Search

PDF - 48K, Table S1. List of utilized primers. Table S2. List of utilized antibodies.

Pancreatic ductal adenocarcinoma is one of the deadliest carcinomas and is characterized by highly tumorigenic and metastatic cancer stem cells (CSC). CSCs evade available therapies, which preferentially target highly proliferative and more differentiated progenies, leaving behind CSCs as a putative source for disease relapse. Thus, to identify potentially more effective treatment regimens, we screened established and new compounds for their ability to eliminate CSCs in primary pancreatic cancer (stem) cells in vitro and corresponding patient-derived pancreatic cancer tissue xenografts in vivo. Intriguingly, we found that in vitro treatment with the antimalarial agent chloroquine significantly decreased CSCs, translating into diminished in vivo tumorigenicity and invasiveness in a large panel of pancreatic cancers. In vivo treatment in combination with gemcitabine was capable of more effectively eliminating established tumors and improved overall survival. The inhibitory effect of chloroquine was not related to inhibition of autophagy, but was due to inhibition of CXCL12/CXCR4 signaling, resulting in reduced phosphorylation of ERK and STAT3. Furthermore, chloroquine showed potent inhibition of hedgehog signaling by decreasing the production of Smoothened, translating into a significant reduction in sonic hedgehog-induced chemotaxis and downregulation of downstream targets in CSCs and the surrounding stroma. Our study demonstrates that via to date unreported effects, chloroquine is an effective adjuvant therapy to chemotherapy, offering more efficient tumor elimination and improved cure rates. Chloroquine should be further explored in the clinical setting as its success may help to more rapidly improve the poor prognosis of patients with pancreatic cancer. Mol Cancer Ther; 13(7); 1758–71. ©2014 AACR.

PDF - 3180K, Figure S1. Chloroquine does not affect differentiated cancer cells. Figure S2. Chloroquine eliminates pancreatic cancer stem cells. Figure S3. Autophagy is not mediating the effect of chloroquine on primary PDAC cells. Figure S4. Chloroquine has no effect on cell migration of non-CSCs Figure S5 - Chloroquine displays negative in vitro effect on the Shh signaling. Figure S6. Inhibitory in vivo effect of chloroquine on stromal hedgehog signaling and expansion of the stroma.

PDF - 147K, Supplementary methods including related references.

The carious lesion is a bacteria caused destruction of tooth mineralized matrices marked by concurrent tissue reparative and immune responses in the dental pulp. While major molecular players in tooth pulp decay have been uncovered, a detailed map of the molecular and cellular landscape of the diseased pulp is still missing. Here we used single-cell RNA sequencing analysis, to generate a comprehensive single-cell atlas of the carious human dental pulp tissue. Our data demonstrated modifications in various cell clusters of the carious pulp, such as immune cells, mesenchymal stem cells (MSC) and fibroblasts, when compared to the healthy dental pulp. These changes include upregulation of genes encoding extracellular matrix (ECM) components and the enrichment of the fibroblast cluster with myofibroblasts. Assessment of the Fibronectin fibres’ mechanical strain showed a significant tension reduction in the carious human pulp, compared to the healthy one. Collectively, the present data demonstrate molecular, cellular and biomechanical alterations in the carious pulp tissue, indicative of extensive ECM remodelling and reminiscent of fibrosis observed in other organs.

Continuous growth of the mouse incisor teeth is due to the life-long maintenance of epithelial stem cells (SCs) in their niche called cervical loop (CL). Several signaling factors regulate SC maintenance and/or their differentiation to achieve organ homeostasis. Previous studies indicated that Hedgehog signaling is crucial for both the maintenance of the SCs in the niche, as well as for their differentiation. How Hedgehog signaling regulates these two opposing cellular behaviors within the confinement of the CL remains elusive. In this study, we used in vitro organ and cell cultures to pharmacologically attenuate Hedgehog signaling. We analyzed expression of various genes expressed in the SC niche to determine the effect of altered Hedgehog signaling on the cellular hierarchy within the niche. These genes include markers of SCs (Sox2 and Lgr5) and transit-amplifying cells (P-cadherin, Sonic Hedgehog, and Yap). Our results show that Hedgehog signaling is a critical survival factor for SCs in the niche, and that the architecture and the diversity of the SC niche are regulated by multiple Hedgehog ligands. We demonstrated the presence of an additional Hedgehog ligand, nerve-derived Desert Hedgehog, secreted in the proximity of the CL. In addition, we provide evidence that Hedgehog receptors Ptch1 and Ptch2 elicit independent responses, which enable multimodal Hedgehog signaling to simultaneously regulate SC maintenance and differentiation. Our study indicates that the cellular hierarchy in the continuously growing incisor is a result of complex interplay of two Hedgehog ligands with functionally distinct Ptch receptors. Stem Cells 2019;37:1238–1248

