Ensaios clinicos tem demonstrado que a imunossupressao em altas doses (IAD) seguida de transplante autologo de celulas tronco hematopoeticas (TACTH) e capaz de suprimir a atividade inflamatoria em pacientes com doencas autoimunes (DAIs) e induzir remissoes clinicas prolongadas nesses pacientes, porem os mecanismos de acao do TACTH ainda nao estao bem esclarecidos. O racional dessa terapia baseia-se na eliminacao das celulas autorreativas pela IAD e na reconstituicao de um sistema imunologico novo e tolerante apos o transplante a partir dos precursores hematopoeticos. O objetivo deste trabalho foi avaliar a reconstituicao imunologica em pacientes com diabetes mellitus tipo 1 (DM1, N=21) e pacientes com esclerose multipla (EM, N=37) sequencialmente apos o TACTH, e correlacionar os dados imunologicos com a resposta clinica dos pacientes ao transplante. Os pacientes com EM e DM1 foram divididos em dois grupos com base na resposta clinica apos o transplante: respondedores (EM-R; N=22) e nao-respondedores (EM-NR; N=15); livres de insulina por periodo maior ou igual a 3 anos (DM13 anos; N=11) e livres de insulina por periodo menor que 3 anos (DM1<3 anos; n=10); e acompanhados clinica e imunofenotipicamente por seis anos. Em relacao ao periodo pre-transplante, todos os grupos de pacientes com DM1 e EM apresentaram: 1, diminuicao do numero absoluto de celulas T CD3+ praticamente em todos os periodos pos-transplante avaliados, indicando uma intensa linfopenia decorrente da IAD; 2, aumento acentuado do numero de linfocitos T CD8+ e a diminuicao dos linfocitos T CD4+, resultando na inversao da razao CD4:CD8 durante todo o seguimento pos-transplante avaliado; 3, aumento significativo no primeiro ano apos o transplante das subpopulacoes de celulas T CD8+ de memoria central CD27+CD45RO+ e memoria efetora CD27- CD45RO+; 4, normalizacao dos numeros de linfocitos T CD4+ e CD8+ naive CD27+CD45RO- somente cinco anos apos o transplante, enquanto o numero de celulas T CD4+CD45RA+CD31+ recem- emigrantes do timo manteve-se abaixo dos valores pre-transplante durante todo o periodo avaliado, demonstrando que durante os seis anos de seguimento apos a IAD/TACTH predominaram mecanismos timo-independentes de reconstituicao imunologica; 5, normalizacao dos numeros de linfocitos B CD19+ entre dois a tres meses pos-transplante. O grupo de pacientes com DM1 que obteve melhor resposta clinica apos o tratamento com IAD/TACTH (DM13anos) apresentou, em comparacao ao periodo pre-transplante, numero diminuido de celulas T CD3+ (linfopenia) em varios periodos pos-transplante, numero aumentado de celulas T CD8+CD28- supressoras no primeiro ano pos-transplante principalmente, numero diminuido de celulas T CD4+ de memoria efetora nos periodos 2 a 9 meses pos-transplante e numero aumentado de celulas T reguladoras CD4+CD25hiFOXP3+ pos-transplante. O grupo de pacientes com EM com melhor resposta clinica apos o tratamento com IAD/TACTH (EM-R) apresentou, em comparacao ao periodo pre-transplante, numero diminuido de celulas T CD3+ (linfopenia) em varios periodos pos-transplante e numeros aumentados de celulas T reguladoras CD4+CD25hiFOXP3+ e CD8+CD28- supressoras nos primeiros tres anos pos-transplante. Vale ressaltar que os pacientes com DM1 e EM que apresentaram melhor resposta clinica permaneceram linfopenicos por maiores periodos de tempo apos o transplante. Desse modo, esse estudo revelou que a resposta terapeutica dos pacientes com DM1 e EM ao TACTH depende de uma linfopenia persistente, alem do aumento de celulas T reguladoras e supressoras e diminuicao de celulas T CD4+ de memoria apos o transplante. / Clinical trials have shown that high-dose immunosuppression (HDI) followed by autologous hematopoietic stem cell transplantation (AHSCT) is able to suppress the inflammatory activity in patients with autoimmune diseases (AID) and induce prolonged clinical remissions in these patients, but the mechanisms of action of AHSCT are still not well understood. The rationale of this therapy is based on the elimination of autoreactive cells by HDI and on the reconstitution of a new tolerant immune system after transplantation from hematopoietic precursors. The aim of this study was to evaluate the immune reconstitution in patients with type 1 diabetes mellitus (T1D, N=21) and patients with multiple sclerosis (MS, N=37) sequentially after the AHSCT, and correlate the immunological data with the clinical response of these patients to the transplant. Patients with MS and T1D were divided into two groups based on clinical response following transplantation: response (MS-R, N=22) and non-response (MS-NR, N=15); insulin-free for a period longer or equal to 3 years (T1D3 years, n=11) and insulin-free for less than 3 years (T1D<3 years, n=10); and accompanied clinical and immunophenotypically by six years. Regarding the pre-transplant period, all groups of patients with T1D and MS showed: 1, decreased absolute number of CD3+ T cells in virtually all post-transplant periods evaluated, indicating an intense lymphopenia resulting from HDI; 2, sharp increase in the number of CD8+ T lymphocytes and decreased CD4+ T lymphocytes, resulting in inversion of CD4:CD8 ratio throughout the follow-up post-transplant evaluation; 3, a significant