Dental Epithelial Stem Cells Research Articles

The miR-200 family is required for ectodermal organ development through the regulation of the epithelial stem cell niche.

The murine lower incisor ectodermal organ contains a single epithelial stem cell (SC) niche that provides epithelial progenitor cells to the continuously growing rodent incisor. The dental stem cell niche gives rise to several cell types and we demonstrate that the miR‐200 family regulates these cell fates. The miR‐200 family is highly enriched in the differentiated dental epithelium and absent in the stem cell niche. In this study, we inhibited the miR‐200 family in developing murine embryos using new technology, resulting in an expanded epithelial stem cell niche and lack of cell differentiation. Inhibition of individual miRs within the miR‐200 cluster resulted in differential developmental and cell morphology defects. miR‐200 inhibition increased the expression of dental epithelial stem cell markers, expanded the stem cell niche and decreased progenitor cell differentiation. RNA‐seq. identified miR‐200 regulatory pathways involved in cell differentiation and compartmentalization of the stem cell niche. The miR‐200 family regulates signaling pathways required for cell differentiation and cell cycle progression. The inhibition of miR‐200 decreased the size of the lower incisor due to increased autophagy and cell death. New miR‐200 targets demonstrate gene networks and pathways controlling cell differentiation and maintenance of the stem cell niche. This is the first report demonstrating how the miR‐200 family is required for in vivo progenitor cell proliferation and differentiation.

Stem cells in pulmonary alveolar regeneration.

The lungs are constantly exposed to the external environment and are therefore vulnerable to insults that can cause infection and injury. Maintaining the integrity and barrier function of the lung epithelium requires complex interactions of multiple cell lineages. Elucidating the cellular players and their regulation mechanisms provides fundamental information to deepen understanding about the responses and contributions of lung stem cells. This Review focuses on advances in our understanding of mammalian alveolar epithelial stem cell subpopulations and discusses insights about the regeneration-specific cell status of alveolar epithelial stem cells. We also consider how these advances can inform our understanding of post-injury lung repair processes and lung diseases.

β-Catenin/Lin28/let-7 regulatory network determines type II alveolar epithelial stem cell differentiation phenotypes following thoracic irradiation.

The contribution of type II alveolar epithelial stem cells (AEC II) to radiation-induced lung fibrosis (RILF) is largely unknown. Cell differentiation phenotypes are determined by the balance between Lin28 and lethal-7 microRNA (let-7 miRNA). Lin28 is activated by β-catenin. The aim of this study was to track AEC II phenotypes at different phases of injury following thoracic irradiation and examine the expression of β-catenin, Lin28 and let-7 to identify their role in AEC II differentiation. Results showed that coexpression of prosurfactant protein C (proSP-C, an AEC II biomarker) and HOPX (homeobox only protein X, an AEC I biomarker) or vimentin (a differentiation marker) was detected in AEC II post-irradiation. The protein expression levels of HOPX and proSP-C were significantly downregulated, but vimentin was significantly upregulated following irradiation. The expression of E-cadherin, which prevents β-catenin from translocating to the nucleus, was downregulated, and the expression of β-catenin and Lin28 was upregulated after irradiation (P < 0.05 to P < 0.001). Four let-7 miRNA members (a, b, c and d) were upregulated in irradiated lungs (P < 0.05 to P < 0.001), but let-7d was significantly downregulated at 5 and 6 months (P < 0.001). The ratios of Lin28 to four let-7 members were low during the early phase of injury and were slightly higher after 2 months. Intriguingly, the Lin28/let-7d ratio was strikingly increased after 4 months. We concluded that β-catenin contributed to RILF by promoting Lin28 expression, which increased the number of AEC II and the transcription of profibrotic molecules. In this study, the downregulation of let-7d miRNA by Lin28 resulted in the inability of AEC II to differentiate into type I alveolar epithelial cells (AEC I).

