L’Osteoprotégérine est une cytokine soluble qui joue un rôle clé dans le métabolisme osseux et est impliquée dans la réponse immunitaire et l’hématopoïèse. Elle est associée à la dysfonction endothéliale et semble intervenir dans l’angiogenèse. Cette cytokine constituerait en fait, un trait d’union entre le tissu osseux et vasculaire. Son rôle dans la formation de la matrice osseuse est aujourd’hui bien élucidé mais son implication dans la vascularisation reste à établir. L’OPG est rapidement libérée par l’endothélium dans des conditions inflammatoires et est donc en mesure d’intervenir dans le processus de revascularisation initié par les cellules progénitrices endothéliales (PECs). Au cours de cette étude, nous avons tenté de comprendre le rôle joué par cette cytokines dans la néovascularisation induite in vitro, par une sous population de PECs appelées ECFCs (endothelial colony-forming cells), et sur la formation des néovaisseaux in vivo.Nous avons montré qu’elle agit sur la « souchitude » des cellules CD34+, potentialise les propriétés proangiogènes des ECFCs in vitro, et participe au processus angiogénique in vivo. L’OPG agit sur les ECFCs via le syndécanne-1, inhibe leur adhésion à la matrice extracellulaire, favorise leur migration et leur tubulogenèse via la voie SDF-1/CXCR4, et potentialise leur adhésion à l'endothélium activé. Les effets observés sont corrélés à la libération du SDF-1, une activation des voies de signalisation ERK1/2, Akt et mTOR et à une activation de l’intégrine αVβ3. Par ailleurs, nous avons montré que l'OPG potentialise l’effet proangiogène du FGF-2 in vivo. Elle participe également au développement tumoral et à la dissémination des métastases, probablement via l'inhibition de l'apoptose des cellules tumorales, mais aussi par la promotion de l'angiogenèse tumorale. / Osteoprotegerin is a key regulator of bone metabolism involved in the immune response, hematopoiesis, and endothelial dysfunction. It seems to be implicated in angiogenesis and may represent a link between bone and vascular system. Although its role in bone is well recognized, its involvement in vasculature remains to be established. In inflammatory conditions, OPG is constitutively released by endothelial cells and smooth muscle cells, and therefore is able to participate in blood vessels formation induced by endothelial progenitor cells (EPCs). In this study we attempted to determine, in vitro the precise role of OPG in angiogenesis process induced by a subpopulation of EPCs called “endothelial colony-forming cells” (ECFCs), and on neovessel formation in vivo.We found that OPG causes phenotype changes of ECFCs via the activation of different molecular pathways targeting cell clonogenicity, differentiation, proliferation, migration and adhesion. Our results suggest that OPG may interact with ECFCs through its binding to syndecan-1, to induce an anti-adhesive effect and thereby promoting ECFCs migration through a SDF-1/CXCR4 dependant pathway and the ERK1/2, Akt and the mTOR pathways activation. OPG can intervene on the autocrine effect of ECFCs by inducing their adhesion to activated endothelium and their tubulogenesis, and potentiate their paracrine effects by inducing SDF-1 release. Alternatively, it can promote ECFCs survival, probably, in a αVβ3 integrin-dependent manner. In vivo, OPG potentiates FGF-2 proangiogenic effects and may participate in tumour growth, invasion and metastasis, possibly through inhibition of tumour cell apoptosis but also by promoting tumour angiogenesis.

