ES cells-derived ectomesenchymal cells for tooth engineering.

Recent progresses in stem cell biology and tissue engineering allow considering the possible development of new therapies for compensating the dental tissue losses associated with traumas, pathologies or ageing. The possibility of generating a tooth by mimicking development through reassociations between dental epithelial cells and ectomesenchymal cells derived from the neural crest (NC) has been demonstrated in the mouse. In the search of cell sources to be used for a human transfer, pluripotent stem cells could represent a good alternative. Our study thus focuses on obtaining, ectomesenchymal cells from pluripotent ES cells, capable of promoting tooth histomorphogenesis, when reassociated with a competent dental epithelium. To this end, two ES differentiation protocols, using cyclopamine or a combination of FGF2 and BMP4, have been developed and tested for their capacity to generate such cells. The differentiated ES cells were characterized by quantitative RT-PCR. Both protocols led the cells to acquire in 10 days a mesenchymal-like cell morphology. Rapidly after induction, the cells loose their expression of pluripotent genes while sequentially activating typical NC specifiers. However, the kinetics of gene activation differed between the 2 protocols. Interestingly, Twist, a gene whose expression in the NC is associated with a commitment towards an ectomesenchymal fate, is only activated under the influence of FGF2 and BMP4. Reassociation experiments with a competent epithelium will allow testing the odontogenic potential of the differentiated ES cells. These experiments performed in the mouse system should allow defining a strategy for obtaining odontogenic competent human cells. Les progrès en matière de biologie de cellules souches et d’ingénierie tissulaire permettent d’envisager le développement de nouvelles thérapies pour pallier les pertes de tissus dentaires consécutives à des traumatismes, des situations pathologiques ou au vieillissement. La possibilité de générer une dent en mimant le développement, par réassociations entre cellules dentaires épithéliales et mésenchymateuses dérivées des crêtes neurales (CN), a été démontrée chez la souris. Dans la recherche de ressources cellulaires utilisables pour un transfert chez l’homme, les cellules souches pluripotentes pourraient constituer une alternative. Notre but est d’obtenir à partir de ces dernières, des cellules ectomésenchymateuses capables d’interagir avec un épithélium dentaire pour promouvoir l’histomorphogenèse d’une dent. Pour cela, deux protocoles de différenciation de cellules ES, utilisant la cyclopamine ou une combinaison de FGF2/BMP4, ont été mis au point. Les cellules induites ont été caractérisées par PCR quantitative. Les deux protocoles de différenciation amènent les cellules à acquérir en 10 jours, une morphologie de type mésenchymateux. Après induction, l’expression des gènes de pluripotence chute de façon drastique alors que celle des gènes spécificateurs de CN est activée. Toutefois, la cinétique varie selon le protocole. Le gène Twist, dont l’expression dans les CN est associée à un engagement vers l’ectomésenchyme, n’est activé significativement que sous l’action de FGF2/BMP4. Des expériences de réassociations avec un épithélium dentaire sont en cours pour évaluer le potentiel odontogène des cellules ES différenciées. A terme, ces approches menées chez la souris devraient permettre de définir une stratégie pour l’obtention de cellules compétentes humaines.Recent progresses in stem cell biology and tissue engineering allow considering the possible development of new therapies for compensating the dental tissue losses associated with traumas, pathologies or ageing. The possibility of generating a tooth by mimicking development through reassociations between dental epithelial cells and ectomesenchymal cells derived from the neural crest (NC) has been demonstrated in the mouse. In the search of cell sources to be used for a human transfer, pluripotent stem cells could represent a good alternative. Our study thus focuses on obtaining, ectomesenchymal cells from pluripotent ES cells, capable of promoting tooth histomorphogenesis, when reassociated with a competent dental epithelium. To this end, two ES differentiation protocols, using cyclopamine or a combination of FGF2 and BMP4, have been developed and tested for their capacity to generate such cells. The differentiated ES cells were characterized by quantitative RT-PCR. Both protocols led the cells to acquire in 10 days a mesenchymal-like cell morphology. Rapidly after induction, the cells loose their expression of pluripotent genes while sequentially activating typical NC specifiers. However, the kinetics of gene activation differed between the 2 protocols. Interestingly, Twist, a gene whose expression in the NC is associated with a commitment towards an ectomesenchymal fate, is only activated under the influence of FGF2 and BMP4. Reassociation experiments with a competent epithelium will allow testing the odontogenic potential of the differentiated ES cells. These experiments performed in the mouse system should allow defining a strategy for obtaining odontogenic competent human cells. Les progrès en matière de biologie de cellules souches et d’ingénierie tissulaire permettent d’envisager le développement de nouvelles thérapies pour pallier les pertes de tissus dentaires consécutives à des traumatismes, des situations pathologiques ou au vieillissement. La possibilité de générer une dent en mimant le développement, par réassociations entre cellules dentaires épithéliales et mésenchymateuses dérivées des crêtes neurales (CN), a été démontrée chez la souris. Dans la recherche de ressources cellulaires utilisables pour un transfert chez l’homme, les cellules souches pluripotentes pourraient constituer une alternative. Notre but est d’obtenir à partir de ces dernières, des cellules ectomésenchymateuses capables d’interagir avec un épithélium dentaire pour promouvoir l’histomorphogenèse d’une dent. Pour cela, deux protocoles de différenciation de cellules ES, utilisant la cyclopamine ou une combinaison de FGF2/BMP4, ont été mis au point. Les cellules induites ont été caractérisées par PCR quantitative. Les deux protocoles de différenciation amènent les cellules à acquérir en 10 jours, une morphologie de type mésenchymateux. Après induction, l’expression des gènes de pluripotence chute de façon drastique alors que celle des gènes spécificateurs de CN est activée. Toutefois, la cinétique varie selon le protocole. Le gène Twist, dont l’expression dans les CN est associée à un engagement vers l’ectomésenchyme, n’est activé significativement que sous l’action de FGF2/BMP4. Des expériences de réassociations avec un épithélium dentaire sont en cours pour évaluer le potentiel odontogène des cellules ES différenciées. A terme, ces approches menées chez la souris devraient permettre de définir une stratégie pour l’obtention de cellules compétentes humaines

