Le cartilage articulaire est un tissu possédant une faible capacité de réparation intrinsèque. Dès lors, la répétition de traumatismes articulaires induit un microenvironnement propice à la dégradation du cartilage et, in fine, l’émergence de l’arthrose. Les traitements utilisés à l’heure actuelle visent uniquement à soulager la douleur, réduire l’inflammation et la progression de l’arthrose. Ainsi, le traitement des lésions chondrales équines revêt une importance majeure puisque les affections locomotrices constituent la première cause de baisse de performances et d’arrêt prématuré de la carrière du cheval sportif. De plus, le cheval est le modèle animal qui possède le cartilage articulaire le plus semblable à celui de l’Homme et, en conséquence, représente un modèle d’étude pertinent pour les lésions chondrales humaines. Dans ce contexte, notre étude s’est attachée à développer de nouvelles stratégies pour le traitement des lésions chondrales basées sur la différenciation chondrogénique de CSM en vue de produire in vitro un substitut cartilagineux implantable en site articulaire. Ainsi, nous avons d’abord isolé et caractérisé des CSM équines à partir de prélèvements de moelle osseuse (MO) et de sang de cordon ombilical (SCO), puis, nous avons réalisé la différenciation en cultivant les CSM durant 14 jours en hypoxie ou normoxie au sein d’un biomatériau (éponges de collagène de types I/III), en présence de BMP-2 et TGF-β1 et de siRNA ciblant le collagène de type I et HtrA1, molécules atypiques du cartilage hyalin. Bien que ce protocole nous ait permis d’obtenir un substitut cartilagineux riche en marqueurs du cartilage hyalin comme le collagène de type II et l’agrécane, la présence du collagène de type I persistait. Nous avons donc tenté d’optimiser le protocole en allongeant le temps de culture, en utilisant le TGF-β3, et en modifiant la stratégie d’interférence par l’ARN. Cette étape nous a permis de conclure sur l’effet bénéfique de l’allongement de la culture à 28 jours et l’efficacité des facteurs chondrogéniques initialement utilisés. Néanmoins, la stratégie d’interférence par l’ARN demeure encore perfectible. Finalement, nous avons comparé la qualité du substitut cartilagineux obtenu après différenciation en fonction de la source de CSM utilisée. Les CSM de MO semblent les plus adaptées mais le protocole que nous avons utilisé n’est probablement pas le plus efficace pour induire la différenciation des CSM de SCO. Dans une partie complémentaire, bien que ces résultats soient préliminaires, nous avons montré que le sécrétome des CSM pourrait être un formidable outil afin d’améliorer le traitement des lésions chondrales. Dans leur ensemble, les résultats obtenus permettent d’avoir un regard optimiste concernant la mise en place de thérapies cellulaire et tissulaire du cartilage, aussi bien en médecine équine qu’humaine. / Articular cartilage is a tissue with low intrinsic repair abilities. Therefore, repeated traumas lead to cartilage degradation and ultimately, to the emergence of osteoarthritis (OA). Current therapies aim to reduce pain, inflammation and to prevent the progression of OA. Thus, treatment of equine chondral lesions is of major importance since locomotor disorders are the main causes of poor performance and early retirement of the athlete horses. In addition, the horse is an animal model with the most human-like articular cartilage and, therefore, represents the best relevant model to study human chondral lesions and arthropathies. In this context, our study focused on developing new strategies for the treatment of chondral lesions based on the chondrogenic differentiation of equine mesenchymal stem cells (MSC) in order to produce an in vitro neo-synthetized cartilaginous substitute, which could be implantable in the chondral lesion site. Thus, we first isolated and characterized equine MSC derived from bone marrow (BM) and umbilical cord blood (UCB). Then, we have differentiated MSC by culturing them for 14 days in hypoxia or normoxia, in a biomaterial (types I / III collagen sponges), in the presence of BMP-2 and TGF-β1 and siRNA targeting type I collagen and HtrA1, two atypical hyaline cartilage molecules overexpressed in OA. Although this protocol allowed us to obtain a cartilaginous substitute composed of large amounts of hyaline