Based on data from the World Healthcare Organisation, cardiovascular diseases are the primary cause of disease-related death globally, with myocardial infarction (MI) being the most prevalent. If not treated effectively, MI can progress to heart failure (HF). With 70 million prescriptions for HF in 2014 and 515 people in the UK being hospitalised daily with MI, the British Heart Foundation calls for novel robust treatments. Even though cardiac stem cell (CSC) therapy for MI has been under investigation for more than a decade, there still has not been a consensus over the identity of the adult endogenous CSC. Recent clinical trials, using selected Ckit+ cells or the cardiosphere-derived cells (CDCs) have shown moderate results. The aim of this thesis was to develop a digestion-based method for isolation of cardiac progenitor cells (CPCs) from the mouse atria. The resulting "CTs" were isolated by collagenase/trypsin (where their name has resulted from) digestion with a prolonged period step for cell attachment. CTs were compared to isolated CDCs for their marker expression, using RT-PCR and Immunocytochemistry, showing cells with a mesenchymal phenotype which expressed SCA1 and CKIT. The CDCs had more of a fibroblast phenotype with higher Ddr2 and Wt1 expression. Using a TGF-β1 differentiation protocol, the CTs could be differentiated more effectively to a CM lineage than could the CDCs. In addition, Oleic acid (OA) supplementation stimulated the Peroxisome proliferator-activated receptor alpha pathway and led to maturation of the CT cells, both before and after differentiation. The differentiated CTs begin to express Tnnt2, while OA led to Myh7 increase and upregulated their oxidative metabolism. Finally, the CTs were more able to survive under serum-starvation than the CDCs, and transfection with miR-210 could enhance CT survival under these conditions and increased VEGF secretion. By digestion of the whole atria and allowing a prolonged time for attachment, we have developed a novel isolation protocol which generates a cell population containing a range of progenitors. Cells within this population can survive under serum starvation and can be differentiated to a CM lineage, making them a promising therapeutic population.

No description available.

Angiogenic cell therapy is currently being developed as a treatment for coronary artery disease (CAD); however, endothelial dysfunction (ED), commonly found in patients with CAD, impairs the ability for revascularization to occur. We hypothesized that culture on a collagen matrix will improve survival and function of circulating progenitor cells (CPCs) isolated from a mouse model of ED. Overall, ED decreased the expression of endothelial markers in CPCs and impaired their function, compared to normal mice. Culture of CPCs from ED mice on collagen was able to increase cell marker expression, and improve migration and adhesion potential, compared to CPCs on fibronectin. Nitric oxide production was reduced for CPCs on collagen for the ED group; however, CPCs on collagen had better viability under conditions of serum deprivation and hypoxia, compared to fibronectin. This study suggests that a collagen matrix may improve the function of therapeutic CPCs that have been exposed to ED.

Of the thousands of dividing progenitor cells (PCs) generated daily in the adult brain only a very small proportion survive to become mature neurons through the process of neurogenesis. Identification of the mechanisms that regulate cell death associated with neurogenesis would aid in harnessing the potential therapeutic value of PCs. Apoptosis, or programmed cell death, is suggested to regulate death of PCs in the adult brain as overexpression of B-cell lymphoma 2 (Bcl-2), an anti-apoptotic protein, enhances the survival of new neurons. To directly assess if Bcl-2 is a regulator of apoptosis in PCs, this study examined the outcome of removal of Bcl-2 from the developing PCs in the adult mouse brain. Retroviral mediated gene transfer of Cre into adult floxed Bcl-2 mice eliminated Bcl-2 from developing PCs and resulted in the complete absence of new neurons at 30 days post viral injection. Similarly, Bcl-2 removal through the use of nestin-induced conditional knockout mice resulted in reduced number of mature neurons. The function of Bcl-2 in the PCs was also dependent on Bcl-2-associated X (BAX) protein, as demonstrated by an increase in new neurons formed following viral-mediated removal of Bcl-2 in BAX knockout mice. Together these findings demonstrate that Bcl-2 is an essential regulator of neurogenesis in the adult hippocampus.

During spinal cord injury (SCI), glutamate excitotoxicity and astrocytic scar formation can impede repair. In a preliminary study we found that ketamine, a N-methyl-D-aspartate (NMDA) receptor non-competitive antagonist, can contribute to functional recovery post SCI. Therefore, we investigated the cellular basis for this recovery with respect to neural progenitor cells using an in vitro cell culture model. We examined whether ketamine and glutamate influenced the proliferation, differentiation, and migration of differentiating endogenous neural progenitor cells (NPCs) found in the subventricular zone and spinal cord. Our study illustrates that the post functional recovery may have been due to ketamine’s influence on delaying spinal cord NPCs derived astrocyte maturation and migration while increasing radial glial cell migration. These results are promising since ketamine administration may help alleviate some of the adverse affects glutamate has on the NPCs found in the spinal cord following SCI.

