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Le diabète est une maladie associée à une perturbation du métabolisme glucidique et représentent un problème de santé publique majeur. L'importante majorité des patients présente un diabète de type 2.Les complications les plus courantes sont vasculaires. Chez le diabétique, l'angiogenèse est défectueuse et paradoxale. La microangiopathie qui survient lors du diabète modifie le microenvironnement de la moelle osseuse et entraine egalement des problemes de cicatrisation alors qu'une angiogenèse exacerbé est responsable de pathologie telle que la rétinopathie ou la néphropathie diabétique.Les cellules souches mésenchymateuses (CSMs) sont connues pour leur potentiel de différenciation et de libération de facteurs paracrins, qui sont impliqués dans la régénération tissulaire. Les mécanismes qui associent le microenvironnement et les fonctions des CSMs dans un contexte diabétique restent actuellement méconnus. Le diabète peut modifier les caractéristiques des CSMs et le microenvironnement diabétique peut influencer la fonctionnalité de CSMs transplantées tout autant que leurs effets autocrins et/ou paracrins sur les cellules environnantes. L'étude du potentiel de CSMs diabétiques et du microenvironnement diabétique sur de CSMs pourrait alors avoir d'importantes implications cliniques. L'objectif primaire de cette étude est de caractériser l'impact du diabète sur la fonctionnalité et le potentiel angiogènique de CSMs à l'aide du modèle de rat dit Zucker Diabetic Fatty (ZDF) qui développe spontanément le diabète de type 2 et les complications vasculaire qui l'accompagne. / Diabetes is a disease associated with a bad functioning of glucose metabolism and represents a major health issue. The majority of diabetic are type 2 diabetic patients.Main complications in diabetes are vascular. Diabetic patients have paradoxical angiogenesis pathologies. Microangiopathy which occurs during diabetes modifies the microenvironment of bone marrow and lead to problem of wound healing whereas exacerbate angiogenesis is responsible for pathologies such as diabetic retinopathy or nephropathy.Mesenchymal stem cells (MSCs) are known for their differentiation potential and their release of bioactive mediators which are involved in tissular regeneration. The mechanism associated with microenvironment and functions of MSCs in a diabetic context remain elusive. Diabetes can change the characteristics of MSCs and diabetic microenvironment can lead to modifications in functionality of transplanted MSCs as well as modifying their autocrine/paracrine capacity on surrounding cells. The study of diabetic MSCs potential and the diabetic microenvironment on MSCs could have major clinical implications. The primary objective of this study was to characterize the impact of diabetes on functionality and angiogenic potential of MSCs with the help of a rat model, the Zucker Diabetic Fatty (ZDF) which develop spontaneously diabetes type 2 and the whole vascular complication associated with it.

