As Distrofias Musculares Progressivas constituem um grupo de doenças genéticas caracterizadas por uma degeneração progressiva e irreversível da musculatura esquelética. As diferentes abordagens terapêuticas propostas para esse grupo de doenças têm como enfoque restaurar a proteína muscular deficiente por meio da terapia celular ou terapia gênica, ou o tratamento dos sinais e sintomas patológicos do músculo pela administração de fármacos e/ou fatores de crescimento. A combinação de diferentes estratégias pode aumentar a eficiência do reparo muscular. Deste modo, este trabalho tem como objetivo principal avaliar o potencial terapêutico das célulastronco mesenquimais (MSCs) associadas ao fator de crescimento semelhante à insulina (IGF-1) para diferentes tipos de distrofias musculares. Inicialmente avaliamos o potencial miogênico de MSCs humanas de cordão umbilical in vitro. Nossos resultados demonstraram que os fatores solúveis liberados pelo músculo distrófico de camundongos mdx foram capazes de induzir a diferenciação miogênica terminal das células-tronco. Além disso, verificamos que o IGF-1, por si só, é capaz de promover a miogênese de MSCs, com mais eficiência que os protocolos de indução padrões. Ainda nos estudos in vitro, demonstramos que MSCs são capazes de interagir com células musculares de pacientes com Distrofia Muscular de Duchenne (DMD) e restaurar a expressão de distrofina, quando cultivadas em meios suplementados com IGF-1. Frente a estes resultados, prosseguimos com os estudos em modelos animais, in vivo, e demonstramos que as MSCs humanas de cordão umbilical e IGF-1, quando administrados conjuntamente por via sistêmica, são capazes de modular a inflamação, reduzir a fibrose, aumentar o reparo muscular e, consequentemente, promover uma melhora clínica significativa do músculo de camundongos LAMA2dy/2j - modelo murino de Distrofia Muscular Congênita. Cabe ressaltar que as células humanas não foram rejeitadas após administração sistêmica em modelos animais não imunossuprimidos. Esses resultados suportam o potencial uso combinado de MSCs de cordão umbilical humano e IGF-1 no tratamento de distrofias musculares. Contudo, a confirmação destes dados em um modelo animal de grande porte, como os modelos caninos de distrofia muscular, é de extrema importância visando o entendimento dos mecanismos envolvidos no reparo muscular e avaliação de eventuais efeitos adversos, o que pode representar um passo importante para o início dos testes clínicos em pacientes / Progressive muscular dystrophies are a clinically and genetically heterogeneous group of disorders caused by the deficiency or abnormal muscle proteins, resulting in progressive degeneration and loss of skeletal muscle function. Strategies for the development of a muscular dystrophy therapy have focused on the possibility of restoring the defective muscle protein by cell therapy or on delivery of growth factors to treat or ameliorate muscular pathology symptoms. Combining both strategies could be a very useful approach to enhance the efficiency of muscle repair. The aim of this study is to evaluate the therapeutic potential of human mesenchymal stem cells (MSCs) from umbilical cord tissue combined with IGF-1 for muscle regeneration. Firstly, we verified the myogenic potential of these cells in vitro. Our results demonstrated that the soluble factors released from mdx dystrophic muscle were able to promote the myogenic differentiation of MSCs. Moreover, we showed that IGF-1 is capable of enhancing considerably the myogenesis of human MSCs from UC in vitro. More interestingly, we showed that IGF-1 enhances the interaction of MSCs and DMD muscle cells in coculture and the restoration of dystrophin expression. Subsequently, our in vivo studies revealed that the association of IGF-1 and MSCs markedly reduced muscle inflammation and fibrosis, and significantly improved muscle strength in LAMA2dy/2j mice, a murine model for congenital muscular dystrophy. It is important to point out that human cells are not rejected even in xenotransplants without immunosuppression. In summary, our results suggest that a combinatorial strategy of both IGF-1 and MSCs could enhance the efficiency of muscle repair and, therefore, should be further tested as a potential therapeutic approach in muscular dystrophies. However it is important to repeat the experiments on canine dystrophic model (GRMD; Golden Retriever Muscular Dystrophy) - in order to enhance our knowledge about the mechanism involved in muscle repair and monitor any eventual long-term side effects, which could represent an important point to start the clinical trial in patients

