The Notch-mediated circuitry in the evolution and generation of new cell lineages: the tooth model.

The Notch pathway is an ancient, evolutionary conserved intercellular signaling mechanism that is involved in cell fate specification and proper embryonic development. The Jagged2 gene, which encodes a ligand for the Notch family of receptors, is expressed from the earliest stages of odontogenesis in epithelial cells that will later generate the enamel-producing ameloblasts. Homozygous Jagged2 mutant mice exhibit abnormal tooth morphology and impaired enamel deposition. Enamel composition and structure in mammals are tightly linked to the enamel organ that represents an evolutionary unit formed by distinct dental epithelial cell types. The physical cooperativity between Notch ligands and receptors suggests that Jagged2 deletion could alter the expression profile of Notch receptors, thus modifying the whole Notch signaling cascade in cells within the enamel organ. Indeed, both Notch1 and Notch2 expression are severely disturbed in the enamel organ of Jagged2 mutant teeth. It appears that the deregulation of the Notch signaling cascade reverts the evolutionary path generating dental structures more reminiscent of the enameloid of fishes rather than of mammalian enamel. Loss of interactions between Notch and Jagged proteins may initiate the suppression of complementary dental epithelial cell fates acquired during evolution. We propose that the increased number of Notch homologues in metazoa enabled incipient sister cell types to form and maintain distinctive cell fates within organs and tissues along evolution.

Human dental pulp stem cells exhibit enhanced properties in comparison to human bone marrow stem cells on neurites outgrowth.

Mesenchymal stem cells (MSCs) have the capacity to self-renew and differentiate into specific cell types and are, therefore, key players during tissue repair and regeneration. The use of MSCs for the regeneration of tissues in vivo is increasingly being explored and already constitutes a promising alternative to existing clinical treatments. MSCs also exert paracrine and trophic functions, including the promotion of innervation that plays fundamental roles in regeneration and in restoration of the function of organs. Human bone marrow stem cells (hBMSCs) and human dental pulp stem cells (hDPSCs) have been used in studies that aimed at the repair and/or regeneration of bone or other tissues of the craniofacial complex. However, the capabilities of hBMSCs and hDPSCs to elicit the growth of specific axons in order to reestablish functional innervation of the healing tissues are not known. Here, we compared the neurotrophic effects of hDPSCs and hBMSCs on trigeminal and dorsal root ganglia neurons using microfluidic organs-on-chips devices. We found that hDPSCs express significantly higher levels of neurotrophins than hBMSCs and consequently neurons cocultured with hDPSCs develop longer axons in the microfluidic co-culture system when compared to neurons cocultured with hBMSCs. Moreover, hDPSCs elicited the formation of extensive axonal networks and established close contacts with neurons, a phenomenon not observed in presence of hBMSCs. Taken together, these findings indicate that hDPSCs constitute a superior option for restoring the functionality of damaged craniofacial tissues, as they are able to support and promote extensive trigeminal innervation.

The Versatile Roles of Nerve Growth Factor in Neuronal Attraction, Odontoblast Differentiation, and Mineral Deposition in Human Teeth.

Nerve growth factor (NGF) is an important molecule for the development and differentiation of neuronal and non-neuronal cells. Here we analyze by immunohistochemistry the distribution of NGF in the dental pulp mesenchyme of embryonic and functional human teeth. In the dental pulp of both embryonic and healthy functional teeth, NGF is mainly expressed in the odontoblasts that are responsible for dentine formation, while in functional teeth NGF is also expressed in nerve fibers innervating the dental pulp. In injured teeth, NGF is expressed in the newly formed odontoblastic-like cells, which replace the dying odontoblasts. In these teeth, NGF expression is also upregulated in the intact odontoblasts, suggesting a role for this molecule in dental tissue repair. Similarly, in cultures of human dental pulp cells, NGF expression is strongly upregulated during their differentiation into odontoblasts as well as during the mineralization process. In microfluidic devices, release of NGF from cultured human dental pulp cells induced neuronal growth from trigeminal ganglia toward the NGF secreting cells. These results show that NGF is closely linked to the various functions of odontoblasts, including secretory and neuronal attraction processes.

Notch in Head and Neck Cancer.