increase, in the first year after transplantation, of CD8+ T central memory CD27+CD45RO+ and effector memory CD27-CD45O+ cell subpopulations; 4, normalization of CD4+ and CD8+ T naive CD27+CD45RO- lymphocytes numbers only five years after transplantation, whereas the number of CD4+CD45RA+CD31+ T cells newly emigrants from the thymus remained below the values pre- transplant during the study period, showing that during the six years of follow-up after the HDI/AHSCT mechanisms thymus-independent immune reconstitution were predominant; 5, normalization of CD19+ B lymphocytes numbers in two to three months post-transplant. The group of patients with DM1 that had the best clinical response after treatment with IAD/AHSCT (T1D 3years) showed, in comparison with the pre-transplant period, decreased number of CD3+ T cells (lymphopenia) at various times after transplantation, increased CD8+CD28- suppressor T cells numbers in the first year post-transplant, decreased number of CD4+ effector memory in periods of 2 and 9 months post-transplant and increased number of CD4+CD25hiFOXP3+ regulatory T cells after transplantation. The group of MS patients with better clinical response after treatment with IAD/AHSCT (MS-R) showed, in comparison to the pre-transplant period, decreased number of CD3+ T cells (lymphopenia) at various times after transplantation and increased numbers of CD4+CD25hiFOXP3+ regulatory Tcell and CD8+CD28- suppressor in the first three years post- transplant. It is noteworthy that patients with T1D and MS which showed better clinical response remained lymphopenic for longer periods of time after transplantation. Thus, this study revealed that the therapeutic response of patients with T1D and MS depend on a AHSCT to persistent lymphopenia, and increased regulatory and suppressor T cells and decreased number of CD4+ effector memory after transplantation.

A sepse é uma doença relacionada como a presença de infeção junto a uma resposta inflamatória sistêmica; sua fisiopatologia envolve uma rede complexa de citocinas e mediadores inflamatórios que causam a injúria de diversos tecidos. Na atualidade são muitas as tentativas para diminuir a mortalidade, porém até agora, não existe uma estratégia específica para tratar a doença. As células-tronco mesenquimais da geleia de Wharton do cordão umbilical (CTM-GW) são conhecidas por expressar genes e fatores envolvidos na angiogênese e imunomodulação. Nós usamos o modelo de ligadura e punção do ceco (LPC) para analisar o papel da CTM-GW em disfunção orgânica relacionada à sepse. Foi utilizada a citometria de fluxo para avaliar o fenótipo das células isoladas. Dividimos ratos Wistar em grupos: sham (operação simulada); LPC; e LPC + CTM (106 CTM-GW i.p., 6 horas após LPC). Às 24 h pós-LPC, foram avaliadas a função renal, hepática e outras variáveis do estudo. As CTM-GW foram negativas para CD3, CD34, CD45 e HLA-DR, enquanto eles foram positivos para CD73, CD90 e CD105. O tratamento com CTM na sepse reduziu a mortalidade, melhorou a filtração glomerular (aferido pelo clearance de inulina), função tubular, reduziu a lesão hepática e demostrou uma ação anti-inflamatória. O tratamento também apresentou um efeito anti-apoptótico e protetor do tecido renal e do endotélio, mediante a regulação da expressão de VEGF, AQP2 e eNOS. Em conclusão as CTM-GW diminuem a injúria renal e hepática, portanto, pode desempenhar um papel protetor na sepse / Sepsis is a disease related to the presence of infection with a systemic inflammatory response. The pathophysiology involves complex cytokine and inflammatory mediator networks that cause injury to various tissues. Currently, there are many attempts to reduce mortality, but so far, there is no specific strategy for treating the disease. Human umbilical cord Wharton\'s jelly-derived mesenchymal stem cells (hWJ-MSCs) are known to express genes and factors involved in angiogenesis and immunomodulation. We used a cecal ligation and puncture (CLP) model to analyze the role of hWJ-MSCs in sepsis-related organ dysfunction. We used flow cytometry to evaluate hWJ-MSC phenotypes. We divided Wistar rats into groups: sham (sham-operated); CLP; and CLP+MSC (106 WJ-MSCs, i.p., 6 h after CLP). At 24 h post-CLP, we evaluated renal function, liver and other variables. hWJ-MSCs were negative for CD3, CD34, CD45 and HLA-DR, whereas they were positive for CD73, CD90 and CD105. In sepsis, treatment with MSC reduced mortality, improved glomerular filtration rate (measured by inulin clearance), tubular function, reduced liver damage and decreased the inflammatory markers. The treatment also showed an anti-apoptotic effect and protected the renal tissue and endothelium by up-regulation the expression of VEGF, AQP2 and eNOS. In conclusion, hWJ-MSCs decrease renal and hepatic injury, therefore, may play a protective role in sepsis

