L’activation du système complément, qui se produit à la suite d’une infection ou d’un trauma, génère de puissants signaux moléculaires capables d’initier la réaction inflammatoire. Parmi ces signaux, le fragment actif C5a permet de recruter sur le site lésé les cellules qui expriment son récepteur (le C5aR/CD88). Bien que le C5aR/CD88 soit initialement connu pour être exprimé par les cellules inflammatoires, il est établi que de nombreuses cellules non immunitaires expriment ce récepteur indiquant son implication dans d’autres processus. Nos résultats ont permis de démontrer que l’activation du système du complément au niveau de la pulpe dentaire est réalisée non seulement à partir des protéines plasmatiques mais aussi des protéines synthétisées par les fibroblastes pulpaires. Ainsi, l’activation locale du système du complément, produit à la suite d’une infection, d’un trauma ou de l’application de biomatériaux, génère du C5a qui induit la migration des cellules progénitrices. Ce travail démontre pour la toute première fois l’implication du fragment actif C5a dans le recrutement de progénitrices pulpaires, étape clef au processus de régénération pulpo-Dentinaire. Ces travaux pourraient donc constituer une piste sérieuse dans l’établissement de nouvelles thérapies permettant de cibler les cellules progénitrices au cours du processus de régénération. / After tissue injury or infection, Complement activation provides powerful signals initiating the inflammatory reaction. These events are mediated by biologically active fragments such as C5a which attracts cells expressing its receptor (C5aR/CD88) to the injury site. Besides inflammatory cells as the main C5aR-Expressing cells, various tissue cells have been reported to express this receptor suggesting its involvement in other processes. In order to investigate the possible relationship between complement activation and pulp regeneration, we investigated Complement activation in the dental pulp and progenitor cell migration from their perivascular niches to the pulp injury site to initiate the regeneration process.Our results indicate that complement activation in the dental pulp is the result of both plasma and fibroblast secreted complement proteins. Thus upon local complement activation, which can occur after pathological injury or biomaterials application, C5a induces pulp progenitors’ migration which is critical in initiating the regenerative processes. To our knowledge, this is the first work to demonstrate the involvement of C5a biologically active fragment in the recruitment human pulp progenitor cells. This may provide a useful future therapeutic tool in targeting the progenitor cells in a dentin/pulp regeneration process.