Recent advances in cellular reprogramming have enabled the generation of embryoniclike cells from virtually any cell of the body. These inducible pluripotent stem cells (iPSCs) are capable of indefinite self-renewal while maintaining the ability to differentiate into all cell types. Nowhere will this technology have a greater impact than in the ability to generate disease and patient-specific cell lines. Here we explore the capacity of human iPSCs reprogrammed from peripheral blood endothelial progenitor cells lines to differentiate into atrial myocytes for the study of patient specific atrial physiology. Methods and Results: Late outgrowth endothelial progenitor cells (EPCs) cultured from clinical blood samples provided a robust cell source for genetic reprogramming. Transcriptome analysis hinted that EPCs would be comparatively more amenable to pluripotent reprogramming than the traditional dermal fibroblast. After 6 passages, EPCs were transduced with a doxycycline inducible lentivirus system encoding human transcription factors OCT4, SOX2, KLF4 and Nanog to permit differentiation after removal of doxycycline. The high endogenous expression of key pluripotency transcripts enhanced the ease of iPSC generation as demonstrated by the rapid emergence of typical iPSC colonies. Following removal of doxycycline, genetically reprogrammed EPC-iPSC colonies displayed phenotypic characteristics identical to human embryonic stem cells and expressed high levels of the pluripotent markers SSEA-4, TRA1-60 and TRA1-81. After exposure to conditions known to favor atrial identity, EPC- iPSC differentiating into sheets of beating cardiomyocytes that expressed high levels of several atrial-specific expressed genes (CACNA1H, KCNA5, and MYL4). Conclusions: EPCs provide a stable platform for genetic reprogramming into a pluripotent state using a doxycycline conditional expression system that avoids reexpression of oncogenic/pluripotent factors. Human EPC-derived iPSC can be differentiated into functional cardiomyocytes that express characteristic markers of atrial identity.

La persistance et la récurrence de la réponse inflammatoire dans le foie est la principale cause de développement des lésions hépatiques et de ses complications. Cette réponse immune est caractérisée par le recrutement de cellules inflammatoires, notamment de monocytes/macrophages et de lymphocytes. Elle est aussi impliquée d'une part dans le développement de la fibrose en activant les cellules fibrogéniques du foie, et d'autre part dans les mécanismes de régénération. Récemment, la présence d'une sous-population de lymphocytes T appelés Th17 a été identifiée dans de nombreuses pathologies hépatiques incluant la fibrose. Leur présence a également été observée lors de la régénération hépatique à partir de cellules progénitrices. Ces lymphocytes Th17 sont caractérisés principalement par la production de deux cytokines l'IL-17 et l'IL-22. Alors que le rôle de l'IL-22 a été récemment caractérisé dans la fibrose et la régénération hépatique, le rôle de l'IL-17 nécessitait d'être défini.Les objectifs de ma thèse ont donc été d'étudier le rôle et la régulation de l'IL-17 dans 1) les mécanismes de développement de la fibrose, et 2) son impact sur la régénération hépatique à partir de cellules progénitrices.1) L'équipe a démontré l'implication du système endocannabinoïde dans les maladies chroniques du foie. Ce système est composé de deux types de récepteurs, CB1 et CB2, couplés aux protéines G, et de ligands lipidiques endogènes. Alors que CB1 est majoritairement exprimé dans le cerveau et faiblement dans les tissus périphériques, CB2 est très fortement exprimé par les cellules immunitaires et présente en général des propriétés anti-inflammatoires. Notre hypothèse a donc été que l'activation du récepteur CB2, exprimé par les lymphocytes T régulerait négativement la réponse de type Th17 et le développement de la fibrose hépatique. Dans un modèle murin de fibrose induite par la ligature de la voie biliaire principale, nous avons montré que des souris CB2-/- développent une