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

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., Unlike human teeth, mouse incisors grow throughout life, based on stem and progenitor cell activity. Here the authors generate single cell RNA-seq comparative maps of continuously-growing mouse incisor, non-growing mouse molar and human teeth, combined with lineage tracing to reveal dental cell complexity.

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.

The tooth is an intricate composition of precisely patterned, mineralized matrices and soft tissues. Mineralized tissues include enamel (produced by the epithelial cells called ameloblasts), dentin and cementum (produced by mesenchymal cells called odontoblasts and cementoblasts, respectively), and soft tissues, which include the dental pulp and the periodontal ligament along with the invading nerves and blood vessels. It was perceived for a very long time that teeth primarily serve an esthetical function. In recent years, however, the role of healthy teeth, as well as the impact of oral health on general well-being, became more evident. Tooth loss, caused by tooth decay, congenital malformations (tooth agenesis), trauma, periodontal diseases, or age-related changes, is usually replaced by artificial materials which lack many of the important biological characteristics of the natural tooth. Human teeth have very low to almost absent regeneration potential, due to early loss of cell populations with regenerative capacity, namely stem cells. Significant effort has been made in recent decades to identify and characterize tooth stem cells, and to unravel the developmental programs which these cells follow in order to generate a tooth.

Continuous growth of the rodent incisor is enabled by epithelial and mesenchymal stem cells (ESCs and MSCs) which unceasingly replenish enamel and dentin, respectively, that wear by persistent animal gnawing. Lineage tracing studies have provided evidence that ESCs contribute to all epithelial lineages of the tooth in vivo. Meanwhile, in the mouse incisor, MSCs continuously contribute to odontoblast lineage and tooth growth. However, in vitro manipulation of ESCs has shown little progress, mainly due to lack of appropriate protocol to successfully isolate, culture, expand, and differentiate ESCs in vitro without using the co-culture system. In this chapter we describe the isolation of the Sox2-GFP+ cell population that is highly enriched in ESCs. Isolated cells can be used for various types of analyses, including in vitro culture, single cell-related analyses, etc. Furthermore, we describe ways to obtain populations enriched in the incisor MSCs using FACS sorting of antibody-labeled cells. Easily accessible FACS sorting enables easy and relatively fast isolation of the cells labeled by the fluorescent protein.

Continuous growth of the rodent incisor is enabled by epithelial and mesenchymal stem cells (ESCs and MSCs) which unceasingly replenish enamel and dentin, respectively, that wear by persistent animal gnawing. Lineage tracing studies have provided evidence that ESCs contribute to all epithelial lineages of the tooth in vivo. Meanwhile, in the mouse incisor, MSCs continuously contribute to odontoblast lineage and tooth growth. However, in vitro manipulation of ESCs has shown little progress, mainly due to lack of appropriate protocol to successfully isolate, culture, expand, and differentiate ESCs in vitro without using the co-culture system. In this chapter we describe the isolation of the Sox2-GFP+ cell population that is highly enriched in ESCs. Isolated cells can be used for various types of analyses, including in vitro culture, single cell-related analyses, etc. Furthermore, we describe ways to obtain populations enriched in the incisor MSCs using FACS sorting of antibody-labeled cells. Easily accessible FACS sorting enables easy and relatively fast isolation of the cells labeled by the fluorescent protein. © Springer Science+Business Media, LLC, part of Springer Nature 2019.