cartilage markers such as type II collagen and aggrecan, the presence of type I collagen persisted. We therefore tried to optimize the protocol by extending the culture time, using TGF-β3, and modifying the RNA-interference strategy. We have concluded on the beneficial effect of the lengthening of the culture to 28 days and the effectiveness of the chondrogenic factors initially used. Nevertheless, the RNA-interference strategy still remains perfectible. Finally, we compared the quality of the neo-synthetized cartilaginous substitute according to the source of MSC used. BM-MSC seem to be the most suitable, but the protocol we used is probably not the most effective for inducing UCB-MSC differentiation. In a complementary part, although these results are very preliminary, we have shown that the MSC secretome could be a tremendous tool to improve current therapies of chondral lesions. Overall, the results obtained make it possible to look ahead with optimism, in order to obtain future efficient cartilage tissue engineering therapies, both in equine and human medicines.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22005号 / 医博第4519号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 松原 和夫, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23417号 / 医博第4762号 / 新制||医||1052(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 秀一特定拠点, 教授 妻木 範行, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

iii Abstract Cells have evolved multiple mechanisms of cellular motility ranging from the migration of large cell cohorts to specialized migration of individual cells. The wide range of invasion modes has been exploited by cancer cells to their advantage, which has rendered the metastatic process so difficult to defeat. To allow for a better understanding of cancer invasion plasticity, we have employed studies on cancer cells that adopt the proteolytically active, adhesion-dependent, elongated mesenchymal invasion mode, the protease-independent, low adhesion, rounded amoeboid invasion mode, or combination of both. To study invasion plasticity directly, we have established two model systems of the mesenchymal- amoeboid transition (MAT) that allow for regulated induction of MAT in 3D in vitro environments. Using these systems, MAT was induced in HT1080 fibrosarcoma cells and the acquisition of a motile, invasive amoeboid phenotype was confirmed. We then observed the mesenchymal and amoeboid invasion strategies within 3D collagen in more detail using a digital holographic microscope. Further, HT1080 cells before and after MAT were subject to high throughput proteomic and transcriptomic studies. Comparison of gene expression and protein levels of mesenchymal and amoeboid cells disclosed an inflammatory-like...

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Introduction : Les cellules souche mésenchymateuse (CSM) présentent une puissante activité immunomodulatrice sur les lymphocytes T et les Natural Killer (NK), impliquées dans les réactions allogéniques. Les propriétés immunomodulatrices des CSM dépendent de contacts cellulaires et des facteurs secrétés. Ainsi les exosomes produits par ces cellules pourraient constituer des nouveaux produits thérapeutiques.L’objectif de ce travail est d’étudier, in vitro, l’effet d’exosomes dérivés de CSM sur les lymphocytes B, T et les NK.Méthodologie : Les CSMs utilisées sont issues de foies fœtaux humains. Les exosomes ont été isolés à partir du milieu de culture des CSMs par une série d’ultracentrifugation à 100000g.Résultats : Contrairement aux CSMs qui inhibent la prolifération des lymphocytes T et B, leurs exosomes n'ont pas d'effet sur leur prolifération. Cependant ils inhibent la prolifération, l’activation et la cytotoxicité (expresion CD107a) des NK. Nous avons mit en évidence, par FACS, la présence de TGFbeta; à la surface des exosomes. De plus leur fonction inhibitrice est abrogé en présence d’un anticorps bloquants anti-TGFbeta;. Réciproquement l’exposition de cellules NK à du TGFbeta; inhibe la cytotoxicité et la prolifération de cellules. Enfin, en présence d'exososmes nous avons montré, par IF, une translocation de Smad 2/3 (messager du signal TGFbeta;) dans les noyaux des cellules NK, inhibé par l'ajout d'anticorps anti-TGFbeta;.Conclusion: Ces résultats suggère que les propriétés immunomodulatrices de CSMs sur