fibrose plus importante associée à une réponse inflammatoire de type Th17 augmentée par rapport à des souris sauvages. De plus, l'inhibition de la production de l'IL-17 chez ces souris CB2-/- à l'aide d'un inhibiteur de la différenciation Th17 (la digoxine), permet de réduire le développement de la fibrose. In vitro, l'activation du récepteur CB2 sur les lymphocytes Th17 par un agoniste sélectif, le JWH-133, diminue leur capacité à produire l'IL-17. Par ailleurs, nous avons montré par immunocyto-fluorescence, que l'activation de CB2 inhibe la production de l'IL-17 par une voie STAT5-dépendante. Ainsi, l'immuno-modulation de la réponse inflammatoire associée à la fibrose représente une stratégie anti-fibrosante prometteuse.2) L'infiltration de ces cellules Th17 a également été observée dans de nombreuses hépatopathies associées à une accumulation de cellules progénitrices intra-hépatiques (CPIH), appelées cellules ovales chez les rongeurs. La deuxième partie de ma thèse a donc consisté en l'étude du rôle de l'IL-17 dans la régénération du foie à partir de CPIH. Dans un modèle murin de régénération hépatique à partir de CPIH induit par un régime déficient en choline, et supplémenté en éthionine (CDE), nous avons montré que des souris invalidées pour l'IL-17 (IL-17-/-) présentent une diminution du nombre de CPIH CK19+, par rapport aux souris sauvages. L'invalidation de l'IL-17-/- a également été associée à une réduction du recrutement macrophagique et de la réponse pro-inflammatoire incluant l'IL-6, l'IL-1b et l'IL-27. Parmi ces cytokines, nous avons identifié l'IL-27 comme étant un facteur clé dans la différenciation des CPIH. In vitro, nous avons également montré que la production de l'IL-27 est induite par la stimulation des macrophages par l'IL-17. Ainsi, nos résultats révèlent que l'action combinée de l'IL-17 et de l'IL-27 orchestre la régénération du foie à partir de CPIH en favorisant leur prolifération et leur différenciation en hépatocytes, respectivement. / Persistence and recurrence of inflammatory response in the liver is the major cause of liver damage and its complications. This immune response is characterized by inflammatory cell recruitment, in particular monocytes / macrophages and lymphocytes. It has also been implicated in fibrosis development by activating liver fibrogenic cells, and also in regeneration mechanisms. Recently, a sub population of T lymphocytes called Th17 has been identified in numerous liver pathologies including fibrosis. These cells have also been observed in regenerating livers from progenitor cells. Th17 lymphocytes are mainly characterized by the production of two cytokines, namely IL-17 and IL-22. While the role of IL-22 has been recently described in liver fibrosis and regeneration, the role of IL-17 remained to be studied.The aims of my thesis were to study the role and the regulation of IL-17 in 1) fibrosis development, and 2) its impact on liver regeneration from progenitor cells.1) Our team has demonstrated the implication of the endocannabinoid system in chronic liver disease. This system is composed of two G-protein coupled receptors, CB1 and CB2, and their endogenous lipidic ligands. While CB1 is mainly expressed in the brain and slightly in peripheral tissues, CB2 is strongly expressed by immune cells and generally displays anti-inflammatory properties. Our hypothesis was that CB2 receptor activation, expressed by T lymphocytes, would negatively regulate Th17 response and liver fibrosis development. In a murine fibrosis model induced by bile duct ligation and section, we have showed that CB2-/- mice have an enhanced fibrosis associated with an increased Th17 inflammatory response as compared to wild-type animals. Additionally, IL-17 production inhibition in these CB2-/- mice with an inhibitor of Th17 differentiation (digoxin), reduced fibrosis development. In vitro, CB2 receptor activation on Th17 lymphocytes by a selective agonist, JWH-133, diminished their ability to produce IL-17. Moreover, we demonstrated by immunocyto-fluorescence, that CB2 activation inhibits