Experimental Induction of Odontoblast Differentiation and Stimulation During Preparative Processes

In vivo implantation experiments have shown that ethylenediaminetetraaceticacid(EDTA)-soluble frac tions of dentin stimulate reparative dentinogenesis . When isolated embryonic dental papillae were cultured in the presence of these dentin constituents, odontoblast cytological and functional differentiation could be initiated and maintained in the absence of an enamel organ. These effects were attributed to the presence of TGF-/1- related molecules [TGF-/11 or bone morphogenetic protein -2a (BMP-2a)] which had to be used in combination with an EDT A-soluble fraction of dentin in order to specifically affect competent preodontoblasts . These EDT A-soluble constituents present in dentin could be replaced by heparin or fibronectin which both have been reported to interact with TGF-/1. The association of such defined matrix components with a TGF-/1-related molecule represents a biologically active complex triggering odontoblast functional differentiation. In response to caries, odontoblasts modulate their secretory activity and are stimulated to elaborate reactionary dentin. This might be induced by active molecules such as IGF, TGF-6 or BMP which are liberated from dentin consecutively to the demineralization process. Reparative dentinogenesis is distinct from reactionary dentinogenesis and more complex since it implicates the differentiation of precursor cells present in the dental papilla. The developmental history of these cells is different from that of the physiological predontoblasts in developing teeth. The nature of these stem cells and the mechanism of their induction still remain open questions

Expression dynamics of metalloproteinases during mandibular bone formation: association with Myb transcription factor

As the dentition forms and becomes functional, the alveolar bone is remodelled. Metalloproteinases are known to contribute to this process, but new regulators are emerging and their contextualization is challenging. This applies to Myb, a transcription factor recently reported to be involved in bone development and regeneration. The regulatory effect of Myb on Mmps expression has mostly been investigated in tumorigenesis, where Myb impacted the expression of Mmp1, Mmp2, Mmp7, and Mmp9. The aim of this investigation was to evaluate the regulatory influence of the Myb on Mmps gene expression, impacting osteogenesis and mandibular bone formation. For that purpose, knock-out mouse model was used. Gene expression of bone-related Mmps and the key osteoblastic transcription factors Runx2 and Sp7 was analysed in Myb knock-out mice mandibles at the survival limit. Out of the metalloproteinases under study, Mmp13 was significantly downregulated. The impact of Myb on the expression of Mmp13 was confirmed by the overexpression of Myb in calvarial-derived cells causing upregulation of Mmp13. Expression of Mmp13 in the context of other Mmps during mandibular/alveolar bone development was followed in vivo along with Myb, Sp7 and Runx2. The most significant changes were observed in the expression of Mmp9 and Mmp13. These MMPs and MYB were further localized in situ by immunohistochemistry and were identified in pre/osteoblastic cells as well as in pre/osteocytes. In conclusion, these results provide a comprehensive insight into the expression dynamics of bone related Mmps during mandibular/alveolar bone formation and point to Myb as another potential regulator of Mmp13

Interactions Between Laminin Receptor and the Cytoskeleton During Translation and Cell Motility

Human laminin receptor acts as both a component of the 40S ribosomal subunit to mediate cellular translation and as a cell surface receptor that interacts with components of the extracellular matrix. Due to its role as the cell surface receptor for several viruses and its overexpression in several types of cancer, laminin receptor is a pathologically significant protein. Previous studies have determined that ribosomes are associated with components of the cytoskeleton, however the specific ribosomal component(s) responsible has not been determined. Our studies show that laminin receptor binds directly to tubulin. Through the use of siRNA and cytoskeletal inhibitors we demonstrate that laminin receptor acts as a tethering protein, holding the ribosome to tubulin, which is integral to cellular translation. Our studies also show that laminin receptor is capable of binding directly to actin. Through the use of siRNA and cytoskeletal inhibitors we have shown that this laminin receptor-actin interaction is critical for cell migration. These data indicate that interactions between laminin receptor and the cytoskeleton are vital in mediating two processes that are intimately linked to cancer, cellular translation and migration