A regeneração de defeitos ósseos continua sendo um grande desafio na área de Odontologia e Medicina. É bem estabelecido que células-tronco mesenquimais (CTMs) e osteoblastos (OBs) desempenham um papel crítico na osteogênese, tornando-se candidatos a utilização em procedimentos de terapia celular que visam otimizar o processo de reparação óssea. Porém, pouco se sabe sobre a interação entre CTMs e OBs, e a maioria dos estudos enfatiza o efeito dos OBs sobre CTMs, fazendo com que a influência das CTMs na atividade osteogênica dos OBs continue sendo uma questão desafiadora. Baseados em nossos estudos anteriores, formulamos a hipótese de que a terapia celular que fizesse uso de uma associação de CTMs e OBs poderia ser mais eficaz para o reparo do tecido ósseo do que essas células isoladamente, principalmente como resultado da estimulação de OBs por CTMs. Para tal, foi realizado estudo in vitro para avaliar os efeitos das CTMs sobre os OBs e in vivo para avaliar os efeitos dessas células, isoladamente e combinadas, sobre a reparação óssea. CTMs da medula óssea de rato foram cultivadas em meio de crescimento para manterem-se como CTMs ou em meio osteogênico para diferenciarem-se em OBs. Após alcançar a subconfluência, as células foram cultivadas in vitro em três diferentes condições: (1) co-cultura direta de CTMs e OBs usando três proporções celulares (1:1, 1:2 e 2:1), (2) co-cultura indireta de CTMs e OBs usando insertos e (3) OBs cultivados em meio condicionado por CTMs. Para avaliação das respostas celulares foram realizados ensaios de proliferação celular, atividade de fosfatase alcalina (ALP), formação de matriz mineralizada, expressão gênica de marcadores osteoblásticos, imunolocalização de sialoproteína óssea (BSP) e osteopontina (OPN) e migração celular. Para os experimentos in vivo, as células foram carreadas em esponja de colágeno através de vários ciclos de centrifugação. Após, defeitos ósseos em calvária de rato foram preenchidos com (1) esponja de colágeno sem células, (2) esponja de colágeno com CTMs, (3) esponja de colágeno com OBs e (4) esponja de colágeno com associação de CTMs e OBs. Para avaliação da reparação óssea in vivo após 4 semanas, foram realizadas análises histomorfométricas através de cortes histológicos e microtomografia computadorizada. Os dados foram comparados pelo teste de Kruskal-Wallis e, se necessário pelo teste de Mann-Whitney (p&le;0,05). Foi observado que CTMs têm efeito repressivo sobre a proliferação e as expressões fenotípicas e genotípicas de OBs (P&le;0,05). Em relação ao reparo dos defeitos ósseos, somente naqueles tratados com células observou-se formação óssea predominantemente como ilhotas isoladas e diferenças, principalmente qualitativas, entre os tipos celulares utilizados, com tendência de maior formação óssea em defeitos tratados com OBs em comparação ao uso de CTMs. Com base nos resultados obtidos, pôde-se concluir que as CTMs apresentam efeito inibitório sobre OBs e que a terapia celular com OBs parece ser mais eficaz no reparo do tecido ósseo. / The regeneration of bone defects remains a major challenge in the field of Dentistry and Medicine. It is well known that mesenchymal stem cells (MSCs) and osteoblasts (OBs) play critical roles in osteogenesis, making them promising alternatives to be employed in cell therapy procedures to enhance the process of bone regeneration. Studies about the crosstalk between MSCs and OBs are mainly focused on the effect of OBs on MSCs, thus how MSCs may affect OBs phenotype expression remains a challenging question. Based on our previous studies, we have hypothesized that cell therapy using a combination of MSCs and OBs could be more effective for the bone repair than these cells separately, mainly due to the stimulation of OBs by MSCs. For this, we carried out in vitro experiments to evaluate the effects of MSCs on the OBs and in vivo experiments to assess the effects of these cells either isolated or combined on bone repair. Rat bone marrow MSCs were cultured either in growth medium to keep MSCs features or in osteogenic medium to differentiate into OBs. After reaching subconfluence, cells were grown in vitro in three different conditions: (1) direct coculture of MSCs and OBs using three cell proportions (1:1, 1:2 and 2:1), (2) indirect coculture of MSCs and OBs using transwell porous filters and (3) OBs cultured in MSCs conditioned medium. Cell responses were evaluated by assaying cell proliferation, alkaline phosphatase activity (ALP), mineralized matrix formation, gene expression of osteoblast markers, immunolocalization of bone sialoprotein (BSP) and osteopontin (OPN), and cell migration. For in vivo experiments, cells were seeded into collagen sponge by a centrifugation method. After, calvarial defects were implanted with (1) collagen sponge without cells, (2) collagen sponge with MSCs, (3) collagen sponge with OBs, and (4) collagen sponge and association of MSCs with OBs. To evaluate bone repair at the end of 4 weeks, histomorphometric analyzes were carried out using histological slides and micro-computed tomography. Data were compared by Kruskal-Wallis test and, if appropriated, by Mann-Whtiney test (p&le;0.05). It was observed that MSCs repressed proliferation, phenotypic and genotypic expressions of OBs (P&le;0.05). Bone formation was observed only in cell treated defects as isolated islets and qualitative differences were noticed among cell types, with a tendency of more bone formation in OBs treated defects compared with MSCs ones. Based on these results, we can conclude that MSCs exhibited inhibitory effect on OBs and that cell therapy with OBs seemed to be more effective for bone repair.