Development of teeth depends on the reciprocal interactions between the surface epithelium (ectoderm) and the underlying neural crest-derived mesenchyme. These interactions are facilitated by the conserved signaling pathways, which build a complex network of signals and transcription factors. Tooth development starts at specific and predetermined loci in the oral ectoderm and is described as a morphologically distinct thickening of oral ectoderm, named dental lamina. Cells within the dental lamina invaginate into the underlying mesenchyme, generating placodes that mark the onset of individual tooth development. In the following stages of development, the tooth epithelium buds and folds transitioning through various shapes, including bud, cap, and bell shapes, which also identify the specific stages of tooth development. Although much of the molecular regulation of tooth development has been unraveled, the regulation of the initial stages of tooth development, as well as the cellular mechanisms that govern tooth development remain largely unknown. This review provides a systematic overview of the current knowledge on the molecular and cellular mechanisms that guide initial stages of tooth development and outlines the challenges which temper the progress. Stem Cells 2019;37:26–32

Continuous growth of rodent incisors relies on epithelial stem cells (SCs) located in the SC niche called labial cervical loop (LaCL). Here, we found a population of apoptotic cells residing in a specific location of the LaCL in mouse incisor. Activated Caspase 3 and Caspase 9, expressed in this location colocalized in part with Lgr5 in putative SCs. The addition of Caspase inhibitors to incisors ex vivo resulted in concentration dependent thickening of LaCL. To examine the role of Wnt signaling in regulation of apoptosis, we exposed the LaCL of postnatal day 2 (P2) mouse incisor ex vivo to BIO, a known activator of Wnt/β-catenin signaling. This resulted in marked thinning of LaCL as well as enhanced apoptosis. We found that Wnt/β-catenin signaling was intensely induced by BIO in the mesenchyme surrounding the LaCL, but, unexpectedly, no β-catenin activity was detected in the LaCL epithelium either before or after BIO treatment. We discovered that the expression of Fgf10, an essential growth factor for incisor epithelial SCs, was dramatically downregulated in the mesenchyme around BIO-treated LaCL, and that exogenous Fgf10 could rescue the thinning of the LaCL caused by BIO. We conclude that the homeostasis of the epithelial SC population in the mouse incisor depends on a proper rate of apoptosis and that this apoptosis is controlled by signals from the mesenchyme surrounding the LaCL. Fgf10 is a key mesenchymal signal limiting apoptosis of incisor epithelial SCs and its expression is negatively regulated by Wnt/β-catenin. Stem Cells 2015;33:1670–1681

Tooth agenesis is one of the predominant developmental anomalies in humans, usually affecting the permanent dentition generated by sequential tooth formation and, in most cases, caused by mutations perturbing epithelial Wnt/β-catenin signaling. In addition, loss-of-function mutations in the Wnt feedback inhibitor AXIN2 lead to human tooth agenesis. We have investigated the functions of Wnt/β-catenin signaling during sequential formation of molar teeth using mouse models. Continuous initiation of new teeth, which is observed after genetic activation of Wnt/β-catenin signaling in the oral epithelium, was accompanied by enhanced expression of Wnt antagonists and a downregulation of Wnt/β-catenin signaling in the dental mesenchyme. Genetic and pharmacological activation of mesenchymal Wnt/β-catenin signaling negatively regulated sequential tooth formation, an effect partly mediated by Bmp4. Runx2, a gene whose loss-of-function mutations result in sequential formation of supernumerary teeth in the human cleidocranial dysplasia syndrome, suppressed the expression of Wnt inhibitors Axin2 and Drapc1 in dental mesenchyme. Our data indicate that increased mesenchymal Wnt signaling inhibits the sequential formation of teeth, and suggest that Axin2/Runx2 antagonistic interactions modulate the level of mesenchymal Wnt/β-catenin signaling, underlying the contrasting dental phenotypes caused by human AXIN2 and RUNX2 mutations.

In past decades, morphologic, molecular, and cellular mechanisms that govern tooth development have been extensively studied. These studies demonstrated that the same signaling pathways regulate development of the primary and successional teeth. Mutations of these pathways lead to abnormalities in tooth development and number, including aberrant tooth shape, tooth agenesis, and formation of extra teeth. Here, we summarize the current knowledge on the development of the primary and successional teeth in animal models and describe some of the common tooth abnormalities in humans.