New insight into dental epithelial stem cells: Identification, regulation, and function in tooth homeostasis and repair.

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.

Transcriptional Regulation of Dental Epithelial Cell Fate.

Dental enamel is hardest tissue in the body and is produced by dental epithelial cells residing in the tooth. Their cell fates are tightly controlled by transcriptional programs that are facilitated by fate determining transcription factors and chromatin regulators. Understanding the transcriptional program controlling dental cell fate is critical for our efforts to build and repair teeth. In this review, we describe the current understanding of these regulators essential for regeneration of dental epithelial stem cells and progeny, which are identified through transgenic mouse models. We first describe the development and morphogenesis of mouse dental epithelium in which different subpopulations of epithelia such as ameloblasts contribute to enamel formation. Then, we describe the function of critical factors in stem cells or progeny to drive enamel lineages. We also show that gene mutations of these factors are associated with dental anomalies in craniofacial diseases in humans. We also describe the function of the master regulators to govern dental lineages, in which the genetic removal of each factor switches dental cell fate to that generating hair. The distinct and related mechanisms responsible for the lineage plasticity are discussed. This knowledge will lead us to develop a potential tool for bioengineering new teeth.

RESTRICTIVE HUT LUNG: A CASE OF DOMESTICALLY-ACQUIRED PNEUMOCONIOSIS WITH A SEVERE RESTRICTIVE PATTERN

SESSION TITLE: Medical Student/Resident Occupational and Environmental Lung Disease SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Domestically acquired particulate lung disease (DAPLD) or “hut lung” is a mixed-dust pneumoconiosis that develops secondary to the inhalation of biomass fuels in poorly ventilated domestic settings (1). DAPLD has largely been considered to be an obstructive lung disease, with only a minority of cases reporting mild restrictive or mixed patterns of disease (1). To our knowledge, this is the first case report of DAPLD with a severe restrictive pattern. CASE PRESENTATION: A 76 year old Hispanic woman presented with worsening shortness of breath for one year, associated with reduced exercise tolerance, nonproductive cough, chest tightness, and wheezing. She spends majority of the year in Honduras, but travels to the United States for the summer months. She denied smoking tobacco and occupational exposures, but admitted to using a wood burning stove for cooking while living in Honduras for many years. CXR was notable for bilateral patchy infiltrates. HRCT showed large patchy and confluent areas of ground glass attenuation in association with linear and reticular opacities, and peripheral honeycombing with cystic changes. Small calcified bilateral hilar lymph nodes were also observed. Quantiferon testing was positive, but serial cultures were negative for tuberculosis. PFTs showed a restrictive pattern: FEV1 82%, FVC 59%, FEV1/FVC 95%, TLC 46%, DLCO 7%. Bronchoscopy showed multiple flat anthracotic plaques; transbronchial biopsy of these lesions demonstrated focal chronic inflammation. BAL cytology and cultures were unremarkable. DISCUSSION: Although the prevalence of DAPLD is unknown, inhalation of particulate residue of biomass fuels used for domestic energy is common in several parts of the world (2). Cooking with biomass fuels may be associated with fine particle carbon dispersion (2). Deposition of biomass fine particles along alveolar surfaces and macrophage activation initiates an inflammatory response (2). Discourse on biomass inhalation and tobacco smoking has focused on the development of COPD, however both of these exposures may also result in ILD (3). Type 2 alveolar epithelial stem cells, which may function as both type I and type II alveolar cells, have been proposed to play a central role in predisposing patients with tobacco and biomass exposure, to both COPD and ILD phenotypes (2,3). Tobacco and biomass have been proposed to induce cellular senescence, which allows for genetic remodeling and profibrotic cellular changes (2,3). Predisposition of these stem cells to senescence would make certain patients susceptible to COPD or ILD based on the chosen behavior of the stem cell (3). CONCLUSIONS: While the body of research on the hazards of exposure to biomass continues to grow, the restrictive phenotype of DAPLD remains a rare condition that requires further research. Reference #1: Dulku, G, Gupta, N, Perera, R, et al. 2017. Hut Lung Disease: A Radiological and Pathological Correlation. J Med Imaging Case Rep 1(1): 1-4. Reference #2: Kunal S, Pilaniya V, Shah A. 2016. Bronchial Anthracofibrosis with Interstitial Lung Disease: An Association Yet to be Highlighted. BMJ Case Rep bcr2015213940. Reference #3: Kumar, A, Cherian, SV, Vassallo, R, et al. 2018. Current Concepts in Pathogenesis, Diagnosis, and Management of Smoking-Related Interstitial Lung Diseases. Chest 154(2): 394-408. DISCLOSURES: No relevant relationships by Rose Flores, source=Web Response No relevant relationships by Johnathan Kirupakaran, source=Web Response No relevant relationships by Chioma Madu, source=Web Response No relevant relationships by Alice Yau, source=Web Response

