Дисертації з теми "Culture de cellules souches mésenchymateuses"
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Martin, Céline. "Étude des procédés d’amplification de cellules souches mésenchymateuses humaines." Thesis, Université de Lorraine, 2016. http://www.theses.fr/2016LORR0261/document.
Progress in regenerative medicines over the past ten years have led to an important research mobilisation, but obtaining a sufficient amount of human stem cells remains nonetheless problematic, especially for mesenchymal stem cells (MSC). Hence, this work developed an approach coupling biology and process engineering to identify barriers limiting MSC growth. The study of scaled-up amplification methods was performed using microcarriers and a 200~mL minibioreactors platform. In order to maximise MSC growth in a biochemically controlled environment, a serum free medium development was tested as well. Human MSC as model cell type for cellular therapies have thus been demonstrated as extremely sensitive to freeze/thaw cycles, temperature variations, subject to premature aging and needing a complex medium enriched in multiple growth and adherence factors. Following this study, several pitfalls might be avoided during MSC process scale-up by integrating the cells biology into the bioreactors' process engineering parameters (heat transfer, hydrodamic stress, adhesion surface)
Ni, Xiaofang. "Culture et différenciation cellulaire sur des substrats structurés et dans des dispositifs microfluidiques." Paris 6, 2009. http://www.theses.fr/2009PA066666.
Yang, Jingwei. "Optimisation de modèles de culture 3D pour la différenciation des cellules souches mésenchymateuses : application à la chondrogénèse." Nancy 1, 2006. http://docnum.univ-lorraine.fr/public/SCD_T_2006_0251_YANG.pdf.
Ln this study, we were interested in the reconstruction of cartilage tissue based on human mesenchymal stem cells (hMSCs). These cells were characterized by phenotype analysis (CD 34- /CD45-/CD166+/CDI66+/CD90+/CD73+), then they were pipetted in 3D culture, rnicromass. We analyzed the typical mesenchymal markers (CDI05, CD166 and CD90) by flow cytometry along 21 days of cultu:e. The differentiation of hMSCs was induced in micromass cultures with a serum-free medium containing TGF-β1 (10ng/ml). We investigated the modification of cell phenotype, analyzed metabolic activity and proliferation. Our experimental results showed a decrease of hMSCs phenotype markers and appearance of eÀiracellular matrix. Furthermare, the analysis of cell cycle revealed a decrease of proliferation activity. We have also defined, in the second part of our study, the culture conditions of a biomaterial (alginate gel) used in cartilage tissue engineering. Different viscosities and concentrations of the biomaterial were monitored. CeIl toxicity and cell proliferation were deterrnined. These results allowed us ta define the experimental condition that will be used in future cartilage tissue engineering study, namely in the effect of mechanical compression
Ferrari, Caroline. "Études cinétiques de procédés d'expansion de cellules souches mésenchymateuses cultivées sur microporteurs en systèmes agités." Thesis, Université de Lorraine, 2012. http://www.theses.fr/2012LORR0117/document.
The extensive use of mesenchymal stem cells (MSC) in tissue engineering increases the necessity to improve the expansion performance. This work aimed at studying an efficient expansion process for porcine MSC in agitated mode. First, a culture medium was adapted to the multipotent porcine MSC. Then, various expansion modes and agitation conditions were evaluated with the cells fixed on microcarriers. Cultures on the Cytodex 1 microcarrier enabled to reach a specific growth rate of 0.54 d-1, which was higher than the one observed in static T-flasks (0.31 d-1), with the same culture conditions. In parallel, an innovative counting method was proposed for the automatic enumeration of cells cultivated on Cytodex 1, without passing by a trypsination step. Finally, the operating conditions of the expansion process were studied. Compared to a culture of MSC on non-agitated Cytodex 1 microcarriers, cell aggregation occurred and an apparent decrease in the cell concentration was observed at an agitation rate of 25 and 75 rpm. Moreover, the addition of microcarriers during a 300 h culture, performed in an agitated culture at 25 rpm and in a volume of 200 mL enabled to prolong the cell proliferation without any aggregation, while maintaining the multipotency of the cells. A cell concentration of 3 x 105 cells/mL was obtained, instead of the 1.2 x 105 cells/mL in static flasks with the same culture conditions. An efficient expansion process for porcine MSC under agitated conditions has therefore been proposed
Basciano, Leticia. "L'effet de l'hypoxie sur les conditions de culture des cellules souches mésenchymateuses de la moelle osseuse." Thesis, Nancy 1, 2011. http://www.theses.fr/2011NAN10100/document.
It is now settled that mesenchymal stem cells (MSC), reside in the same microenvironment or niche than hematopoietic stem cells (HSC), within the bone marrow (BM). It is also known that the O2 tension (pO2) of the niche is below 5% as compared to 21% O2 in the air and 12-15% in the arterial blood. As developed in our recent review, this physiological hypoxia protects stem cells from oxidative stress and maintains their multipotential state. Our hypothesis is that MSC cultured in hypoxia should be closer to their physiological condition and therefore more "multipotent". MSC from human BM were cultured et 21% and at 5% pO2. Their morphology, their ability to differentiate into osteocytes and adipocytes, and their transcriptome were compared at different passages. We observed a decrease of proliferation rate in early times in hypoxia, characterized by inhibition of the expression of genes involved in cell replication and cell cycle, and an increase in later passages. Whatever the passage, the genes encoding adhesion molecules and extracellular matrix are stimulated by hypoxia. At later times, the ability of MSC differentiation is stimulated by hypoxia, the cells look to be more immature and show decreased synthesis of mitochondria. Indeed, hypoxia stimulates the synthesis of plasticity genes according to "Gene Ontology" (GO) terms, and of several genes involved in neuronal- and epithelial-cell development. In conclusion, the culture of MSC from BM in hypoxia seems to be more physiological and may be useful for regenerative medicine applications
Berlier, Jessica. "Identification de facteurs favorisant la survie des cellules souches mésenchymateuses humaines carencées en sérum." Doctoral thesis, Universite Libre de Bruxelles, 2016. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/235574.
Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)
info:eu-repo/semantics/nonPublished
Guerrero, Julien. "Devenir des cellules souches mésenchymateuses humaines dans un environnement tridimensionnel : application à l’ingénierie du tissu osseux." Thesis, Bordeaux, 2014. http://www.theses.fr/2014BORD0200/document.
Bone tissue engineering aims to resolve the existing limitations of boneregeneration methods. One of the proposed strategies consists on the association,within a three-dimensional (3D) matrix, with autologous cells able to regenerate afunctional 3D tissue. The purpose of this study was therefore to investigate theimpact of cellular communication, between cells of the stromal compartment andendothelial cells, within the three-dimensional porous matrix made of biodegradablenatural polysaccharides, focusing on bone repair. Our results show that thearchitecture and the nature of the 3D macroporous matrix promotes the guidance ofmesenchymal stems cells, derived from human bone marrow, towards theosteoblastic lineage. Also, that the organization in aggregates, promoted by the 3Dmatrices, stimulated cell communication, evidenced by the formation of GAPjunctions and activity of Connexins 43. We also focused on the function ofPannexines 1 and 3 for the 3D culture in these matrices of polysaccharides. Inconclusion, this work shows that cell-cell interactions play a major role in order toimprove bone tissue regeneration. Also, cellular and experimental data demonstratesthe advantage of using a total fraction of bone marrow cells to promote both boneformation and vascularization
Thepenier, Cédric. "Optimisation d'un procédé de culture d'épiderme autologue : influence d'un feeder humanisé et d'une faible tension d'oxygène." Paris 7, 2014. http://www.theses.fr/2014PA077083.
To enhance the production conditions for cultured epidermal autografts (CEA) for large burns, we sought to study the in vitro effect of a low oxygen level on epidermal growth. We tested this parameter on CEA grown on murin feeder cells (Green's method) as well as human feeder cells. We first could evidence that the optimal feeder density depended on the oxygen level. A feeder density made optimal at 20% 02 could prove inhibitory on keratinocyte growth at 3% 02. At their respective best feeder densities, low oxygen level (3%) led to an average 4,2 fold increase in keratinocyte yield for a same arrest day as compared with 20% culture. This effect proved to be stable on several successive passagings, showing the increase in proliferation did not take place at the expense of tell self renewal. Keratinocytes grown at a low oxygen level kept their ability to form a stratifying epidermis on an in vivo NOD/SCID mouse excisional model. In parallel, the increase in proliferation was also observed when keratinocytes were cultured on human feeder cells, bone marrow mesenchymal stroma' cells and dermal fibroblasts. This effect of a low oxygen tension on keratinocytes appears to be partly direct, as the growth rate of HaCat feederless keratinocytes was enhanced at 3% vs 20% 02. It is also partly an indirect effect, as conditioned medium from murin feeder cells cultured in hypoxia has a more pronounced positive effect on keratinocyte growth than its normoxic counterpart. These preliminary resuits could lead to the modification on the culture protocol currently in use for the majority of CEA grafts for large burns. The expected benefits for the patients, beyond slightly shortening culture time, would include salvaging abortive cultures and bringing less differentiated keratinocytes, a parameter linked with a decrease in fibrotic evolution on murin models
Mohand, Kaci Faïza. "Bioingénierie des cellules souches mésenchymateuses médullaires cultivées en 3D : application au traitement de l’anévrysme de l’aorte abdominale." Thesis, Paris Est, 2012. http://www.theses.fr/2012PEST0079.