Head and neck cancer is a group of neoplastic diseases affecting the facial, oral, and neck region. It is one of the most common cancers worldwide with an aggressive, invasive evolution. Due to the heterogeneity of the tissues affected, it is particularly challenging to study the molecular mechanisms at the basis of these tumors, and to date we are still lacking accurate targets for prevention and therapy. The Notch signaling is involved in a variety of tumorigenic mechanisms, such as regulation of the tumor microenvironment, aberrant intercellular communication, and altered metabolism. Here, we provide an up-to-date review of the role of Notch in head and neck cancer and draw parallels with other types of solid tumors where the Notch pathway plays a crucial role in emergence, maintenance, and progression of the disease. We therefore give a perspective view on the importance of the pathway in neoplastic development in order to define future lines of research and novel therapeutic approaches.

Distinct Expression Patterns of Cxcl12 in Mesenchymal Stem Cell Niches of Intact and Injured Rodent Teeth.

Specific stem cell populations within dental mesenchymal tissues guarantee tooth homeostasis and regeneration throughout life. The decision between renewal and differentiation of stem cells is greatly influenced by interactions with stromal cells and extracellular matrix molecules that form the tissue specific stem cell niches. The Cxcl12 chemokine is a general marker of stromal cells and plays fundamental roles in the maintenance, mobilization and migration of stem cells. The aim of this study was to exploit Cxcl12-GFP transgenic mice to study the expression patterns of Cxcl12 in putative dental niches of intact and injured teeth. We showed that endothelial and stromal cells expressed Cxcl12 in the dental pulp tissue of both intact molars and incisors. Isolated non-endothelial Cxcl12+ dental pulp cells cultured in different conditions in vitro exhibited expression of both adipogenic and osteogenic markers, thus suggesting that these cells possess multipotent fates. Taken together, our results show that Cxcl12 is widely expressed in intact and injured teeth and highlight its importance as a key component of the various dental mesenchymal stem cell niches.

Three-Dimensional Culture Systems for Dissecting Notch Signalling in Health and Disease.

Three-dimensional (3D) culture systems opened up new horizons in studying the biology of tissues and organs, modelling various diseases, and screening drugs. Producing accurate in vitro models increases the possibilities for studying molecular control of cell-cell and cell-microenvironment interactions in detail. The Notch signalling is linked to cell fate determination, tissue definition, and maintenance in both physiological and pathological conditions. Hence, 3D cultures provide new accessible platforms for studying activation and modulation of the Notch pathway. In this review, we provide an overview of the recent advances in different 3D culture systems, including spheroids, organoids, and "organ-on-a-chip" models, and their use in analysing the crucial role of Notch signalling in the maintenance of tissue homeostasis, pathology, and regeneration.

Exploiting teeth as a model to study basic features of signaling pathways.

Teeth constitute a classical model for the study of signaling pathways and their roles in mediating interactions between cells and tissues in organ development, homeostasis and regeneration. Rodent teeth are mostly used as experimental models. Rodent molars have proved fundamental in the study of epithelial-mesenchymal interactions and embryonic organ morphogenesis, as well as to faithfully model human diseases affecting dental tissues. The continuously growing rodent incisor is an excellent tool for the investigation of the mechanisms regulating stem cells dynamics in homeostasis and regeneration. In this review, we discuss the use of teeth as a model to investigate signaling pathways, providing an overview of the many unique experimental approaches offered by this organ. We discuss how complex networks of signaling pathways modulate the various aspects of tooth biology, and the models used to obtain this knowledge. Finally, we introduce new experimental approaches that allow the study of more complex interactions, such as the crosstalk between dental tissues, innervation and vascularization.

Roles of innervation in developing and regenerating orofacial tissues.

The head is innervated by 12 cranial nerves (I-XII) that regulate its sensory and motor functions. Cranial nerves are composed of sensory, motor, or mixed neuronal populations. Sensory neurons perceive generally somatic sensations such as pressure, pain, and temperature. These neurons are also involved in smell, vision, taste, and hearing. Motor neurons ensure the motility of all muscles and glands. Innervation plays an essential role in the development of the various orofacial structures during embryogenesis. Hypoplastic cranial nerves often lead to abnormal development of their target organs and tissues. For example, Möbius syndrome is a congenital disease characterized by defective innervation (i.e., abducens (VI) and facial (VII) nerves), deafness, tooth anomalies, and cleft palate. Hence, it is obvious that the peripheral nervous system is needed for both development and function of orofacial structures. Nerves have a limited capacity to regenerate. However, neural stem cells, which could be used as sources for neural tissue maintenance and repair, have been found in adult neuronal tissues. Similarly, various adult stem cell populations have been isolated from almost all organs of the human body. Stem cells are tightly regulated by their microenvironment, the stem cell niche. Deregulation of adult stem cell behavior results in the development of pathologies such as tumor formation or early tissue senescence. It is thus essential to understand the factors that regulate the functions and maintenance of stem cells. Yet, the potential importance of innervation in the regulation of stem cells and/or their niches in most organs and tissues is largely unexplored. This review focuses on the potential role of innervation in the development and homeostasis of orofacial structures and discusses its possible association with stem cell populations during tissue repair.