Single-Cell RNA-Sequencing From Mouse Incisor Reveals Dental Epithelial Cell-Type Specific Genes.

Dental epithelial stem cells give rise to four types of dental epithelial cells: inner enamel epithelium (IEE), outer enamel epithelium (OEE), stratum intermedium (SI), and stellate reticulum (SR). IEE cells further differentiate into enamel-forming ameloblasts, which play distinct roles, and are essential for enamel formation. These are conventionally classified by their shape, although their transcriptome and biological roles are yet to be fully understood. Here, we aimed to use single-cell RNA sequencing to clarify the heterogeneity of dental epithelial cell types. Unbiased clustering of 6,260 single cells from incisors of postnatal day 7 mice classified them into two clusters of ameloblast, IEE/OEE, SI/SR, and two mesenchymal populations. Secretory-stage ameloblasts expressed Amel and Enam were divided into Dspp + and Ambn + ameloblasts. Pseudo-time analysis indicated Dspp + ameloblasts differentiate into Ambn + ameloblasts. Further, Dspp and Ambn could be stage-specific markers of ameloblasts. Gene ontology analysis of each cluster indicated potent roles of cell types: OEE in the regulation of tooth size and SR in the transport of nutrients. Subsequently, we identified novel dental epithelial cell marker genes, namely Pttg1, Atf3, Cldn10, and Krt15. The results not only provided a resource of transcriptome data in dental cells but also contributed to the molecular analyses of enamel formation.

Glycogen synthase kinase-3β promotes radiation-induced lung fibrosis by regulating β-catenin/lin28 signaling network to determine type II alveolar stem cell transdifferentiation state.

The role of type II alveolar epithelial stem cells (AEC II) for alveolar repair in radiation-induced lung fibrosis (RILF) remains largely unknown, mainly because of AEC II phenotype's spontaneous change in vitro. Cell differentiation status is determined by Lin28 and let-7 miRNAs in see-saw-pattern. Lin28, a repressor of let-7 and a stem cell marker, is activated by β-catenin. The expression of β-catenin is regulated by GSK-3β/TGF-β1 signaling. To understand the true role of AEC II in RILF, we freshly isolated primary AEC II directly from thoracically irradiated lungs. We then explored the expressions of cell phenotype markers and differentiation regulators in these isolated AEC II to analyze the correlation between GSK-3β/TGF-β1/β-catenin signaling pathway, lin28/let-7 balance, and AEC II phenotypes at different injury phases following irradiation. Results showed that isolated single primary cells displayed AEC II ultrastructural features and proSP-C positive. The gene expressions of prosp-c (an AEC II biomarker) and hopx (an AEC I marker) significantly increased in isolated AEC II during injury repair phase (P<.001 and P<.05) but decreased at end-stage of injury, while mesenchymal markers increased in both isolated AEC II and irradiated lungs. mRNA levels of gsk-3β, tgf-β1, and β-catenin increased in all irradiated AEC II, but more pronounced in the second half of injury phase (P<.05-P<.001). Similarly, the expression of lin28 was also significantly elevated in isolated AEC II at the late phase (P<.05-P<.001). Four let-7 miRNAs were significantly upregulated in all irradiated AEC II groups (P<.05-P<.001). The time-dependent and highly consistent uptrends for four lin28/let-7 ratios in sorted AEC II contrasted to downtrends in irradiated lungs. In conclusion, RILF occurred when GSK-3β/TGF-β1 signaling increased β-catenin levels, which led to the augmentation of AEC II population by elevated lin28/let-7 ratio and the transcription of profibrotic cytokines and factors, thereby inducing AEC II to undergo transdifferentiation into mesenchymal cells.