Abdominal aortic aneurysm (AAA) is a degenarative disease of the arterial wall, which is usually treated with a conventional surgery or an andovascular stent. Due to its high morbidity and mortality, the AAA constitutes a major public health concern. The aim of this thesis is to evaluate the imapct of OD culture of mesenchymal stem cells (MSC), in particular on their phenotype, their multipotency, their ability to repair aneurysms in vivo and to acquire a phenotype suitable to the nechanical stress they support in vitro. Optmal culture conditions in a 3D hydrogel of hyaluronic acid preserving the multipotency of MSC in vitro have been established. Under mechanical effects, reproducing those supported by the aortic wall in vivo, 2D and 3D CSM seem to preserve their multipotency. However, under such dynamic conditions, the viability of 3D CSM increases unlike that of 2D CSM. By using a rat xenograft model, the results also show that injection of 2D or 3D CSM, stabilizes the AAA and improves the mechanical strenght of the aneurysmal vessel wall. The study in rat was supplemented by an evaluation of a therapeutic cell-based approach using 3D CSM in the case of chronic false aneurysms of the isthlus in pigs. This step allowed the characterization of 3D CSM and the development of an experimental model in pigs, which allows to consider cell therapy in this model. More genrally, this work contributes to a better understanding of CSM biology and to an improvement of the approaches used in cell therapy and regenerative medicine
Hammoud, Mohammad. "Effet de l’association des basses concentrations d’O2 et des cellules stromales mésenchymateuses sur l’expansion ex vivo des cellules souches et progénitrices hématopoïétiques." Thesis, Besançon, 2012. http://www.theses.fr/2012BESA3008/document.
To optimize at best the hematopoietic engraftment, we suggest in this work to improve the ex vivo expansion conditions by moving them closer to physiology. Indeed, we propose to culture placental CD34+ (HSC/PH) on MSC layer in combination with LO2-C to ensure the amplification of HP together with the maintenance/expansion of HSC. Compared to the single culture and/or atmospheric oxygenation, our experimental model allows a better maintenance of primitive HP (Pre-CFC) and HSC together with a quite good amplification of total cells, CD34+ cells and committed HP despite of lower than control condition. Moreover, exogenous IL-3 shows crucial effect in co-culture at LO2-C (1.5% O2) since its addition better preserves and even increases the number of HSC compared to the CD34+ cells control from D0. We then studied the secretion of soluble factors in culture supernatants and found that IL-6, VEGF and IL-8 were present in larger quantities at LO2-C in both co-culture and MSC culture. Finally, the CD146, CD49a, CD54, CD200 and CD105 membrane antigens appear to be up-regulated in MSCs when incubated at 5% O2. However, the involvement of these factors and antigens in paracrine effect and/or direct cell to cell contact mechanisms at LO2-C requires further investigations. In conclusion, the combination of LO2-C and MSC would be promising in the field of HSC/PH grafts expansion to achieve its main objective of reducing the post-transplant cytopenia period together with maintaining the long-term graft potential
Maillot, Charlotte. "Quantification and impact of microcarrier collisions during mesenchymal stem cell culture in bioreactors." Electronic Thesis or Diss., Université de Lorraine, 2022. http://www.theses.fr/2022LORR0314.
To date, bottlenecks persist concerning deep scientific understanding of how various process parameters will impact the Mesenchymal Stem Cell production. Specifically applied to microcarrier-based expansion processes of WJ-MSC's, very little information is available to characterize the impact of microcarrier concentration on MSC growth and death rates or on critical quality attributes which may have crucial and possibly dangerous clinical impacts. As a result, the following work proposes to rationally describe the impact of particle concentration on MSC growth through a pluri-disciplinary characterization of microcarrier-microcarrier interactions in agitated conditions. In order to do so the biological and physical aspects of this work will be presented. To begin with, a quantitative approach to estimate cell growth and death kinetics caused by microcarrier-microcarrier collisions in both Erlenmeyer Flasks and Spinner Flasks is described. For this, cells were grown at various microcarrier concentrations using two microcarrier types : Cytodex-1 and Synthemax II. Complementary cultures were performed by adding various concentrations of particles with the same size and density as microcarriers in view of providing specific information on how additional particles may impact MSC growth on microcarriers. In addition, elements of MSC characterization were performed for these experiments to understand not only the impact of microcarrier-microcarrier interactions on growth but also on defined elements of cell quality. In parallel, in order to estimate the amount and intensity of microcarrier-microcarrier collisions in a specific tank geometry, experiments were performed using both the attenuation of light by Cytodex-1 microcarriers (to estimate local microcarrier concentration) and the acoustic signal which comes from particles colliding with a hydrophone (to estimate microcarrier-sensor collision frequency and intensity). These experiments provided elements to estimate the amount of particle collisions that MSC's may perceive during specific dynamic and steady phases of cell culture in STR's. Lastly, a bioreactor-based approach to MSC manufacturing will be presented focusing on biological aspects of how particle concentration and agitation impacts MSC growth and quality attributes. For this, various MSC cultures were performed in STRs with varying particle concentrations and agitation strategies. The MSC's produced in these conditions were then characterized to define if certain critical quality attributes could be affected by parameters such as microcarrier concentration and/or agitation
Le, Pape Fiona. "Evaluation de la contribution d'une hémoglobine marine dans la culture cellulaire et dans la cellularisation de substituts osseux et méniscaux par des cellules souches mésenchymateuses." Thesis, Brest, 2016. http://www.theses.fr/2016BRES0002/document.
This work aimed to develop cell culture systems, in 2D and 3D, based on the properties of HEMOXCell®, a marine oxygen carrier. Our approach was articulated in two main parts: the first one dealing with the assessment of the use of HEMOXCell® in the culture of two cellular models, and the second one, exploiting the results obtained for tissue engineering purposes. In this first axis, the dose-response effect of HEMOXCell® in the CHO-S cells and mesenchymal stem cells (MSC) in vitro culture, allowed the identification of optimal working concentrations, which can promote cell viability and proliferation. The CHO-S model has contributed to the establishment of a performance test of the molecule, and encouraged its use for bioproduction stimulation. The tests performed on MSCs were used to validate the harmlessness of the molecule at low doses and the maintenance of "stemness". The idea to associate MSCs with porous scaffolds is a promising approach for tissue engineering applications, but it is confronted to the lack of oxygen in the depth of the substitutes. In the second part of this project, we worked at improving the cellularization of bone and meniscal substitutes, under static and dynamic culture systems, w/ and w/o HEMOXCell®. In parallel, a study was conducted to attempt to characterize the meniscal cells. Analyses of cellularized biomaterials suggest a beneficial effect of HEMOXCell® when used as a differentiation media supplement. This work contributed to improve this oxygen carrier understanding and to extend the field of its potential uses particularly for therapeutic applications
Ben, Azouna Nesrine. "Etude phénotypique et fonctionnelle des cellules souches mésenchymateuses et hématopoïétiques du sang placentaire en comparaison avec la moëlle osseuse ou le sang périphérique adulte." Thesis, Tours, 2012. http://www.theses.fr/2012TOUR3321.
The mesenchymal stem cells (MSC) are adult stem cells which are at the origin of the ostebiastic lignage cells (O), adipocytes (A) and chondrobiasts (C). The MSC are initially found in the bone marrow (BM) but it also exists there in other tissues as the umbilical cord blood (UCB).Endowed with a regenerative potential, MSC can used in diverse degenerative pathologies in a purpose of tissular repair. Besides, their immunosuppressive properties allowed to envisage their use in a purpose of immunomodulation as during the reactions of transplant against the host in allogenic hematopoietic stem cell transplants (HSC). The essential purpose of this work was to compare the characteristics of the MSC derived from the UCB in comparison to those stemming from the bone marrow (BM)
Loncaric, Darija. "Atténuation des oxydations phosphorylantes et induction d'une réponse cellulaire hypoxique : effêt de l'[alpha]-tocophérol-acétate et de miR-210 sur les cellules stromales mesenchymateuses." Thesis, Bordeaux, 2019. http://www.theses.fr/2019BORD0198.