Les cellules souches embryonnaires (ES) et induites à la pluripotence (iPS) portent de grands espoirs pour la médecine régénératrice du fait de leur capacité d’auto-renouvellement et de différenciation. Une question cruciale est de savoir comment ces cellules mettent en place et maintiennent l’épigénome pluripotent. Les cellules ES et iPS ont un cycle cellulaire particulier, avec un temps de doublement rapide, une phase G1 courte et une phase S représentant 60-70% du cycle cellulaire. Au cours de ce projet, nous avons essayé de voir si les chromosomes dans les cellules ES murines et humaines étaient répliqués de façon particulière qui aiderait à maintenir l’état pluripotent.Les chromosomes mammifères sont dupliqués grâce au recrutement de ~20000 origines de réplication qui sont organisés dans des clusters. Ces clusters forment des foyers de réplication qui sont régulés dans le temps pendant la phase S et dans l’espace nucléaire. Certains de ces domaines topologiques changent leur timing de réplication pendant la différenciation ou la reprogrammation. Néanmoins les mécanismes exacts impliqués dans ce processus et leurs conséquences sur l’expression génique ne sont pas connus.Nous avons étudié la dynamique de réplication dans des cellules pluripotentes murines et humaines à l’échelle de molécules individuelles par la technique de peignage moléculaire de l’ADN. Nous avons comparé les vitesses de fourches, les distances inter-origines et la densité de fourches dans des cellules différenciées (MEF) et pluripotentes (mES), ainsi que pendant la différenciation de ces dernières. Les vitesses de fourches de réplication sont légèrement moins élevées dans les cellules souches embryonnaires que dans les fibroblastes (1.8 vs 2.0 kb/min), et les distances inter-origines intra-cluster sont équivalentes. Par contre, la densité globale instantanée de fourches est divisée par deux dans les cellules ES (1 fourche/Mb) par rapport aux fibroblastes. Un résultat similaire est retrouvé dans les cellules souches embryonnaires humaines (H9) comparées aux fibroblastes (BJ).Afin de tester si cette densité de fourches basse est compensée par un allongement de la phase S, nous avons développé une technique basée sur deux marquages aux analogues de la thymidine. Elle permet une mesure de la durée de la phase S sur des populations asynchrones de cellules. Nous avons trouvé que la phase S a la même durée dans les cellules mES et MEF (~8.4h). Une question intéressante est donc comment les cellules ES peuvent répliquer la même quantité de l’ADN, dans la même durée mais en utilisant deux fois moins de fourches ? Nous proposons que la plus faible densité instantanée en origines serait compensée par une fréquence plus élevée de l’activation des foyers de réplication. Cette fréquence élevée pourrait participer au maintien de la structure épigénétique responsable de la pluripotence ou de l’auto-renouvellement. / Embryonic stem (ES) and induced pluripotent stem (iPS) cells have a great potential for regenerative medicine due to their capacity to self-renew indefinitely and to generate multiple cell types, but the key question of how they establish and maintain a pluripotent epigenome is not resolved. Interestingly all ES and iPS cells display a peculiar cell cycle with rapid doubling time, very short G1, and S phase representing 60-70% of the total cell cycle. In this work we tried to see whether chromosomes in mouse and human ES cells are replicated in a special way that might be used to set up the pluripotency state or to define cell identity. Mammalian genomes are duplicated by the firing of ~20,000 replication origins, organized in ~3000 small clusters forming replication foci that are spatially and temporally regulated during S phase. It has been shown that many of these topologically-associated domains change their replication time upon cell differentiation or reprogramming, but the exact mechanisms involved remain poorly understood. Here we used DNA combing to compare fork velocity (FV), local inter-origin distances (IOD) and global instant fork density (GIFD) between pluripotent mouse ES cells and fibroblasts (MEF), as well as during the differentiation of mES cells into embryoid bodies (EB) and neural precursors. We found that FV is slightly reduced (1.8 vs 2.0 kb/min) and IOD basically unchanged in mES compared to MEF. In contrast GIFD, which represents the density of forks active at any moment during S phase, shows a strong reduction from 2 forks/Mb in MEF to 1 fork/Mb in mES cells. We found a similar drop in GIFD in human ES cells (H9) compared to fibroblasts (BJ). To test whether this lower fork density is compensated by an extension of S phase, we developed a dual pulse/chase protocol to measure S-phase length in asynchronous populations by FACS. Using this assay, we found that S-phase length is identical (~8.4 hr) in both mES and MEF cells, despite the GIFD drop in the former. This raises an interesting question: how can ES cells replicate the same amount of DNA, in the same time and with similar fork velocity, but using a 2-fold lower instant fork density? We propose that the lower GIFD (amplitude) is compensated by a higher frequency of replication foci activation, which is not detected by the GIFD pulse protocol. This higher frequency of replication foci activation could play a role in the establishment and/or maintenance of a chromatin structure permissive for pluripotency or self-renewal.