Ectodermal Organ Development Is Regulated by a microRNA-26b-Lef-1-Wnt Signaling Axis.

The developmental role of Lef-1 in ectodermal organs has been characterized using Lef-1 murine knockout models. We generated a Lef-1 conditional over-expression (COEL) mouse to determine the role of Lef-1 expression in epithelial structures at later stages of development after endogenous expression switches to the mesenchyme. Lef-1 over expression (OE) in the oral epithelium creates a new dental epithelial stem cell niche that significantly increases incisor growth. These data indicate that Lef-1 expression is switched off in the dental epithelial at early stages to maintain the stem cell niche and regulate incisor growth. Bioinformatics analyses indicated that miR-26b expression increased coinciding with decreased Lef-1 expression in the dental epithelium. We generated a murine model over-expressing miR-26b that targets endogenous Lef-1 expression and Lef-1-related developmental mechanisms. miR-26b OE mice have ectodermal organ defects including a lack of incisors, molars, and hair similar to the Lef-1 null mice. miR-26b OE rescues the Lef-1 OE phenotype demonstrating a critical genetic and developmental role for miR-26b in the temporal and spatial expression of Lef-1 in epithelial tissues. Lef-1 expression regulates Wnt signaling and Wnt target genes as well as cell proliferation mechanisms, while miR-26b OE reduced the levels of Wnt target gene expression. The extra stem cell compartment in the COEL mice expressed Lef-1 suggesting that Lef-1 is a stem cell factor, which was absent in the miR-26b OE/COEL rescue mice. This is the first demonstration of a microRNA OE mouse model that has ectodermal organ defects. These findings demonstrate that the levels of Lef-1 are critical for development and establish a role for miR-26b in the regulation of ectodermal organ development through the control of Lef-1 expression and an endogenous stem cell niche.

Molecular Insight into Odontogenesis in Hyperglycemic Environment: A Systematic Review.

ABSTRACTDiabetes mellitus is an endocrinal disorder affecting worldwide and the disease incidence is rising alarmingly high. The effects of diabetes on tooth development are explored by limited studies and their molecular insights are very rarely studied. This systematic review is aimed to provide the best scientific literature source on the molecular insights into odontogenesis in hyperglycemic environment caused by diabetes mellitus or by maternal diabetes on the offspring. The literature search was conducted on the databases, namely PubMed, PubMed Central, Cochrane, and Scopus. The original studies exploring the alterations in the molecular pathways of odontogenesis in diabetes mellitus were selected. Data were extracted, chosen, and evaluated by two independent researchers. At the end of thorough data search, four articles were eligible for the review. Three articles brought out the molecular pathways involved in the offspring of gestational diabetes through animal models. Fourth article was an in vitro study, which treated the stem cells in hyperglycemic environment and drafted the molecular pathway. The altered molecular pathways in dental epithelial stem cells (DESCs), dental papilla cells (DPCs), and stem cells from apical papilla were studied and empowered with statistical analysis. Thus with this systematic review, we conclude that apurinic/apyrimidinic endonuclease1 downregulation causing deoxyribonucleic acid hypermethylation and Oct4, Nanog gene silencing, activation of toll-like receptor-4/nuclear factor kappa B (TLR4/NF-κB) pathway are involved in suppressing cell proliferation and accelerated apoptosis in DESCs in high glucose environment. DPCs are suppressed from odonto differentiation by activation of TLR4 signaling and resulting inhibition of SMAD1/5/9 phosphorylation in diabetic condition. NF-κB pathway activation causes decreased cell proliferation and enhanced differentiation in apical papilla stem cells in hyperglycemia. Further studies targeting various stages of odontogenesis can reveal more molecular insight.

