making neurodegenerative diseases probably the one of the major challenges of public health in the 21st century. Neurodegenerative diseases are characterized mainly by a progressive loss of cognitive abilities and patient autonomy related to loss and degeneration of neurons in brain structures. Unfortunately, today, the only treatments available for this type of disease do only relieve the symptoms, they do not treat them, and few clinical trials have been truly convincing to date. Hence, hope lies for these diseases in the development of other therapeutic approaches. As such, dental stem cells could be a promising area of research because of their rapid growth, their great capacity for differentiation into different types of cells (among neuronal ones for some of them) and how easy they can be obtained, without raising ethical issues as for example for embryonic stem cells.

Optimization of culture conditions for the efficient differentiation of mouse-induced pluripotent stem cells into dental epithelial-like cells.

The establishment of a method to derive dental epithelial cells seems to be an important challenge toward realizing the whole tooth regeneration. In order to obtain a source of dental epithelial-like cells, a new methodology has been previously developed by our research group. In the method, induced pluripotent stem cells are cultured in suspension in the presence of neurotrophin-4 to form embryoid bodies followed by further adherent culture of the embryoid bodies in DMEM basal nutrient medium. The present study was directed to improve the efficiency of dental epithelial-like cell production, by focusing on the optimization of initial cell number for the formation of embryoid bodies and the addition of epidermal growth factor as well as its timing. Our results demonstrated that an initial cell number of 1000 cells/drop gives the highest efficiency of dental epithelial-like cell production. It appears that, under this condition, medium deterioration is moderated, and that cell-cell interactions are optimized within embryoid bodies. On the other hand, epidermal growth factor serves to increase the abundance of dental epithelial-like cells when added to the medium together with neurotrophin-4 during embryoid body formation. The promotive effect of epidermal growth factor may involve the transactivation of TrkB, mediated by the effectors of epidermal growth factor receptor signaling.

Dental cell type atlas reveals stem and differentiated cell types in mouse and human teeth.

Understanding cell types and mechanisms of dental growth is essential for reconstruction and engineering of teeth. Therefore, we investigated cellular composition of growing and non-growing mouse and human teeth. As a result, we report an unappreciated cellular complexity of the continuously-growing mouse incisor, which suggests a coherent model of cell dynamics enabling unarrested growth. This model relies on spatially-restricted stem, progenitor and differentiated populations in the epithelial and mesenchymal compartments underlying the coordinated expansion of two major branches of pulpal cells and diverse epithelial subtypes. Further comparisons of human and mouse teeth yield both parallelisms and differences in tissue heterogeneity and highlight the specifics behind growing and non-growing modes. Despite being similar at a coarse level, mouse and human teeth reveal molecular differences and species-specific cell subtypes suggesting possible evolutionary divergence. Overall, here we provide an atlas of human and mouse teeth with a focus on growth and differentiation.

Role of Special AT-Rich Sequence-Binding Protein 2 in the Osteogenesis of Human Dental Mesenchymal Stem Cells.

Dental mesenchymal stem cells (MSCs) are recognized as a critical factor in repair of defective craniofacial bone owing to the multiple differentiation potential, the ability to regenerate distinct tissues, and the advantage that they can be easily obtained by relatively noninvasive procedures. Special AT-rich sequence-binding protein 2 (SATB2) is a nuclear matrix protein, involved in chromatin remodeling and transcriptional regulation, and has been reported to be as a positive regulator of osteoblast differentiation, bone formation, and bone regeneration in MSCs. In this study, we systematically investigated the capability of SATB2 to promote the osteogenic differentiation of periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and stem cells from human exfoliated deciduous teeth (SHED). RNA-seq analysis and quantitative real-time PCR (RT-PCR) revealed that genes regulating osteogenic differentiation were differentially expressed among three cell types and SATB2 was found to be expressed at a relatively high level. When the three cell types overexpressed SATB2 with AdSATB2 infection, alkaline phosphatase (ALP) staining, ALP activity, Alizarin Red S staining, and quantification tended to increase with an increasing infection rate. It showed opposite results after infection with AdsiSATB2. RNA-seq analysis indicated that the expression of downstream osteogenic genes was affected by AdSATB2 infection and quantitative RT-PCR confirmed that nine osteogenic genes (Spp1, Sema7a, Atf4, Ibsp, Col1a1, Sp7, Igfbp3, Dlx3, and Alpl) were upregulated, to various extents, following SATB2 overexpression. In addition, quantitative PCR results indicated that SATB2 affected the expression of MSC markers. These results suggested an important role of SATB2 in the osteogenesis of PDLSCs, DPSCs, and SHED. Further research is warranted to investigate SATB2-mediated regulation of osteogenic differentiation and to evaluate the therapeutic use of SATB2 for the regeneration of defective craniofacial bone tissue.