NK pourraient dépendre de TGFβ présenté ou associé aux exosomes. / Introduction: Mesenchymal stem cells (MSCs) are powerful immunomodulators regulating the function of B and T lymphocytes and natural killers cells (NK) involved in allogeneic reactions. Their immunomodulatory properties depend on cell contact and secretion factors produced by MSCs. Thus exosomes produced by these cells could provide new therapeutic tools.Objective: The objective of this work is to study the effect of MSC derived exosomes in vitro on B and T lymphocytes and NK cells.Method: MSCs used for this study are isolated from human fetal liver. Exosomes were isolated from MSC culture medium by a serie of ultracentrifugation at 100000g.Results: MSCs inhibit the proliferation of T lymphocytes. Unlike MSCs, their exosomes do not abrogate the proliferation of T and B cells. However they inhibit the proliferation, activation and cytotoxicity (CD107a expression) of NK cells. By FACS analysis we showed a surface expression of TGFb; by exosomes. Inhibition of NK cells activation by exosomes is altered by a neutralizing anti-TGFb; antibody. Contrary when NK cells are cultured with TGFb; the same effect qs exosomes is demonstrated. By IF, we found a nuclear translocation of Smad 2/3 (TGFb; signal transducer) in NK cells cultured with exosomes, which is inhibited by the qddition of anti-TGFb; antibody.Conclusion: These results suggest that the immunomodulatory properties of MSCs on NK could depend on exosome presentation or association with TGFb;.

Introduction : L’efficacité de la greffe d’îlots n’est plus à démontrer mais elle reste l’objet de recherches pour améliorer la qualité et la survie des îlots greffés souvent fragilisés par la destruction enzymatique de leur microenvironnement lors de la procédure d’isolement. Dans ce contexte, les cellules stromales mésenchymateuses (CSM) d’origine pancréatique représentent un outil intéressant par leurs propriétés d’immunomodulation et par leur capacité de sécrétion de facteurs du microenvironnement. L’objectif de ce travail est d’évaluer l’effet des cellules stromales pancréatiques humaines sur les cellules β humaines.Méthodes : Des îlots humains purifiés ont été maintenus en culture pendant plusieurs jours. Les cellules adhérentes se formant en périphérie de l’îlot ont été sélectionnées et immortalisées. Ces nouvelles cellules « human islet-derived stromal cells » (hISC) ont ensuite été caractérisées pour déterminer leur profil mésenchymateux Nous avons ensemencé des cellules β humaines (lignée EndoC-βH1 ou cellules primaires) sur du milieu conditionné de hISC (hISC-CM) utilisé comme support de culture. L’adhérence, la survie, la prolifération, l’insulinosécrétion des cellules β cultivées sur le hISC-CM ont été mesurées et comparées à un support contrôle : la poly-L-lysine.Résultats : Les hISC présentent un profil phénotypique et transcriptomique très proche des CSM issues de la moelle osseuse. D’un point de vue fonctionnel, les hISC présentent une capacité d’immunomodulation. Elles expriment et sécrètent des protéines matricielles connues pour être présentes autour et à l’intérieur des îlots humains tels que les collagènes de type I, IV et VI, la laminine et la fibronectine. Au contact du hISC-CM les cellules EndoC-βH sur adhèrent st s’étalent. Le hISC-CM augmente l’expression du marqueur de prolifération PCNA et améliore la survie et la fonction des cellules EndoC-βH1. D’un point de vue mécanistique, l’interaction cellules β/hISC-CM active la phosphorylation de FAK (focal adhesion kinase) et ERK (extracellular signal-regulated kinases). A l’interface de cette interaction, la sous-unité β1 de l’intégrine est impliquée dans les effets observés du hISC-CM sur l’adhérence et la fonction des cellules β.Conclusion : Nos travaux démontrent l’intérêt prometteur des hISC en tant que cellules de soutien des cellules β humaines par la sécrétion de protéines matricielles pancréatiques. Ces résultats ouvrent de nouvelles perspectives pour le maintien des îlots en culture et leur conditionnement dans un microenvironnement plus physiologique permettant ainsi de préserver leur qualité fonctionnelle lors de la greffe. / Introduction : The efficacy of islet transplantation is well established. However, the procedure still needs improvements in the quality of grafted