E-cadherin regulates the behavior and fate of epithelial stem cells and their progeny in the mouse incisor.

Stem cells are essential for the regeneration and homeostasis of many organs, such as tooth, hair, skin, and intestine. Although human tooth regeneration is limited, a number of animals have evolved continuously growing teeth that provide models of stem cell-based organ renewal. A well-studied model is the mouse incisor, which contains dental epithelial stem cells in structures known as cervical loops. These stem cells produce progeny that proliferate and migrate along the proximo-distal axis of the incisor and differentiate into enamel-forming ameloblasts. Here, we studied the role of E-cadherin in behavior of the stem cells and their progeny. Levels of E-cadherin are highly dynamic in the incisor, such that E-cadherin is expressed in the stem cells, downregulated in the transit-amplifying cells, re-expressed in the pre-ameloblasts and then downregulated again in the ameloblasts. Conditional inactivation of E-cadherin in the cervical loop led to decreased numbers of label-retaining stem cells, increased proliferation, and decreased cell migration in the mouse incisor. Using both genetic and pharmacological approaches, we showed that Fibroblast Growth Factors regulate E-cadherin expression, cell proliferation and migration in the incisor. Together, our data indicate that E-cadherin is an important regulator of stem cells and their progeny during growth of the mouse incisor.

Noggin null allele mice exhibit a microform of holoprosencephaly.

Holoprosencephaly (HPE) is a heterogeneous craniofacial and neural developmental anomaly characterized in its most severe form by the failure of the forebrain to divide. In humans, HPE is associated with disruption of Sonic hedgehog and Nodal signaling pathways, but the role of other signaling pathways has not yet been determined. In this study, we analyzed mice which, due to the lack of the Bmp antagonist Noggin, exhibit elevated Bmp signaling. Noggin(-/-) mice exhibited a solitary median maxillary incisor that developed from a single dental placode, early midfacial narrowing as well as abnormalities in the developing hyoid bone, pituitary gland and vomeronasal organ. In Noggin(-/-) mice, the expression domains of Shh, as well as the Shh target genes Ptch1 and Gli1, were reduced in the frontonasal region at key stages of early facial development. Using E10.5 facial cultures, we show that excessive BMP4 results in reduced Fgf8 and Ptch1 expression. These data suggest that increased Bmp signaling in Noggin(-/-) mice results in downregulation of the hedgehog pathway at a critical stage when the midline craniofacial structures are developing, which leads to a phenotype consistent with a microform of HPE.

BMPs and FGFs target Notch signalling via jagged 2 to regulate tooth morphogenesis and cytodifferentiation.

The Notch signalling pathway is an evolutionarily conserved intercellular signalling mechanism that is essential for cell fate specification and proper embryonic development. We have analysed the expression, regulation and function of the jagged 2 (Jag2) gene, which encodes a ligand for the Notch family of receptors, in developing mouse teeth. Jag2 is expressed in epithelial cells that give rise to the enamel-producing ameloblasts from the earliest stages of tooth development. Tissue recombination experiments showed that its expression in epithelium is regulated by mesenchyme-derived signals. In dental explants cultured in vitro, the local application of fibroblast growth factors upregulated Jag2 expression, whereas bone morphogenetic proteins provoked the opposite effect. Mice homozygous for a deletion in the Notch-interaction domain of Jag2 presented a variety of severe dental abnormalities. In molars, the crown morphology was misshapen, with additional cusps being formed. This was due to alterations in the enamel knot, an epithelial signalling structure involved in molar crown morphogenesis, in which Bmp4 expression and apoptosis were altered. In incisors, cytodifferentiation and enamel matrix deposition were inhibited. The expression of Tbx1 in ameloblast progenitors, which is a hallmark for ameloblast differentiation and enamel formation, was dramatically reduced in Jag2(-/-) teeth. Together, these results demonstrate that Notch signalling mediated by Jag2 is indispensable for normal tooth development.

Kidney capsule transplantation of chemically treated embryonic murine tooth germs.