Dental Epithelial Stem Cells as a Source for Mammary Gland Regeneration and Milk Producing Cells In Vivo.

The continuous growth of rodent incisors is ensured by clusters of mesenchymal and epithelial stem cells that are located at the posterior part of these teeth. Genetic lineage tracing studies have shown that dental epithelial stem cells (DESCs) are able to generate all epithelial cell populations within incisors during homeostasis. However, it remains unclear whether these cells have the ability to adopt alternative fates in response to extrinsic factors. Here, we have studied the plasticity of DESCs in the context of mammary gland regeneration. Transplantation of DESCs together with mammary epithelial cells into the mammary stroma resulted in the formation of chimeric ductal epithelial structures in which DESCs adopted all the possible mammary fates including milk-producing alveolar cells. In addition, when transplanted without mammary epithelial cells, DESCs developed branching rudiments and cysts. These in vivo findings demonstrate that when outside their niche, DESCs redirect their fates according to their new microenvironment and thus can contribute to the regeneration of non-dental tissues.

INCREASED SOX2-POSITIVE CELLS IN BRAF(V600E) MUTATED AMELOBLASTOMAS

Objective SRY related HMG box gene 2 (SOX2) is a transcription factor expressed in embryonic and adult stem cells. SOX2 positive dental epithelial stem cells have been shown to give rise to all dental epithelial cell lineages. Increased SOX2 expressing cells has been reported in ameloblastic carcinomas than ameloblastomas, which might indicate SOX2 contributes to the pathogenesis of ameloblastic neoplasms. Recent and our previous studies have shown high frequency of BRAF(V600E) mutation in ameloblastomas. Interestingly, recent studies have reported that BRAF mutation is associated with the expression of SOX2 in colorectal cancers. Here, we investigated if SOX2-positive cell component is expanded in BRAF(V600E) mutated than wild type ameloblastomas. Methods Fifty-five formalin fixed paraffin embedded ameloblastoma tissue sections were used for macro-dissection of tumor component, DNA extraction and SOX2 immunohistochemistry. Sanger sequencing was further performed to detect the BRAF(V600E) mutation. The correlation between SOX2 positive cell numbers and BRAF status in ameloblastomas was evaluated by T-test. Results Among 55 ameloblastoma cases, forty-eight cases harbored BRAF(V600E) mutation. SOX2 positive cells were found in all cases regardless of BRAF status with average 22.3% SOX2 positive cells in ameloblastomas. BRAF(V600E) mutated ameloblastoma cases showed significantly more Sox2-positive cells (24.5%) than in wild type (6.6%) (p Conclusion SOX2 positive cells were found in all ameloblastomas and BRAF(V600E) mutated ameloblastomas showed significantly more SOX2-positive cells. The results suggested BRAF(V600E) mutation may contribute to the expansion of SOX2 positive cell compartment.

Effect of canonical NF-\u03baB signaling pathway on the differentiation of rat dental epithelial stem cells