Pitx2-Sox2-Lef1 interactions specify progenitor oral/dental epithelial cell signaling centers.

Epithelial signaling centers control epithelial invagination and organ development, but how these centers are specified remains unclear. We report that Pitx2 (the first transcriptional marker for tooth development) controls the embryonic formation and patterning of epithelial signaling centers during incisor development. We demonstrate using Krt14Cre /Pitx2flox/flox (Pitx2cKO ) and Rosa26CreERT/Pitx2flox/flox mice that loss of Pitx2 delays epithelial invagination, and decreases progenitor cell proliferation and dental epithelium cell differentiation. Developmentally, Pitx2 regulates formation of the Sox2+ labial cervical loop (LaCL) stem cell niche in concert with two signaling centers: the initiation knot and enamel knot. The loss of Pitx2 disrupted the patterning of these two signaling centers, resulting in tooth arrest at E14.5. Mechanistically, Pitx2 transcriptional activity and DNA binding is inhibited by Sox2, and this interaction controls gene expression in specific Sox2 and Pitx2 co-expression progenitor cell domains. We demonstrate new transcriptional mechanisms regulating signaling centers by Pitx2, Sox2, Lef1 and Irx1.

Novel strategies for expansion of tooth epithelial stem cells and ameloblast generation.

Enamel is secreted by ameloblasts derived from tooth epithelial stem cells (SCs). Humans cannot repair or regenerate enamel, due to early loss of tooth epithelial SCs. Contrarily in the mouse incisors, epithelial SCs are maintained throughout life and endlessly generate ameloblasts, and thus enamel. Here we isolated Sox2-GFP+ tooth epithelial SCs which generated highly cellular spheres following a novel in vitro strategy. This system enabled analysis of SC regulation by various signaling molecules, and supported the stimulatory and inhibitory roles of Shh and Bmp, respectively; providing better insight into the heterogeneity of the SCs. Further, we generated a novel mouse reporter, Enamelin-tdTomato for identification of ameloblasts in live tissues and cells, and used it to demonstrate presence of ameloblasts in the new 3D co-culture system of dental SCs. Collectively, our results provide means of generating 3D tooth epithelium from adult SCs which can be utilized toward future generation of enamel.

Sonic Hedgehog Signaling and Tooth Development.

Sonic hedgehog (Shh) is a secreted protein with important roles in mammalian embryogenesis. During tooth development, Shh is primarily expressed in the dental epithelium, from initiation to the root formation stages. A number of studies have analyzed the function of Shh signaling at different stages of tooth development and have revealed that Shh signaling regulates the formation of various tooth components, including enamel, dentin, cementum, and other soft tissues. In addition, dental mesenchymal cells positive for Gli1, a downstream transcription factor of Shh signaling, have been found to have stem cell properties, including multipotency and the ability to self-renew. Indeed, Gli1-positive cells in mature teeth appear to contribute to the regeneration of dental pulp and periodontal tissues. In this review, we provide an overview of recent advances related to the role of Shh signaling in tooth development, as well as the contribution of this pathway to tooth homeostasis and regeneration.

Molecular and cellular mechanisms of tooth development, homeostasis and repair.