In this thesis, we combined approaches of single-cell cultures, flow-cytometry, energetic metabolismanalysis and molecular genetics in order to get insight in the effects of α-Tocopherol-Acetate (α-TOA)on Mesenchymal Stromal Cells (MStroC) and their functional subpopulations (mesenchymal stem and progenitor cells). The other aim was to test a miR-210 molecule with respect to its potential use as hypoxia mimicking molecule. After defining MStroC population heterogeneity and concluding that the first passage population is convenient for further experiments, we demonstrated that α-TOA exhibits a positive effect on the maintenance of high proliferative capacity of mesenchymal stem cells. This effect could be associated with an attenuation of electron transport chain (ETC) activity, which, on the other hand could explain moderate increase in the level of mitochondrial Reactive Oxygen Species (mtROS) we detected. The increase in mtROS level could be associated with a decreased HIF-1 alpha protein degradation in MStroC exposed to α-TOA. Although we did not detect a compensatory increase in glycolysis, the observed phenomena depict part of a complex cellular response to the low O2 that is demonstrated to be related with maintenance of stem cell primitiveness. The exact mechanism remains to be elucidated as well as its translational potential. In addition, we provided new evidences that miR-210 is integral part in MStroC response to low O2. In the study, we showed increased in miR-210 expression in a short-term (up to 24 hours) and after extended (up to 72 hours) MStroC exposed to low O2. Moreover, we demonstrated that this micro- RNA could be regulated by both HIF-1 and HIF-2 transcriptional factors, suggesting it as integral part of MStroC response to low O2. So far, our data suggest that miR-210 is worthy to be considered as good hypoxia mimicking molecule
Loison-Robert, Ludwig. "Cellule souche gingivale : origine et multipotence." Thesis, Paris Est, 2016. http://www.theses.fr/2016PESC0083/document.
Gingiva is a natural regeneration model thanks to its "ad integrum" healing capability. Gingival fibroblasts are the main actors of this property. These cells, the main cellular component of the gingival connective tissue, regulate the inflammatory responses and healing process. This tissue contains, like many others, mesenchymal stem cells; which also partly explain these regenerative abilities. Moreover, as the gingiva is abundant and easily accessible, the use of these stem cells may interest cell therapy or in vitro model tissues responses. In this work, we demonstrated that Stem Cells Derived from Human Gingiva (SCHG) have common properties with neural crest adult stem cells. These cells can be called "stem cells" for their ability to self-renew, adhere to plastic and to differentiate. First, we have shown that the method and the culture products used for isolation of gingival fibroblasts from gingival biopsy had an influence on the obtained cells. Secondly, an analysis of in vitro clonal populations of gingival fibroblasts has shown that gingival fibroblasts are composed of subpopulations that express specific markers of stem cells and neural crests. In addition to their embryological origin, the study of their multipotency was also characterized after expansion and depending on the used additives. Finally, two examples of using these cells and dental pulp stem cells as a model to study the in vitro biocompatibility of biomaterials have been developed, mimicking oral mucosa or dentin reactions (reparative or reactional)
Sion, Caroline. "Development of an optimized perfused-continuous process of culture of human umbilical cord mesenchymal stem cells (hMSC) grown on innovative adhesion supports." Electronic Thesis or Diss., Université de Lorraine, 2020. http://www.theses.fr/2020LORR0113.
Mesenchymal stem cells (MSCs) show great interest in cellular therapies. Their various characteristics such as their immuno-modulatory properties, their ability to differentiate, and also the secretion of factors, are numerous and promising for new clinical treatments for diseases where few therapies are proposed or have few efficiencies. The doses to be injected for significant results must be repeated and generally contain high quantities of cells (106 cells kg-1 per patient approx). Large scale production methods must be implemented to meet the demand, and in the least costly way possible. In this PhD work, the main objective was to develop a scalable process adapted to these support-dependent cells. For this end, a first study allowed to understand part of the mechanisms of interaction of cells with their growth supports, the microcarriers. The adhesion time but also the cell migrations between microcarriers were characterized and evaluated. A strategy of fed-batch mode strategy with microcarriers addition at specific times in the culture was also proposed. Following this, the second part of the study of this work was to determine the efficiency on larger scale expansion process (1.5 L), using of innovative microcarriers developed by the partner teams of the ‘ImprovesStem’ European project. Several microcarriers candidates with chemically modified surface proved to be promising for the expansion of Wharton’s jelly stem cells. Finally, in the last part of the thesis, an innovative process based on the removal of empty microcarriers, avoiding the risk of deleterious frictions between highly concentrated microcarriers was proposed. Moreover, an on-line monitoring of viable cell concentration was carried-out in the stirred tank bioreactor. Innovative commercial microcarriers, soluble under the action of enzymes, were used in this last part of the study. An improvement of the expansion factor (by a factor of 1.5) was obtained in this continuous-perfused mode of culture in the stirred bioreactor. In addition, these enzymatically-soluble commercial microcarriers allowed for an excellent detachment yield, essential to consider their use in cell therapy
Loubière, Céline. "Characterization and impact of the hydrodynamics on the performance of umbilical-cord derived stem cells culture in stirred tank bioreactors." Electronic Thesis or Diss., Université de Lorraine, 2018. http://www.theses.fr/2018LORR0220.
Mesenchymal stem cells (MSC) are becoming increasingly involved in the regenerative medicine field, particularly to treat diseases that are not effectively curable with the current therapies. Two scientific barriers are nevertheless responsible for MSC use and commercialization limitations. On one side, large amounts of cells are needed to reach the high cell dose requirements. On the other side, cells being the final product themselves, directly injected into the patient, their quality have to be controlled (stem cell phenotype, differentiation capability). MSC cultivation on microcarriers in a stirred bioreactor seems to meet these challenges. However, a precise knowledge about the impact of the technologies and the hydrodynamics generated, on the physiological cell response, is necessary to improve the scale-up of MSC cultures in bioreactors. In this context, present work is dedicated to the study of the impact of the agitation mode (orbital or mechanical) on the cell attachment, expansion and detachment on various microcarrier types, in the case of MSC derived from the Wharton’s jelly (WJ-MSC) of umbilical cords. To quantify more precisely cell distribution and expansion on microcarriers, an automatic and in situ counting method was developed, which need no detachment step. This allowed the identification of commercial microcarriers suitable for WJ-MSC cultures, which were then compared to home-made microcarriers, synthesized by a partner laboratory, in terms of cell attachment and expansion, and detachment efficiency. In parallel to these works, the impact of the impeller design on the microcarrier suspension in stirred tank bioreactors was investigated. Based on a dimensional analysis and CFD simulations, it resulted in the establishment of two models relating the minimal agitation rate to ensure all particle suspension (Njs) with the impeller geometrical characteristics (design, size, off-bottom clearance) and the material properties of both the solid and the liquid phases. CFD models validation allowed then to develop a strategy to optimize the geometrical configuration of an impeller, dedicated to MSC cultures on microcarriers in a minibioreactor. Parameters characterizing the hydromechanical stress encountered by the solid phase were wisely chosen and integrated into CFD simulations. Based on a design of experiments, and the hydrodynamics data recovered from simulations, response surfaces were built and a multiobjective optimization was achieved in order to determine the geometry minimizing the particle stress, and also by adhered cells. WJ-MSC cultures in minibioreactors equipped with impellers displaying various geometries were finally validated, with a preliminary comparison of the impact of these geometries on the cell expansion
Ishac, Nicole. "Comment deux lignées cellulaires stromales mésenchymateuses humaines récapitulent in vitro le microenvironnement hématopoïétique ? : Intérêt en ingénierie." Thesis, Tours, 2015. http://www.theses.fr/2015TOUR4038/document.
Hematopoiesis occurs in a hypoxic microenvironment or niche in which hematopoietic stem cells (HSCs) are in close contact with mesenchymal stromal cells. Cellular interactions as well as microenvironmental factors such as reactive oxygen species are crucial for the maintenance of normal and leukemic HSCs. Developing an in vitro human culture system that closely mimcs marrow physiology is therefore essential to study the niche. Here, we present a model using two human stromal cell lines, HS-27a and HS-5. Previously poorly described in the literature, we have further characterized both of these cell lines. The first objective was to assess the quality of HS-27a and HS-5 niches by investigating their cellular, molecular and functional characteristics. Our results clearly show that HS-27a cells display features of a “quiescent” niche whereas HS-5 cells rather represent a “proliferative” niche. The second objective was to engineer a hematopoietic niche where the oxidative metabolism is optimized for the expression of an antioxidant protein, glutathione peroxidase 3 (GPx3). The originality of this work is the use of a non-viral gene transfer system by using the transposon piggyBac. This strategy was achieved by delivering a DNA plasmid carrying the gene of interest, and an mRNA source of transposase, the enzyme which catalyzes the transgene integration. Functionally, GPx3 was shown to be a key regulator for sustaining hematopoietic homeostasis by maintaining immature progenitor cells. For the first time, an original non-viral gene transfer has been used to create an in vitro hematopoietic niche that recapitulates the complexity of normal and leukemic microenvironment. This niche not only provides a platform to identify regulatory factors controlling medullary cells, but may also help in the development of targeted therapeutic strategies
Reppel, Loïc. "Potentialité des cellules stromales de la gelée de Wharton en ingénierie du cartillage." Thesis, Université de Lorraine, 2014. http://www.theses.fr/2014LORR0164/document.