BackgroundNuclear factor-κB (NF-κB), an important transcription factor, participates in many physiological and pathological processes such as growth, differentiation, organogenesis, apoptosis, inflammation, and immune response, including tooth development. However, it is still unknown whether NF-κB participates in the regulation of dental epithelial stem cells (DESCs) in postnatal rat incisors. Here, we investigated the specific differentiation regulatory mechanisms of the canonical NF-κB signaling pathway in DESCs and provided the mechanism of cross-talk involved in DESC differentiation.MethodsAfter adding the activator or inhibitor of the NF-κB signaling pathway, Western blot and quantitative real-time PCR were used to analyze the expressions of amelogenesis-related genes and proteins and canonical transforming growth factor-β (TGF-β) signaling. In addition, we used amelogenesis induction in vitro by adding the activator or inhibitor of the NF-κB signaling pathway to the amelogenesis-induction medium, respectively. Recombinant TGF-β was used to activate the TGF-β pathway, and SMAD7 siRNA was used to downregulate the expression of SMAD7 in DESCs.ResultsWe found that the expression of amelogenesis-related genes and proteins as well as TGF-β signaling were downregulated, while SMAD7 expression was increased in NF-κB-activated DESCs. In addition, NF-κB-inhibited DESCs exhibited opposite results compared with NF-κB-activated DESCs. Furthermore, the canonical NF-κB signaling pathway suppressed the canonical TGF-β-SMAD signaling by inducing SMAD7 expression involved in the regulation of DESC differentiation.ConclusionsThese results indicate that the canonical NF-κB signaling pathway participated in the regulation of DESC differentiation, which was through upregulating SMAD7 expression and further suppressing the canonical TGF-β-SMAD signaling pathway.

New in vivo microRNA biotechnology reveals specific roles for the miR‐200 family in dental stem cell maintenance

Extracellular signaling as well as transcription factor and epigenetic regulation are well described mechanisms controlling stem cell differentiation. The regulation of these processes by microRNAs (miRs) is recognized as an important biological process that can be coopted for future stem cell‐based therapies. Previously, our group has found the miR‐200 family is highly expressed in differentiated tooth cell types, but absent in dental epithelial stem cell (DESC) precursors. Inhibition of the miR‐200 family in mice using a novel Plasmid‐based miR Inhibitor System (PMIS) demonstrates that the miR‐200 family controls DESC maintenance and compartmentalization.ObjectivesTo investigate the mechanisms by which the miR‐200 family promotes the differentiation of ameloblasts and other mature tooth cell types during tooth morphogenesis, and how inhibition of the family maintains stemness.MethodsWe generated mice expressing two different PMIS constructs. One inhibitor construct is specific for the seed sequence of miR‐200a, and ‐141, while the other inhibitor is specific for the seed sequence found in miR‐200b,‐200c and ‐429. Crossing these mice allows the functional inhibition of the entire miR‐200 family (double inhibitor mice).ResultsDouble inhibitor mice are neonatal lethal and have poorly differentiated lower incisors. The teeth are smaller and lack markers of mature odontoblasts and ameloblasts. Several transcription factors marking the stem cell niche of the lower incisor, including Sox2, have expanded expression domains, and BrdU labeling of proliferating cells demonstrated that miR‐200 inhibition leads to increased epithelial cell proliferation outside of the stem cell niche. Rnf126, an E‐3 ubiquitin ligase that promotes P21 degradation and a direct target of miR‐200c, is expressed at higher levels in the proliferating cells leading to the loss of P21 protein. Although more epithelial cells are proliferating, the lower incisors are shorter due to an increase in a caspase independent, programmed cell death pathway.ConclusionsThe miR‐200 family is required for the appropriate differentiation of DESCs and the family functions by reducing the expression of transcription factors promoting stemness outside of the stem cell niche compartment of the lower incisor. Overall, craniofacial development is affected due to a lack of cell differentiation.Support or Funding InformationUniversity of Iowa College of DentistryNIH, NIDCRT90 training grant 2T90DE023520‐06This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dental Epithelial Stem Cells Express the Developmental Regulator Meis1.