IL-17 production in a STAT5-dependent manner. Thus, the modulation of the inflammatory response associated with fibrosis is a promising strategy against fibrosis.2) Th17 cell infiltration has also been observed in liver disease associated with liver progenitor cell (LPC) accumulation, called oval cells in rodents. The second part of my thesis was aimed to study IL-17 role in liver regeneration from LPC. In a murine model of liver regeneration from LPC induced by a choline deficient and éthionine supplemented diet (CDE), we have demonstrated that IL-17 deficient mice (IL-17-/-) have a reduced number of CK19+ LPC as compared to wild-type animals. IL-17 deficiency was also associated with a decrease in macrophage recruitment and reduced pro-inflammatory response including IL-6, IL-1b and IL-27. Among these cytokines, we identified IL-27 as a key factor in LPC differentiation. In vitro, we also showed that IL-27 production is induced after IL-17-induced macrophage activation. Thereby our results revealed that the combined action of IL-17 and IL-27 leads to LPC driven liver regeneration by enhancing their proliferation and their differentiation towards hepatocytes, respectively.

Células-tronco humanas derivadas da polpa dental (hDPSCs) e células-tronco humanas derivadas de tecido adiposo (AhSCs) são células multipotentes capazes de diferenciação osteogênica in vitro e in vivo, e promissoras fontes de células para a engenharia de tecido ósseo, dada a sua facilidade de expansão, isolamento e diferenciação. É de grande interesse compreender qual é o melhor tipo celular para diferenciação osteogênica, assim, o objetivo deste estudo foi comparar o potencial de diferenciação osteogênica in vitro e in vivo entre hDPSCs e hASCs. Foram isoladas e estabelecidas seis populações de células-tronco de hDPSCs (entre 7-12 anos) e seis da hASC (de indivíduos com idade entre 30-49 anos). Após a indução in vitro, a diferenciação osteogênica foi comprovado através das colorações de fosfatase alcalina (9 dias) e vermelho de alizarina (14 e 21 dias). A quantificação da mineralização da matriz após 21 dias de diferenciação osteogênica revelou 2,24 mais ossificação das hDPSCs em relação às hASCs. Para realizar o experimento in vivo, foram triados seis biomateriais para verificar qual melhor biomaterial para o nosso modelo, defeito crítico em calvária de Ratos Wistar não imunossuprimidos, com três amostras de hDPSCs. Após 45 dias, CellCeram(TM) exibiu a melhor neoformação óssea in vivo, e foi selecionado para comparar os potenciais osteogênicos in vivo entre hDPSCs e hASCs. Células (10e6) foram associadas a discos de 4,5 mm CellCeram(TM), grupo controle foi realizado através do transplante do biomaterial livre de células. Neoformação óssea foi mensurada 45 dias após a cirurgia através da coloração histológica de hematoxilina / eosina. A formação óssea total foi quantificada através da análise de imagens de todas as ilhas de ossificação. A associação entre hDPSCs e CellCeram(TM) promoveu 7,24 vezes mais neoformação óssea quando comparado com a associação entre esse mesmo material e hASCs (p <0,0001). A utilização de células-tronco adultas para regeneração óssea é uma ótima abordagem para uso terapêutico, e calcular ou predizer o potencial osteogênico das células utilizadas é extremamente importante e necessário para futura aplicação em novas estratégias de bioengenharia de tecido ósseo / Human dental pulp stem cells (hDPSCs) and human adipose-derived stem cells (AhSCs) are multipotent cells capable of undergoing osteogenesis in vitro and in vivo, and promising cell-source populations for bone tissue engineering given their easiness of isolation, expansion and differentiation. It is of great interest to understand which is the best cell type for osteogenic differentiation, thus the aim of this study is to compare the in vitro and the in vivo osteogenic differentiation potentials between DPSCs and ASCs. We isolated six stem cell populations from DPSCs (aged 7-12 years) and six from ASCs (from subjects aged 30-49 years) and cell culture was