Introdução: A colocação de próteses através da junção esofagogástrica propicia o refluxo gastroesofágico. Objetivos e métodos: Avaliação da paliação da disfagia, manifestações broncoaspirativas, parâmetros nutricionais e qualidade de vida de 30 pacientes com tumores avançados da junção esofagogástrica tratados por prótese metálica auto-expansível com válvula anti-refluxo de 2001 a 2004. Resultados: Melhora da disfagia e regurgitação. Sem alteração na tosse, eructação, pirose, parâmetros nutricionais e qualidade de vida. Conclusões: A prótese com válvula anti-refluxo foi eficaz na paliação da disfagia, impediu a ocorrência de manifestações broncoaspirativas, não interferiu nos parâmetros nutricionais e qualidade de vida / Background: Placement of stents through the gastroesophageal junction can cause reflux. Objective and methods: Evaluate dysphagia palliation, aspiration symptoms, nutritional parameters and quality of life in 30 patients treated for advanced gastroesophageal junction tumors with a self-expanding metal stent with anti-reflux valve from 2001 to 2004. Results: Dysphagia and regurgitation symptoms improved after placement. There was no changes in heartburn, cough, belching symptoms and also no improvement in nutritional parameters or quality of life. Conclusions: Antireflux valve self-expanding stents was effective in dysphagia palliation, avoided aspiration symptoms and did not affect nutritional parameters or quality of life

A placenta é um anexo embrionário que tem atraído grande interesse como fonte de células-tronco para medicina regenerativa, devido à plasticidade fenotípica de alguns dentre os vários tipos celulares isolados a partir deste tecido. Apesar de terem a mesma origem, não fazem parte do embrião, portanto o uso da placenta como fonte de células embrionárias não provoca debates éticos. Uma característica que vale a pena mencionar, é que a placenta está envolvida na manutenção da tolerância do feto pelo organismo materno, pois contém células que apresentam propriedades imunomoduladoras. Por fim, o tecido placentário é disponibilizado após o parto e é geralmente descartado. Estas características tornam esse tecido de grande interesse para protocolos de terapia celular, tanto que tem surgido bancos de célulatronco de placenta humana. Alguns trabalhos demonstraram plasticidade de células extraídas da placenta, porém existe ainda a necessidade de se definir melhor a região de coleta e os métodos de extração e isolamento dessas células. Nosso grupo estabeleceu a cultura de células derivadas da região do trofoblasto extraviloso (TEV) de placenta humana a termo, que são as células responsáveis pelos mecanismos de imunotolerância materno-fetal. As células TEV apresentam os marcadores de pluripotencia Oct-4 e Nanog, e, portanto, podem reter mesmo extraídas da placenta a termo, alguma plasticidade celular, caracterizando-as como células-tronco. Entretanto, nossa experiência no cultivo destas células mostrou que existem limitações relacionadas ao tempo de cultivo celular e capacidade de proliferação das TEV, que certamente fornece argumentos sólidos para limitar o seu uso em protocolos de terapia celular em medicina regenerativa. / The placenta is attached embryo that has attracted great interest as a source of stem cells for regenerative medicine due to phenotypic plasticity of some of the various cell types isolated from this tissue. Despite having the same origin, they are not part of the embryo, so the use of placenta as a source of embryonic cells does not provoke ethical debates. A feature worth mentioning is that the placenta is involved in maintaining tolerance of the fetus by the mother, because it contains cells that have immunomodulatory properties. Finally, the placental tissue is present after birth and is usually discarded. These characteristics make this fabric of great interest for cell therapy protocols, which has emerged both banks of stem cells from human placenta. Some studies have demonstrated plasticity of cells extracted from the placenta, but there is still a need to better define the catchment area and the methods of extraction and isolation of these cells. Our group has established a culture of cells derived from the extravillous trophoblast region (TEV) from human placenta at term, which are the cells responsible for the mechanisms of maternalfetal immunotolerance. TEV cells have the pluripotency markers Oct-4 and Nanog, and therefore can retain, even extracted from the placenta at term, some cellular plasticity, characterizing them as stem cells. However, our experience in growing these cells showed that there are limitations related to the time of cell culture and proliferation capacity, which certainly provides strong arguments for limiting their use in cell therapy protocols in regenerative medicine.

Les lésions cérébrales induisent une mort neuronale associée à des déficits fonctionnels importants. Afin de pallier aux capacités limitées de régénération spontanée des neurones du système nerveux central adulte, nous avons évalué, dans un modèle de lésion par aspiration du cortex moteur chez la souris adulte, le potentiel de stratégies de remplacement cellulaire par la transplantation de neurones embryonnaires ou dérivés de cellules souches, et la mobilisation des cellules souches endogènes présentes dans la zone sous-ventriculaire (ZSV). L'efficacité des neurones greffés dépend de leur capacité à adopter un phénotype neuronal approprié et à établir des projections spécifiques vers l'hôte. Nous avons montré que les cellules embryonnaires transplantées immédiatement après la lésion dans le cortex moteur lésé se différencient en neurones matures corticaux et envoient des projections appropriées vers les cibles du cortex moteur. Nous avons montré qu'introduire un délai d'une semaine entre la lésion du cortex moteur et la transplantation augmente la vascularisation et la prolifération des cellules transplantées, ainsi que la densité des projections qu'elles développent. Par ailleurs, nous avons étudié la possibilité de générer des neurones corticaux à partir de cellules souches humaines comme source alternative de neurones à transplanter. Enfin, nous avons montré que la lésion du cortex moteur induit une augmentation de la prolifération cellulaire et de la neurogenèse dans la ZSV, et favorise la migration des neuroblastes de la ZSV vers le site de lésion. / Damage to the adult motor cortex can lead to severe deficits in motor function. One strategy for overcoming the generally limited capacity of the mature central nervous system to regenerate axons in response to cell loss is cell replacement based therapies. We studied brain repair strategies in a mouse model of motor cortex aspiration lesion by using transplantation of embryonic neurons or stem cells-derived neurons and by evaluating the potential of endogenous stem cells found in the subventricular zone. Neuronal transplantation efficacy depends on the capacity of the transplanted cells to developp into appropriate neuronal phenotype and establishment of specific connections. We have shown that embryonic cells grafted immediately after lesion into the lesioned motor cortex develop into mature neurons with appropriate phenotype and establish projections towards appropriate targets. We have shown that introducing a delay of one week between motor cortex lesion and transplantation enhances graft vascularization, grafted cells proliferation and the density of transplant-to-host projections. Besides, we have studied the possibility to generate cortical neurons from human stem cells as an alternative source of neurons for transplantation. Finally, recruitment of endogenous stem cells found in the SVZ was examined in a mouse model of cortical lesion. We have shown that motor cortex injury increases cellular proliferation and neurogenesis in the SVZ and the migration of neuroblasts near the lesion site via blood vessels and astrocytes assisted migration.