MEIS1 is a key developmental regulator of several organs and participates in stem cell maintenance in different niches. However, despite the murine continuously growing incisor being a well described model for the study of adult stem cells, Meis1 has not been investigated in a dental context. Here, we uncover that Meis1 expression in the tooth is confined to the epithelial compartment. Its expression arises during morphogenesis and becomes restricted to the mouse incisor epithelial stem cell niche, the labial cervical loop. Meis1 is specifically expressed by Sox2+ stem cells, which give rise to all dental epithelial cell lineages. Also, we have found that Meis1 in the incisor is coexpressed with potential binding partner Pbx1 during both embryonic and adult stages. Interestingly, Meis2 is present in different areas of the forming tooth and it is not expressed by dental epithelial stem cells, suggesting different roles for these two largely homologous genes. Additionally, we have established the expression patterns of Meis1 and Meis2 during tongue, hair, salivary gland and palate formation. Finally, analysis of Meis1-null allele mice indicated that, similarly, to SOX2, MEIS1 is not essential for tooth initiation, but might have a role during adult incisor renewal.

Microdissection and Isolation of Mouse Dental Epithelial Cells of Continuously Growing Mouse Incisors.

Mouse incisors are regenerative tissues, which grow continuously throughout life and are good model for the study of epithelial stem cells. The study of dental epithelial stem cells allows investigation of a variety of basic biological processes in the context of the stem cells. The ability to analyze dental epithelial stem cells in vitro has emerged as a powerful tool to understand how teeth are constructed and the signaling pathways that regulate ameloblast developmental processes. Here, we describe in detail our protocols for the culture of dental epithelial stem cells and the production of the cell lines. These techniques allow us to reproduce the differentiation process of ameloblasts and estimate the effect of specific genes ex vivo, as well as are a tool for studies on the mechanisms of normal and abnormal amelogenesis. They may also be applied to studies on other aspects of developmental biology and regenerative medicine using stem cells.

The transcription factor NKX2-3 mediates p21 expression and ectodysplasin-A signaling in the enamel knot for cusp formation in tooth development

Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme. During tooth development, tooth cusps are regulated by precise control of proliferation of cell clusters, termed enamel knots, that are present among dental epithelial cells. The interaction of ectodysplasin-A (EDA) with its receptor, EDAR, plays a critical role in cusp formation by these enamel knots, and mutations of these genes is a cause of ectodermal dysplasia. It has also been reported that deficiency in Nkx2-3, encoding a member of the NK2 homeobox family of transcription factors, leads to cusp absence in affected teeth. However, the molecular role of NKX2-3 in tooth morphogenesis is not clearly understood. Using gene microarray analysis in mouse embryos, we found that Nkx2-3 is highly expressed during tooth development and increased during the tooth morphogenesis, especially during cusp formation. We also demonstrate that NKX2-3 is a target molecule of EDA and critical for expression of the cell cycle regulator p21 in the enamel knot. Moreover, NKX2-3 activated the bone morphogenetic protein (BMP) signaling pathway by up-regulating expression levels of Bmp2 and Bmpr2 in dental epithelium and decreased the expression of the dental epithelial stem cell marker SRY box 2 (SOX2). Together, our results indicate that EDA/NKX2-3 signaling is essential for enamel knot formation during tooth morphogenesis in mice.

A basic study on self-reconstitution of alveolar epithelium-like cells by tissue stem cells in mouse lung.

In recent research on regenerative medicine, three-dimensional (3D) tissue reconstruction using the induced pluripotent stem cell (iPS cell) differentiated cells has attracted attention. In this study, mouse lungs at 1.5, 10, and 20d old were subjected to enzyme treatment, and aggregates formed in serum-free suspension culture (3D-culture) were observed. The number of aggregates formed was the highest in 1.5d. The cell aggregates in which the interior of the aggregate is filled and form small vacuoles and the organoid-like aggregates having a relatively large vacuole inside and forming the alveolar-like structure were observed. At 1.5d, the formation ratio of the organoid-like aggregates was the highest and aggregate size was small at 20d. For the cell aggregates derived from 1.5d, positive cells of SSEA-1, CD29, CD90, CD105, alveolar epithelial stem cell marker of SP-C, and Sca-1 were observed in the center. In the cell aggregates derived from 10d, the expression level of 1.5d each protein markers and OCT4 gene of transcription factor was decreased, and furthermore, markers were hardly observed in the organoid-like aggregates derived from 10d. In addition, cells surrounding the vacuole of organoid-like aggregate obtained over 10d differentiated into periodic acid-Schiff (PAS), podoplanin-positive cells. When the formed cell aggregates were dispersed, cell aggregates and organoid-like aggregates were reformed. Comparing 3D-culture and adhesion culture (2D-culture), SP-C expression of 10d of cells was maintained. Expression of markers of undifferentiated markers and alveolar tissue stem cells decreased when cell aggregates were cultured with the addition of fetal bovine serum.