The tooth provides an excellent system for deciphering the molecular mechanisms of organogenesis, and has thus been of longstanding interest to developmental and stem cell biologists studying embryonic morphogenesis and adult tissue renewal. In recent years, analyses of molecular signaling networks, together with new insights into cellular heterogeneity, have greatly improved our knowledge of the dynamic epithelial-mesenchymal interactions that take place during tooth development and homeostasis. Here, we review recent progress in the field of mammalian tooth morphogenesis and also discuss the mechanisms regulating stem cell-based dental tissue homeostasis, regeneration and repair. These exciting findings help to lay a foundation that will ultimately enable the application of fundamental research discoveries toward therapies to improve oral health.

New Insights into the Control of Cell Fate Choices and Differentiation by Retinoic Acid in Cranial, Axial and Caudal Structures.

Retinoic acid (RA) signaling is an important regulator of chordate development. RA binds to nuclear RA receptors that control the transcriptional activity of target genes. Controlled local degradation of RA by enzymes of the Cyp26a gene family contributes to the establishment of transient RA signaling gradients that control patterning, cell fate decisions and differentiation. Several steps in the lineage leading to the induction and differentiation of neuromesodermal progenitors and bone-producing osteogenic cells are controlled by RA. Changes to RA signaling activity have effects on the formation of the bones of the skull, the vertebrae and the development of teeth and regeneration of fin rays in fish. This review focuses on recent advances in these areas, with predominant emphasis on zebrafish, and highlights previously unknown roles for RA signaling in developmental processes.

Developmental plasticity of epithelial stem cells in tooth and taste bud renewal.

In Lake Malawi cichlids, each tooth is replaced in one-for-one fashion every ∼20 to 50 d, and taste buds (TBs) are continuously renewed as in mammals. These structures are colocalized in the fish mouth and throat, from the point of initiation through adulthood. Here, we found that replacement teeth (RT) share a continuous band of epithelium with adjacent TBs and that both organs coexpress stem cell factors in subsets of label-retaining cells. We used RNA-seq to characterize transcriptomes of RT germs and TB-bearing oral epithelium. Analysis revealed differential usage of developmental pathways in RT compared to TB oral epithelia, as well as a repertoire of genome paralogues expressed complimentarily in each organ. Notably, BMP ligands were expressed in RT but excluded from TBs. Morphant fishes bathed in a BMP chemical antagonist exhibited RT with abrogated shh expression in the inner dental epithelium (IDE) and ectopic expression of calb2 (a TB marker) in these very cells. In the mouse, teeth are located on the jaw margin while TBs and other oral papillae are located on the tongue. Previous study reported that tongue intermolar eminence (IE) oral papillae of Follistatin (a BMP antagonist) mouse mutants exhibited dysmorphic invagination. We used these mutants to demonstrate altered transcriptomes and ectopic expression of dental markers in tongue IE. Our results suggest that vertebrate oral epithelium retains inherent plasticity to form tooth and taste-like cell types, mediated by BMP specification of progenitor cells. These findings indicate underappreciated epithelial cell populations with promising potential in bioengineering and dental therapeutics.

A Regenerative Endodontic Approach in Mature Ferret Teeth Using Rodent Preameloblast-conditioned Medium.

BACKGROUND: This study evaluated the effectiveness of a regenerative endodontic approach to regenerate the pulp tissue in mature teeth of ferret. The presence of odontoblast-like cells in the newly-formed tissue of teeth treated with or without preameloblast-conditioned medium was evaluated based on morphological criteria. MATERIALS AND METHODS: Twenty-four canines from six ferrets were treated. The pulp was removed, and the apical foramen was enlarged. After inducing the formation of a blood clot, a collagen sponge with or without preameloblast-conditioned medium was placed underneath the cementoenamel junction. The samples were analyzed at the eighth week of follow-up. RESULTS: Vascularized connective tissue was observed in 50% of teeth, without differences between groups. The tissue occupied the apical third of the root canals. Odontoblast-like cells were not observed in any group. CONCLUSION: Revitalization of mature teeth is possible, at least in the apical third of the root canal. Further experimental research is needed to produce more reliable outcomes.