Orientador: Dimas Tadeu Covas / Banca: José Orlando Bordin / Banca: Carmino Antônio de Souza / Banca: Orlando Castro e Silva Júnior / Banca: Aparecida Maria Fontes / Resumo: Alguns trabalhos realizados recentemente relatam que as células-tronco mesenquimais (CTM) podem ser induzidas à aquisição de marcadores hepatocíticos pelo transplante em modelos animais de dano hepático, ou pelo cultivo in vitro com fatores de crescimento e citocinas. O presente trabalho teve por objetivo avaliar o comportamento das CTM frente à indução da diferenciação hepatocítica. As CTM foram isoladas da medula óssea de quatro doadores saudáveis, caracterizadas e submetidas ao protocolo de indução à diferenciação hepatocítica in vitro e in vivo. As células induzidas in vitro apresentaram mudanças na sua morfologia, mostrando a morfologia semelhante à do hepatócito, porém, o perfil imunofenotípico não foi modificado. As células induzidas também não apresentaram o aumento dos transcritos de albumina, citoqueratina 18 e citoqueratina 19 quando analisadas por RT-PCR em tempo real, e não alteraram a expressão de albumina, citoqueratina 18 e alfafetoproteína como demonstrado por imunofluorescência. Quando analisadas in vivo, as CTM demonstraram o potencial migratório para o tecido hepático danificado de camundongos imunodeficientes. Em conjunto, os resultados sugerem que as CTM da medula óssea não são capazes de se diferenciar em hepatócitos quando estimuladas in vitro pela metodologia utilizado neste trabalho, mas são capazes de migrar para o tecido hepático danificado in vivo, o que sugere o seu papel no reparo do fígado. A contribuição para o reparo pode estar associada com o efeito parácrino dessas células. / Abstract: Some recently works have been reported that mesenchymal stem cells (MSC) can be induced to the acquisition of hepatocytic markers for the transplant in animal models of liver damage, or for the in vitro culture with growth factors and cytokines. The present work aim is to evaluate the behavior of the MSC in front of the induction of the hepatocytic differentiation. The MSC was isolated from the bone morrow of 4 normal donators, characterized and submitted to the protocol of in vitro and in vivo induction of hepatocytic differentiation. The in vitro induced cells showed morphology changes acquiring hepatocytes-like morphology. However, the immunophenotypic profile of those cells was not modified. The induced cells did not present increase of the albumin, cytokeratin 18 and cytokeratin 19 transcripts, when analyzed by real time RTPCR. The expression of albumin, cytokeratin 18 and alpha foetoprotein was also not modified as demonstrated by immunofluorescence. In vivo, the MSC have demonstrated the migratory potential for the damaged liver of immunodeficient mice. Together, the results suggest that the bone morrow MSC are not capable of in vitro hepatocytic differentiating according to the approach in this work, but are capable to homming into damaged hepatic tissue in vivo. This migration capacity suggests their role in the repair mechanisms. / Doutor

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 / 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

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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;.

. / 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]

Immunosuppressive drugs have been used for many years for the treatment of autoimmune diseases and post-transplantation treatment. While these drugs have a lot of advantages, they also show several undesirable side effects. The most common side effects are higher blood pressure, lowered renal function and susceptibility to infections. Therefore, in recent years there has been a demand for other medical approaches that do not exhibit the above-mentioned adverse effects. Among one of the newly tested approaches is the application of mesenchymal stem cells (MSCs), which possess several advantages such as immunomodulatory abilities, safety and relatively easy isolation, however, stem cell use alone has not yet provided sufficiently strong immunomodulation. Only a small part of research of MSCs is focused on their use in the combination with immunosuppressive therapy. Therefore, in my thesis I focused on the model which allows to reduce the dose of immunosuppressive drugs in the combination with MSCs. Combined therapy is more advantageous than both monotherapies thanks to lower dosages of these drugs used. It enables to decrease negative side effects of immunosuppressive drugs, when combined with MSCs to provide sufficient immunomodulation in comparison to classical therapy. The aim of my work was to...

Maintaining of homeostasis is essential for the survival of the organism. Stress disturbs the homeostasis and prepares the organism for mental or physical stress. During the stress situation, the endogenous stress factors are released. Through these factors stress affects tissue regeneration, the immune system and other metabolic processes. Chronic stress impacts many parts of body and mind and has a negative effect on these processes. Acute stress has the opposite effect. Mesenchymal stem cells (MSCs) participate in regenerative processes and modulate the immune system. Therefore, it can be assumed that stress will affect on MSCs. The aim of this study was to investigate the effect of stress factors, norepinephrine and corticosterone on the properties and function of MSCs in acute and chronic stress model. In our study, stress factors did not affect the morphology, vitality and differentiation of MSCs. However, the metabolic activity of MSCs was reduced regardless of the duration of their action. The action of stress factors also affected the production of some immunologically relevant molecules and proteins. Unfortunately, the results did not show a clear effect of stress factors on the lymphocyte modulation by MSCs. Key words: mesenchymal stem cells, catecholamines, adrenergic receptors,...

Infectious diseases are the worldwide leading cause of morbidity and mortality. Sepsis is the major cause of death in infectious diseases. It is one of the most serious and also one of the most difficult treatable conditions of contemporary medicine. Sepsis is the main cause of death in intensive care units. Causal therapy of sepsis does not yet exist. With a far better understanding of patho/physiological mechanism of sepsis, it is possible to model new preclinical experiments to verify the efficiency and security of new therapeutic procedures. Large animal experiments in progressive sepsis, with the use of domestic porcine, play a vital role. Long-standing experience with this model and similarity to human facilitate the realization of more complex experiments with potential for the relevant translation of results into the subsequent clinical studies on human subjects. The objective of this doctoral dissertation was to assess on the clinically relevant model: 1) the efficiency and security of extracorporeal membrane oxygenation in the event of vasoplegic septic shock; and 2) the benefit of two innovative therapeutic approaches to treatment of sepsis: a) the intravenous administration of mesenchymal stem cells; and b) the activation of the neuro-inflammatory reflex through the vagus nerve stimulation.