Bivalency of Shh Modulates Cell Fate Decision of Dental Epithelium Stem Cells

Rodent incisor grows throughout whole life of the animal and owns every stage of dental epithelium development, thus it provides an ideal model to study the fate decision of dental epithelial stem cells (DESCs) as well as to understand the mechanisms of tooth regeneration. Bivalency, characterized as concurrence of H3K27me3 and H3K4me3, manages key developmental genes transcript in some organs, however no evidences answer whether it works on enamel organ development. Here using mouse incisor models, we first of time reveal the dynamic alterations of bivalency during DESCs differentiation, which is dominated via H3K27me3 rather than H3K4me3. Ezh2 is identified to catalyze this H3K27me3 change, then determining cell fate of DESCs. Ezh2 defecient DESCs show impaired stemness but activated amelogenic gene amelogenin (Amelx). Conversely, elevating Ezh2 maintains pluripotency and represses Amelx, which is rescued by deleting SET trimethylase activity domain in Ezh2. Mechanistically, Ezh2 is validated to catalyze H3K27me3 within promoter of Shh in a methytransferase activity dependent manner, thus determining Shh transcript. In vitro silencing Ezh2 results in activation of Shh expression, increasing the recruitment of GLI1 within Amelx promoter, however, Ezh2 overexpression represses such Shh signaling. In vivo bivalency related Shh activation occurs in pre-ameloblasts (PAB), which directly triggers Ptch1/Gli1 signaling to enhance the enrichment of Gli1 within promoter of Amelx, finally opens the door of differentiation. These results bring light to brand new insights on role of bivalency in enamel organ formation and cell fate choices of stem cell. Funding Sources: This work was supported by National Natural Science Foundation of China (NSFC grant 81470711&81771033 to LWZ), preeminent youth fund of Sichuan province (2016JQ0054 to LWZ), Sichuan Province Science and Technology Innovation Team Program (2015TD0011 to YL, 2017TD0016 to Y.Q). Conflicts of Interest: The authors declare no potential conflicts of interest with respect to the authorship and publication of this article. Ethics Approval Statement: All mouse experiments were conducted in accordance with protocols approved by ethical committees of the West China School of Stomatology, Sichuan University and State Key Laboratory of Oral Diseases.

Bioengineered Tooth Buds Exhibit Features of Natural Tooth Buds

Tooth loss is a significant health issue currently affecting millions of people worldwide. Artificial dental implants, the current gold standard tooth replacement therapy, do not exhibit many properties of natural teeth and can be associated with complications leading to implant failure. Here we propose bioengineered tooth buds as a superior alternative tooth replacement therapy. We describe improved methods to create highly cellularized bioengineered tooth bud constructs that formed hallmark features that resemble natural tooth buds such as the dental epithelial stem cell niche, enamel knot signaling centers, transient amplifying cells, and mineralized dental tissue formation. These constructs were composed of postnatal dental cells encapsulated within a hydrogel material that were implanted subcutaneously into immunocompromised rats. To our knowledge, this is the first report describing the use of postnatal dental cells to create bioengineered tooth buds that exhibit evidence of these features of natural tooth development. We propose future bioengineered tooth buds as a promising, clinically relevant tooth replacement therapy.