ABSTRACT
Human-induced pluripotent stem cells (hiPSCs) offer a promising source for generating dental epithelial (DE) cells. Whereas the existing differentiation protocols were time-consuming and relied heavily on growth factors, herein, we developed a three-step protocol to convert hiPSCs into DE cells in 8 days. In the first phase, hiPSCs were differentiated into non-neural ectoderm using SU5402 (an FGF signaling inhibitor). The second phase involved differentiating non-neural ectoderm into pan-placodal ectoderm and simultaneously inducing the formation of oral ectoderm (OE) using LDN193189 (a BMP signaling inhibitor) and purmorphamine (a SHH signaling activator). In the final phase, OE cells were differentiated into DE through the application of Purmorphamine, XAV939 (a WNT signaling inhibitor), and BMP4. qRT-PCR and immunostaining were performed to examine the expression of lineage-specific markers. ARS staining was performed to evaluate the formation of the mineralization nodule. The expression of PITX2, SP6, and AMBN, the emergence of mineralization nodules, and the enhanced expression of AMBN and AMELX in spheroid culture implied the generation of DE cells. This study delineates the developmental signaling pathways and uses small molecules to streamline the induction of hiPSCs into DE cells. Our findings present a simplified and quicker method for generating DE cells, contributing valuable insights for dental regeneration and dental disease research.
Subject(s)
Cell Differentiation , Epithelial Cells , Induced Pluripotent Stem Cells , Morpholines , Purines , Pyrimidines , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/drug effects , Cell Differentiation/drug effects , Epithelial Cells/metabolism , Epithelial Cells/cytology , Epithelial Cells/drug effects , Tooth/cytology , Ectoderm/cytology , Ectoderm/metabolism , Cells, Cultured , Bone Morphogenetic Protein 4/metabolism , Bone Morphogenetic Protein 4/pharmacology , Pyrazoles/pharmacology , Signal Transduction/drug effects , Small Molecule Libraries/pharmacologyABSTRACT
Ameloblasts are the specialized dental epithelial cell type responsible for enamel formation. Following completion of enamel development in humans, ameloblasts are lost and biological repair or regeneration of enamel is not possible. In the past, in vitro models to study dental epithelium and ameloblast biology were limited to freshly isolated primary cells or immortalized cell lines, both with limited translational potential. In recent years, large strides have been made with the development of induced pluripotent stem cell and organoid models of this essential dental lineage - both enabling modeling of human dental epithelium. Upon induction with several different signaling factors (such as transforming growth factor and bone morphogenetic proteins) these models display elevated expression of ameloblast markers and enamel matrix proteins. The advent of 3D bioprinting, and its potential combination with these advanced cellular tools, is poised to revolutionize the field - and its potential for tissue engineering, regenerative and personalized medicine. As the advancements in these technologies are rapidly evolving, we evaluate the current state-of-the-art regarding in vitro cell culture models of dental epithelium and ameloblast lineage with a particular focus toward their applicability for translational tissue engineering and regenerative/personalized medicine.
Subject(s)
Ameloblasts , Bioprinting , Organoids , Regeneration , Humans , Organoids/cytology , Organoids/metabolism , Ameloblasts/metabolism , Ameloblasts/cytology , Tooth/cytology , Tooth/growth & development , Animals , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Tissue Engineering/methods , Epithelium/metabolism , Epithelium/growth & development , Printing, Three-Dimensional , Models, BiologicalABSTRACT
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.
Subject(s)
Calcinosis , Mesenchymal Stem Cells , Neoplasm Proteins , Tooth , Animals , Rats , Calcinosis/metabolism , Receptor, Nerve Growth Factor/metabolism , Tooth/cytology , Tooth/growth & development , Tooth/metabolism , Transcription Factors/metabolism , Neoplasm Proteins/metabolismABSTRACT
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)
Subject(s)
Humans , Stem Cells , Tissue Engineering , Mesenchymal Stem Cells/physiology , Periodontal Ligament/physiology , Regeneration/physiology , Tooth/cytology , Tooth Germ/physiology , Biocompatible Materials/therapeutic use , Bone Regeneration/physiology , Dental Pulp/physiology , Tissue Scaffolds , COVID-19/therapyABSTRACT
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.
Subject(s)
Epithelial Cells/metabolism , Histone-Lysine N-Methyltransferase/deficiency , Histone-Lysine N-Methyltransferase/genetics , Myeloid-Lymphoid Leukemia Protein/deficiency , Myeloid-Lymphoid Leukemia Protein/genetics , Tooth/metabolism , Wnt Signaling Pathway/genetics , Animals , CDC2 Protein Kinase/metabolism , Cell Cycle/genetics , Cell Line , Cell Proliferation/genetics , Cyclin D1/metabolism , Epithelial Cells/cytology , Histones/metabolism , Lithium Chloride/pharmacology , Mice , Mice, Inbred ICR , Molar/metabolism , Tooth/cytology , Wnt Signaling Pathway/drug effectsABSTRACT
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).
Subject(s)
Cell Separation/methods , Dental Pulp/cytology , Periodontium/cytology , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Cell Culture Techniques , Cells, Cultured , Humans , Tooth/cytologyABSTRACT
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.
Subject(s)
Culture Media, Conditioned/metabolism , Dental Pulp/metabolism , Regeneration/physiology , Stem Cells/cytology , Tooth/cytology , Animals , Cell Differentiation/physiology , Cell Proliferation/physiology , Cells, Cultured , Humans , Mice, Nude , Phosphatidylinositol 3-Kinases/metabolismABSTRACT
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.
