A regeneração de defeitos ósseos continua sendo um grande desafio na área de Odontologia e Medicina. É bem estabelecido que células-tronco mesenquimais (CTMs) e osteoblastos (OBs) desempenham um papel crítico na osteogênese, tornando-se candidatos a utilização em procedimentos de terapia celular que visam otimizar o processo de reparação óssea. Porém, pouco se sabe sobre a interação entre CTMs e OBs, e a maioria dos estudos enfatiza o efeito dos OBs sobre CTMs, fazendo com que a influência das CTMs na atividade osteogênica dos OBs continue sendo uma questão desafiadora. Baseados em nossos estudos anteriores, formulamos a hipótese de que a terapia celular que fizesse uso de uma associação de CTMs e OBs poderia ser mais eficaz para o reparo do tecido ósseo do que essas células isoladamente, principalmente como resultado da estimulação de OBs por CTMs. Para tal, foi realizado estudo in vitro para avaliar os efeitos das CTMs sobre os OBs e in vivo para avaliar os efeitos dessas células, isoladamente e combinadas, sobre a reparação óssea. CTMs da medula óssea de rato foram cultivadas em meio de crescimento para manterem-se como CTMs ou em meio osteogênico para diferenciarem-se em OBs. Após alcançar a subconfluência, as células foram cultivadas in vitro em três diferentes condições: (1) co-cultura direta de CTMs e OBs usando três proporções celulares (1:1, 1:2 e 2:1), (2) co-cultura indireta de CTMs e OBs usando insertos e (3) OBs cultivados em meio condicionado por CTMs. Para avaliação das respostas celulares foram realizados ensaios de proliferação celular, atividade de fosfatase alcalina (ALP), formação de matriz mineralizada, expressão gênica de marcadores osteoblásticos, imunolocalização de sialoproteína óssea (BSP) e osteopontina (OPN) e migração celular. Para os experimentos in vivo, as células foram carreadas em esponja de colágeno através de vários ciclos de centrifugação. Após, defeitos ósseos em calvária de rato foram preenchidos com (1) esponja de colágeno sem células, (2) esponja de colágeno com CTMs, (3) esponja de colágeno com OBs e (4) esponja de colágeno com associação de CTMs e OBs. Para avaliação da reparação óssea in vivo após 4 semanas, foram realizadas análises histomorfométricas através de cortes histológicos e microtomografia computadorizada. Os dados foram comparados pelo teste de Kruskal-Wallis e, se necessário pelo teste de Mann-Whitney (p≤0,05). Foi observado que CTMs têm efeito repressivo sobre a proliferação e as expressões fenotípicas e genotípicas de OBs (P≤0,05). Em relação ao reparo dos defeitos ósseos, somente naqueles tratados com células observou-se formação óssea predominantemente como ilhotas isoladas e diferenças, principalmente qualitativas, entre os tipos celulares utilizados, com tendência de maior formação óssea em defeitos tratados com OBs em comparação ao uso de CTMs. Com base nos resultados obtidos, pôde-se concluir que as CTMs apresentam efeito inibitório sobre OBs e que a terapia celular com OBs parece ser mais eficaz no reparo do tecido ósseo. / The regeneration of bone defects remains a major challenge in the field of Dentistry and Medicine. It is well known that mesenchymal stem cells (MSCs) and osteoblasts (OBs) play critical roles in osteogenesis, making them promising alternatives to be employed in cell therapy procedures to enhance the process of bone regeneration. Studies about the crosstalk between MSCs and OBs are mainly focused on the effect of OBs on MSCs, thus how MSCs may affect OBs phenotype expression remains a challenging question. Based on our previous studies, we have hypothesized that cell therapy using a combination of MSCs and OBs could be more effective for the bone repair than these cells separately, mainly due to the stimulation of OBs by MSCs. For this, we carried out in vitro experiments to evaluate the effects of MSCs on the OBs and in vivo experiments to assess the effects of these cells either isolated or combined on bone repair. Rat bone marrow MSCs were cultured either in growth medium to keep MSCs features or in osteogenic medium to differentiate into OBs. After reaching subconfluence, cells were grown in vitro in three different conditions: (1) direct coculture of MSCs and OBs using three cell proportions (1:1, 1:2 and 2:1), (2) indirect coculture of MSCs and OBs using transwell porous filters and (3) OBs cultured in MSCs conditioned medium. Cell responses were evaluated by assaying cell proliferation, alkaline phosphatase activity (ALP), mineralized matrix formation, gene expression of osteoblast markers, immunolocalization of bone sialoprotein (BSP) and osteopontin (OPN), and cell migration. For in vivo experiments, cells were seeded into collagen sponge by a centrifugation method. After, calvarial defects were implanted with (1) collagen sponge without cells, (2) collagen sponge with MSCs, (3) collagen sponge with OBs, and (4) collagen sponge and association of MSCs with OBs. To evaluate bone repair at the end of 4 weeks, histomorphometric analyzes were carried out using histological slides and micro-computed tomography. Data were compared by Kruskal-Wallis test and, if appropriated, by Mann-Whtiney test (p≤0.05). It was observed that MSCs repressed proliferation, phenotypic and genotypic expressions of OBs (P≤0.05). Bone formation was observed only in cell treated defects as isolated islets and qualitative differences were noticed among cell types, with a tendency of more bone formation in OBs treated defects compared with MSCs ones. Based on these results, we can conclude that MSCs exhibited inhibitory effect on OBs and that cell therapy with OBs seemed to be more effective for bone repair.