Subcapsular transplantation of developing tissues and organs into the richly vascularized murine kidney provides the necessary trophic support, thus ensuring proper completion of their growth.1,2,3 Here, we provide a protocol for kidney capsule transplantation that allows the full differentiation of embryonic teeth previously exposed to chemicals. We describe steps for dissection and in vitro culture of embryonic teeth, followed by transplantation of tooth germs. We then detail harvesting of kidneys for further analysis. For complete details on the use and execution of this protocol, please refer to Mitsiadis et al.4.

Extracellular matrix remodelling in dental pulp tissue of carious human teeth through the prism of single-cell RNA sequencing.

Carious lesions are bacteria-caused destructions of the mineralised dental tissues, marked by the simultaneous activation of immune responses and regenerative events within the soft dental pulp tissue. While major molecular players in tooth decay have been uncovered during the past years, a detailed map of the molecular and cellular landscape of the diseased pulp is still missing. In this study we used single-cell RNA sequencing analysis, supplemented with immunostaining, to generate a comprehensive single-cell atlas of the pulp of carious human teeth. Our data demonstrated modifications in the various cell clusters within the pulp of carious teeth, such as immune cells, mesenchymal stem cells (MSC) and fibroblasts, when compared to the pulp of healthy human teeth. Active immune response in the carious pulp tissue is accompanied by specific changes in the fibroblast and MSC clusters. These changes include the upregulation of genes encoding extracellular matrix (ECM) components, including COL1A1 and Fibronectin (FN1), and the enrichment of the fibroblast cluster with myofibroblasts. The incremental changes in the ECM composition of carious pulp tissues were further confirmed by immunostaining analyses. Assessment of the Fibronectin fibres under mechanical strain conditions showed a significant tension reduction in carious pulp tissues, compared to the healthy ones. The present data demonstrate molecular, cellular and biomechanical alterations in the pulp of human carious teeth, indicative of extensive ECM remodelling, reminiscent of fibrosis observed in other organs. This comprehensive atlas of carious human teeth can facilitate future studies of dental pathologies and enable comparative analyses across diseased organs.

Notch Signaling Pathway in Tooth Shape Variations throughout Evolution.

Evolutionary changes in vertebrates are linked to genetic alterations that often affect tooth crown shape, which is a criterion of speciation events. The Notch pathway is highly conserved between species and controls morphogenetic processes in most developing organs, including teeth. Epithelial loss of the Notch-ligand Jagged1 in developing mouse molars affects the location, size and interconnections of their cusps that lead to minor tooth crown shape modifications convergent to those observed along Muridae evolution. RNA sequencing analysis revealed that these alterations are due to the modulation of more than 2000 genes and that Notch signaling is a hub for significant morphogenetic networks, such as Wnts and Fibroblast Growth Factors. The modeling of these tooth crown changes in mutant mice, via a three-dimensional metamorphosis approach, allowed prediction of how Jagged1-associated mutations in humans could affect the morphology of their teeth. These results shed new light on Notch/Jagged1-mediated signaling as one of the crucial components for dental variations in evolution.

Exosomes as Carriers for Notch Molecules.

Exosomes are extracellular vesicles involved in cell-to-cell communication as well as extrusion of biological material. Using dental pulp stem cells culture as a model, we hereby describe a method for the packaging of Delta-like 4 (DLL4), a representative Notch ligand, into newly generated exosomes. We then provide methods of analysis to confirm the presence of Notch proteins and transcripts internalization and transport via exosomes.

Adam10-dependent Notch signaling establishes dental epithelial cell boundaries required for enamel formation.

The disintegrin and metalloproteinase Adam10 is a membrane-bound sheddase that regulates Notch signaling and ensures epidermal integrity. To address the function of Adam10 in the continuously growing incisors, we used Keratin14 Cre/+;Adam10 fl/fl transgenic mice, in which Adam10 is conditionally deleted in the dental epithelium. Keratin14 Cre/+;Adam10 fl/fl mice exhibited severe abnormalities, including defective enamel formation reminiscent of human enamel pathologies. Histological analyses of mutant incisors revealed absence of stratum intermedium, and severe disorganization of enamel-secreting ameloblasts. In situ hybridization and immunostaining analyses in the Keratin14 Cre/+;Adam10 fl/fl incisors showed strong Notch1 downregulation in dental epithelium and ectopic distribution of enamel-specific molecules, including ameloblastin and amelogenin. Lineage tracing studies using Notch1 CreERT2 ;R26 mT/mG mice demonstrated that loss of the stratum intermedium cells was due to their fate switch toward the ameloblast lineage. Overall, our data reveal that in the continuously growing incisors the Adam10/Notch axis controls dental epithelial cell boundaries, cell fate switch and proper enamel formation.