La transplantation cellulaire apparaît aujourd’hui comme une thérapie prometteuse pour certaines formes graves d’insuffisance cardiaque réfractaire aux traitements classiques. Nous avons tenté dans ce travail d’améliorer la thérapie cellulaire en agissant sur deux paramètres : la perte et la survie des cellules. Dans une première étape, nous avons comparé les effets de deux méthodes de couverture épicardique des cellules via des biomatériaux cellularisés (feuilles de cellules et une matrice de gélatine) par rapport à ceux des injections conventionnelles dans le myocarde. Nous avons choisi de transplanter des cellules souches musculaires pour une étude de preuve de concept. Une amélioration de la fonction contractile au bout de 1 mois associée à une amélioration de la rétention cellulaire, une augmentation du nombre de vaisseaux sanguins et une diminution du pourcentage de la fibrose ont été enregistrées dans les groupes de biomatériaux cellularisés par rapport au groupe des injections de cellules. Nous avons tenté de confirmer les bénéfices de la couverture épicardique avec un autre type cellulaire. Nous avons choisi d’utiliser des cellules souches stromales d’origine adipeuse (ADSC pour Adipose Derived Stroma/Stem Cells). Toutefois, et malgré les bénéfices fonctionnels apportés lors des greffes, les feuilles d’ADSC étaient difficilement maniables lors de la chirurgie. De plus, les ADSC ont été incapables de générer de nouveaux cardiomyocytes s’intégrant électriquement et mécaniquement dans le tissu receveur. L’objectif de « régénération » myocardique requiert l’apport de cellules ayant un potentiel de différenciation cardiaque et devrait donc pouvoir être atteint avec des cellules souches embryonnaires (CSE). Nous avons choisi de travailler avec ce type cellulaire en raison de la possibilité de dériver, à partir de ces cellules, de véritables progéniteurs des cardiomyocytes. Pour limiter les conditions hypoxiques de l’environnement ischémique, nous avons co-transplanté les progéniteurs cardiaques dérivés des CSE humaines avec des ADSC afin d’en exploiter les propriétés trophiques, et d’optimiser ainsi la survie du greffon. Les deux populations cellulaires ont été transférées sur le myocarde infarci via, une matrice de gélatine (GELFILM™) qui représente de meilleures propriétés mécaniques. Lors de cette étude, nous avons remarqué une préservation contre le remodelage ventriculaire à court (1mois) et long (6mois) terme chez les animaux greffés avec les patchs co-ensemencés par rapport aux animaux recevant le patch seul et l’étude des patchs composites a montré la présence de cellules humaines in vitro mais pas in vivo, probablement à cause d’une maîtrise insuffisante du rejet. Notre travail a donc consisté en une analyse systématique de plusieurs paramètres fondamentaux de thérapie cellulaire (méthode de transfert des cellules, limitation des conditions de mort cellulaire après greffes, choix du type cellulaire). Les résultats obtenus valident l’emploi des matrices cellularisées déposées sur l’épicarde. Il convient maintenant d’optimiser la nature du biomatériau, les conditions de culture des progéniteurs cardiaques avec des cellules trophiques dans le but d’améliorer la survie du greffon. / Cell transplantation has emerged as a promising therapy for some kind of severe heart failure refractory to conventional treatment. We have attempted in this work to improve cell therapy by acting on two parameters: the loss and cell survival. In a first step, we compared the effects of two methods of epicardial deposition of cells via natural biomaterials (Cell sheet and gelatin matrix) compared to conventional cells injections into the myocardium. We chose to transplant muscle stem cells for a proof of concept study. Improved contractile function after 1 month associated with improved cell retention, an increased number of blood vessels and a decrease in the percentage of fibrosis were recorded in the groups of cellularized biomaterials compared with injections group. We attempted to confirm the benefits of the epicardial cover with another cell type. We chose to use stromal stem cells from adipose origin as ADSC for Adipose Derived Stromal / Stem Cells. However, despite the functional benefits provided after transplantation, ADSC sheets were hardly manipulated during surgery. In addition, ADSC were unable to generate new cardiomyocytes electrically and mechanically integrated into the host tissue. The objective of "regeneration" requires the input of contractile cells with potential of cardiac differentiation that should be achieved with embryonic stem cells (ESC). We chose to work with this cell type because of the possibility of deriving of cardiac progenitor from these cells. To reduce the hypoxic conditions of the ischemic environment, we co-transplanted cardiac progenitor derived from human embryonic stem cells with ADSC in order to develop the trophic properties, and thus optimize the graft survival. Both cell populations were transferred to the infarcted myocardium using a gelatin matrix (GELFILM ™) which represents better mechanical properties than the cell sheet. In this study, we observed a significant functional preservation against negative ventricular remodeling in the short (1 month) and long (6 months) term in animals grafted with co-seeded patches compared to animals receiving only the patch. The study of composite patches showed the presence of human cells in vitro but not in vivo, probably because of inadequate control of rejection. Our work has involved a systematic analysis of several basic parameters of cell therapy (method of cell transfer, reduce of cell death after transplantation, choice of cell type…). The results validate the use of cellularized matrix deposited on the epicardium. In conclusion, to improve graft survival, it is necessary to optimize the nature of the biomaterial, the culture conditions of cardiac progenitor cells.