L'incidence des maladies cardiovasculaires d'origine athéromateuse constitue un problème majeur en santé publique et malgré le développement de techniques curatives endovasculaires, la chirurgie demeure nécessaire chez de nombreux patients. La faible disponibilité des vaisseaux naturels, autologues ou non, et les limites mécaniques et biologiques des substituts artificiels pour le remplacement des vaisseaux de petit calibre, imposent le recours à une nouvelle science : l'ingénierie vasculaire. Ce concept a émergé et évolué depuis quelques années. Il pourrait permettre de proposer de nouveaux types de substituts vasculaires synthétiques et/ou biologiques, en particulier grâce à l'utilisation de cellules souches, ouvrant d'intéressantes perspectives dans le domaine de l'ingénierie vasculaire. Le but de ce travail a été d'obtenir de manière fiable et reproductible des cellules à phénotype endothélial mature à partir de cellules souches mésenchymateuses (CSMs) issus de la gelée de Wharton (GW) du cordon ombilical et de la moelle osseuse (MO). Cependant, la différenciation de ces cellules nécessite une fonctionnalisation de la surface de culture, et notre groupe a démontré l'avantage des films multicouches de polyélectrolytes, constitués de PAH (hydrochlorure de poly(allylamine)) et de PSS (poly(styrène sulfonate)), sur l'adhésion, la prolifération et la différenciation cellulaire. Les cellules ont été cultivées sur films (PAH-PSS)4 ou sur collagène de type I (témoin), en présence de facteurs de croissance angiogéniques. La différenciation en cellules endothéliales (CEs) a été suivie par l'expression des marqueurs endothéliaux (PCR et western blot), et la fonctionnalité par leur capacité à incorporer les lipoprotéines acétylées (Ac-LDLs) ainsi que la capacité à produire du monoxyde d'azote et à exprimé le facteur von Willebrand (vWF). Après 14 jours de stimulation, seules les CSM-GWs étaient différenciées en CEs fonctionnelles démontrant l'intérêt de combiner l'utilisation des CSM-GWs et des films (PAH-PSS)4 dans le domaine de l'ingénierie vasculaire / The incidence of cardiovascular disease remains a major public health problem. Despite the development of endovascular therapies, surgical treatment is necessary for many patients. The low availability of natural vessels, autologous or not, and the mechanical and biological limits of artificial substitutes, led to the use of a new domain: vascular engineering. In recent years, the concept has emerged and evolved. He could afford to offer new types of synthetic vascular substitutes and / or biological, in particular through the use of stem cells, offering interesting perspectives in the field of vascular engineering. The purpose of this study was to obtain a reliable and reproducible protocol to generate functional endothelial cells (ECs) from mesenchymal stem cells (MSCs) derived from the umbilical cord Wharton's jelly (WJ) and bone marrow (BM). Nevertheless, their differentiation into vascular cells needs a culture surface functionalization; our group demonstrated the potential use of polyelectrolyte multilayer made of poly(allylamine hydrochloride): PAH, and poly(styrene sulfonate): PSS, in promoting cells adhesion, proliferation and differentiation. Cells were cultured on (PAH-PSS)4 films or collagen type I (used as control), in the presence of angiogenic growth factors. Cells differentiation into EC was followed through the expression of endothelial markers (PCR and western blot); cell functionality was checked through their ability to incorporate acetylated LDL (Low Density Lipoprotein), to produce NO (Nitric oxide) and to express the von Willebrand factor (vWF). After 14 days of stimulation, only WJ-MSCs were able to generate functional ECs demonstrating the potential of combining WJ-MSCs and (PAH-PSS)4 films in vascular tissue engineering field

Parmi les facteurs de risque environnementaux de l'arthrose, la consommation de tabac occupe une place importante, mais reste encore controversée. Parmi les 4000 composés présents dans la cigarette, la nicotine est l'une des molécules les plus actives physiologiquement. Au cours de ce travail, nous avons étudié l'impact de la nicotine sur les chondrocytes humains et la prolifération et la différenciation chondrogénique des cellules souches mésenchymateuses de la gelée de Wharton (CSM-GW). Nous avons trouvé que la nicotine aux concentrations utilisées n’a pas d’effet sur la prolifération cellulaire, mais induit une augmentation de l’expression de la métalloproteinase matricielle (MMP13) dans les chondrocytes humains. Ces données suggèrent que la nicotine a un effet pro-catabolique sur les chondrocytes humains, en stimulant la dégradation des composants matriciels. Chez des patients arthrosiques fumeurs, les voies de synthèse et de dégradation des composants matriciels sont plus activées dans les chondrocytes par rapport aux non fumeurs. De plus, la nicotine inhibe la prolifération et la migration cellulaire de CSM-GW, et présente un effet délétère sur la chondrogénèse de CSM-GW. Elle stimule la réaction inflammatoire et la différenciation hypertrophique. Nous avons montré, pour la première fois, l'expression du nicotinic acetylcholine receptor (nAChR), en particulier de la sous unité α7 dans les CSM-GW aux niveaux transcriptionnel et traductionnel. L’effet délétère de la nicotine sur les CSM-GW serait probablement médiée par la sous unité α7 nAChR, De façon intéressante, l’α-Bungarotoxin (α-BTX), inhibiteur spécifique de α7 nAChR, peut réverser cet effet partiellement. En conclusion, nous suggérons que la nicotine pourrait altérer l’ontogenèse du cartilage, et induire potentiellement l’augmentation de la prévalence de l’arthrose chez l’adulte / Among the environmental risk factors for osteoarthritis (OA), tobacco consumption features prominently but is still controversial today. Among the 4,000 compounds present in cigarette smoke, nicotine is one of the most physiologically active molecules. The aim of the study is to measure the impact of nicotine on human chondrocytes and on the proliferation and chondrogenic differentiation of Wharton’s Jelly stem cells (WJ-MSC). We found that nicotine at the concentrations used had no effect on the proliferation of primary human OA chondrocytes, but induced