Nogo-A Regulates the Fate of Human Dental Pulp Stem Cells toward Osteogenic, Adipogenic, and Neurogenic Differentiation.

Human teeth are highly innervated organs that contain a variety of mesenchymal stem cell populations that could be used for cell-based regenerative therapies. Specific molecules are often used in these treatments to favorably modulate the function and fate of stem cells. Nogo-A, a key regulator of neuronal growth and differentiation, is already used in clinical tissue regeneration trials. While the functions of Nogo-A in neuronal tissues are extensively explored, its role in teeth still remains unknown. In this work, we first immunohistochemically analyzed the distribution of Nogo-A protein in the dental pulp of human teeth. Nogo-A is localized in a variety of cellular and structural components of the dental pulp, including odontoblasts, fibroblasts, neurons and vessels. We also cross-examined Nogo expression in the various pulp cell clusters in a single cell RNA sequencing dataset of human dental pulp, which showed high levels of expression in all cell clusters, including that of stem cells. We then assessed the role of Nogo-A on the fate of human dental pulp stem cells and their differentiation capacity in vitro. Using immunostaining, Alizarin Red S, Nile Red and Oil Red O staining we showed that Nogo-A delayed the differentiation of cultured dental pulp stem cells toward the osteogenic, adipogenic and neurogenic lineages, while addition of the blocking anti-Nogo-A antibody had opposite effects. These results were further confirmed by qRT-PCR, which demonstrated overexpression of genes involved in osteogenic (RUNX2, ALP, SP7/OSX), adipogenic (PPAR-γ2, LPL) and neurogenic (DCX, TUBB3, NEFL) differentiation in the presence of the anti-Nogo-A antibody. Conversely, the osteogenic and adipogenic genes were downregulated by Nogo-A. Taken together, our results show that the functions of Nogo-A are not restricted to neuronal cells but are extended to other cell populations, including dental pulp stem cells. We show that Nogo-A regulates their fates toward osteogenic, adipogenic and neurogenic differentiation, thus indicating its potential use in clinics.

Future dentistry: cell therapy meets tooth and periodontal repair and regeneration.

Cell-based tissue repair of the tooth and - tooth-supporting - periodontal ligament (PDL) is a new attractive approach that complements traditional restorative or surgical techniques for replacement of injured or pathologically damaged tissues. In such therapeutic approaches, stem cells and/or progenitor cells are manipulated in vitro and administered to patients as living and dynamic biological agents. In this review, we discuss the clonogenic potential of human dental and periodontal tissues such as the dental pulp and the PDL and their potential for tooth and periodontal repair and/or regeneration. We propose novel therapeutic approaches using stem cells or progenitor cells, which are targeted to regenerate the lost dental or periodontal tissue.

The genetic basis of craniofacial and dental abnormalities.

The embryonic head development, including the formation of dental structures, is a complex and delicate process guided by specific genetic programs. Genetic changes and environmental factors can disturb the execution of these programs and result in abnormalities in orofacial and dental structures. Orofacial clefts and hypodontia/ oligodontia are examples of such abnormalities frequently seen in dental clinics. An insight into the mechanisms and genes involved in the formation of orofacial and dental structures has been gradually gained by genetic analysis of families and by the use of experimental vertebrate models such as the mouse and chick models. The development of novel clinical therapies for orofacial and dental pathological conditions depends very much on a detailed knowledge of the molecular and cellular processes that are involved in head formation.

New Scenarios in Pharmacological Treatments of Head and Neck Squamous Cell Carcinomas.

Head and neck squamous cell carcinoma (HNSCC) is one of the most frequent types of cancer with a lethal outcome in half of the diagnosed cases. Mostly, HNSCC develops in the oral cavity, and its development is associated with tobacco and areca nut/betel quid usage, alcohol consumption, and HPV infection. Oral squamous cell carcinoma, as other head and neck cancers, presents a high degree of intratumor heterogeneity, which makes their treatment difficult, and directly correlates with drug resistance. Since the classical treatments for HNSCC oftentimes do not resolve the clinical picture, there is great need for novel therapeutic approaches, models for drug testing, and new drug delivery systems.