Immunophenotyping Reveals the Diversity of Human Dental Pulp Mesenchymal Stromal Cells In vivo and Their Evolution upon In vitro Amplification

International audienceMesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31 − cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31 − DP cell population contained 1.4% of CD56 + , 1.5% of CD146 + , 2.4% of CD271 + and 6.3% of MSCA-1 + cells but very few Stro-1 + cells (≤1%). CD56 + , CD146 + , CD271 + , and MSCA-1 + cell subpopulations expressed various levels of these markers. CD146 + MSCA-1 + , CD271 + MSCA-1 + , and CD146 + CD271 + cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56 + , CD271 + , MSCA-1 + , and Stro-1 + cells, and MSCA-1 by 15-30% of CD56 + , CD146 + , CD271 + , and Stro-1 + cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by <1% of the expanded cell populations. Interestingly, the percentage of CD56 + cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146 + CD56 + , MSCA-1 + CD56 + , and CD146 + MSCA-1 + cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products

Caractérisation de la dextrane-saccharase DSR-E de Leuconostoc mesenteroides NRRL B-1299 et application à la synthèse de composés prébiotiques

La dextrane-saccharase DSR-E de L. mesenteroides NRRL B-1299, appartenant à la famille 70 des glycoside-hydrolases, se distingue sur le plan fonctionnel par sa capacité à synthétiser à la fois des liaisons glucosidiques de type α-1,6 et α-1,2 (ces dernières étant rares dans la nature). Cette propriété est mise en évidence par la synthèse d'un dextrane hautement ramifié à partir de saccharose, et est conservée lors de la production de glucooligosaccharides (GOS) en présence de maltose. Sur le plan structural, DSR-E est unique car elle possède deux domaines catalytiques (CD1 et CD2), séparés par un domaine de liaison au glucane (GBD). De plus, CD2 se distingue du fait de la présence de peptides originaux situés dans les régions catalytiques, habituellement très conservées. L'objectif de ce travail de thèse était la compréhension des rôles respectifs du CD1 et du CD2 dans la catalyse et la spécificité de l'enzyme. Pour y parvenir, plusieurs formes délétées du gène dsrE ont été clonées et exprimées chez E. coli. La caractérisation structurale des produits (dextrane ou GOS) synthétisés par les enzymes correspondantes a révélé que le CD1 est spécifique de la synthèse de liaisons α-1,6 alors que le CD2 ne synthétise que des liaisons de type α-1,2. Ces études de structure/fonction ont permis de générer la forme GBD-CD2, nouvelle transglucosidase exempte de la capacité de polymérisation, et hautement spécialisée dans la synthèse de liaisons α-1,2. Cette enzyme est capable de produire 90% (p/p) d' α-1,2 GOS, alors que dans le cas de l'activité sauvage, ceux-ci sont produits dans les mêmes proportions que les α-1,6 GOS (p/p). Les α-1,2 GOS possèdent des propriétés prébiotiques et préviennent l' " intolérance au glucose ", symptôme précoce du diabète de type II. Concernant la synthèse de dextrane, le CD2 est responsable de la formation de points de ramification, selon un mécanisme ping-pong bi-bi. De plus, l'action concertée des deux domaines catalytiques, produits de façon dissociée et liés au GBD, permet de synthétiser une nouvelle structure de polymère en peigne, présentant 50% de liaisons α-1,2. - The activity of the dextransucrase DSR-E from L. mesenteroides NRRL B-1299, belonging to the glycoside-hydrolase family 70, is highly original, catalysing the syntheses of both α-1,6 and α-1,2 glucosidic linkages. This property is shown by the production, from sucrose, of a highly branched dextran, and also by the syntheses of glucooligosaccharides (GOS) in the presence of maltose. Moreover, DSR-E is unique in its enzymatic family, possessing two catalytic domains (CD1 and CD2), separated by a long Glucan Binding Domain (GBD). In order to understand the role of each domain, several truncated forms of DSR-E were produced in E. coli. The structural characterization of the products revealed that CD1 is specific for the synthesis of α-1,6 linkages, CD synthesizing only α-1,2 linkages. The newly constructed GBD-CD2 is a transglucosidase highly specialized for the synthesis of α-1,2 linkages, able to produce 90% (p/p) of α-1,2 GOS, whereas the native activity leads to the synthesis of 50% of α-1,6 GOS (w/w) and 50% of α-1,2 GOS (p/p). α-1,2 GOS possess a prebiotic activity and prevent the glucose sensitivity, first symptom of diabetes. Moreover, CD2 is responsible for the formation of α-1,2 ramification points into the dextran structure, following a ping-pong bi-bi kinetic model. Finally, the concerted action of the two catalytic domains, dissociated but linked to GBD, permits the synthesis of a novel structure of glucan, possessing 50% of α-1,2 linkages, reassembling a comb

Intracellular transit of a yeast protease is rescued by trans-complementation with its prodomain

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Intracellular transit of a yeast protease is rescued by trans-complementation with its prodomain

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Présence en France de Pezizella ericae Read, champignon endomycorhizogène des Ericacées horticoles

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La variation des seuils de nuisibilite des mauvaises herbes. Resultats experimentaux

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Synthesis of dextrans with controlled amounts of α-1,2 linkages using the transglucosidase GBD-CD2

International audienceGBD-CD2 is an α-1,2 transglucosidase engineered from DSR-E, a glucansucrase naturally produced by Leuconostoc mesenteroides NRRL B-1299. This enzyme catalyses from sucrose, the α-1,2 transglucosylation of glucosyl moieties onto α-1,6 dextran chains. Steady-state kinetic studies showed that hydrolysis and transglucosylation reactions occurred at the early stage of the reaction in the presence of 70 kDa dextran as acceptor and sucrose. The transglucosylation reaction catalysed by GBD-CD2 follows a Ping Pong Bi Bi mechanism with a high kcat value of 970 s-1. The amount of the synthesised α-1,2 side chains was found to be directly dependent on the initial molar ratio [Sucrose]/[Dextran]. Dextrans with controlled α-1,2 linkage contents ranging from 13% to 40% were synthesised. The procedure resulted in the production of dextrans with the highest content of α-1,2 linkages ever reported

From the gold-catalysed benzylation of arenes to the regio- and stereoselective synthesis of procyanidins dimers

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Cloning, sequencing and amplification of the alkaline extracellular protease (XPR2) gene of the yeast Yarrowia lipolytica

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