Cancer stem cells (CSCs) are thought to drive tumor growth, metastasis and chemoresistance. Although surface markers such as CD133 and CD44 have been successfully used to isolate CSCs, their expression is not exclusively linked to the CSC phenotype and is prone to environmental alteration. We identified cells with an autofluorescent subcellular compartment that exclusively showed CSC features across different human tumor types. Primary tumor-derived autofluorescent cells did not overlap with side-population (SP) cells, were enriched in sphere culture and during chemotherapy, strongly expressed pluripotency-associated genes, were highly metastatic and showed long-term in vivo tumorigenicity, even at the single-cell level. Autofluorescence was due to riboflavin accumulation in membrane-bounded cytoplasmic structures bearing ATP-dependent ABCG2 transporters. In summary, we identified and characterized an intrinsic autofluorescent phenotype in CSCs of diverse epithelial cancers and used this marker to isolate and characterize these cells.

Pancreatic ductal adenocarcinoma is one of the deadliest carcinomas and is characterized by highly tumorigenic and metastatic cancer stem cells (CSC). CSCs evade available therapies, which preferentially target highly proliferative and more differentiated progenies, leaving behind CSCs as a putative source for disease relapse. Thus, to identify potentially more effective treatment regimens, we screened established and new compounds for their ability to eliminate CSCs in primary pancreatic cancer (stem) cells in vitro and corresponding patient-derived pancreatic cancer tissue xenografts in vivo. Intriguingly, we found that in vitro treatment with the antimalarial agent chloroquine significantly decreased CSCs, translating into diminished in vivo tumorigenicity and invasiveness in a large panel of pancreatic cancers. In vivo treatment in combination with gemcitabine was capable of more effectively eliminating established tumors and improved overall survival. The inhibitory effect of chloroquine was not related to inhibition of autophagy, but was due to inhibition of CXCL12/CXCR4 signaling, resulting in reduced phosphorylation of ERK and STAT3. Furthermore, chloroquine showed potent inhibition of hedgehog signaling by decreasing the production of Smoothened, translating into a significant reduction in sonic hedgehog-induced chemotaxis and downregulation of downstream targets in CSCs and the surrounding stroma. Our study demonstrates that via to date unreported effects, chloroquine is an effective adjuvant therapy to chemotherapy, offering more efficient tumor elimination and improved cure rates. Chloroquine should be further explored in the clinical setting as its success may help to more rapidly improve the poor prognosis of patients with pancreatic cancer. Mol Cancer Ther; 13(7); 1758–71. ©2014 AACR.

Frozoni M, Balic A, Sagomonyants K, Zaia AA, Line SRP, Mina M. A feasibility study for the analysis of reparative dentinogenesis in pOBCol3.6GFPtpz transgenic mice. International Endodontic Journal, 45, 907–914, 2012. Abstract Aim To examine the feasibility of using the pOBCol3.6GFPtpz [3.6-green fluorescent protein (GFP)] transgenic mice as an in vivo model for studying the biological sequence of events during pulp healing and reparative dentinogenesis. Methodology Pulp exposures were created in the first maxillary molar of 12–16-week-old 3.6-GFP transgenic mice with CD1 and C57/Bl6 genetic background. Direct pulp capping on exposed teeth was performed using mineral trioxide aggregate followed by restoration with a light-cured adhesive system (AS) and composite resin. In control teeth, the AS was placed in direct contact with the pulp. Animals were euthanized at various time points after pulp exposure and capping. The maxillary arch was isolated, fixed and processed for histological and epifluorescence analysis to examine reparative dentinogenesis. Results Analysis of teeth immediately after pulp exposure revealed absence of odontoblasts expressing 3.6-GFP at the injury site. Evidence of reparative dentinogenesis was apparent at 4 weeks in 3.6-GFP mice in CD1 background and at 8 weeks in 3.6-GFP mice with C57/Bl6 background. The reparative dentine with both groups contained newly formed atubular-mineralized tissue resembling a dentine bridge and/or osteodentine that was lined by cells expressing 3.6-GFP as well as 3.6-GFP expressing cells embedded within the atubular matrix. Conclusion This study was conducted in a few animals and did not allow statistical analysis. The results revealed that the 3.6-GFP transgenic animals provide a unique model for direct analysis of cellular and molecular mechanisms of pulp repair and tertiary dentinogenesis in vivo. The study also shows the effects of the capping material and the genetic background of the mice in the sequence and timing of reparative dentinogenesis.