A hematopoese tem sido bem descrita em modelos murinos nas últimas décadas, contudo, trabalhos demonstrando os mecanismos da hematopoese em humanos ainda são escassos. A derivação da primeira linhagem de células-tronco embrionárias humanas (CTEhs) em 1998, gerou novas perspectivas tanto para o estudo da hematopoese na tentativa de mimetizar o que ocorre naturalmente durante o desenvolvimento embrionário, quanto para a aplicação clínica das células hematopoéticas obtidas a partir da diferenciação dessas células. Contudo, apesar de inúmeros trabalhos terem demonstradoa obtenção de células hematopoéticas a partir de CTEhs, os protocolos têm gerado quantidades variáveis de células, com baixa eficiência e com propriedades funcionais de células primitivas. Desse modo, este trabalho procurou estabelecer um modelo próprio de diferenciação de CTEhs-H1 em células progenitoras hematopoéticas para que estas pudessem ser melhor caracterizadas e obtidas de forma mais eficiente. Para isto, foi desenvolvido um sistema de diferenciação baseado no co-cultivo da linhagem de CTEh-H1 com fibroblastos de embrião de camundongo (MEFs), em meio de diferenciação suplementado soro fetal bovino (SFB) e citocinas e fatores de crescimento hematopoéticos em baixas concentrações. Como resultado, o desenvolvimento do presente trabalho permitiu o estabelecimento de um método para geração de populações mistas de células enriquecidas em CPHs positivas para o marcador CD45, o qual mostrou ser coexpresso com outros marcadores hematopoéticos (CD31, CD43, CD71 e CD38), e células hematopoéticas maduras positivas para marcadores mielóide-específicos (235a, CD14, CD15, CD16) e com características morfológicas típicas. Foi demonstrado que as células obtidas expressavam genes relativos ao sistema hematopoético (CD45, CD31, runx1, tal1, lmo2, prom1, CD34 e notch1), e possuíam potencial clonogênico in vitro da ordem de 1/574 células plaqueadas. Em adição, corroboramos os achados de que as células hematopoéticas apresentam duas origens distintas: a partir do endotelio hemogênico e a partir de células com propriedades hemangioblásticas independentes do endotélio hemogênico. / Hematopoiesis has been well described in murine models in recent decades, however, studies demonstrating the mechanisms of hematopoiesis in humans are still scarce. The first human embryonic stem cells line (hESCs) derived in 1998, has generated new perspectives about the study of hematopoiesis as in attempting to mimic what naturally occurs during embryonic development, as for clinical application of hematopoietic cells obtained from the differentiation of these cells. However, although numerous studies have shown the production of hematopoietic cells derived from hESCs, the protocols have generated varying quantities of cells with low efficiency and functional properties of primitive stem cells. Thus, this study sought to establish our own model for hESC-H1 differentiation in hematopoietic progenitor cells so that they could be better characterized and obtained more efficiently. For this way, we developed a differentiation system based on co-culture of hESC-H1 line with inactivated mouse embryonic fibroblasts (MEFs) in differentiation medium supplemented with fetal calf serum (FCS) and cytokines and hematopoietic growth factors in low concentrations. As a result, the development of this study allowed the establishment of a method for generation of mixed population of cells enriched in hematopoietic progenitor cells positive for the marker CD45, which proved to be co-expressed with other hematopoietic markers (CD31, CD43, CD71 and CD38), and mature hematopoietic cells positive for myeloid-specific markers (235a, CD14, CD15, CD16) and morphological characteristics typical. It was shown that these cells expressed genes related to the hematopoietic system (CD45, CD31, runx1, TAL1, LMO2, prom1, CD34 and NOTCH1), and had clonogenic potential in vitro of 1/574 plated cells. In addition, we corroborate the findings that hematopoietic cells have two distinct origins: they can arise as from an hemogenic endothelium as from cells with hemangioblastic properties by an hemogenic endothelium-independent way.

O saco vitelino é o único anexo embrionário presente em todas as espécies dos embriões vertebrados, répteis, aves e mamíferos. Em mamíferos domésticos o saco vitelino é inicialmente grande, pois nestas espécies ele é transitório. Após a implantação, surge no mesênquima lateral à notocorda agrupamentos de células, denominados ilhotas sanguíneas, que representam os progenitores dos sistemas vascular e hematopoético: os hemangioblastos. Os hemangioblastos centrais das ilhas sanguíneas formam as primeiras células-tronco hematopoéticas, enquanto os hemangioblastos periféricos se diferenciam em angioblastos, os precursores dos vasos sanguíneos. O desenvolvimento inicial da atividade hematopoética no saco vitelino conduz a hipótese de que esse tecido é o local primário de desenvolvimento hematopoético e que as células-tronco derivadas dele semeiam os outros sítios intraembriônicos. Foi possível observar nas análises microscópicas que realmente existe uma relação entre ambas linhagens. Nas análises de expressão gênica, alguns genes expressos pelo hemangioblasto apresentaram alta expressão nas análises D+0 e outros genes também específicos do hemangioblasto, porém em estágios secundários de diferenciação como os encontrados na região aórtica, a nível de endotélio hemogênico apresentaram altos níveis de expressão após 3 dias em cultivo. Concluímos portanto, que o saco vitelino por ser o local primário de formação das células sanguíneas e endoteliais nos estágios iniciais da embriogênese, por serem primitivas e, portanto não expressarem marcadores de células maduras na sua superfície, tornam estas células uma importante fonte de células-tronco relevante para a Terapia Celular para hemofilia e muitas outras doenças humanas. / The yolk sac is the single attachment embryo present in all species of vertebrate embryos, reptiles, birds and mammals. In domestic mammals the yolk sac is initially large, since these species it is transient. After implantation, appears in the lateral mesenchyme to the notochord cell clusters, called \"blood islands\" that represent the progenitors of vascular and hematopoietic systems: the hemangioblasts. The central islands hemangioblasts form the first blood hematopoietic stem cells, while peripheral hemangioblasts, the angioblastic differentiate into the precursors of blood vessels. The initial development of the yolk sac hematopoietic activity leads to the hypothesis that this tissue is the primary site of development and that hematopoietic stem cells derived from them sow other intraembryos sites. It was observed in the microscopic analysis that there is indeed a relationship between the two lineages. In the analysis of gene expression, some genes expressed by hemangioblasts showed high expression in D+0 and other specific genes also hemangioblasts, but in secondary stages of differentiation as found in the aortic region, the level of hemogenic endothelium showed high levels of expression after 3 days in culture. We therefore conclude that the yolk sac to be the primary site of formation of blood and endothelial cells in the early stages of embryogenesis, for its cells be primitive and therefore do not express markers of mature cells on the surface, these cells become an important source of cells relevant to stem cell therapy for hemophilia and many other human diseases.