islets, often weakened by the loss of their surrounding tissue during the isolation process. In this respect, mesenchymal stromal cells (MSC) represent an interesting tool as they have immunomodulatory and anti-inflammatory properties and are known to secrete proteins involved in creating a favorable microenvironment. This work aims to investigate the effect of human pancreatic stromal cells on human β-cells.Methods : We characterized the mesenchymal profile of cells, previously immortalized in our lab from human islets of Langerhans adherent cells, hereafter named hISC (human islet-derived stromal cells). We seeded human β-cells (EndoC-βH1 cell line or primary β-cells) on hISC-conditioned medium (hISC-CM) used as coating of Petri dishes. We assessed spreading, survival, proliferation and glucose-induced insulin secretion of β-cells cultured on hISC-CM as compared to poly-L-lysine coating.Results : Phenotypic and transcriptomic profiles of hISC are close to bone-marrow MSC. The hISC have an immunomodulation capacity. They express and secrete extracellular matrix proteins known to be present around and within human islet such as types I, IV and VI collagens, laminin and fibronectin. EndoC-βH1 seeded on hISC-CM adhere and spread on cell culture surface. We show that hISC-CM has positive effects on EndocC-βH1 proliferation, survival and glucose-induced insulin secretion, as compared to poly-L-lysine. From mechanistic point of view, hISC-CM induces FAK (focal adhesion kinase) and ERK (extracellular signal-regulated kinases) phosphorylations. The β1-integrin subunit is involved in both adhesion and increased insulin secretion of β cells induced through hISC-CM.Conclusion : Our work demonstrates a promising interest of hISC as support cells for human β-cells by scaffolding factors secretion. It opens new perspectives for conditioning human β-cells in a more physiological microenvironment to preserve their functional quality before and after transplantation.

La migration cellulaire est un phénomène essentiel au développement, à l'immunité et à la cicatrisation. Pourtant, l'activation des programmes de migration en dehors des situations physiologiques peut avoir des effets néfastes. Par exemple, la migration cellulaire permet aux cellules d'une tumeur primaire d'envahir de nouveaux territoires et d'installer des tumeurs secondaires ou métastases. Lorsqu'une migration cellulaire est initiée à partir d'un tissu épithélial, ces cellules doivent acquérir des caractéristiques mésenchymateuses. Pour cela, elles diminuent leur adhérences cellule-cellule, perdent leur polarité apico-basale, réorganisent leur cytosquelette, changent d'adhérence à la matrice et modifient la composition et l'organisation de la matrice. C'est ce qu'on appelle la transition épithélio-mésenchymateuse (TEM). La famille des Métalloprotéinases Matricielle (MMP) est connue pour participer au remodelage de la matrice. Les MMPs sont au nombre de 25 et sont sécrétées ou membranaires. L'une de ces MMP membranaires est MMP14 ou MT1-MMP. Elle participe à la migration physiologique et pathologique via la dégradation de composants de la matrice. Elle dégrade également des protéines non matricielles sécrétées ou membranaires. De plus, MMP14 agit indépendamment de son activité catalytique en régulant par exemple l'activation de petites GTPases, de voies de signalisation et en contrôlant l'expression de gênes. Cependant, beaucoup d'études sur MMP14 ont été faites in vitro et ex vivo et il n'est pas clair si toutes les fonctions de MMP14 sont retrouvées in vivo. Plus spécifiquement les fonctions possibles de MMP14 dans la TEM et la migration in vivo sont encore mal définies. Nous proposons d'utiliser les crêtes neurales (CN) de l'embryon de poulet comme modèle pour étudier MMP14 au cours de la TEM et de la migration in vivo. Les CN sont des cellules embryonnaires retrouvées dans la partie dorsale du tube neural. Les CN réalisent une TEM pour quitter le tube neural avant de parcourir de longues distances et donner de nombreux types cellulaires. Les CN se séparent en deux populations, les CN céphaliques retrouvées dans la tête et les CN troncales dans le reste de l'embryon. Ces deux populations de CN réalisent des TEM différentes, avec une TEM rapide et massive pour les CN céphaliques et plus lente et en continue