Transgenic mouse lines in which GFP expression is under the control of tissue- and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stage of differentiation along a lineage. In the present study, we used primary cell cultures derived from the dental pulp from pOBCol3.6GFP and pOBCol2.3GFP transgenic mice as a model to develop markers for early stages of odontoblast differentiation from progenitor cells. We analyzed the temporal and spatial expression of 2.3-GFP and 3.6-GFP during in vitro mineralization. Using FACS to separate cells based on GFP expression, we obtained relatively homogenous subpopulations of cells and analyzed their dentinogenic potentials and their progression into odontoblasts. Our observations showed that these transgenes were activated before the onset of matrix deposition and in cells at different stages of polarization. The 3.6-GFP transgene was activated in cells in early stages of polarization, whereas the 2.3-GFP transgene was activated at a later stage of polarization just before or at the time of formation of secretory odontoblast.

The continuous growth of rodent incisors requires the presence of stem cells capable of generating ameloblasts and odontoblasts. While epithelial stem cells giving rise to ameloblasts have been well-characterized, cells giving rise to the odontoblasts in incisors have not been fully characterized. The goal of this study was to gain insight into the potential population in dental pulps of unerupted and erupted incisors that give rise to odontoblasts. We show that pulps from unerupted incisors contain a significant mesenchymal-stem-cell (MSC)-like population (cells expressing CD90+/CD45-, CD117+/CD45-, Sca-1+/CD45-) and few CD45+ cells. Our in vitro studies showed that these cells displayed extensive osteo-dentinogenic potential, but were unable to differentiate into chondrocytes and adipocytes. Dental pulps from erupted incisors displayed increased percentages of CD45+ and decreased percentages of cells expressing markers of an MSC-like population. Despite these differences, pulps from erupted incisors also displayed extensive osteo-dentinogenic potential and inability to differentiate into chondrocytes and adipocytes. These results provide evidence that continuous generation of odontoblasts and dentin on the labial and lingual sides of unerupted and erupted incisors is supported by a progenitor population and not multipotent MSCs in the dental pulp.

Lyme disease is caused by transmission of the spirochete Borrelia burgdorferi from ticks to humans. Although much is known about B. burgdorferi replication, the routes and mechanisms by which it disseminates within the tick remain unclear. To better understand this process, we imaged live, infectious B. burgdorferi expressing a stably integrated, constitutively expressed GFP reporter. Using isolated tick midguts and salivary glands, we observed B. burgdorferi progress through the feeding tick via what we believe to be a novel, biphasic mode of dissemination. In the first phase, replicating spirochetes, positioned at varying depths throughout the midgut at the onset of feeding, formed networks of nonmotile organisms that advanced toward the basolateral surface of the epithelium while adhering to differentiating, hypertrophying, and detaching epithelial cells. In the second phase of dissemination, the nonmotile spirochetes transitioned into motile organisms that penetrated the basement membrane and entered the hemocoel, then migrated to and entered the salivary glands. We designated the first phase of dissemination “adherence-mediated migration” and provided evidence that it involves the inhibition of spirochete motility by one or more diffusible factors elaborated by the feeding tick midgut. Our studies, which we believe are the first to relate the transmission dynamics of spirochetes to the complex morphological and developmental changes that the midgut and salivary glands undergo during engorgement, challenge the conventional viewpoint that dissemination of Lyme disease–causing spirochetes within ticks is exclusively motility driven.

We have examined and compared the effects of various differentiation-inducing media on mineralization, cell morphology and expression of pOBCol3.6GFP (3.6-GFP) in primary dental pulp cultures derived from 3.6-GFP transgenic mice. Our results show that media containing ascorbic acid only could not induce mineralization in primary dental pulp cultures. On the other hand, media containing ascorbic acid and β-glycerophosphate induced formation of mineralized matrix-containing dentin. The amount of mineralized matrix was increased by addition of dexamethasone. Cells treated with ascorbic acid and β-glycerophosphate were fibroblast like and cells treated with dexamethasone were cuboidal. In all culture conditions, high levels of 3.6-GFP were expressed in areas of mineralization