Mesenchymal Stromal/Stem Cells from human Wharton’s jelly (WJ-MSC) are an abundant and interesting source of stem cells for applications in cell and tissue engineering. Their fetal origin confers specific characteristics compared to Mesenchymal Stromal/Stem Cells isolated from human bone marrow (BM-MSC). First, the aim of this work is to optimize WJ-MSC culture conditions for their subsequent clinical use. We focus on the influence of oxygen concentration during monolayer expansion on several parameters to characterize MSC. The results are compared to those obtained with BM-MSC. Our work distinguishes WJ-MSC from BM-MSC in terms of proliferation and adipogenic differentiation. Considering our results, hypoxia during cell expansion is an important parameter to take into account regarding proliferation potential but also chondrogenic differentiation potential. The influence of obstetric factors on WJ-MSC characteristics is also explored. In cartilage tissue engineering context, the second phase of the project is to induce cell differentiation into chondrocytes by seeding them in Alginate/Hyaluronic Acid hydrogel scaffold, and during 28 days. The results obtained are compared to those obtained with BM-MSC. After 4 weeks of culture, WJ-MSC are able to adapt to their environment and express specific cartilage-Related genes and matrix proteins such as type 2 collagen, which is found more expressed after differentiation fromWJ-MSC, than from BM-MSC
Loubière, Céline. "Characterization and impact of the hydrodynamics on the performance of umbilical-cord derived stem cells culture in stirred tank bioreactors." Thesis, Université de Lorraine, 2018. http://www.theses.fr/2018LORR0220/document.
Mesenchymal stem cells (MSC) are becoming increasingly involved in the regenerative medicine field, particularly to treat diseases that are not effectively curable with the current therapies. Two scientific barriers are nevertheless responsible for MSC use and commercialization limitations. On one side, large amounts of cells are needed to reach the high cell dose requirements. On the other side, cells being the final product themselves, directly injected into the patient, their quality have to be controlled (stem cell phenotype, differentiation capability). MSC cultivation on microcarriers in a stirred bioreactor seems to meet these challenges. However, a precise knowledge about the impact of the technologies and the hydrodynamics generated, on the physiological cell response, is necessary to improve the scale-up of MSC cultures in bioreactors. In this context, present work is dedicated to the study of the impact of the agitation mode (orbital or mechanical) on the cell attachment, expansion and detachment on various microcarrier types, in the case of MSC derived from the Wharton’s jelly (WJ-MSC) of umbilical cords. To quantify more precisely cell distribution and expansion on microcarriers, an automatic and in situ counting method was developed, which need no detachment step. This allowed the identification of commercial microcarriers suitable for WJ-MSC cultures, which were then compared to home-made microcarriers, synthesized by a partner laboratory, in terms of cell attachment and expansion, and detachment efficiency. In parallel to these works, the impact of the impeller design on the microcarrier suspension in stirred tank bioreactors was investigated. Based on a dimensional analysis and CFD simulations, it resulted in the establishment of two models relating the minimal agitation rate to ensure all particle suspension (Njs) with the impeller geometrical characteristics (design, size, off-bottom clearance) and the material properties of both the solid and the liquid phases. CFD models validation allowed then to develop a strategy to optimize the geometrical configuration of an impeller, dedicated to MSC cultures on microcarriers in a minibioreactor. Parameters characterizing the hydromechanical stress encountered by the solid phase were wisely chosen and integrated into CFD simulations. Based on a design of experiments, and the hydrodynamics data recovered from simulations, response surfaces were built and a multiobjective optimization was achieved in order to determine the geometry minimizing the particle stress, and also by adhered cells. WJ-MSC cultures in minibioreactors equipped with impellers displaying various geometries were finally validated, with a preliminary comparison of the impact of these geometries on the cell expansion
Carpentier, Benoît. "Développement, caractérisation et optimisation d'un bioréacteur dédié à la production accélérée de substituts osseux biohybrides implantables." Compiègne, 2009. http://www.theses.fr/2009COMP1827.
Bone tissue engineering aims at producing implantable bone substitutes capable of promoting bone reconstruction and healing. The aim of the present work was to develop, characterize and optimize a bioreactor-based system dedicated to the production of such substitutes. In order to favor cell invasion and vessel ingrowth post-implantation, 80-200 um biphasic calcium 3 phosphate granules were chosen as a biomaterial. The most appropriate configuration (bioreactor geometry, biomaterial arrangement) for culturing cells on microparticles was determined by means of experimental and numerical studies. Using glass microbeads with similar diameters, monolayers within plane-parallel bioreactor chambers appear to be the most favorable environment. The culture of MC3T3 cells on monolayers of granules under both static and dynamic conditions allowed us to define the appropriate setting for promoting bone tissue formation and to evaluate the impact of fluid flow. The optimal conditions were applied to rat bone marrow mesenchymal cells, showing very satisfying cell proliferation and matrix synthesis. Fluid flow seems to reduce matrix synthesis and promote cell differentiation and bone mineralization. Bone tissue sheets were successfully obtained after 5 weeks, proving the potential of the concept for the production of bone substitutes under sterile and reproducible conditions. The “bone tissue sheet concept” seems promising, owing to the easiness of the “scaling-up” phase necessary to produce clinically relevant volumes. Due to their adaptability to various defect geometries and potential for promoting bone formation, such substitutes appear to be valuable candidates for medical use
Lalande, Charlotte. "Développement d'un nouveau produit d'ingenierie tissulaire osseuse à base de polymères et de cellules souche du tissu adipeux." Thesis, Bordeaux 2, 2011. http://www.theses.fr/2011BOR21853/document.
Bone tissue engineering may associate osteoprogenitor cells to a tridimensional scaffold that can promote tissue reconstruction in order to replace bone grafting strategies whose limitations are well known. This study aims to develop a new tissue-engineered construct for bone regeneration constituted by i) a tridimensional polysaccharide-based scaffold, ii) adult stem cells extracted from human adipose tissue and identify the best culture conditions needed to develop a functional construct for clinical use. Our results show that this macroporous scaffold offers, without any osteoinductive factors, a suitable architecture and composition for driving osteoblastic differentiation of ADSCs especially when placing the tissue-engineered construct in dynamic conditions, thanks to cell aggregate conformation promoting cell-to-cell interactions. Thanks to ADSCs labeling, the tissue-engineered construct can be tracked in vivo in a non invasive way by magnetic resonance imaging (MRI), after their subcutaneous implantation. Results evidenced that this scaffold behaves as a cell carrier for of holding in its own cell fraction and delivering another fraction to the site of implantation for inducing a better tissue regeneration process. Finally, a serum free medium meeting standards GMPs (Good Manufacturing Practices) has been developed for inducing ADSCs osteoblastic differentiation as a first step towards clinical application.In conclusion, this polysaccharide-based scaffold associated with ADSCs, cultured under low fluid flow in a new bioreactor device, could be a relevant and promising tissue engineered construct for bone tissue engineering applications
Bouacida-Boucherma, Amina. "Caractérisation des cellules souches mésenchymateuses natives." Thesis, Tours, 2014. http://www.theses.fr/2014TOUR3304.
Native mesenchymal stem cells were found tore perivascular cells with pericyte features. This suggests that pericyte phenotype is crucial for the stenness of MSC. We cultured MSC from bone marrow upon in vitro conditions (EGM2 versus standard mediums). They all express MSC, markers and character. Cells cultivated into ECM2 were found to be more immature than cells obtained from standard conditions (expressed OCT4, NANOG and SOX2), with high neuronal and engraftment potential
Roubeix, Christophe. "Intérêt des cellules souches mésenchymateuses dans la thérapie du glaucome." Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066535/document.
Glaucoma is a sight-threatening retinal neuropathy associated with elevated intraocular pressure (IOP) due to degeneration and fibrosis of the trabecular meshwork (TM). Glaucoma medications aim to reduce IOP without targeting the specific TM pathology, which could explain treatment failure observed in some cases. Bone-marrow mesenchymal stem cells (MSCs) are used today in various clinical studies to treat various degenerative processes. Here, we investigated the potential of MSC therapy in an ocular hypertension model. We demonstrated a rapid and long-lasting in vivo effect of MSC transplantation that significantly reduced IOP in hypertensive eyes induced by episcleral vein cauterization (EVC). MSCs were found located to the ciliary processes and the TM and are able to survive at these places. Enumeration of retinal ganglion cells (RGCs) on whole flat-mounted retina highlighted a protective effect of MSCs on RGC death. In vitro, the effect of MSC-conditioned medium (MSC-CM) on both the primary human trabecular meshwork (hTM) and RGCs showed that MSC-CM promotes: (i) hTM survival by activating the antiapoptotic pathway, Akt, (ii) hTM decontractibility as analyzed by the decrease in myosin phosphorylation and (iii) inhibition of TGF-β2-dependent profibrotic phenotype acquisition in hTM, (iiii) RGC survival and neuritic outgrowth in vitro. Finally, MSCs injection in the ocular anterior chamber in a rat model of ocular hypertension provides a neuroprotective effect in the glaucoma pathophysiology directly on RGC and indirectly via TM protection. These results originally demonstrate that MSCs represent promising tool for treating ocular hypertension and retinal cell degeneration
Detante, Olivier. "Thérapie cellulaire par cellules souches mésenchymateuses humaines après ischémie cérébrale." Grenoble, 2010. http://www.theses.fr/2010GRENV008.