pour les CN troncales. Même si ces TEM sont différentes, elles présentent une diminution des jonctions cellulaires, une perte de la polarité apico-basale, un changement d'adhérence à la matrice et une réorganisation de la matrice. Une particularité des CN troncales est la localisation du noyau en position basale de l'épithélium juste avant la sortie du tube neural. Plusieurs substrats de MMP14 sont retrouvés dans la TEM et la migration des CN et une étude a montré par PCR la présence de l'ARNm de MMP14 dans les CN céphaliques de poulet. L'objectif de la thèse est d'explorer la fonction de MMP14 au cours de la TEM et de la migration des CN. Nous avons montré que MMP14 est exprimée dans les deux populations de CN au cours de la TEM et de la migration. / Cell migration is an essential event during embryonic development, immunity and wound healing. Furthermore, the activation of migration program in non-physiologic conditions can have side effects. For example, cell migration promotes invasion of primary tumor cells in new territories and the formation of secondary tumors or metastasis. When an epithelial tissue initiates migration, epithelial cells need to gain mesenchymal attributes. To this end, they decrease their cell-cell adhesions, loss their apico-basal polarity, reorder their cytoskeleton, change their matrix adhesions and modify the matrix composition and organization. This event is named epithelial-mesenchymal transition (EMT). The family of Matrix Metalloproteinase (MMP) is known to reshape the matrix. MMP family is composed of 25 members which are secreted or linked to the membrane. One of the membrane-bound MMP is MMP14 or MT1-MMP. MMP14 is known to promote physiological and pathologic cell migration by inducing degradation of numerous matrix components. MMP14 cleaves also non-matrix proteins which are secreted or membrane-bound. Moreover, MMP14 can act independently of its catalytic activity for example in the regulation of small GTPases, signaling pathway and in gene expression control. However, the vast majority of MMP14 related studies were conducted in vitro or ex vivo and it is not clear whether some of its functions occur in vivo. More specifically, MMP14's putative functions in EMT and migration are still ill-defined. We propose to use the Neural Crest (NC) of chick embryo as model to study MMP14 during in vivo EMT and migration. NC is an embryonic cell population located in the dorsal part of the neural tube. NC cells realize an EMT to leave the neural tube before performing a long-distance migration and producing a myriad of cell types as neurons, bones and cartilages of the face and pigment cells. NC cells are divided in two populations, the cephalic NC in embryo's head and the trunk NC in the posterior part. The cephalic NC perform a fast and massive EMT while the trunk NC's EMT is slower and continuous. Although the EMT are different, they conserve common characteristics with a decrease of cell junctions, a loss of the apico-basal polarity, a change of matrix adherence and a rearrangement of the matrix. One particularity of trunk NC is the epithelium basal position of the nucleus just prior their exit from the neural tube. Many MMP14's substrates are found during NC EMT and migration and a study suggested by PCR that chick cephalic NC express MMP14 mRNA. The goal of this thesis is to explore the function of MMP14 during chick NC EMT and migration. Our results show that MMP14 is expressed by the two populations of NC during EMT and migration. Moreover, MMP14 cell localization changes from apical to basal during EMT. Loss of function experiments show that MMP14 is needed for NC EMT. Our rescues with various MMP14 versions indicate that: 1/ the cytoplasmic domain is not essential, 2/ the extracellular domain is needed and 3/ the catalytic activity is not required for EMT. MMP14 is involved in the control of cell junctions by a switch between cadherin-6B and cadherin-7 but not in the remodeling of the matrix during NC EMT. We have also showed that MMP14 is necessary for the change of cell polarity during EMT. Furthermore, we have showed that MMP14 is needed for the formation of matrix adherence. In conclusion, our study shows that MMP14 is involved in NC EMT and migration and that NC are a good model to investigate MMP14 function in vivo.