Reciprocal interactions between epithelial and mesenchymal tissues play a fundamental role in the morphogenesis of teeth and regulate all aspects of tooth development. Extensive studies on mouse tooth development over the past 25 years have uncovered the molecular details of the signaling networks mediating these interactions (reviewed by JussilaThesleff, 2012; Lan, Jia,Jiang, 2014). Five conserved signaling pathways, namely, the Wnt, BMP, FGF, Shh, and Eda, are involved in the mediation of the successive reciprocal epithelial-mesenchymal cross talk which follows the general principle of morphogenetic interactions (Davidson, 1993). The pathways regulate the expression of transcription factors which confer the identity of dental epithelium and mesenchyme. The signals and transcription factors are integrated in complex signaling networks whose fine-tuning allows the generation of the variation in tooth morphologies. In this review, we describe the principles and molecular mechanisms of the epithelial-mesenchymal interactions regulating successive stages of tooth formation: (i) the initiation of tooth development, with special reference to the shift of tooth-forming potential from epithelium to mesenchyme; (ii) the morphogenesis of the tooth crown, focusing on the roles of epithelial signaling centers; (iii) the differentiation of odontoblasts and ameloblasts, which produce dentin and enamel, respectively; and (iv) the maintenance of dental stem cells, which support the continuous growth of teeth.

Epithelial morphogenesis generates the shape of the tooth crown. This is driven by patterned differentiation of cells into enamel knots, root-forming cervical loops and enamel-forming ameloblasts. Enamel knots are signaling centers that define the positions of cusp tips in a tooth by instructing the adjacent epithelium to fold and proliferate. Here we show that the forkhead-box transcription factor Foxi3 inhibits formation of enamel knots and cervical loops and thus the differentiation of dental epithelium. Conditional deletion of Foxi3 (Foxi3 cKO) led to fusion of molars with abnormally patterned shallow cusps. Foxi3 was expressed in the epithelium, and its expression was reduced in the enamel knots and cervical loops as well as in ameloblasts. Bmp4, a known inducer of enamel knots and dental epithelial differentiation, downregulated Foxi3 in wild-type teeth. Using genome-wide gene expression profiling we showed that in Foxi3 cKO there was an early upregulation of differentiation markers, such as p21, Fgf15 and Sfrp5. Different signaling pathway components that are normally restricted to the enamel knots were expanded in the epithelium, and Sostdc1, a marker of the intercuspal epithelium was missing. These findings indicated that the activator-inhibitor balance regulating cusp patterning was disrupted in Foxi3 cKO. In addition, early molar bud morphogenesis and, in particular, formation of the suprabasal epithelial cell layer were impaired. We identified Keratin-10 as a marker of suprabasal epithelial cells in teeth. Our results suggest that Foxi3 maintains dental epithelial cells in an undifferentiated state and thereby regulates multiple stages of tooth morphogenesis.

Structured Abstract Authors – Balic A, Mina M Background – In recent years there has been increasing progress in identifying stem cells from adult tissues and their potential application for tooth replacement/regeneration. Our previous in vivo studies show that pOBCol3.6GFP and pOBCol2.3GFP transgenic animals provide a unique model to gain insight into progenitor/stem cells in the dental pulp capable of giving rise to odontoblasts. Objectives – To characterize the behavior of dental pulp cells derived from pOBCol3.6GFP animals in vitro. Experimental design – Primary cultures were established from the coronal portions of the pulps isolated first molars from 5-day-old pOBCol3.6GFP heterozygous mice and grown for 21 days. In these cultures proliferation, clonogenic capacity, activation of 3.6-GFP and mineralization were examined. Results – Our observations show that dental pulp cells derived from 3.6-GFP contain a population of proliferative, clonogenic cells with the ability to mineralize. We also show the stage specific activation/upregulation of 3.6-GFP in primary cultures derived from dental pulp. In these cultures, expression of Col1a1-3.6-GFP occurs prior to the appearance of mineralized nodules and is unregulated in mineralized nodules. Conclusions – Col1a1-GFP transgenes appear to fulfill many of the requirements of a marker gene for cell lineage studies in intact tooth and primary cultures derived from dental pulp.