Stroke is the leading cause of acquired adult disability. Improving brain plasticity after stroke represents an important therapeutic strategy. Cell therapy favours functional recovery after cerebral ischemia in rodent models. The pioneer clinical studies did not reproduce this benefit for patients due to a limited number of studied patients. Ln our preclinical studies, we observed a good tolerance and a functional benefit of the intracerebral (at the acute phase) and intravenous (IV) (at the subacute phase) administration of clinical-grade human mesenchymal stem cells (hMSC). The hMSC survival (during several weeks) and their differentiation into neurons or astrocytes are very limited and can not explain alone the functional benefit. Among different mechanisms of action of hMSC (neurotrophic and/or proangiogenic effects, immunomodulation. . . ), we showed by MRI an early microvascular effect. We also showed that hMSC can be labeled for MRI and microscopy by iron microparticles without altering cell properties. This cell labeling is useful to detect and follow hMSC grafted into the brain but is insufficient to follow the IV injected hMSC. To assess the biodistribution ofIV injected hMSC, we conducted a nuclear imaging study. This experiment showed that the hMSC are attracted to cerebral ischemic lesion in the first hours following their injection. Ln paraIlel, we developed a phase 2 clinical trial. We do not know yet the best route of administration, the best dose and the optimal delay of the graft. The cell therapy optimization needs the development of translational projects with experimental studies linked to clinical trials. Thus cell therapy could become an efficient treatment for stroke
Goldman, Orit. "Modèle de différenciation de cellules endothéliales à partir des cellules souches embryonnaires humaines." Paris 7, 2009. http://www.theses.fr/2009PA077060.
The development of the vascular System takes place through two pathways: vasculogenesis and angiogenesis. Endothelial cells play a central role in the formation and function of blood vessels. They take place in the inner wall of ail vessels and constitute a dynamic barrier for the exchanges between blood and tissues. In addition, the endothelial cells are highly specialized. For example, the endothelial cells of blood-brain barrier (BBB) restrict the passage of molecules and cells into the central nervous System. Vasculogenesis can be studied in vitro through the model of human embryonic stem cells (hES). HES cells can generate ail three embryonic layers: mesoderm, endoderm and ectoderm. They form spontaneously in culture compact structures called embryoïd bodies (EBs). The EBs treatment with a combination of cytokines leads to the differentiation into endothelial and hematopoietic precursor, confirming the existence of a stem cell common to both Systems. The production of endothelial cells from hES is stimulated by BMP4, which accelerates the time to onset of endothelial cells emergence (CD144/KDR) and the number of cells involved in this differentiation process. In the presence of BMP4, we highlight a population of endothelial progenitors, which differentiate into functional endothelial cells. We currently operate the model to obtain endothelial cells with specialized functions, such as endothelial cells of the BBB. Such endothelial cells express the proteins of tight junctions, adhesion molecules. During development, these characteristics are acquired and maintained by contact with astrocytes. For this reason, we co-culture the endothelial cells derived from hES with astrocytes. In parallel, we have tried to isolate more immature endothelial progenitors by using a combination of markers expressed on post-natal endothelial progenitors. BMP4 boosted differentiating hES cells were sorted with CD133/KDR markers. However, put into culture, these cells generate a population of cells expressing markers of both mesenchymal cells (vimentin and ASMA) and epithelial cells (CK8, CK18 and CK19). In culture, these cells are able to differentiate into chondrocytes, adipocytes and osteoblasts. Their differentiation into epithelial cells is currently under investigation. We suggest that these cells are in epithelial-mesenchymal transition (EMT), and we are currently working on that hypothesis. These studies have shown that hES cells can give access a broad spectrum of differentiation from immature cells to cells that have acquired specialized functions. With regard to endothelial cells and mesenchymal cells, this property may have uses in pharmacology and, further, will open the way to regenerative medicine
Sottile, Virginie. "Cellules souches mésenchymateuses de la moe͏̈lle osseuse : différenciation adipogénique et ostéogénique." Nice, 2001. http://www.theses.fr/2001NICE5650.
Osteoporosis is characterized by decreased bone mass associated with increased marrow fat content. Osteoblasts and adipocytes arise from a common precursor cell present in the bone marrow stromal, the mesenchymal stem cell (MSC). The regulatory events governing the commitment of this precursor cell to either lineage are intensely investigated. To determine the effect of osteogenic factors on adipogenesis, we have treated the mesenchymal precursor cell line 3T3-L1 with BMP2 (Bone Morphogenetic Protein-2), a potent inducer of bone formation. Although BMP2 did not affect adipogenesis on its own, is strongly stimulated adipogenic differentiation in synergy with the PPARgamma agonist BRL49653. Likewise, when BRL49653 was combined with a classical osteogenic treatment, no inhibition of the osteogenic response could be detected. Our data suggest that osteoblasts and adipocytes do not systematically develop at the expense of each that osteoblasts and adipocytes do not systematically develop at the expense of each other, but follow a common differentiation pathway that can be positively modulated by both BMPs and PPARgamma activators. We also identified human trabecular bone as a new source of mesenchymal precursor cells. Based on clonal expansion experiments followed by osteogenic, chondrogenic and adipogenic differentiation assays, we could conclude that cultures prepared from trabecular bone fragments have mesenchymal stem cell characteristics in vitro
Potier, Esther. "Viabilité et fonctionnalité des cellles souches mésenchymateuses humaines dans un environnement ischémique." Paris 7, 2007. http://www.theses.fr/2007PA077106.
Local supply of mesenchymal stem cells (MSCs) has been proposed for the repair of damaged tissues. However, upon transplantation, MSCs are far from the vasculature and experience nutrient and oxygen starvation, which may affect cell viability and functionality. The first part of this thesis aimed to assess in vitro the effects of serum and oxygen deprivation on the human MSC survival rates. The MSC death rates were not affected by a 48h hypoxia (≤1% O2), but were increased (13%) by FBS starvation (1% FBS). Most importantly, long-term (120h) hypoxia combined with serum deprivation resulted in the complete death of MSCs (99%). The effects of a 48h hypoxia on MSC functionality were then investigated. When leading to limited stimulation of angiogenic factor secretion (from all the angiogenic factors tested only VEGF was increased 2-fold), hypoxia results in persistent down-regulation of MSC bone forming potential (persistent down-regulation of Runx2, osteocalcin and type I collagen expression). The last part of this thesis described a method to enhance the MSC angiogenic factor expression in order to accelerate vascularization. For this purpose, MSCs were treated with DFX, a chemical inducer of HIF-1, a transcription factor involved in the expression of several angiogenic factors. Under normoxia, DFX exposure resulted in up-regulation of bFGF (20-fold) and VEGF (4-fold) expression. Under hypoxia, DFX induced a 50% increase in VEGF secretion. These results suggest that DFX-exposed MSCs could be used to supply angiogenic factors to promote the establishment of a vascular network providing nutrients and oxygen to the transplanted cells
Maria, Alexandre. "Evaluation de l'effet thérapeutique des cellules souches mésenchymateuses dans la sclérodermie systémique." Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT015/document.
Systemic sclerosis (SSc) is a rare intractable disease characterized by skin fibrosis and autoimmunity. Diffuse and rapidly progressive SSc (d-SSc) is associated with life-threatening involvements such as lung fibrosis, where there still is an unmet medical need. Displaying immunomodulatory and antifibrotic properties, mesenchymal stem cells (MSC) are an attractive cure for SSc. In this study, we aim at evaluating the therapeutic potential of MSC in a preclinical model of d-ScS.Patients and Methods: We evaluated the effects of MSC infusion in the murine model of ScS-HOCl, based on repeated injections of hypochlorite (HOCl). We compared several approaches using MSC in a preventive and curative approach, in syngeneic, allogeneic and xenogeneic settings, and using MSC isolated from adipose tissue (ASC) or bone marrow (BM-MSC).Results: ScS-HOCl is close to d-ScS phenotype, leading to skin and lung fibrosis, together with anti-topoisomerase 1 antibody production. We show beneficial effects of a preventive or curative injection of syngeneic MSCs, reducing tissue fibrosis. Fibrosis reduction following MSC treatment involves immunosuppressive effects, tissue remodeling via metalloprotease activation, and anti-oxydant activity. Similar benefits are observed in allogeneic and xenogeneic settings. ASC display greater immunosuppressive and remodeling capacities than BM-MSCs.Discussion and conclusion: Our study demonstrates the anti-fibrotic effects of MSCs in a relevant preclinical model of ScS, mimicking diffuse and rapidly progressive ScS. Pleiotropic capabilities of MSCs, combining anti-inflammatory, remodeling and antioxidant properties, are promising for future clinical applications in this disease.Keywords: systemic sclerosis, mesenchymal stem cells, HOCl
Pers, Yves-Marie. "Effet thérapeutique des cellules souches mésenchymateuses dans l'arthrose : mécanismes et translation clinique." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT045.