an increase in the expression of matrix metalloproteases (MMP13). It unravels the catabolic effects of nicotine in the joint by stimulating matrix degradation. This result suggests a pro-catabolic effect of nicotine in the joint by stimulating matrix degradation. In smokers, the synthesis as well as the degradation pathways in chondrocytes are stimulated, when compared to no smokers. In addition, the cell proliferation and migration of WJ-MSC were significantly impaired by nicotine, and it also had an adverse effect on the chondrogenesis of WJ-MSC by stimulating the inflammatory response and hypertrophic differentiation. We have shown, for the first time, the expression of the nicotinic acetylcholine receptor (nAChR), in particular that of the α7 subunit in the WJ-MSC at the transcriptional and translational levels. The adverse effect of nicotine on WJ-MSC was probably mediated by α7 nAChR. Interestingly, α-Bungarotoxin (α-BTX), a specific inhibitor of α7 nAChR, could partially reverse this effect. In conclusion the results show that nicotine has an adverse effect on the ontogenesis of cartilage, and potentially induces an increase in the prevalence of osteoarthritis in adults

Les vésicules extracellulaires nanométriques (nEVs) issues de cellules souches mésenchymateuses (CSMs) et les nanovésicules synthétiques sont au centre de nombreuses recherches pour le développement de nouvelles stratégies thérapeutiques en médecine régénérative. La mise en place d’une méthode standardisée pour isoler les nEVs à partir de milieu conditionné de CSMs et de pouvoir les caractériser a été nécessaire. Nous nous sommes concentrés sur leur taille qui se situe entre 30 et 150 nm ainsi que la présence de certains de leur marqueurs membranaires (CD9, CD63 et CD81). Durant ce travail, deux méthodes d’isolement ont été testées. Les résultats obtenus par les analyses physiques (Nanosight®, microscopie électronique à transmission) et biologiques (cytométrie en flux) des différents échantillons ont permis de standardiser la méthode d’isolement des nEVs par centrifugations et ultracentrifugations successives. Ensuite, nous nous sommes intéressés à l’utilisation de ces nEVs sécrétées par les CSMs en culture cellulaire. Il a été mis en évidence que des interactions existent entre ces nEVs et des cellules endothéliales (CEs) in vitro. Ces interactions vont entraîner des modifications dans le comportement cellulaire des CEs en augmentant leur potentiel de formation de réseaux vasculaires. En parallèle de ces travaux sur les nEVs, une étude a été réalisée sur l’utilisation de nanovésicules synthétiques, des nanoliposomes (NLPs), élaborées à partir de lécithine d’agro-ressource (saumon) comme transporteur de TGF-ß1 pour une application en médecine régénérative. Après leur caractérisation physico-chimique, cette étude préliminaire a montré que ces NLPs ne présentent pas de cytotoxicité pour les CSMs in vitro. Il existe un potentiel important d’utilisation des nEVs de CSMs ainsi des NLPs pour développer de nouvelles stratégies innovantes en thérapie « cell-free » dans le domaine de la médecine régénérative / Nanoscale extracellular vesicles (nEVs) derived from mesenchymal stem cells (MSCs) and synthetic nanovesicles are at the centre of many research studies for the development of new therapeutic strategies in regenerative medicine. A standardized method was used to isolate nEVs from conditioned media of CSMs and to characterize them. We focused on their size with a range of 30 to 150 nm and the presence of some of their membrane markers (CD9, CD63 and CD81). During this work, two isolation methods were tested. The results obtained by the physical (Nanosight®, transmission electron microscopy) and biological (flow cytometry) analyses of the different samples allowed to standardize the method of isolation of the nEVs by successive centrifugation and ultracentrifugation. Then, we studied the use of these nEVs derived from MSCs in cell culture. Interactions between these nEVs and endothelial cells (ECs) have been demonstrated in vitro. These interactions lead to changes in the cellular behaviour of ECs by increasing their potential to form vascular networks. In parallel of this work on nEVs, we studied the use of synthetic nanovesicles, called nanoliposomes (NLPs) prepared from agro-resource derived lecithin (salmon) as TGF-β1 transporters for applications in regenerative medicine. After their physicochemical characterization, this preliminary study showed that these NLPs do not exhibit cytotoxicity for MSCs in vitro. There is an important potential for the use of nEVs derived from MSCs as well as NLPs to develop new cell-free therapy innovative strategies in the field of regenerative medicine

La thérapie cellulaire et les médicaments de thérapie innovante sont des solutions prometteuses pour la régénération des tissus ou organes présentant des défauts fonctionnels ou organiques. Avant le stade de l'insuffisance cardiaque terminale (stade IV NYHA) suite à un infarctus du myocarde, l'implantation d'un patch de fibrine cellularisé avec des progéniteurs myocardiques sur le site de nécrose de l'infarctus, est l'une des perspectives qui permettrait de régénérer un muscle cardiaque fonctionnel et apparait comme étant une alternative nouvelle avec notamment un essai clinique de phase I en cours (ESCORT : NCT02057900). Cependant, cette thérapie innovante présente de réelles contraintes, parmi lesquelles, un protocole nécessitant, i) une utilisation pour la production de cellules progénitrices myocardiques CD15+, de DMSO, de sérum foetal bovin, de trypsine porcine, de fibroblastes murins pouvant être la source d'une contamination chimique ou microbiologique, ii) une caractérisation importante des cellules produites, pour déterminer leur viabilité, leur pureté, leur état de différenciation, iii) d'implanter le patch de fibrine cellularisé dans un délai limité avant l'obtention des résultats de stérilité et d'endotoxines, iv) d'inciser le péricarde et de former une poche, geste chirurgical très invasif, afin d'implanter le patch cellularisé. Avec l'objectif de limiter ces contraintes et de renforcer la sécurisation pharmaceutique de ce médicament de thérapie innovante, les différents axes de ce travail ont porté sur i) l'ajout, juste