Reciprocal interactions between epithelial and mesenchymal tissues play a fundamental role in the morphogenesis of teeth and regulate all aspects of tooth development. Extensive studies on mouse tooth development over the past 25 years have uncovered the molecular details of the signaling networks mediating these interactions (reviewed by Jussila & Thesleff, 2012; Lan, Jia, & Jiang, 2014). Five conserved signaling pathways, namely, the Wnt, BMP, FGF, Shh, and Eda, are involved in the mediation of the successive reciprocal epithelial-mesenchymal cross talk which follows the general principle of morphogenetic interactions (Davidson, 1993). The pathways regulate the expression of transcription factors which confer the identity of dental epithelium and mesenchyme. The signals and transcription factors are integrated in complex signaling networks whose fine-tuning allows the generation of the variation in tooth morphologies. In this review, we describe the principles and molecular mechanisms of the epithelial-mesenchymal interactions regulating successive stages of tooth formation: (i) the initiation of tooth development, with special reference to the shift of tooth-forming potential from epithelium to mesenchyme; (ii) the morphogenesis of the tooth crown, focusing on the roles of epithelial signaling centers; (iii) the differentiation of odontoblasts and ameloblasts, which produce dentin and enamel, respectively; and (iv) the maintenance of dental stem cells, which support the continuous growth of teeth.

Pancreatic ductal adenocarcinoma is one of the most aggressive and deadliest carcinomas worldwide. Despite modest response rates and extremely low cure rates, gemcitabine-based therapies still represent standard of care. Importantly, chemotherapeutic agents such as gemcitabine target mainly highly proliferative and differentiated cells, while cancer stem cells (CSCs) as the most aggressive and chemoresistant cells in the tumor are regularly spared. CSCs are capable of self-renewing and giving rise to more differentiated progenies, representing the bulk of cancer cells. Thus, CSCs represent the root of the tumor and are considered the source of disease relapse. To actually achieve long-term progression-free survival, treatment regimens should also target CSCs, either by directly eliminating them or inducing their differentiation rendering them more susceptible to chemotherapy. Intriguingly, our recent studies revealed that the anti-malaria drug chloroquine preferentially targets CD133+ CSCs by depleting their number and self-renewal ability translating into drastically reduced in vivo tumorigenic activity. Even more importantly, chloroquine virtually abolished invasiveness of the metastatic CSC sub-population that co-expresses CD133 and CXCR4 . Thus, out data suggest that repurposing chloroquine as a CSC-targeting agent could represent a valuable and imminently available adjuvant therapy for patients with pancreatic ductal adenocarcinoma.

Emerging evidence suggests that stem cells play a crucial role not only in the generation and maintenance of different tissues, but also in the development and progression of malignancies. For the many solid cancers, it has now been shown that they harbor a distinct subpopulation of cancer cells that bear stem cell features and therefore, these cells are termed cancer stem cells (CSC) or tumor-propagating cells. CSC are exclusively tumorigenic and essential drivers for tumor progression and metastasis. Moreover, it has been shown that pancreatic ductal adenocarcinoma does not only contain one homogeneous population of CSC rather than diverse subpopulations that may have evolved during tumor progression. One of these populations is called migrating CSC and can be characterized by CXCR4 co-expression. Only these cells are capable of evading the primary tumor and traveling to distant sites such as the liver as the preferred site of metastatic spread. Clinically even more important, however, is the observation that CSC are highly resistant to chemo- and radiotherapy resulting in their relative enrichment during treatment and rapid relapse of disease. Many laboratories are now working on the further in-depth characterization of these cells, which may eventually allow for the identification of their Achilles heal and lead to novel treatment modalities for fighting this deadly disease.

Terminal differentiation of odontoblasts from dental papilla is a long process involving several intermediate steps and changes in the transcriptional profile and expression of proteins secreted by cells in the odontoblast lineage. Transgenic mouse lines in which GFP expression is under the control of tissue- and stage specific promoters have provided powerful experimental tools for identification and isolation of cells at specific stages of differentiation along a lineage. Our previous studies showed utilization of pOBCol3.6GFP and pOBCol2.3GFP animals for identification of odontoblasts at early and late stages of polarization respectively. In the present study we used the DMP1-GFP transgenic animal as an experimental model to examine its expression during the differentiation of odontoblasts from progenitor cells in vivo and in vitro. Our observations showed that DMP1-GFP transgene is first activated in secretory/functional odontoblasts engaged in secretion of predentin and then transiently expressed at high levels in newly differentiated odontoblasts. Expression of DMP1-GFP was down-regulated in highly differentiated odontoblasts. The temporal and spatial pattern of expression of DMP1-GFP transgene closely mimics the expression of endogenous DMP1. This transgenic animal will facilitate studies of gene expression and biological functions in secretory/functional odontoblasts.