Mesenchymal Stem Cells (MSCs) are stromal cells present in a number of different tissue types. In addition to their ability to differentiate into multiple lineages (chondrocytes, adipocytes and osteoblasts), MSCs also display immunosuppressive properties. Whilst these mechanisms are far from fully understood, their immunosuppressive capacity has recently been shown to be modulated by miRNAs. OA is the most common form of joint diseases without curative treatment and mainly characterized by the degradation of articular cartilage, with subchondral bone alterations and synovial inflammation. MSC might provide therapeutic potential for treatment of OA.Here, we showed that an autologous injection of adipose-derived MSC (ASC) into an osteoarthritic joint improved pain and function levels in patients. We underscored the systemic immune tolerance induced following intra-articular injections of ASCs. Finally, we investigated the miRNA expression profile of human MSCs upon their stimulation by peripheral blood mononuclear cells. We identified miR-29a and PSAT1 as new candidates to regulate immunosuppressive activity mediated by MSCs
Jego, Chloé. "Le rôle des cellules souches mésenchymateuses médullaires dans la leucémie myélomonocytaire chronique." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS377/document.
Chronic myelomonocytic leukemia (CMML, is a rare myeloid hemopathy of the elderly. Clinical, genetic and molecular characteristics of the disease are well-known. The highly heterogeneous expression of the disease can’t be solely explained by genetic heterogeneity of the leukemic clone. Epigenetic alterations obviously play an important role. However, the role of extrinsic factors from the medullar microenvironment in CMML physiopathology is still poorly understood. The hematopoietic niche hosts a lot of bi-directionnal interactions between cells. Two non-exclusive schemes of primary and secondary alterations of the niche can be proposed. First postulate implies that the emergence of a hematopoietic clone alters its environment. The second one supposes that the first event causing the emergence of a clonal hemopathy is an alteration of the environment. My PhD work consisted of studying medullar alterations in patients and their impact on CMML physiopathology upon 2 axes: 1) to set up a murine model of human hematopoietic niche reconstitution 2) to caracterise mesenchymal stem cells from CMML patient ex vivo. During the first part of my PhD, I adapted a model published in 2016 to CMML. This model of human MSC graft in immunodeficient mice proved to be hardly reproducible. During the second part, I analysed of CMML patients MSC. I identified an excessive production of IGFBP2 (Insuline-like Growth Factor Binding Protein 2) probably secondary to an epigenetic disregulation. Single cell RNA sequencing revealed a restriction of MSC heterogeneity of which only a fraction produces IGFBP2. Finally, I showed that IGFBP2 favors myeloid progenitors differenciation towards monocytic lineage. IGFBP2 could therefore contribute to the amplification of CMML characteristic monocytosis.To conclude, CMML goes along with modifications of hematopoietic niche cells, some of which produce excessive amounts of IGFBP2. Investigation on the origin of this alteration and its significance in disease progression should allow to evaluate the potential interest of its neutralization for therapeutic strategies
Monsarrat, Paul. "Cellules souches, médecine régénérative et régénération parodontale." Thesis, Toulouse 3, 2016. http://www.theses.fr/2016TOU30031/document.
The first part of this work introduces a new concept of analysis of clinical trial records and the dynamics of their evolution, both thematic and temporal. This concept has been applied to regenerative medicine, showing the lack of correlation between the source of stem cells and the fields of application. The stomatognathic diseases are few involved in clinical trials for stem cells therapy. Yet periodontitis, immuno-infectious diseases responsible for the destruction of the tooth supporting tissues, are a major public health issue. While the authors agree on the responsibility of the immune and microbial ecology in the pathophysiology of the disease, the reasons for dysbiosis, individual susceptibilities, are still unclear. Graft of mesenchymal stromal cells (MSCs) would return to homeostasis by promoting the activation of endogenous MSCs. The second part of this work shows that periodontitis were potentially associated with 57 systemic diseases; the clinical trials registry of the World Health Organization have been analyzed. The efficacy and safety of the use of MSCs for periodontal regeneration in animal models have also been demonstrated. Yet the models suffered from methodological problems, periodontal lesions are few representative of the pathophysiology. This second part thus provides data on the effectiveness of ASC (CSM from adipose tissue) to improve quantitative and qualitative regeneration of periodontal supporting tissues in a mouse model where periodontal lesions were generated by repeated administration of parodonto-pathogenic bacteria. It is therefore a model whose pathophysiology is closer to that found in humans. Finally, the second part demonstrates broad antibacterial spectrum of ASC whose effect is both direct (macrophage-like effect) and indirect (via the secretion of antibacterial factors)
Nakhle, Jean. "Transfert de mitochondries des cellules souches mésenchymateuses aux cellules souches de glioblastome : effets sur le métabolisme et la résistance à la chimiothérapie." Thesis, Montpellier, 2020. http://www.theses.fr/2020MONTT036.
Glioblastomas are heterogeneous tumors with high metabolic plasticity. Their poor prognosisis linked to glioblastoma stem cells (GSCs) which provide resistance to therapy, in particularto temozolomide (TMZ). It is worsened with mesenchymal stem cells (MSCs) recruitment. Weshow that, following tunneling nanotubes interactions, MSCs transfer mitochondria to GSCs.We found that MSC mitochondria modify the metabolic response of GSCs to TMZ and increasethe production of metabolites linked to the TCA cycle, pentose phosphate and pyrimidinesynthesis pathways, which was linked to enhanced GSC survival. A RNA-seq analysis revealedthat MSC mitochondria also disrupt the GSC transcriptional response to TMZ and lead to theexpression of genes related to cell cycle progression. Together, our data show that themitochondria transfers that originate from cells of the tumor microenvironment can modifythe response of cancer cells to therapy, at both at the levels of cellular metabolism and geneexpression
Leroy, Adrien. "Ingénierie tissulaire du ligament : association de copolymères dégradables et de cellules souches mésenchymateuses." Phd thesis, Université Montpellier II - Sciences et Techniques du Languedoc, 2013. http://tel.archives-ouvertes.fr/tel-01022842.
Massoudi, Ali. "Les cellules souches mésenchymateuses du tissu adipeux humain : étude de leur potentiel myogénique." Nice, 2007. http://www.theses.fr/2007NICE4012.
Our laboratory has isolated from human infant adipose tissue, a mesenchymal stem cell population, named hMADS cells (human Multipotent Adipose-Derived Stem cells). HMADS cells exhibit at the single-cell level fundamental stem cell properties such self-renewal and multipotentiality. In addition, mesenchymal stem cells may have an intrinsic capacity to differentiate into supplementary cell types in culture or after transplantation. Previously we have shown that hMADS cells were able to restore dystrophin expression after transplantation in mdx mice. In the current study, we investigated the mechanism of skeletal muscle phenotype acquisition by hMADS cells. We first tested various culture conditions to induce an intrinsic myogenic program in hMADS cells. Myotube formation and muscle determination/differentiation factors, i. E. Pax7, myf-5, MyoD and myogenin were assessed. No myogenic conversion could be detected under these conditions. In contrast, when co-cultured with myoblasts, hMADS cells reproducibly contributed to hybrid myotube formation, expressed several muscle differentiation markers and had the ability to restore dystrophin expression in human dystrophic background. Interestingly, this myogenic conversion was not associated by the expression of key transcription factors for muscle determination/differentiation. Therefore, our results clearly demonstrated that acquisition of myogenic identity by hMADS cells did not occur via a bona fide determination/differentiation process. We propose a model of myogenic conversion of human mesenchymal stem cells derived from adipose tissue in which, only after fusion, transcription factors of myoblasts were able to program hMADS genome toward the skeletal muscle lineage
Chagraoui, Jalila. "Microenvironnement hématopoïétique du foie foetal : caractérisation et relation avec les cellules souches mésenchymateuses." Paris 7, 2003. http://www.theses.fr/2003PA077022.
Ferrand, Jonathan. "Helicobacter pylori dans un modèle de carcinogenèse gastrique impliquant les cellules souches mésenchymateuses." Thesis, Bordeaux 2, 2009. http://www.theses.fr/2009BOR21627/document.
Helicobacter pylori infection is found in about half of the world population and can induce several gastrointestinal pathologies including gastric adenocarcinoma. An animal model recently led to the hypothesis of a cellular origin for the cancer initiating cells after Helicobacter infection. Bone marrow-derived mesenchymal stem cells (MSC) are believed to be recruited in the gastric mucosa in order to repair the damages due to infection, by an epithelial differentiation. Gastric carcinoma may rise from MSC reconstituted gastric glands. This study aimed to analyze, by sequential in vitro approaches, the different steps allowing tumor initiation including MSC recruitment by infected epithelial cells, epithelial differentiation of MSC, and cancer marker appearance after H. pylori infection. We first demonstrated that H. pylori infected epithelial cells may recruit MSC by a secretion of cytokines. We then showed that MSC fuse with gastric epithelial cells leading to MSC-derived epithelial cells. Finally, we studied the interaction between H. pylori and epithelial cells providing a preliminary explanation for cancer initiation. Our results allow a better understanding of gastric adenocarcinoma pathophysiology and will be helpful for the understanding of other cancers in which the role of MSC as cancer initiating cells is suspected
Laydi, Fatima Ezzahra. "Effet de la nature des biomatériaux sur la différenciation des cellules souches mésenchymateuses." Thesis, Université de Lorraine, 2013. http://www.theses.fr/2013LORR0165/document.