avant l'implantation, d'une étape de cryoconservation des cellules dans un milieu sans sérum et sans DMSO, mais avec des agents cryoprotectants de qualité pharmaceutique. L'avantage apporté par la cryoconservation étant de rendre possible une production par lot, et la réalisation des contrôles sans contrainte de temps avant l'implantation, ii) la vectorisation des cellules par une encapsulation dans des microsphères formant une suspension injectable et permettant une implantation directement au travers du péricarde et immédiatement après la décongélation, iii) l'utilisation de polymères bioadhésifs afin de maintenir les microsphères au site d'implantation. Dans un premier temps, ce travail a permis d'identifier l'alginate de sodium de faible viscosité à 1,2% comme polymère pour réaliser l'encapsulation à l'aide d'une buse vibrante de 120 µm de diamètre. La nature et la concentration d'agents cryoprotectants ont également été définies. Les agents cryoprotectants ont été sélectionnés parmi les oses (glucose, saccharose, tréhalose), les polyols (glycérol, mannitol, sorbitol) et l'urée, à une concentration permettant d'atteindre une osmolarité totale de 500 mOsm/L pour abaisser le point de congélation de l'eau. Enfin le chitosane de faible viscosité à 0,5% a été utilisé comme polymère bioadhésif de surface pour maintenir les propriétés mécaniques et la forme des microsphères après la congélation. Dans un second temps, une évaluation biologique a permis de mesurer l'impact des étapes du procédé d'encapsulation et de cryoconservation, sur des cellules souches mésenchymateuses humaines utilisées comme modèle. Il a ainsi été possible d'optimiser le protocole ce qui a eu pour effet d'augmenter la viabilité, évaluée après encapsulation et congélation par une analyse en cytométrie de flux avec le 7AAD, de moins de 5% à environ 35%. / Cell therapy and advanced therapy medicinal products are promising solutions for the regeneration of tissues or organs with functional or organic defects. Before the terminal heart failure stage (stage IV NYHA) following a myocardial infarction, the implantation of a cellularized fibrin patch with myocardial progenitors at the location of the infarct necrosis, is one of the perspectives that would allow a functional heart muscle to regenerate and appears to be a new alternative, in particular, with an ongoing Phase I clinical trial (ESCORT : NCT02057900). However, this innovative therapy presents real constraints, among which, a protocol requiring, i) the use for the production of CD15+ myocardial progenitor cells of, DMSO, bovine fetal serum, porcine trypsin, and murine fibroblasts which may be the source of chemical or microbiological contamination, ii) an important characterization of the produced cells, to determine their viability, purity, and state of differentiation, iii) to implant the cellularized fibrin patch within a limited time frame before getting the results of sterility and endotoxins, iv) to incise the pericardium and to form a pouch, a very invasive surgical gesture, in order to implant the cellularized patch inside. With the objective of limiting these constraints and strengthening the pharmaceutical safety of this innovative therapy medication, different axes of this work have focused on i) the addition, just before the implantation, of a step of cryopreservation of the cells in a medium without serum and without DMSO, but with pharmaceutical-grade cryoprotectants. The advantages of cryopreservation is to allow production in batches, and controls to be carried out without time constraints before the implantation, ii) the vectorization of the cells by encapsulation in microspheres forming an injectable suspension and allowing direct implantation through the pericardium immediately after thawing, iii) the use of bioadhesive polymers to maintain the microspheres at the location of the implantation. This study initially enabled to identify a low-viscosity sodium alginate at 1.2% as a polymer being used for the encapsulation with the use of a vibrating nozzle which diameter is of 120 µm. The nature and the concentration of the cryoprotectants have also been defined. The cryoprotectants were selected from oses (glucose, sucrose, trehalose), polyols (glycerol, mannitol, sorbitol) and urea, at a concentration which achieves a total osmolarity of 500 mOsm/L in order to lower the freezing point of water. Finally, low viscosity chitosan at 0.5% was used as a bioadhesive polymer at the surface of the microspheres to maintain their shapes and mechanical properties after freezing. In a second step, a biological evaluation allowed to measure the impact of the encapsulation and the cryopreservation processes, on human mesenchymal stem cells used as a model. It was thus possible to optimize the protocol, which in return increased the viability ; evaluation made after encapsulation and freezing by a flow cytometry analysis with 7AAD ; from less than 5% to about 35%.

The heterogeneous nature of acute myeloid leukemia (AML) worsens the results of patients treated with standard therapy. Understanding the processes of leukemogenesis can contribute to identification of more appropriate treatment. Family of DLX genes (Distal-less homeobox), belonging to the homeobox genes, are associated with haematological malignancies and solid tumors. In the analysis of expression data, the low level of the DLX1 gene was associated with a worse prognosis of patients with AML. In this work we studied phenotypic changes of cell lines with different expression of the DLX1 gene. We silenced the DLX1 gene in AML cell line (sh cells) and compared it to the parental line with higher expression of DLX1 (NSC cells). By cell cycle analysis and apoptosis assays in vitro and in vivo, we have observed the arrest of sh cells in the G0 phase and a lower number of apoptotic cells. Differences were found when measuring the absolute number of cells in time. In in vitro conditions there were less sh cells, in in vivo environment there was significantly higher number of sh cells engrafted in comparison to NSC cells. Further results have shown that sh cells have lower levels of pro-apoptotic proteins and exhibit a higher level of TGF-β targeting PAI-1 gene that activates replicative senescence. We...