Subject(s)
Stem Cells/cytology , Tooth/cytology , Animals , Biomarkers/metabolism , Bone Regeneration , Humans , Organoids/cytology , OsteogenesisABSTRACT
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.
Subject(s)
Epithelial Cells/cytology , Induced Pluripotent Stem Cells/cytology , Tooth/cytology , Cell Differentiation , Cell Line , Cell Lineage , Humans , Odontogenesis , Tooth/growth & developmentABSTRACT
Tissue regeneration in dentistry has demonstrated impressive progress over during the last decades compared to other medical sciences [...].
Subject(s)
Cells/metabolism , Tissue Engineering/methods , Tooth/metabolism , Humans , Tooth/cytologyABSTRACT
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.
Subject(s)
Epithelial Cells/cytology , Epithelial Cells/metabolism , Signal Transduction , Tooth/cytology , Transforming Growth Factor beta1/metabolism , Animals , Cell Proliferation , Epithelium/metabolism , Ligands , Mice , Phosphorylation , Receptors, Transforming Growth Factor beta/metabolism , Smad Proteins/metabolism , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
Neural Stem Cells/physiology , Neurodegenerative Diseases/therapy , Regenerative Medicine/trends , Stem Cells/physiology , Tooth/cytology , Animals , Cell Differentiation , Humans , Nerve Regeneration/physiology , Neural Stem Cells/cytology , Neurogenesis/physiology , Regenerative Medicine/methods , Stem Cell Transplantation/methods , Stem Cell Transplantation/trends , Stem Cells/cytology , Tissue Banks/trends , Tissue Culture Techniques/methods , Tissue Culture Techniques/trendsABSTRACT
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.
Subject(s)
Cell Culture Techniques/methods , Cell Differentiation , Epithelial Cells/cytology , Induced Pluripotent Stem Cells/cytology , Tooth/cytology , Animals , Biomarkers/metabolism , Cell Count , Embryoid Bodies/cytology , Epidermal Growth Factor/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Mice , Receptor, trkB/metabolism , Signal Transduction/drug effectsABSTRACT
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.
Subject(s)
Cell Differentiation , Stem Cells/cytology , Tooth/cytology , Tooth/growth & development , Adolescent , Adult , Animals , Cell Differentiation/genetics , Epithelial Cells , Female , Gene Expression Regulation, Developmental , Genetic Heterogeneity , Humans , Incisor/cytology , Incisor/growth & development , Male , Mesoderm/cytology , Mesoderm/growth & development , Mesoderm/metabolism , Mice , Mice, Inbred C57BL , Models, Animal , Molar/cytology , Molar/growth & development , Odontoblasts , Young AdultABSTRACT
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.
Subject(s)
Matrix Attachment Region Binding Proteins/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Osteogenesis , Tooth/cytology , Transcription Factors/metabolism , Adolescent , Biomarkers/metabolism , Cell Differentiation/genetics , Dental Pulp/cytology , Female , Gene Expression Profiling , Gene Expression Regulation , Gene Silencing , Humans , Male , Matrix Attachment Region Binding Proteins/genetics , Osteogenesis/genetics , Periodontal Ligament/cytology , Reproducibility of Results , Tooth Exfoliation , Tooth, Deciduous/cytology , Transcription Factors/geneticsABSTRACT
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.
Subject(s)
Epithelial Cells/metabolism , Homeodomain Proteins/metabolism , Lymphoid Enhancer-Binding Factor 1/metabolism , SOXB1 Transcription Factors/metabolism , Signal Transduction , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Differentiation , Cell Proliferation , Dental Enamel/metabolism , Embryo, Mammalian/metabolism , Epithelial Cells/cytology , Gene Expression Regulation, Developmental , Hedgehog Proteins/metabolism , Homeodomain Proteins/genetics , Lymphoid Enhancer-Binding Factor 1/genetics , Mice , Mice, Knockout , Odontogenesis , SOXB1 Transcription Factors/genetics , Stem Cell Niche , Stem Cells/cytology , Stem Cells/metabolism , Tooth/cytology , Tooth/growth & development , Tooth/metabolism , Transcription Factors/deficiency , Transcription Factors/genetics , YAP-Signaling Proteins , Homeobox Protein PITX2ABSTRACT
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.
Subject(s)
Ameloblasts/cytology , Cell Differentiation , Epithelial Cells/cytology , Stem Cells/cytology , Tooth/cytology , Ameloblasts/metabolism , Animals , Cells, Cultured , Coculture Techniques , Epithelial Cells/metabolism , Mice , Mice, Inbred C57BL , Signal Transduction , Stem Cells/metabolism , Tooth/metabolismABSTRACT
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.
Subject(s)
Hedgehog Proteins/metabolism , Odontogenesis/physiology , Signal Transduction/physiology , Tooth/growth & development , Animals , Dental Enamel/cytology , Dental Enamel/growth & development , Dental Pulp/growth & development , Epithelium/metabolism , Epithelium/pathology , Homeostasis , Humans , Mesenchymal Stem Cells , Tooth/cytology , Tooth Root/cytology , Tooth Root/growth & development , Zinc Finger Protein GLI1/metabolismABSTRACT
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.
Subject(s)
Homeostasis , Odontogenesis/genetics , Regeneration/physiology , Tooth/cytology , Tooth/metabolism , Animals , Humans , Morphogenesis , Signal TransductionABSTRACT
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.