Purpose In spite of intense research efforts, pancreatic ductal adenocarcinoma remains one of the most deadly malignancies in the world. We and others have previously identified a subpopulation of pancreatic cancer stem cells within the tumor as a critical therapeutic target and additionally shown that the tumor stroma represents not only a restrictive barrier for successful drug delivery, but also serves as a paracrine niche for cancer stem cells. Therefore, we embarked on a large-scale investigation on the effects of combining chemotherapy, hedgehog pathway inhibition, and mTOR inhibition in a preclinical mouse model of pancreatic cancer. Experimental Design Prospective and randomized testing in a set of almost 200 subcutaneous and orthotopic implanted whole-tissue primary human tumor xenografts. Results The combined targeting of highly chemoresistant cancer stem cells as well as their more differentiated progenies, together with abrogation of the tumor microenvironment by targeting the stroma and enhancing tissue penetration of the chemotherapeutic agent translated into significantly prolonged survival in preclinical models of human pancreatic cancer. Most pronounced therapeutic effects were observed in gemcitabine-resistant patient-derived tumors. Intriguingly, the proposed triple therapy approach could be further enhanced by using a PEGylated formulation of gemcitabine, which significantly increased its bioavailability and tissue penetration, resulting in a further improved overall outcome. Conclusions This multimodal therapeutic strategy should be further explored in the clinical setting as its success may eventually improve the poor prognosis of patients with pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinomas contain a subset of exclusively tumorigenic cancer stem cells (CSCs), which are capable of repopulating the entire heterogeneous cancer cell populations and are highly resistant to standard chemotherapy. Here we demonstrate that metformin selectively ablated pancreatic CSCs as evidenced by diminished expression of pluripotency-associated genes and CSC-associated surface markers. Subsequently, the ability of metformin-treated CSCs to clonally expand in vitro was irreversibly abrogated by inducing apoptosis. In contrast, non-CSCs preferentially responded by cell cycle arrest, but were not eliminated by metformin treatment. Mechanistically, metformin increased reactive oxygen species production in CSC and reduced their mitochondrial transmembrane potential. The subsequent induction of lethal energy crisis in CSCs was independent of AMPK/mTOR. Finally, in primary cancer tissue xenograft models metformin effectively reduced tumor burden and prevented disease progression; if combined with a stroma-targeting smoothened inhibitor for enhanced tissue penetration, while gemcitabine actually appeared dispensable.

Mice lacking both Prx1 and Prx2 display severe abnormalities in the mandible. Our analysis showed that complete loss of Prx gene products leads to growth abnormalities in the mandibular processes evident as early as embryonic day (E) 10.5 associated with changes in the survival of the mesenchyme in the medial region. Changes in the gene expression in the medial and lateral regions were related to gradual loss of a subpopulation of mesenchyme in the medial region expressing eHand. Our analysis also showed that Prx gene products are required for the initiation and maintenance of chondrogenesis and terminal differentiation of the chondrocytes in the caudal and rostral ends of Meckel's cartilage. The fusion of the mandibular processes in the Prx1/Prx2 double mutants is caused by accelerated ossification. These observations together show that, during mandibular morphogenesis, Prx gene products play multiple roles including the cell survival, the region-specific terminal differentiation of Meckelian chondrocytes and osteogenesis.

SummaryNodal and Activin belong to the TGF-β superfamily and are important regulators of embryonic stem cell fate. Here we investigated whether Nodal and Activin regulate self-renewal of pancreatic cancer stem cells. Nodal and Activin were hardly detectable in more differentiated pancreatic cancer cells, while cancer stem cells and stroma-derived pancreatic stellate cells markedly overexpressed Nodal and Activin, but not TGF-β. Knockdown or pharmacological inhibition of the Nodal/Activin receptor Alk4/7 in cancer stem cells virtually abrogated their self-renewal capacity and in vivo tumorigenicity, and reversed the resistance of orthotopically engrafted cancer stem cells to gemcitabine. However, engrafted primary human pancreatic cancer tissue with a substantial stroma showed no response due to limited drug delivery. The addition of a stroma-targeting hedgehog pathway inhibitor enhanced delivery of the Nodal/Activin inhibitor and translated into long-term, progression-free survival. Therefore, inhibition of the Alk4/7 pathway, if combined with hedgehog pathway inhibition and gemcitabine, provides a therapeutic strategy for targeting cancer stem cells.