In tissue engineering, biomaterials, cells and the induction of cell differentiation are factors to be studied. The aim of this study is to know the effect of biomaterials composition and mechanical properties on the differentiation of mesenchymal stem cells from bone marrow. At first, we studied the effect of a protein biomaterial (collagen type I) supplemented with hydroxyaptatite (HAP) particles. We found that the addition of HAP improves the mechanical properties of the biomaterial and conditione cell differentiation towards osteoarticular lineages. In a second step, we studied the effect of biomaterial composed of alginate supplemented with hyaluronic acid or HAP particles, using an experimental design to select suitable matrices for biological study based on their mechanical properties. We found that the components of this biomaterial have an effect on elasticity of the latter and the differentiation of mesenchymal stem cells. In conclusion, this study shows that mesenchymal stem cells are sensitive to the composition of the biomaterial and its mechanical properties
Uzunhan, Yurdagül. "Effet des cellules souches mésenchymateuses dans les altérations épithéliales alvéolaires induites par l'hypoxie." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCD103/document.
Non communiqué
Colombier, Pauline. "Médecine régénératrice du disque intervertébral : intérêt des cellules souches mésenchymateuses et pluripotentes induites." Nantes, 2016. https://archive.bu.univ-nantes.fr/pollux/show/show?id=5e5379b6-d687-4fba-8c30-14e03b8982bd.
Intervertebral discs (IVD) are composed of a central part named Nucleus pulposus (NP). This Nucleus pulposus is populated by notochord cells (NCT), which are considered as NP cell progenitors and nucleopulpocytes I (NPCytes), responsible for extracellular matrix synthesis. Both cell types are derived from the embryonic notochord. The loss of NCT and NPCytes early in life is associated with first signs of disc degeneration. Thus, the supplementation of the degenerative NP with human stem cells- derived NCT and NPCytes could be a promising approach for the regenerative medicine of IVD. In this context, this work aimed at the establishment of a notochordal differentiation protocol from human induced pluripotent stem cells (iPSC). Molecular and cellular mechanisms involved in the formation of the notochord in mice embryos were transposed to iPSC technology. To address whether NPCytes can be generated, we investigated the abilities of human mesenchymal stem cells to differentiate toward nucleopulpogenic lineage. In summary, we have shown that NCT and I NPCytes can be generated by controlling I human stem cells differentiation. In addition, molecular mechanisms driving both differentiation processes have beendescribed. Finally, these findings contributed to the regenerative medicine of IVD, by generating cells potentially able to restore NP homeostasisand function
Perrot, Pierre. "Ostéosarcome, cellules souches mésenchymateuses, tissu adipeux : interactions dans des approches de reconstruction tissulaire." Nantes, 2010. https://archive.bu.univ-nantes.fr/pollux/show/show?id=4849d2c3-47e3-42ac-9154-21dc22cf949a.
The response to standard treatment for osteosarcoma (chemotherapy and surgery) is insufficient. The mesenchymal stem cells (MSCs) or osteoblast precursors may be used as vectors to produce anti-bone resorption molecules like osteoprotegerin (OPG) or receptor activator of NF-kB (RANK) soluble form (Rank -Fc). Due to their properties to differentiate into osteoblats, these cells could also participate in the reconstruction of bone tissue often damaged near the tumor site. One of the most controversial issues concerns the pro-tumor or anti-tumor effect of MSCs depending on either MSC origin or tumor type. On osteosarcoma, our results show either a local pro-tumor effect or a pro-metastatic effect. The MSCs seem to play a central role in the microenvironment of tumor cells. They may be responsible for late local recurrence or metastatic developments delayed. We report a clinical case of late local recurrence of osteosarcoma 13 years after the initial disease and 18 months after lipofilling. The autologous fat injection causes a major modification of the microenvironment of potential quiescent tumor cells. The intake of fat cells and Ad-CSM secreting multiple cytokines may influence the tumor progression and metastasis, this which leads to the question of the safety of lipofilling in a post-neoplasic context
Mouiseddine, Moubarak. "Utilisation des cellules souches mésenchymateuses humaines dans le traitement des atteintes tissulaires radio-induites." Versailles-St Quentin en Yvelines, 2008. http://www.theses.fr/2008VERS0038.
Ionising radiation can induce toxic effects on body. They provoke physiological modifications of tissues and organs which can be lethal. Total body irradiation or local abdominal irradiation can induce serious complications. Intestine is the first tissue concerned by these side effects. Radiation induces malabsorption of the intestine and lost of it integrity. Radio-induced physiopatological effects on intestine could lead to distant effects on other tissues and organs such as liver. The actual treatments have a limited efficiency or are not adapted to gastro-intestinal damages. Indeed, in this type of lesions, the heterogeneous systems which are concerned and the gravity of lesions complicate the medical care. Our purpose is to show that cell therapy using human mesenchymal stem cells (MSC) constitutes resolution in this type of illness. Our works show that MSC are multipotent and have heterogeneous expression of molecules. These cells are able to establish its selves in many organs and tissues after injection into irradiated mouse model. Thus we have shown that MSC can prevent the small intestine from radio-induced damages. Indeed we demonstrate that through their action on gut, MSC can indirectly restore hepatic integrity
Selmani, Zohair. "Investigation au niveau de la caractérisation phénotypique et fonctionnelle des cellules souches mésenchymateuses natives et cultivées de moelle osseuse." Besançon, 2008. http://www.theses.fr/2008BESA3003.
During the previous decade our knowledge of mesenchymal stem cells (MSC) bas been stimulated by their application in regenerative medecine. Today we can distinguish two types of MSC : fresh cells (MSCn), which are enriched directly from bone marrow and resemble more closely to in vivo MSC ; the second type (MSCc) can be obtained after isolation and in vitro expansion of MSCn. Here we have characterized MSCn and investigated the immunosuppressive potential of MSCc. - Due to our ability to isolate MSCn by immunomagnetic separation using an antibody against CD49a (highly conserved protein between species), we have been able to characterize MSCn from different species showing that they express common stem cell markeTs such as CD133, c-kit and Sca-1 (murine). - Secondly, we showed that in contrast to endothelial progenitors (PE), MSCn are not mobilized into the peripheral blood of patients treated by statins. - Lastly, we demonstrated for the first time that MSCc express the immunosuppressive molecule HLA-G5. This molecule is implicated in the induction of regulatory T-cells, is able also to both inhibit the proliferation of allogeneic T cells and prevent NK cells lysis. Moreover, these properties were shown to be dependent on cell contact and IL-10 expression. - ln summary this work shed light on the functional and phenotypic properties of mesenchymal stem cells
Fioretti, Florence. "Contribution à l'étude de cellules humaines mésenchymateuses à des fins de reconstruction tissulaire." Paris 5, 2007. http://www.theses.fr/2007PA05M001.
In this work, conducted mainly on three dimensional culture (collagen lattice), we demonstrated that human dermal and gingival fibroblasts as well as mesenchymal stem cells (MSCs) from human bone marrow were able to remodel the matrix in which they were incorporated. MSCs, dermal and gingival fibroblasts act in a similar way concerning macromolecules expression and synthesis (collagen types I,III, fibrillin-1), metalloproteinases (MMP-1, MMP-2, MMP-9) and tissue inhibitor of metalloproteinases (TIMP-1, TIMP2). Furthermore, in our hands, MSCs adopt a fibroblasts / myofibroblasts like phenotype, and thus can be considered a useful cellular source to build in vitro dermal or gingival equivalent tissue. Such a tissue equivalent after keratinocytes seeding could be beneficial with parents after trauma for exemple large burns or with patients suffering from epidermolysis for whom tissular lesions wear down the vital prognosis. Lastly, these bioengineered tissues containing appropriate cell number characteristic of a given tissue are a powerful tool for studying molecular and cellular mecanisms bring into play for exemple during phases of tissue regeneration after wound healin
Larguech, Gaithallah. "Effets de la Laminarine sur les cellules souches mésenchymateuses : impact sur la différentiation chondrogénique." Thesis, Orléans, 2017. http://www.theses.fr/2017ORLE2018.
Mesenchymal stems cells (MSCs) are a population of multipotent cells residing in several readily available adult tissue compartments, thus allowing for their ex vivo expansion. MSCs have a reliable potential for differentiation (plasticity) into cells of the mesodermal lineage (chondrocytes, osteoblasts, adipocytes). Bone marrow-derived MSCs have been a focus of stem cell research in light of their relative ease of isolation and expansion and of their high potential for differentiation. Herein, the aim of the present PhD is to explore the potential of a β-glucan (laminarin) on Mesenchymal stem cell proliferation and differentiation for future benefit for osteoarthritis treatment. MSCs were cultured in MSC growth and chondrogenic differentiation mediums. Cells viability and apoptosis were explored by cell count, MTT assays and Annexin V staining. In addition, Analysis of the specific protein of cell proliferation was performed by western blott. Furthermore, mRNA and protein expression of specifics markers for MSCs and chondrocytes were studied using qPCR and immunofluorescence. Our results demonstrated that stimulation of MSC with laminarin at a dose of 1 mg/ml in either basic growth culture or chondrogenesis inhibited cell proliferation without inducing their apoptosis. Furthermore, under chondrogenic culture conditions, laminarin at a similar dose prevented the differentiation of MSC into chondrocytes
Che, Thi Cam Ha. "Effets des cellules souches mésenchymateuses sur la cancérogenèse colique chimio-induite chez le rat." Paris 7, 2010. http://www.theses.fr/2010PA077147.