Introduction: Vascular injury is the crucial initiating event in atherosclerosis and is universal following percutaneous coronary intervention. The cellular response to this injury largely determines vessel outcome. Endothelial progenitor cells (EPCs) and their progeny, late outgrowth endothelial cells (EOCs) are thought to play an important role in this process and characterising this role would be valuable in better understanding vascular injury and repair. Methods: The radial artery in the context of transradial cardiac catheterisation was examined as a model of vascular injury with characterisation of structural injury, longitudinal function and EPC populations. To examine the role of late outgrowth endothelial cells a method for GMP-compliant cell culture and labelling with F18Fluorodeoxyglucose was developed with a view to conducting a cell-tracking study of human administration. Results: Radial artery function was reduced following transradial cardiac catheterisation with recovery over a period of three months. There was no correlation between recovery of arterial function and EPC populations as defined by conventional surface markers. A research grade protocol for EOC culture was successfully translated to a GMP-compliant process producing a viable, phenotypically homogeneous EOC product. Cells were successfully labelled with F18Fluorodeoxyglucose and whilst proliferation was reduced, acute viability and function were not compromised. Conclusion: The radial artery in the context of transradial cardiac catheterisation is a useful model of vascular injury and repair although recovery of vascular function does not appear to be influenced by EPC populations. GMP-compliant culture and labelling of EOCs is feasible and will allow examination of the physiology of these cells in vivo in man.

Les adipocytes (cellules spécialisées dans le stockage des graisses) sont formés à partir de cellules immatures appelées cellules progénitrices lors du processus d'adipogenèse. Chez l'homme, les différentes étapes de ce processus sont mal connues ainsi que les signaux qui le régulent. La première partie de mon travail de thèse a eu pour but de caractériser la cellule intermédiaire entre la cellule progénitrice et l'adipocyte : le préadipocyte. La deuxième partie a consisté à évaluer le rôle des protéines morphogénétiques de l'os (ou BMP), des inducteurs de l'adipogenèse décrits chez la souris, dans l'adipogenèse humaine. Nous avons montré que les BMP2, 4 et 7 sont produites dans le tissu gras humain et BMP7 est modulée par l'obésité. Les BMP2 et 4 induisent l'adipogenèse des cellules progénitrices humaines mais seule la BMP7 permet la production d'adipocytes particuliers " beiges " décrits pour consommer les lipides et produire de la chaleur. Ces travaux affinent nos connaissances sur les mécanismes impliqués dans l'expansion du tissu gras et permettront d'élaborer des stratégies pour lutter contre le développement des pathologies liées à l'obésité. / Adipocytes (cells specialized in fat storage) arise from immature cells, called progenitor cells, during the process of adipogenesis. In human, the different stages of adipogenesis are not well defined as well as the signals involved in adipogenic modulation. The first part of my thesis work aimed to characterize the intermediate cell state between progenitor cell and mature adipocyte: the preadipocyte. The second part aimed to evaluate the role of bone morphogenetic proteins (BMPs) in human adipogenesis. In mice, BMP2 and BMP4 induce classical adipogenesis whereas BMP7 leads to the production of "brite" adipocytes with the capacity to use lipids to produce heat. We have shown that BMP2, 4 and 7 are produced in human fat depots and BMP7 is modulated by obesity. BMP2 and 4 induce classical adipogenesis and BMP7 only induces brite adipogenesis from human progenitor cells. These works improve our knowledge about the mechanisms involved in the expansion of fat depot and may allow the identification of new strategies to fight against the development of obesity-associated pathologies.