A desnutrição proteico-energética (DPE) provoca anemia e leucopenia decorrente da redução de precursores hematopoéticos e comprometimento da produção de mediadores indutores da hematopoese, bem como alterações estruturais e ultra-estruturais na matriz extracelular medular. A hematopoese ocorre em nichos medulares distintos - endosteal e perivascular - que modulam os processos de diferenciação, proliferação e auto-renovação da célula tronco hematopoética (CTH). As células tronco mesenquimais (CTM) tem um papel importante na formação destes nichos, através da sua diferenciação nos diversos tipos celulares que os compõe. Adicionalmente, a CTM pode modular a função de outras células, como a CTH e a célula endotelial (CE) medular, através da liberação de diversos fatores de crescimento e citocinas. As CE expressam proteínas que regulam a diferenciação e movimentação das CTH na MO. Há sinais que a CTM pode ser a precursora da CE medulares, pois in vitro a CTM pode se diferenciar em CE-like. Desta forma, a CTM é um ponto chave no estudo das alterações causadas pela DPE no nicho perivascular e sobre a regulação da hematopoese. Neste trabalho, investigamos se a DPE afeta a diferenciação in vitro da CTM medular em CE-like e avaliamos se essas células apresentam diferentes capacidades em produzir alguns mediadores regulatórios da hematopoese (CXCL-12, SCF, Ang-1, IL-11, GM-CSF e TFG-β), bem como possíveis alterações no perfil de expressão gênica de marcadores de função das CTM e CE-like. Utilizamos camundongos C57BL/6 machos, divididos em grupos Controle e Desnutrido, sendo que o grupo Controle recebeu ração normoprotéica (12% caseína) e o grupo Desnutrido recebeu ração hipoprotéica (2% caseína), ambos durante 5 semanas. Após este período, os animais foram eutanasiados, foi realizada a avaliação nutricional e hematológica, caracterizando a DPE. As CTM foram isoladas, caracterizadas e diferenciadas in vitro em CE-like, o que foi evidenciado pela maior expressão gênica de NT5E, FLT1, KDR, PECAM1 e VCAM1. Avaliamos a expressão dos genes CDH5, CSPG4, LEPR, NES, CSF1, CSF2, CSF3, MCAM, PROM1, ANGPT1, CXCL12, ENG, IGF1, IL3, IL11, KITL, TGFB1, WNT3A, WNT5A, ICAM1, PDGFB1 e VWF. Encontramos alterações causadas pela DPE na expressão gênica e quantificação de CXCL-12, SCF e Ang-1, os quais mostraram que as células avaliadas do grupo Desnutrido encontram-se em um estado \"pró-proliferativo\", em um esforço para restabelecer a hematopoese na DPE. Entretanto, foi observado neste trabalho e nos demais trabalhos do grupo que há hipoplasia medular na DPE e, portanto, pode-se inferir que as alterações hematopoéticas observadas na DPE não são ocasionadas por alterações na síntese de SCF, CXCL-12 ou Ang-1. / Protein-energy malnutrition (PEM) causes anemia and leukopenia as it reduces hematopoietic precursors, impairs the production of mediators that induce hematopoiesis and alters structural and ultrastructural changes in bone marrow (BM) extracellular matrix. Hematopoiesis occurs in distinct BM niches - endosteal and perivascular - which modulate the processes of differentiation, proliferation and self-renewal of hematopoietic stem cell (HSC). Mesenchymal stem cells (MSC) play an important role in the formation of these niches through their differentiation in several cell types that compose them. Additionally, MSC can modulate the function of other cells, such as HSC and endothelial cells (EC), through the release of several growth factors and cytokines. The EC express proteins that regulate the differentiation and migration of HSC in the BM. MSC seem to be the precursor of medullary EC because in vitro MSC can differentiate into EC-like cells. Thus, MSC are a key point in the study of changes caused by DPE on the perivascular niche and on the regulation of hematopoiesis. In this study, we investigated whether PEM would affect BM-MSC in vitro differentiation into EC-like cells and evaluated whether these cells would have distinct capacities of producing some regulatory mediators of hematopoiesis (CXCL- 12, SCF, Ang-1, IL-11, GM -CSF and TFG-β), as well as analyzed possible changes in the gene expression profile of MSC function and EC-like cells related markers. C57BL/6 mice were divided into Control and Malnourished groups, which received for 5 weeks, respectively, a normal protein diet (12% casein) and a low protein diet (2% casein). After this period, animals were euthanized, nutritional and hematological evaluations were performed, featuring the PEM. MSC were isolated, characterized and differentiated in vitro into EC-like cells, which were evidenced by increased gene expression of NT5E, FLT1, KDR, PECAM1 and VCAM1. The expression of CDH5, CSPG4, LEPR, NES, CSF1, CSF2, CSF3, MCAM, PROM1, ANGPT1, CXCL12, ENG, IGF1, IL3, IL11, KITL, TGFB1, Wnt3a, WNT5A, ICAM1, PDGFB1 and VWF genes was also evaluated. Changes caused by PEM on gene expression and quantification of CXCL-12, SCF and Ang-1 were found, indicating that tested cells from the Malnourished group were in a \"pro-proliferative\" state in an effort to restore hematopoiesis. However, our results are in accordance to the literature regarding bone marrow hypoplasia as a consequence of PEM. Therefore, we infer hematopoietic changes observed in this work are not related to changes in the synthesis of SCF, 12 CXCL-12 or Ang-1.