established. After in vitro induction the populations were able to undergo osteogenic differentiation, as evidenced by alkaline phosphatase (9 days) and alizarin red S (14 and 21 days) stainings. Quantification of matrix mineralization after 21 days of osteogenic differentiation revealed an enhancement of 2.24-fold increase between hDPSCs and hASCs differentiation. To perform the in vivo experiment, we promoted a screening of six scaffolds to find out which would be best scaffold to our model, a calvarial critical-sized defect in Wistar non-immunosuppressed rats, with three different culture samples of hDPSCs. After 45 days, CellCeram(TM) displayed the best in vivo bone neoformation, and was used to compare the in vivo osteogenic potentials between hDPSCs and hASCs. Cells (10e6) were associated to 4.5 mm CellCeram(TM) discs, and control groups were performed transplanting the biomaterial free of cells. Bone healing was measured through histological hematoxylin/eosin staining 45 days after surgery. Newly formed bone was also evaluated by total bone island surface quantification through image analysis. The association between hDPSCs and CellCeram(TM) induced a mean of 7.24 times more bone formation when compared to the association between this same material and hASCs (p<0.0001). The use of adult stem cells for bone regeneration is a robust therapeutic option, and calculate or predicts the osteogenic potential of the cell used are extremely important and necessary to future application, and translation to new strategies in bone tissue engineering

O Corpo Ciliar (CC) é uma fonte de células tronco da retina de animais adultos, mas sua ativação permanece desconhecida. GTPases Rho são proteínas que reorganizam do citoesqueleto de actina, regulam vias de sinalização e transcrição gênica, sobrevivência celular e proliferação. Neste trabalho, investigamos a expressão das GTPases Rho nas células do CC e seu efeito na regulação do ciclo celular. As GTPases RhoA, RhoB e Rac1 foram expressas nas células do CC e sua ativação pelo ácido lisofosfatidico (LPA) aumentou a expressão dos genes progenitores retinianos Pax6 e Chx10. A inibição das proteínas por Toxina A de Clostridium difficile aumentou a proliferação no CC e potencializou o efeito proliferativo dos fatores de crescimento. A inibição especifica destas proteínas diminuiu a expressão dos transcritos de p21cip, p27kip, p16INK4a e p19INK4d e aumentou de Ki67, CiclinaA e D1. O estudo da via de Wnt indicou que Rac1 regulou os genes de componentes da degradação de -catenina e Lef1. Concluímos que a inativação das GTPases Rho induziu a proliferação das células progenitoras retinianas localizadas no CC e regulou a via de Wnt. Sua ativação induziu o perfil de célula progenitora, sugerindo uma nova ferramenta para o mecanismo de reparo retiniano. / Ciliary Body (CB) is a potential source of stem cells in the adult retina, but its activation is still unknown. Rho GTPases play a role in actin-based cytoskeleton reorganization, regulate signaling pathways and gene transcription, cell survival and cell proliferation. In this study we investigated the expression of Rho GTPases in CB cells and their role on cell cycle regulation. The GTPases RhoA, RhoB and Rac1 were present in CB cells and the activation by lysophosphatidic acid (LPA) increased the expression of the progenitor genes Pax6 and Chx10. The inhibition by Clostridium difficile Toxin A increased the proliferation of CB cells and potentiated the proliferative effect of growth factors. The specific inhibition decreased the expression of p21cip, p27kip, p16INK4a and p19INK4d as well as increased Ki67, cyclinA and D1 transcripts. The Wnt pathway study indicated that Rac1 regulated -catenin degradation genes components and Lef1. Taken together, the inactivation of Rho GTPases stimulated the proliferation of progenitor cells located in CB as well as regulate the Wnt signaling pathway. The proteins activation was correlated to progenitor profile induction. These different mechanisms may provide a potential new approach on retinal repair.