Conventional medical treatments fail to address the underlying problems associated with the damage inflicted by a coronary event. Thus, the long-term prognosis of patients admitted for heart failure is disheartening, with reported survival rates of 25 percent. Recent advances in stem cell research highlight the potential benefits of autologous stem cell transplantation for stimulating repair in heart tissue. However, a majority of those suffering from cardiovascular diseases are older adults whose autologous cells no longer possess optimum functional capacity. Additional work is needed to identify the optimal cell types or conditions that will promote cardiovascular regeneration across all age groups. A pretreatment, such as short-term hypoxia, and concurrent implementation of a novel progenitor, such as those that co-express Isl-1 and c-Kit, may enhance the results reported in clinical trials completed to date. However, the effects of short-term hypoxia in this novel cell type are unknown and warrant investigation in vitro. Cloned adult and neonatal Isl-1+ c-Kit+ human cardiovascular progenitor cells were characterized and expanded for study. Populations from both age groups were preconditioned using short-term hypoxia (1% O2 for six hours) and, to identify shifts in gene expression, compared to their respective control (21% O2 at 37 °C) via qRT-PCR. Flow cytometry and western blot analysis was utilized to measure phosphorylation of Akt. Progression through the cell cycle was also analyzed by flow cytometry. Cellular function was evaluated by the use of a TUNEL assay and Transwell® invasion assay. Hypoxia-mediated alterations of a genetic or functional nature in Isl-1+ c-Kit+ human cardiac progenitors are clearly age-dependent. Although both age groups accrued benefit, the neonatal progenitors procured significantly greater improvements. Short-term hypoxia significantly elevated Akt phosphorylation in neonatal Isl-1+ c-Kit+ human cardiac progenitors. Benefits afforded to both age groups by hypoxic pretreatment included significant upregulation of pro-survival transcripts, and enhanced invasion capabilities in vitro. Therefore, prior to transplantation, hypoxic preconditioning may improve the ability of transplanted stem cells to home towards damaged areas of the heart and support cardiac regeneration in vivo.

The discovery of neural stem cells (NSC) within the adult mammalian brain continues to fuel optimism regarding the ability of potential regenerative medicine applications to provide enhanced functional recovery from brain injuries. The adult NSC population is maintained within a complex microenvironment, referred to as the niche, where a unique cellular and extracellular environment maintains and regulates the NSC population and their progeny, enabling ongoing neurogenesis throughout adulthood. Characterization of how NSC interact with the extracellular environment and other cell subpopulations is an active area of research that will generate fundamental design parameters for biomaterial and tissue engineering strategies for neural tissue repair. A major obstacle to further progress is the lack of access to purified populations of primary NSC, a challenge which became the focus of this thesis. To address this obstacle, experimental methods were developed and optimized for isolating neural stem and progenitor cells (NSPC) from the adult NSC niche with fluorescence activated cell sorting (FACS). These methods were enhanced by the incorporation of a fluorescent reporter mouse driven by the gene Sox2, a neural stem cell associated transcription factor, which allowed NSPC enrichment within the Sox2+ population. The FACS based research approach was further developed to include additional surface antigens allowing isolation of NSPC at over 34% purity. The highly enriched population of NSPC was subjected to vital dye cell cycle analysis leading to the observation that an active and quiescent fraction exists within the NSPC pool that is delineated by β1-integrin expression. Access to enriched primary adult NSPC will lead to more a more accurate understanding of NSC dynamics with implications in fundamental biological research as well as biomaterials and tissue engineering.