L’endothélium joue un rôle primordial dans le développement et le maintien des multiples fonctions vasculaires. Il est ainsi largement impliqué dans des situations pathologiques comme les maladies cardio-vasculaires. La description de marqueurs endothéliaux circulants a permis une exploration non invasive de l'endothélium. Notre équipe s’est intéressée principalement aux cellules endothéliales circulantes (CEC), dont le taux reflète la lésion ou l’activation de l’endothélium, et aux progéniteurs endothéliaux circulants (PEC), marqueurs de régénération endothéliale. La découverte en 1997 par Asahara de la présence chez l’adulte de ces PEC, participant à la formation de nouveaux vaisseaux par vasculogenèse, a ouvert de nouvelles perspectives, notamment pour la thérapie cellulaire des pathologies ischémiques. Ce travail a consisté à développer les méthodes d’étude de ces cellules dans plusieurs contextes. Tout d’abord, nous avons exploré l’utilité de ces marqueurs dans la physiopathologie de l’hypertension artérielle pulmonaire (HTAP). Puis nous avons analysé le potentiel de mobilisation des progéniteurs endothéliaux à partir de la paroi vasculaire lors d’une ischémie locale chez des volontaires sains dans le cadre du développement d’un produit de thérapie cellulaire autologue. Une partie de ce projet a été de mettre en place et d’optimiser les techniques d’étude de ces marqueurs. Les CEC ont été quantifiées par immunoséparation magnétique (IMS), technique mise au point en 1992 (Dignat-George 1992) et transférée dans notre laboratoire. La quantification des PEC a été réalisée par cytométrie en flux et par culture cellulaire. En culture, deux types de PEC sont décrits : les PEC précoces, dont l’origine est monocytaire et pour lesquels la culture est déjà standardisée, et les « Endothelial Colony Forming Cells » (ECFC), seules cellules présentant des caractéristiques de cellules endothéliales progénitrices et pouvant être proposées comme produit de thérapie cellulaire. Nous avons optimisé la quantification des ECFC en culture en étudiant l’effet de diverses matrices et de la densité d’ensemencement des cellules mononucléées issues du sang total sur l’obtention de ces cellules et leurs propriétés angiogènes. La dysfonction endothéliale a été décrite comme un élément central dans le développement de l’HTAP dont le diagnostic repose sur la mesure de la pression artérielle pulmonaire par cathétérisme cardiaque droit. En l’absence de marqueur biologique non invasif dans cette maladie, nous avons quantifié les CEC et les progéniteurs circulants dans deux études. Une étude réalisée chez des patients adultes a montré une augmentation spécifique des CEC dans l’HTAP et non dans l’hypertension pulmonaire thromboembolique chronique. Ainsi les CEC semblent être le reflet des lésions endothéliales pulmonaires et non de la sévérité clinique des patients. L’autre étude a montré l’intérêt de la quantification des CEC dans la prise en charge thérapeutique des enfants souffrant d’HTAP secondaire à une cardiopathie congénitale, dont les formes irréversibles présentaient des taux élevés de CEC. Nous avons ainsi défini un nouveau marqueur non invasif à utilité diagnostique et pronostique. Les PEC sont des cellules rares dans le sang circulant, difficiles à expandre, et dont les essais de mobilisation médullaire se sont révélés insuffisants. L’hypothèse récente d’une réserve vasculaire des progéniteurs endothéliaux nous a conduits à étudier l’effet d’un processus d’ischémie locale sur la mobilisation de ces cellules chez des volontaires sains. Deux groupes d'âge ont été inclus afin d'évaluer l'impact du vieillissement sur la méthode de mobilisation étudiée. Malgré un effet de cette ischémie sur la dilatation endothéliale cette méthode n’a pas permis de mobiliser significativement les PEC issus de la paroi endothéliale, quel que soit l'âge des sujets. A l’inverse, l’hypoxie a eu un effet délétère sur les capacités angiogènes des ECFC. / The endothelium plays a key role in the development and the homeostasis of vascular functions. It is also well involved in pathological situations like cardiovascular diseases. Thanks to the description of circulating endothelial markers, non invasive study of the endothelium is now possible. Our group was particularly interested in circulating endothelial cells (CECs), the level of which reflects an endothelial activation or lesion, and to circulating endothelial progenitors cells (EPCs), markers of endothelial repair. EPC description by Asahara in 1997 in adult blood, involved in new blood vessel formation by vasculogenesis, offered new perspectives, specially for cell therapy in ischemic diseases. This work consisted in the development of methods to study these markers in different contexts. First, we explored the interest of these markers in the physiopathology of pulmonary arterial hypertension (PAH). Then we evaluated endothelial progenitors mobilization from the vascular wall by a local ischemia process in healthy volunteers, in the perspective of an autologous cell therapy product development. One part of this project was the implementation and optimization of the methods to study CEC and EPC. CEC were quantified by magnetic immunoseparation. This technique was developped in 1992 by F. Dignat-George's group and transferred