Peripheral nerve injuries occur with high incidence and often result in profound and permanent impact on the life of patients and on healthcare expenditure. Schwann cells (SC) play a promoting role in peripheral nerve regeneration providing physical and neurotrophic support that aids axon re-growth. However, these beneficial properties are not exploitable in nerve tissue engineering due to the difficulties in SC harvesting and expansion in culture. Adult stem cells derived from bone marrow (BM-MSC) and from adipose tissue (ASC) can be differentiated in SC-like cells and be used as SC substitutes in bioengineered nerve conduits for the improvement of peripheral nerve regeneration. Pharmacological intervention approaches for the treatment of nerve injury are still not clinically available. Nevertheless, γ-Aminobutyric acid (GABA) receptors have been recently suggested as a putative target for such purpose. GABA is the main inhibitory neurotransmitter of the adult brain and interacts with two different receptor types. However, both GABA-A and GABA-B receptor types are functionally expressed also in SC, where they are involved in the regulation of SC physiology and in the development of the peripheral nervous system (PNS).The aim of this thesis was to characterise the GABAergic system of BM-MSC and ASC differentiated into a SC-like phenotype and to evaluate changes in the expression levels following differentiation. Moreover, the effect of specific GABA receptor ligands on cell proliferation and neurotrophic potential of differentiated stem cells were assessed. Using reverse transcriptase polymerase chain reaction, western blot analysis and immunohistochemistry we demonstrated that adult stem cells express several subunits of both GABA-A and GABA-B receptor systems such as GABA-B1a, GABA-B1b and GABA-B2, as well as GABA-A α2 and GABA-A β3. Expression levels and cellular localisation were comparable with adult and neonatal SC cultures used as positive controls, and protein expression levels for some of the subunits changed following glial differentiation. Interestingly, stimulation of GABA receptors with specific agonists influenced stem cell proliferation in two opposite ways. Baclofen, a GABA-B receptor agonist decreased proliferation of SC and differentiated ASC (dASC), but not of SC-like BM-MSC (dBM-MSC). By contrast, muscimol, a GABA-A receptor agonist, increased proliferation in SC and in both dASC and dBM-MSC. This suggests that GABAergic signalling could be a potential player in the mechanisms regulating stem cell differentiation and proliferation as reported in SC. Finally, baclofen treatments on SC and dASC modulated the expression levels and the release of the neurotrophins BDNF and NGF, which are key actors in the processes involved with peripheral nerve regeneration. Although further studies will be needed to clarify the role of GABA receptors in the PNS, the presence of functional GABA receptors on SC-like adult stem cells could represent an exploitable pharmacological target to modulate stem cell physiology and improve their neurotrophic potential for peripheral nerve regeneration.

The niche in which stem cells reside is involved in the regulation of stem cell fate, such as differentiation and self-renewal, by providing ECM proteins, growth factors, cell-cell interactions and balancing chemical factors such as the level of oxygen and pH. ECM proteins are involved in maintaining stemness of stem cells and in regulating differentiation via integrin-mediated signalling. Following the interaction of ECM proteins with integrins, integrins cluster and interact with large complexes of signalling proteins. These adhesion complexes have been reported to contain at least 150 proteins, which have been termed the adhesome. Adhesion complex proteins interact with the actin cytoskeleton and signalling pathways to play an essential role in stem cell fate. The hypothesis in this study was that the interaction of stem cells via integrin receptors with ECM proteins, lead to changes in the abundance or composition of adhesion complexes, which potentially activates signalling pathways involved in either maintaining or differentiation of stem cells. In this study, three principal advances have been made:First, a method was developed using ligand-coated magnetic beads for the isolation of integrin-associated complexes from pluripotent human embryonic stem cells (hESCs). The isolated integrin-associated complexes from hESCs were analysed by proteomic methods, which led to the detection of key integrin-associated adhesion proteins such as talin, vinculin, alpha actinin 4, filamin B, filamin C and zyxin. Second, isolation of integrin-associated complexes from multipotent MSCs was performed using a method based on “de-roofing” MSCs from FN or PDL coated plastic dishes, leading to the detection of key adhesome components by mass spectrometry. Ontological analysis of proteins enriched on FN demonstrated the enrichment of adhesion complexes. Third, following the induction of multipotent MSCs into early adipogenic MSCs and the isolation of integrin-associated complexes from early adipogenic MSCs and undifferentiated MSCs, core adhesome components were identified in induced and non-induced MSCs, with induction hypothesised to cause putative changes in the FN-induced adhesome network. Also, the level of adhesion complexes increased significantly in MSCs on FN upon induction into adipocytes compared to non-induced MSCs on FN and versus the control as shown by bioinformatics analysis. This data led to the hypothesis that upon induction of MSCs into adipocytes the abundance of proteins in integrin-associated complexes or the number of adhesion complexes increases.In conclusion, in this study two biochemical affinity methods were developed for the isolation of integrin-associated complexes from hESCs and MSCs, using ligand coated magnetic beads and ligand-coated plastic dishes. The development of these methods led to the isolation of adhesion-related proteins from pluripotent hESCs and differentiated MSCs and the detection of a pattern of changes in the abundance of adhesion related proteins in differentiated MSCs incubated on FN. The development of methods for the isolation of adhesion related complexes from stem cells can lead to a better understanding of the role of adhesion in differentiation and maintenance of pluripotency in stem cells. A better understanding of adhesion could have future implications in obtaining pure populations of undifferentiated stem cells for cell-based therapies and differentiated cells for the use in tissue engineering and repair.