Regenerative medicine is a rapidly developing field, merging engineering and biological life sciences to create biological replacements for damaged tissue and organ function. Development of cellular based therapies has the potential of curing present untreatable diseases and conditions, such as diabetes. The identification of protein expression patterns, that guide undifferentiated cells to different lineages, can provide important information about the progression of cellular differentiation at various stages. This research project utilizes proteomics and in vitro live-cell microscopy to investigate two distinct cellular systems: (1) the signaling pathways of calmodulin (CaM) in the differentiation of a human glioblastoma cell line; and (2) the effect of islet neogenesis associated protein (INGAP) on human islet-derived progenitor cells (hIPCs). Using a proteomic readout with a long term live-cell imagining approach, it was hypothesized that highly specific binding proteins of a CaM-mutant, and proteins in hIPCs perturbed by INGAP, could be identified and studied in vitro, characterizing specific signaling pathways which control the function of CaM in brain tumour cells and the mechanism(s) of INGAP in islet-derived progenitor cells. This thesis presents the utility of a proteomics and an in vitro cell microscopy approach to investigate therapeutic proteins, such as INGAP, on cell culture systems. The results have established the limitations and the utility of DIGE, differential binding of a CaM-mutant versus calcium-CaM, and the cell specific uptake feasibility of using the TAT-binding domain. In the hIPC system, proteomic, phenotypic, motility, proliferation and nuclear effects of INGAP were determined. Specifically, hIPCs exposed to INGAP had 50% decrease in average nuclear speed, the translocation of two identified proteins caldesmon and tropomyosin and INGAP was found to bind specifically to hIPCs. However, hIPCs had no changes in insulin specific hormone expression.

No description available.

In der vorliegenden Arbeit konnten erstmals der TGF-β-Signalweg und dessen Smad- Signalmoleküle innerhalb der MPCs nachgewiesen werden. Dieser Nachweis erfolgte sowohl auf zellulärer und Gewebeebene als auch auf Gen- und Proteinebene. Zusätzlich konnte auf Gen- und Proteinebene gezeigt werden, dass die Signalmoleküle Smad2, Smad3 und Smad4 in MPCs aus gering erkranktem Meniskusgewebe eine vermehrte Expression aufweisen im Vergleich zu den MPCs aus hochgradig erkranktem Meniskusgewebe. Diese Erkenntnis weist auf eine mögliche protektive Funktion des TGF- β-Signalwegs während degenerativer Prozesse im Meniskusgewebe hin. Um die Effekte des TGF-β-Signalwegs und dessen Smad-Signalmoleküle genauer zu verstehen und besser beschreiben zu können, wurde eine Überexpression der Smad-Signalmoleküle innerhalb von MPCs durchgeführt und die Auswirkungen dieser auf die Kollagen I- und Kollagen II-Synthese genauer beleuchtet. Infolgedessen konnte sowohl eine vermehrte Kollagen I-Synthese als auch eine vermehrte Kollagen II-Synthese festgestellt werden. Dies bestätigt die Annahme, dass dem TGF-β-Signalweg und dessen Smad-Signalmolekülen eine zentrale, protektive Funktion während der Meniskusdegeneration zukommt. Durch die vermehrte Synthese von Matrixkomponenten wird den Degenerationsprozessen innerhalb des Meniskusgewebes entgegengewirkt. Ein nicht degenerierter bzw. ein regenerierter Meniskus besitzt eine biomechanische Schutzfunktion für das Kniegelenk und wirkt somit einer Kniegelenkarthrose entgegen. In Zukunft könnte der TGF-β-Signalweg  einen möglichen Ansatzpunkt für therapeutische Behandlungen bei Meniskusläsionen darstellen. Da Meniskusdefekte häufig direkt mit einer Osteoarthrose im Kniegelenk assoziiert sind, spielt die durch den TGF-β-Signalweg induzierte Regeneration von Meniskusgewebe auch in der Prävention der Osteoarthrose eine zentrale Rolle.