Introdução: A fibrose pulmonar idiopática (FPI) é definida como um tipo de doença fibrosante intersticial crônica de etiologia desconhecida limitada aos pulmões e que apresenta padrão histológico de pneumonia intersticial usual. A prevalência de FPI é estimada em, aproximadamente, 20/100.000 em homens e em 13/100.000 em mulheres, sendo que a idade média do diagnóstico é 67 anos e a sobrevivência média é 2 a 5 anos. Estima-se que 5 milhões de pessoas sejam afetadas em todo o mundo. O tratamento clínico atual está associado com melhora parcial e transitória, com resultados duvidosos ou insatisfatórios. Na abordagem cirúrgica da FPI, tem destaque o transplante pulmonar, cuja realização é rara, devido à escassez de doadores e à limitação das equipes capazes de realizar tais procedimentos. A terapia celular é uma alternativa terapêutica com grande potencial de aplicabilidade na fibrose pulmonar. Objetivos: Utilizar um modelo de fibrose pulmonar induzida pela bleomicina em ratos para investigar os efeitos da terapia com células-tronco mesenquimais (CTM) e o meio condicionado no remodelamento pulmonar com objetivo de elucidar o mecanismo de ação das CTM e meio condicionado, na reversão da fibrose pulmonar. Para tanto, buscamos: 1) padronizar a cultura de células-tronco mesenquimais e meio de cultura; 2) caracterizar o modelo experimental de fibrose pulmonar induzida pela bleomicina por microscopia óptica antes e após o tratamento com CTM e meio de cultura; 3) avaliar a expressão de biomarcadores sorológicos (Fibrinogênio, Fator von Willebrand e PDGF); 4) quantificar a expressão de proteínas relacionadas ao estresse oxidativo (NOS), citocinas pró-inflamatórias e pró-fibróticas (IL-17 e TGF-beta) e pró-angiogênicas (VEGF e endotelina) por imuno-histoquímica; e 5) quantificar a deposição de fibras do colágeno I e V por imunofluorescência. Materiais e Métodos: Utilizou-se um total de 44 ratos Wistar machos albinos com peso médio de 250-300g e 8 semanas de idade. Quatro grupos experimentais foram compostos de 10 animais, que participaram do experimento divididos em três momentos: D0, D10 e eutanásia (D14 ou D21). Em D0, foi realizada a instilação orotraqueal de bleomicina na dose de 1,5 U/kg; em D10, foi realizada a infusão em veia caudal de células-tronco mesenquimais na dose de 106 CTM/Kg ou 200 ?l de meio condicionado. Para o preparo das CTM, foi obtido, em média, 1,2 g de tecido adiposo, procedida a dissociação com colagenase tipo I, sendo que a contagem média de células foi de 3,05 x 106 células linfomononucleares/g de tecido adiposo. Estas células foram cultivadas durante 21 dias em meio Knockout DMEM-F12 suplementado com 10% de soro fetal bovino. Os seguintes três critérios foram utilizados para comprovar o perfil das células-tronco mesenquimais: aderência plástica, expressão de CD90 por citometria de fluxo ( > 90%) e capacidade de diferenciação em três linhagens mesodérmicas. Em D10, um pool destas células alogênicas foi infundido intravenosamente, na veia caudal, a uma concentração de 1 x 106 células/ animal num volume de 200 ul de solução salina. Em D14 ou D21, os animais foram eutanasiados e analisados quanto ao peso e conforme análises microscópico-laboratoriais. As análises histológicas foram realizadas por dois especialistas diferentes em estudo duplo-cego. Os parâmetros de ganho de peso e recuperação microscópica do tecido pulmonar foram analisados em cada grupo. Resultados: Nossos dados mostram que as células-tronco mesenquimais oriundas do tecido adiposo abdominal de ratos Wistar tiveram seu perfil fenotípico, capacidade de adesão plástica e diferenciação em 3 linhagens mesodérmicas estabelecidas inequivocamente, conforme estabelecido internacionalmente pela ISSCR. As células-tronco mesenquimais e o meio condicionado induziram: a recuperação clínica após tratamento; a reversão da inflamação e de fibrose pulmonar induzida pela bleomicina; a reversão da arteriopatia no parênquima pulmonar distal; a redução das concentrações de fibrinogênio, fator von Willebrand e PDGF (marcadores sorológicos); a diminuição da expressão de enzimas oxidantes; a diminuição da expressão de endotelina e a modulação da expressão da proteína de remodelamento (IL-17), da ativação dos fibroblastos TGF-B e da síntese de colágeno. Conclusão: Neste estudo, comprovamos que as terapias com células-tronco mesenquimais derivadas do tecido adiposo e com o meio condicionado foram eficazes na modulação dos processos inflamatórios e fibrogênicos no modelo induzido por bleomicina, agindo na ativação miofibroblástica, e, também, na restauração tecidual e endotelial. Além disso, o meio condicionado se mostrou tão eficiente quanto as células-tronco propriamente ditas, no efetivo remodelamento pulmonar, devendo ser considerado como uma proposta terapêutica viável e inovadora / Introduction: Idiopathic pulmonary fibrosis (IPF) is defined as a type of chronic interstitial fibrosing disease of unknown etiology limited to the lungs and presenting a histological pattern of usual interstitial pneumonia. The prevalence of IPF is estimated at approximately 20/100,000 in men and 13/100,000 in women, with the mean age of diagnosis being 67 years and the average survival is 2 to 5 years. It is estimated that 5 million people are affected worldwide. The current clinical treatment is associated with partial and transient improvement, with dubious or unsatisfactory results. In the surgical approach to IPF, pulmonary transplantation is a prominent feature, which is rare because of the scarcity of donors and the limitation of the teams capable of performing such procedures. Cell therapy is a therapeutic alternative with great potential for applicability in pulmonary fibrosis. Objectives: To use a model of bleomycin-induced lung fibrosis in rats to investigate the effects of mesenchymal stem cell (MSC) therapy and conditioned medium on lung remodeling in order to elucidate the mechanism of action of MSC and conditioned medium, in the reversion of pulmonary fibrosis. To do so, we aim to: 1) standardize the culture of mesenchymal stem cells