No description available.

Mechanical properties of cells, mainly defined by their cytoskeleton, are closely related to cell function and can be measured with a dual-beam laser trap (optical stretcher). Functional changes, which go hand in hand with changes of the cytoskeleton, also occur during differentiation of stem cells. This suggests monitoring differentiation by the changing compliance of the cells. During the course of my PhD I measured the compliance of three different types of stem cells before and after differentiation and was able to detect differences in some of the cell types. In order to relate rheological experiments to cell migration as a further example of functional change I investigated the migration behavior of cells that showed different compliance and found differences in migration. I was additionally able to show an altered migration behavior after I actively changed the mechanical behavior of one cell type using cytoskeletal drugs. These migration experiments have been carried out in 2D and 3D migration assays. Furthermore, the influence of the stiffness of the surrounding material on the migration behavior has been investigated. After relating functional changes to changes in compliance, I studied which mechanisms can be used to actually influence cell compliance and investigated the effect of cytoskeletal stabilizers or destabilizers as well as drugs acting on molecular motors. The effect of the surrounding temperature has been considered as well. Finally, I developed a new version of the optical stretcher measurement tool, which enables cell sorting and drug screening using a monolithic glass chip. With the results presented in this thesis I relate mechanical compliance to the cytoskeleton and specific cellular functions. I deliver insights how mechanical changes in cells can be used to identify and follow functional changes and how this knowledge can help to interfere with such functions, specifically in pathologies correlated to these functions. My modified optical stretcher would be developed to screen the effects of drugs on cell compliance and to sort cells with different mechanical properties. Such drug screening and cell sorting will offer diagnostic treatment options for various pathologies.

No description available.

Cryopresevation is the process of cooling biological materials to low sub-zero temperatures for storage purposes. Numerous medical and technical applications, such as hematopoeitic stem cell transplantation and sperm banking, sometimes require the use of cryopreserved cells. Cryopreservation, however, can induce cell injury and reduce the yields of viable functional cells. Ice recrystallization is a mechanism of cryopreservation injury, but is rarely addressd in strategies to optimize cell cryopreservation. The results from this thesis demonstrate an association between the potency of carbohydrate-mediated ice recrystallization inhibition used in the cryopreservation of umbilical cord blood and recovery of viable non-apoptotic cells and hematopoietic progenitor function. Furthermore, increased numbers of apoptotic cells in hematopoeitic stem cell grafts were associated with reduced hematopoietic function and delayed hematopoietic recovery in patients undergoing blood stem cell transplantation. These findings provide a basis for pursuing further studies assessing ice recrystallization inhibition as a strategy for improving cell cryopreservation.

Pax3 and Pax7 belong to a family of conserved transcription factors that play important and diverse roles in development. In the embryo, they carry out similar roles in neural and somite development, but Pax7 fails to compensate for critical functions of Pax3 in the development of limb musculature. Conversely, in the adult, Pax7 is necessary for the maintenance and survival of muscle satellite cells, whereas Pax3 cannot effectively fulfill these roles in the absence of Pax7. To identify the unique roles of Pax7 in adult muscle cells, we have analyzed global binding of Pax3 and Pax7 by ChIP-Seq. Here, we show that despite highly homologous DNA-binding domains, the majority of binding sites are uniquely recognized by Pax7 and are enriched for homeobox motifs. Genes proximal to conserved, unique Pax7 binding sites cluster into specific functional groups which may reflect the unique biological roles of Pax7. Combining Pax7 binding sites with gene expression data, we describe the regulatory networks directed by Pax7 and show that Pax7 binding is associated with positive gene regulation. Moreover, we show Myf5 is a direct target of Pax7 and identify a novel binding site in the satellite cell control region upstream of Myf5.