in our laboratory. EPC were quantified by flow cytometry and cell culture. Two types of EPC are described in culture: the early EPC, which originate from monocyte lineage and which culture is standardized, and the « Endothelial Colony Forming Cells » (ECFC), the only cells presenting endothelial progenitor cell properties and which use as a cell therapy product can be considered. ECFC quantification by culture was optimized by assessment of the impact of diverse matrices and seeding concentrations of mononuclear cells isolated from whole blood, on ECFC commitment and their angiogenic properties. Endothelial dysfunction was described as a central element in the development of PAH, which diagnosis is based on the use of right heart catheterization. Due to the lack of noninvasive marker for this disease, CEC and circulating progenitors were quantified in two studies. One of them realized in adult patients showed a specific increase of CEC in PAH and not in post-embolic PH. CEC would then reflect the presence of specific endothelial lesions and not the clinical state of the patients. The other study demonstrated the interest of CEC quantification in the therapeutic care of children with PAH secondary to congenital heart disease, for whom patients in irreversible state had a higher level of CEC. We then defined a new noninvasive biomarker.that can be used for the diagnosis and prognosis of PAH. EPC are rare events in whole blood, difficult to expand and for which, mobilization protocols revealed insufficient. The recent hypothesis of a vascular reservoir for endothelial progenitor led us to study the effect of a local ischemia procedure on the mobilization of these cells in healthy volunteers. Two age groups were included to assess the impact of aging on this procedure. Despite a significant endothelial dilation with the local ischemia, no EPC were mobilized, whatever the age group. Ischemia even altered ECFC angiogenic properties.

La thérapie cellulaire est une envisagée pour traiter des pathologies cardiaques ou squelettiques basée sur la médecine régénérative. Les progéniteurs cellulaires classiquement utilisés (myoblastes ou cellules mésenchymateuses) n'ont démontré qu'une efficacité limitée. Dans ce contexte, notre laboratoire a identifié une nouvelle catégorie de progéniteurs, sur la base de leur activité enzymatique aldéhyde déshydrogénase (ALDH) mise en évidence par un substrat fluorescent, l'Aldéfluor, et en association avec le marqueur CD34. Les ALDH sont impliquées dans le métabolisme et la détoxification des aldéhydes, et constituent un nouveau marqueur des cellules souches. Ce travail de thèse a permis de mieux caractériser les progéniteurs myogéniques (ALDH+/CD34-) et non myogéniques (ALDH+/CD34+), dans différents contextes physiopathologiques. Leur présence dans différents muscles de primates humains ou non humains, leur persistance au cours du vieillissement naturel ou lors d'atteinte par la dystrophie musculaire de Duchenne (DMD) chez l'Homme et dans des modèles animaux, suggèrent une utilisation possible des cellules ALDH+/CD34- pour des développements thérapeutiques ultérieurs. L'étude phénotypique révèle que des marqueurs transmembranaires sont associés à des sous-populations de cellules ALDH myogéniques ou non myogéniques dont la comparaison permettra de proposer de meilleurs candidats de thérapie cellulaire. En parallèle, les caractérisations histologiques et cytologiques ont identifié des sous-populations exprimant des isoenzymes et les analyses d'expressiongénique réalisées ex vivo et en culture suggèrent que certaines sont impliquées dans l'homéostasie musculaires. / Cell therapy is a regenerative medicine strategy considered for the treatment of cardiac or skeletal muscle diseases. The cellular progenitors used to date (myoblasts or mesenchymal stem cells) provided mitigated success, thus mandating the identification and characterization of new categories of progenitors. Our laboratory has identified new populations of progenitors, based on their Aldehyde Deshydrogenase activity (ALDH) detectable using the fluorescent substrate Aldefluor, associated with the expression of the CD34 marker. ALDH are involved in metabolism and detoxification of aldehydes, they play important roles in cell survival and differentiation and are considered a new marker of stem cells. The present project allowed characterizing extensively the myogenic (ALDH+/CD34-) and non myogenic (ALDH+/CD34+) progenitors, in several physiopathological contexts and animal models. The presence of ALDH+/CD34- cells in distinct muscle groups in Human and non-human Primates, their persistence through natural ageing and despite the ongoing degenerative process observed in Duchenne muscular dystrophy in Human patients and animal models suggest their future use for therapeutic applications. The phenotypic characterization indicated that membrane markers are associated to myogenic or non myogenic sub-populations of ALDH cells. The comparison on their efficacies in vito and in vivo will allow proposing new candidates for cell therapy. In parallele, histological and cytological analysis identified cell populations expressing isoenzymes The analysis of gene expressions suggested that, at least, some of them are involved in muscle homeostasis in situ or in vitro.