The aim of this work was to evaluate in an animal model the harmlessness of cell therapy for tissue repair in a cancer environment. For that purpose, we induced colon carcinogenesis by intrarectal instillations of MNNG (N-Methyl-N'-Nitro-N-Nitrosoguanidine) in the rat. The MNNG induce an increase in the thickness of colon mucosa, as well as of the content in MCP-1, IL-6 and flbronectin. We have characterized the tumors (1) in vivo by endoscopy and PET scanning, (2) after autopsy by histology and immunohistology and ELIS A assay of proteins. In this model, we studied the influence of mesenchymal stem cells (MSC) obtained from bone marrow of rats transgenic for fluorescent protein GFP. MSC were injected intraveinously 4 and 6 weeks after initiating MNNG treatment of rats. The MSC-GFP were traced by immunofluorescence and identified in the chorion of colonic epithelium 6 days after injection, but not thereafter. Thirty-two weeks after MNNG treatment, the number of rats bearing tumors was significatively lower in the MSC-treated batch, as compared to MNNG alone. This resuit was confirmed after 52 weeks. On the other hand, MSC injections increased the effect of MNNG on the thickness of mucosa, especially epithelium, suggesting an intensification of tissue repair process after the attack by the carcinogen. The results suggest an early but ongoing action of MSC. Our hypothesis is that MSC contribute to thé restoration of a favourable micro-environment after mucosa lesion by MNNG therefore slowing cancer development
Gindraux, Florelle. "Application des cellules souches mésenchymateuses dans la réparation osseuse : étude expérimentale et potentiel thérapeutique." Besançon, 2007. http://www.theses.fr/2007BESA3703.
Mesenchymal stem cells (MSC) are able to generate mesenchyme differentiated cells : osteoblasts, adipocytes, bone marrow (BM), stromacytes,. . . MSC are found in adult's BM, adipose tissue, muscle. . . This study consists elaborating a cell therapy product for bone repair by associating a " stem cell potential " to a biomaterial. This work, initialized at Besançon ITBC (Ingénierie et Biologie Cellulaire et Tissulaire) under the direction of Pr Hervé et Dr Deschaseaux, consisted to study MSC from BM, predilection tissue. We have directly selected MSC from human BM by using an immunomagnetic cell sorting to facilitate in vitro characterization. We have then adapted this cell sorting to rodent BM in order to study in vivo osteogenic potential MSC in : i) Tissue inflammation model : syngenic administration of MSC by intravenous infusion in lethal irradiated mice ; ii) Osteogenic model (subcutaneous implantation) : association of sorted MSC to a bi-phasic synthetic hydroxyapatite/tricalcium phosphate ceramic (Triosite®, Zimmer, Biomatlante). Results have shown that selected MSC were able to homing into irradiated mice BM and to endure three months after administration. Osteogenic results have shown that biphasic ceramic did not allow efficient bone reconstruction. The study, secondly performed at the TBF company (Tissue Bank of France, Mions (69)) under the direction of Drs Barnouin and Laganier, consisted to select and osteo-differentiated MSC from human adipose tissue, tissue available in unlimited quantity and whose collection generates little or no morbidity. The MSC were cultivated on viroinactived, lyophilized and radiosterilized human bone allograft (Phoenix®, TBF) in osteoinduction medium. Results have shown that this bone substitute was efficiently tolerated by MSC which were able to locally generate a mineralized tissue. We have then evaluated the osteogenic potential of MSC cultivated on bone allograft in osteoinduction conditions and intramusculary implanted in nude rat (xenograft). Results have shown that human MSC were able to produce type I collagen preferentially localized in osteoblastic tissue. To elaborate a cell therapy protocol for bone repair, MSC were selected from rabbit adipose tissue and cultivated on bone allograft in osteoinduction conditions and implanted in a bone cranial defect. Results have shown osteoblastic tissue only in the defect filled with bone allograft and MSC in comparison with the defect filled with bone allograft alone. This study allowed us to choose adipose tissue as MSC source and bone allograft as an efficient support for bone repair by tissue engineering. A bone repair model associating MSC from adipose tissue and bone allograft is currently developed in the large animal model (sheep) with Dr Petite
Margossian, Talar. "Caractérisation des cellules souches mésenchymateuses du sang placentaire et de la gelée de Wharton." Thesis, Université de Lorraine, 2013. http://www.theses.fr/2013LORR0030/document.
Stem cells are the hopes for cell therapy and tissue engineering. MSCs from fetal tissue (umbilical cord blood and WJ), which are a source of embryonic epiblast grow relatively faster comparing to other adult sources. The culture condition can affect cell behavior. In our study, we explored the effect of oxygen concentration on the expansion, immunophenotyping, and differentiation of these cells. The aim of this work is to identify the optimal method for isolation of MSCs derived from fetal tissue. Given the low rate of success in the isolation of MSCs from cord blood, we headed to WJ-MSCs. We have determined in siu, the specific markers expressed in the WJ and in the perivascular region. Studies on the morphology growth kinetics, and phenotypic expression of surface makers of MSCs isolated from WJ were made over a long period (7 passages) in different culture conditions. We have shown that WJ is composed of an abundant extracellular matrix rich in collagen and glycasominoglycans and have variable phenotype depending from their localization in the jelly. This tissue is able to provide a large amount of MSCs (6.7x105 Cs/cm of cord) that maintain a constant morphology. Finally, regardless of the passage, the oxygen concentration does not effect on the phenotype of the cells. In contrast, a low oxygen concentration during expansion appears to decrease the doubling time of MSCs, promote chondrogenesis and inhibit osteogenic differentiation. Finally, whatever the culture conditions, adipogenic differentiation of WJ-MSC seems difficult to obtain
Gallay, Nathalie. "Etude du rôle du microenvironnement médullaire sur la prolifération, le cycle cellulaire, l'apoptose et la migration des cellules de leucémies aiguës myéloïdes." Tours, 2007. http://www.theses.fr/2007TOUR3302.
Acute myeloblastic leukemia is characterized by uncontrolled proliferation within the bone marrow (BM) of malignant myeloid progenitors arrested in their maturation process. BM microenvironment is composed of stromal cells, endothelial cells and extracellular matrix (ECM) and is attended to play a role in leukemia cell survival and drug resistance. We studied interactions of leukemic cells with mesenchymal stem cells (MSC), BM endothelial cells and ECM proteins (fibronectin). We showed that close contact with MSC protects leukemic cells from drug-induced apoptosis by promoting quiescence, with a role of α4 integrin in this process. We demonstrated that CD31 and CD38 are involved in the control of the retention or dissemination of leukemic cells. These results give new insights about the mechanisms of drug resistance induced by marrow microenvironment, and provide new therapeutic strategies consisting in disturbing interactions of leukemic cells with their supportive microenvironment
Lavenus, Sandrine. "Études des interactions entre cellules souches et surfaces implantaires nanostructurées." Nantes, 2010. https://archive.bu.univ-nantes.fr/pollux/show/show?id=2d0946e5-0bbf-466c-a5b0-5c6e5d88f147.
Metal implants allow nowadays prosthetic rehabilitations with high clinical success due to their surface properties. Some studies have shown that surface properties such as roughness, wettability and chemistry changed the adhesion and differentiation of cells, and thereby, the integration of implant in tissues. Understanding of the interactions between cells and implant surfaces is essential in the field of tissue engineering and biomaterials. Attachment, adhesion and spreading of cells establish the first step of interaction between cells and surfaces and, so the quality of this step determined the cell capacity to proliferate and differentiate on implant surface. In this context, the aim of this study was to study the adhesion and differentiation of human mesenchymal stem cells (hMSC) on nanostructured surface. In the first part, the adhesion, proliferation and differentiation of hMSC, osteoblasts and gingival fibroblasts were compared on substrates with similar surface roughness and wettability, but different chemistries. Secondly, nanostructured titanium surface were realized and characterized. Titanium vapor deposition was performed on polycarbonate membranes with pores of 50, 200 or 400 nm of diameter. Anodisation also allowed obtaining a regular surface with pores of 30, 50 and 100 nm of diameter. In the last part of this work, the adhesion and osteoblastic differentiation of hMSC were studied on these nanostructured surfaces. Cell adhesion and differentiation have been investigated using staining, immunostaining, image analysis and gene expression. Finally, histomorphometric analysis of anodized implant after 1 and 3 weeks of implantation in rat tibia allowed the characterization of osteointegration. The characterization of surface properties and biological study of different cell type on nanostructured surface was necessary to understand the behaviour of cells and so, the consequence for the osteointegration