and conditioned medium; 2) characterize the experimental model of lung fibrosis induced by bleomycin by optical microscopy before and after treatment with MSC and conditioned medium; 3) to evaluate the expression of serological biomarkers (Fibrinogen, Factor von Willebrand and PDGF); 4) to quantify the expression of proteins related to oxidative stress (NOS), pro-inflammatory and pro-fibrotic (IL-17 and TGF-beta) and pro-angiogenic cytokines (VEGF and endothelin) by immunohistochemistry; and 5) to quantify the deposition of collagen I and V fibers by immunofluorescence. Materials and methods: A total of 44 male albino Wistar rats weighing 250-300g and 8 weeks of age were used. Four experimental groups were composed of 10 animals, which participated in the experiment divided in three moments: D0, D10 and euthanasia (D14 or D21). In D0, orotracheal instillation of bleomycin at the dose of 1.5 U/ kg was performed; in D10 caudal vein infusion of mesenchymal stem cells at the dose of 106 MSC/ kg or 200 ul of conditioned medium was performed. To prepare the MSC, a mean of 1.2 g of adipose tissue was obtained, dissociated with type I collagenase and the mean cell count was 3.05 x 106 lymphomonuclear cells / g of adipose tissue. These cells were cultured for 21 days in DMEM-F12 Knockout medium supplemented with 10% fetal bovine serum. The following three criteria were used to prove the profile of mesenchymal stem cells: plastic adherence, expression of CD90 by flow cytometry ( > 90%) and differentiation capacity in three mesodermal lines. In D10, a pool of these allogeneic cells was infused intravenously into the caudal vein at a concentration of 1 x 106 cells / animal in a volume of 200 ul of saline. In D14 or D21 the animals were euthanized and analyzed for weight and microscopic-laboratory analyzes. Histological analyzes were performed by two different specialists in a double-blind study. The parameters of weight gain and microscopic recovery of lung tissue were analyzed in each group. Results: Our data show that the mesenchymal stem cells derived from the abdominal adipose tissue of Wistar rats had their phenotypic profile, plastic adhesion capacity and differentiation in 3 mesodermal lines established unequivocally, as established internationally by ISSCR. Mesenchymal stem cells and conditioned medium induced: clinical recovery after treatment; bleomycin-induced reversal of inflammation and pulmonary fibrosis; the reversal of arteriopathy in the distal pulmonary parenchyma; the reduction of fibrinogen, von Willebrand factor and PDGF concentrations (serologic markers); decreased expression of oxidizing enzymes; the reduction of endothelin expression and the modulation of the expression of the remodeling protein (IL-17), the activation of TGF-B fibroblasts and the synthesis of collagen. Conclusion: In this study we demonstrated that therapies with mesenchymal stem cells derived from adipose tissue and conditioned medium were effective in the modulation of inflammatory and fibrogenic processes in the bleomycin-induced model, acting to modulate myofibroblastic activation and also in tissue restoration and endothelial cells. In addition, the conditioned medium proved to be as efficient as the stem cells themselves, in effective pulmonary remodeling, and should be considered as a viable and innovative therapeutic proposal

O tecido ósseo possui potencial regenerativo e capacidade em restaurar completamente sua estrutura e função original. Há situações em que o organismo não consegue por si só, a reparação desejada dos defeitos ósseos. Vários métodos são propostos para a reparação de defeitos ósseos, entre eles, o uso de diferentes tipos de enxertos os quais demonstram capacidade em promover a formação óssea. Por muitos anos o osso autógeno foi considerado a referência padrão como enxerto ósseo, devido as suas vantagens biológicas e potencial osteogênico. Entretanto, por este material apresentar limitações, estimulou-se a pesquisa para um substituto ósseo ideal para o enxerto ósseo autogênico. O advento de um novo biomaterial xenogênico, como o osso bovino, que se comporta como promotor de reparação e é portador de fatores de indução óssea parece representar o futuro da reconstrução de defeitos ósseos. Mas pelo pequeno potencial de regeneração óssea produzida pelos enxertos alógenos e xenógenos, os pesquisadores tem utilizado a bioengenharia tecidual óssea para reconstrução de defeitos ósseos. Diante destas informações, este estudo teve como objetivo quantificar histomorfometricamente a reparação óssea após o enxerto de uma associação de osso autógeno e/ou osso bovino composto (Gen-Mix) associados a células- tronco mesenquimais em defeitos ósseos produzidos pela extração dental de ratos. 108 ratos foram separados em 6 grupos : Controle (c) - o defeito ósseo foi preenchido só por sangue; Osso autógeno (oa) - o defeito ósseo foi preenchido por sangue e osso autógeno; Gen-Mix (G-mix) o defeito ósseo foi preenchido por osso bovino composto (osso medular e cortical), liofilizado, desproteinizado, desmineralizado com aglutinante de colágeno bovino, na forma de grânulos de 0,25 a 1.0 mm (Gen-Mix, Baumer, Mogi Mirim, SP, Brasil); Célula-tronco (ctr)o defeito ósseo foi preenchido por sangue e células- tronco obtidas da medula óssea;Osso autógeno + células-tronco(oa+ctr)- o defeito foi preenchido pela associação destes dois compostos; Gen-Mix + células-tronco (G-mix+ctr)- o defeito foi preenchido pela associação dos dois produtos. Os animais foram sacrificados nos períodos de 7, 21 e 42 dias pós-cirurgia (n=6 por grupo) e as amostras teciduais foram processadas para a obtenção de secções finas (5 µ) e coradas com HE. Através de um sistema de análise de imagens se estimou a fração de volume do osso trabecular (%) nas vizinhanças do enxerto no interior do alvéolo. Os resultados histológicos mostraram que os materiais enxertados apresentaram uma osteointegração progressiva e sem reação de corpo estranho. A histometria revelou que o grupo enxertado com G-mix sozinho ou associado as célula- tronco produziu menor