. / Articular cartilage is made up of dense, connective tissue localized at the junction of several locations in the skeleton. It covers the surface of the joints to ensure that bones can move. It is an avascular tissue that is not innervated and is composed primarily of a single cell type, the chondrocyte, which synthesizes an abundant extracellular matrix (ECM). Osteoarthritis (OA), a degenerative disease of articular cartilage, is characterized by the degradation of the ECM, associated with increased secretion of matrix metalloproteinases (MMPs) and aggrecanases. In addition, the OA process induces chondrocyte dedifferentiation characterized at least in part by increased synthesis of type I collagen, an atypical isoform in articular cartilage. Moreover, due to the poor intrinsic healing capacity of articular cartilage, there is currently no treatment to restore the chondrocyte phenotype and, in the most advanced stages of OA, the joint must be replaced with a prosthesis, requiring surgery. Therefore, various drug and surgical treatments have been developed in an attempt to prevent the destruction of cartilage which, in light of their relative success, then lead to new, improved therapeutic strategies. One of the most promising approaches is the cartilage tissue engineering based on the procedure described by Brittberg using autologous chondrocyte implantation (ACI). Applied in the earliest stages of OA or chondral lesions, ACI is based on the use of chondrocytes from a healthy, non-bearing region of the diseased joint. The cells are then amplified in monolayer culture and then re-implanted in the lesion. However, amplification of autologous chondrocytes in two-dimensional culture mimics, at least in part, some of the characteristics of the OA process and is accompanied by cell dedifferentiation leading to the formation of nonfunctional fibrocartilage. The numerous pharmaceutical approaches and surgical techniques developed to repair cartilage lesions have revealed their limitations. Ideally, traumatic cartilage lesions should be treated earlier to prevent OA and postpone prosthetic surgery. In the interest of preventing OA, cartilage cell therapy has proven to be a pivotal approach for repairing damaged tissue. Cell therapy consists not only in filling the cartilage lesion with healthy chondrocytes, but also in reconstituting the structure, the physico-chemical properties and the functionality of the hyaline matrix. The transplantation of autologous chondrocytes is the foundation of cell therapy and cartilage tissue engineering and there have been several generations of ACI, each improving on the previous one. However, even the most recent ACI techniques are showing limitations and consequently, research efforts are now focused on improving this technique in order to obtain, after amplification, a differentiated and stable chondrocyte phenotype. This is to be achieved by using new types of biomaterials that can fill more important lesions, molecules and growth factors to better control the chondrogenic differentiation and more suitable cell sources that avoid morbidity at the donor site as it is the case with articular chondrocytes. Today, MSCs hold much promise for biomedical research because they are able to recapitulate many tissues, including cartilage. However, for future advances in the field of regeneration and tissue engineering it is important to know the exact nature of these cells. With this goal, in this work, we first fully characterized 4 categories of serum free amplified mesenchymal stem cells extracted from adipose tissue (AT), bone marrow (BM), dental pulp (DP) and Wharton’s jelly (WJ) of the umbilical cord. The cells were characterized in terms of efficiency of isolation, amplification kinetics and according to an extensive immunophenotyping using flow cytometry... [etc]

Tumour cell invasion is one of the most critical steps in malignant progression. It includes a broad spectrum of mechanisms, including both individual and collective cell migration, which enables them to spread towards adjacent tissue, and form new metastases. Understanding the mechanisms of cell spreading, and invasion, is crucial for effective anticancer therapy. Two modes of individual migration of tumour cells have been established in a three-dimensional environment. Mesenchymally migrating cells use proteases to cleave collagen bundles, and thus overcome the ECM barriers. Recently described protease-independent amoeboid mode of invasion has been discovered in studies of cancer cells with protease inhibitors. During my PhD study, I have focused on determining the molecular mechanisms involved in amoeboid invasion of tumour cells. We have examined invasive abilities in non-metastatic K2 and highly metastatic A3 rat sarcoma cell lines. We have shown that even though highly metastatic A3 rat sarcoma cells are of mesenchymal origin, they have upregulated Rho/ROCK signalling pathway. Moreover, A3 cells generate actomyosin-based mechanical forces at their leading edges to physically squeeze through the collagen fibrils by adopting an amoeboid phenotype. Amoeboid invasiveness is also less dependent on...