Introduction.Le fibroblaste gingival (FG) est la cellule majoritaire de la gencive. Cette dernière fait face constamment aux agressions physico-chimiques, infectieuses et thermiques. L'une des caractéristiques de la gencive est sa réparation quasi-parfaite suite à une lésion ponctuelle. Ce n'est pas le cas pour d'autres tissus comme la paroi aortique. L'anévrisme aortique (AA) est un affaiblissement de la paroi aortique provoqué par une sécrétion exhaustive de métalloprotéases (MMPs) et en particulier de MMP-9. Il en résulte une dilatation de l'artère. Dans un modèle d'anévrisme de lapin, Durand et al (2012) avait montré que le FG pouvait ralentir, voire réparer un anévrisme. Dans notre étude, nous avons mis en place un modèle de coculture FG/AA d'origine humaine.Chez l'homme, la localisation de la pathologie peut être au niveau abdominal (Anévrisme Aortique Abdominale : AAA) ou thoracique (Anévrisme Aortique Thoracique : AAT). Etant donné que leur étiologie sont différentes, nous avons souhaité savoir s'il existait des différences selon les lésions. Cela nous permettrait en effet de mieux appréhender la prise en charge. Nous avons réalisé une étude comparative histo et physiopathologique entre les AAA et AAT. L'une des différences soulevée, est la présence d'un facteur infectieux au niveau des AAA. C'est un élément à prendre en compte pour une thérapie cellulaire et ainsi nous avons mis en culture des FG en présence de LPS, une endotoxine bactérienne.De plus pour approfondir notre travail sur l'utilisation du FG dans la thérapie cellulaire, nous avons initié une étude sur la plasticité de la sous-population souche des FG en étudiant, notamment leur orientation en cellules vasculaires (cellules endothéliales).Résultats/discussionLe FG, grâce à sa secrétion de TIMP-1, contribue à l'inhibition de la MMP-9 anévrismale. La sécrétion de MMP-9 est plus importante dans les lésions avec athérome (AAA) que celles sans athérome (AAT dans notre étude). Ceci est en corrélation avec la dégradation qui est plus importante dans les AAA que dans les AAT. La MMP-9 est une protéine sécrétée entre autre par les cellules inflammatoires. Une inflammation est présente dans les AAA et pas dans les lésions thoraciques. Ceci pourrait expliquer la différence de sécrétion de MMP-9 et donc de dégradation. Concernant l'origine de cette inflammation, nous avons recherché une cause infectieuse. Porphyromonas gingivalis (Pg) qui est une bactérie importante dans le développement de la parodontite (maladie inflammatoire des tissus de soutien de la dent) a été détectée dans les AAA. Une relation pathologique existerait entre la parodontite et l'AAA mais l'étude devrait être plus poussée pour connaître le mécanisme physiopathologique de ce phénomène. Toutefois, en ce qui concerne la thérapie cellulaire, le LPS qui est une endotoxine du Pg, n'affecte pas la capacité du FG à secréter du TIMP-1.En plus de la possibilité du FG à neutraliser la MMP-9 anévrismale, nous avons souhaité savoir si le FG avait des compétences de différentiation en cellule vasculaire. Un début d'exploration de la plasticité cellulaire de la souche multipotente de FG en cellule endothéliale, donnent des résultats préliminaires encourageants.Conclusion. Le FG pourrait être une cellule prometteuse pour une thérapie cellulaire de l'anévrisme aortique mais des explorations plus poussées sont encore nécessaires pour une telle application. / IntroductionGingival fibroblast (GF) is the main cell in gingiva which is constantly facing infectious, thermal and physico-chemical attacks. When a lesion occurs, the repair of gingiva is almost perfect. It is not the case for other tissues as the aortic wall. The aortic aneurysm (AA) is a pathologic expansion of aorta due to a weakening of the wall with an exhaustive secretion of metalloproteinases (MMPs) and particularly of MMP-9. In an aneurysm rabbit model, Durand and al (2012) have showed that GF could slow down or repair the aneurysm. In our study, we have established a co-culture model of human GF and human AA.For human, the location of the aortic disease may be at abdominal level (Abdominal Aortic Aneurysm: AAA) and thoracic level (Thoracic Aortic Aneurysm: TAA). Since the aetiologies are different, we wondered if histo and physiopathologic differences would existe between the both. It is impotant to know that for better supporting the disease. One of the difference between AAA and TAA is the presence of an infectious factor in AAA. This is an element to consider for cell therapy, so we studied the behavior of GF in presence of an endotoxin, the LPS.In addition, to further our work on the use of GF in cell therapy, we have initiated a study of the plasticity of the GF multipotente subpopulation including the differentiation into vascular cells (endothelial cell in particular).Results/DiscussionThanks to its TIMP-1 secretion, GF could contribute to the inhibition of MMP-9 activity in aneurysm. The secretion of MMP-9 in AA with atheroma (AAA) is highter than in TAA (without atheroma in our study). It is correlated to the degradation of AAA which is more important than the degradation of TAA. Inflammatory cells may secrete MMP-9. Inflammation is present in AAA and not in TAA. This, could explain the highter secretion of MMP-9 in abdominal lesion and also the degradation which is more important in AAA than in TAA. As for the origin of this inflammation, we researched an infectious factor. We isolated Porphyromonas gingivalis (Pg) in AAA, which might trigger or aggravate inflammation. This is an important bacterium in the development of periodontitis (inflammatory disease of the tissues supporting the tooth). A pathological relationship may exist between periodontitis and the AAA. The study should be further to know the pathophysiology of AAA related to Pg. But as regards the cell therapy, LPS, which is an endotoxin of Pg would not affect the secretion of TIMP-1 by the GF.In addition to its abilities to inhibate MMP-9 in aneurysm, we wondered if GF would be able to differentiate into vascular cell. An early exploration of GF multipotent subpopulation plasticity reveals a possible opportunity to go further in a the cell therapy.Conclusion.GF might be a promising cell for treating aortic aneurysm but further explorations are still necessary for its application.