formação de osso, ao passo que, o osso autógeno sozinho ou associado ás células-tronco foi superior em volume de tecido ósseo em relação aos demais grupos. Conclui-se que o osso autógeno e o Gen-mix isoladamente ou associados ás célula- tronco foram biologicamente compatíveis desenvolvendo osteointegração progressiva e que o osso autógeno foi superior no processo de reparação do defeito ósseo. Estes resultados ainda sugerem que a associação das células-tronco aos enxertos ósseos acelerou a neoformação óssea, principalmente quando associadas ao osso autógeno. / Bone tissue has a regenerative potential as well as a capacity to fully restore its original structure and function. There are situations in which the body cannot repair in a proper way bone defects by itself. Several methods are proposed for repairing bone defects, among them there is the use of different types of grafts that demonstrates the capacity to promote bone formation. For many years, autogenous bone was considered the standard reference as bone grafts, due to its biological advantages and osteogenic potential. However, due to these material limitations, an ideal bone substitute research was encouraged for an autogenous bone graft. The advent of new xenogenic biomaterials, such as bovine bone, that behaves as a repair promoter and also has an induction bone factors, seems to represent the future reconstruction of bone defects. Researchers have used the bioengineered bone tissue for reconstruction of bone defects, due to the short potential produced by xenogeneic and allogenic grafts for bone regeneration. As the previous information show, this study aimed to quantify the histomorphometric bone healing after grafting a combination of autogenous bone and / or bovine bone composite (Gen-Mix) associated with mesenchymal stem cells in bone defects produced by tooth extraction in rats. 108 rats were divided into 6 groups: control (c) - the bone defect was filled only by blood, autogenous bone (oa) - the bone defect was filled with blood and autogenous bone graft; Gen-Mix (G-mix) bone defect were filled by bovine bone composite (marrow and cortical bone), lyophilized, deproteinized, demineralized with bovine collagen binder, in form of 0.25 to 1.0 mm granules (Gen-Mix, Baumer, Mogi Mirim, SP, Brazil); stem cells (ctr), the bone defect was filled with blood and stem cells obtained from bone marrow, autogenous bone + stem cells (oa + ctr) - the defect was filled by the association of these two compounds; Gen-Mix + stem cells ( G-mix + ctr) - the defect was filled by the association of both products. The animals were sacrificed at 7, 21 and 42 days post-surgery (n = 6 per group) and tissue samples were processed to obtain thin sections (5 µ) and stained with HE. The fraction of trabecular bone volume (%) in the vicinity of the graft inside the alveoli was estimated through an image analysis system. Histological findings showed that the grafted material has a progressive osseointegration and no foreign body reaction. The histometric analysis revealed that the group grafted with G-mix alone or associated with stem cells produced less bone formation, while the autogenous bone alone or associated with stem cells was higher in volume of bone tissue in relation to other groups. We conclude that autogenous bone and Gen-mix alone or associated with stem cells are biologically compatible developing progressive osseointegration and the autogenous bone graft was superior in the bone defect repair process. These results also suggested that the association of stem cells to bone grafts accelerated the bone formation, especially when combined with autogenous bone.

Human tissue banks are a potential source of cellular material for the emerging cellbased therapy industry; umbilical cord tissue (UCT) private banking is increasing in such facilities as a source of mesenchymal stem cells for future therapeutic use. However, early handling of UCT is relatively uncontrolled due to the clinical demands of the birth environment and subsequent transport logistics. It is therefore necessary to develop extraction methods that are robust to real world operating conditions,rather than idealised operation. This will be critical for all processes using primary tissue or cell sources. The research work undertaken in this PhD project was initiated by the collaboration with one of the leading private cord blood banks in the UK and later driven by the prospect of expanding the cell therapy and business potential of the bank. The investigation described in this thesis has focused on: - Developing an extraction method for human mesencymal stem cells (hMSCs) from UCT. - Understanding and minimizing the noticed variability in cell yield extracted from UCT by mapping the operating environment and assessing the risk factors before empirically determining their effect on the process. - Establishing the necessary process controls in the production of high quality hMSCs, through a series of wet experiments, targeted at narrowing down the sources of variability down to sub-process level. - Finding a novel method for assessing the cell content and viability of cords prior to processing. Therefore, helping the tissue processing facility to predict the risk of suboptimal cell yield from a given cord tissue section and processing method, given different operating ranges. - Determining the tissue storage requirements and isolation method with acceptable risk of adequate cell recovery. - Characterization of cells extracted from UCT via different extraction methods and comparison to primary cells extracted from other tissue sources. - Investigation of cryopreservation method for UCT. The result of this work provides a solid example of the type of data and analysis that will be required to inform a Quality-by-Design type approach for cell product development and manufacture. It will help tissue processing facilities and banks to predict the probability of cell yields from tissue sections given different operating ranges, and to aid and inform the experimental approach of others.