A osteogênese imperfeita (OI) é caracterizada como uma desordem genética na qual uma osteopenia generalizada leva a baixa estatura, fragilidade óssea excessiva e deformidades ósseas graves. As células mesenquimais estromais multipotentes (CTMs) são precursores presentes na medula óssea adulta capazes de se diferenciar em osteoblastos, adipócitos e mioblastos que passaram a ter grande importância como fonte terapia celular. O objetivo do presente estudo foi analisar o perfil de expressão gênica durante a diferenciação osteogênica a partir de células mesenquimais estromais multipotentes da medula óssea obtidas de pacientes diagnosticados com Osteogênese Imperfeita e de indivíduos controle. Foram coletadas amostras de três indivíduos normais e cinco amostras de pacientes portadores de Osteogênese Imperfeita. As células mononucleares (CMN) foram isoladas para a obtenção de células mesenquimais que foram expandidas até a terceira passagem quando iniciou-se o estímulo para diferenciação osteogênica. Também foram realizadas análises para contagem de CFU-F e para quatro das cinco amostras de pacientes portadores de OI, o número de CFU-F observado foi inferior ao geralmente encontrado para amostras de doadores normais. Foram coletadas células para análises de imunofenotipagem celular por citometria de fluxo e o RNA foi extraído originando a amostra denominada T0. As garrafas restantes tiveram suas células estimuladas para diferenciação osteogênica. Após um dia em cultura com estímulo, mais uma garrafa teve o RNA de suas células extraído (T1), e o mesmo procedimento foi realizado nos dias 2 (T2), 7 (T7), 12 (T12), 17 (T17) e 21 (T21). Todas as amostras demonstraram possuir potencial de diferenciação in vitro em osteoblastos e adipócitos. A imunofenotipagem de células mesenquimais foi realizada e as amostras de todos os pacientes apresentaram perfil imunofenotípico compatível com trabalhos anteriores. Foram identificadas mutações nos genes COL1A1 e/ou COL1A2 responsáveis pelo desenvolvimento da doença para quatro dos cinco pacientes avaliados. Para o paciente portador de Osteogênese Imperfeita e Síndrome de Bruck a região codificadora do gene PLOD2 também foi seqüenciada, porém não foram encontradas mutações. A análise da expressão gênica foi realizada pela técnica de microarranjos e foram identificados vários genes com expressão diferencial. Alguns genes com importância fundamental na diferenciação osteoblástica apresentaram menor expressão nas amostras dos pacientes portadores de OI, sugerindo um menor comprometimento das CTMs desses pacientes com a linhagem osteogênica. Outros genes também tiveram sua expressão diferencial confirmada por PCR em Tempo Real. Foi observado um aumento na expressão de genes relacionados a adipócitos, sugerindo um aumento da diferenciação adipogênica em detrimento à diferenciação osteogênica. A expressão das variantes do gene PLOD2 mostrou-se diferencial entre amostras normais, de OI e do paciente portador de Síndrome de Bruck. Também foi evidenciada uma expressão diferencial do microRNA 29b, um microRNA com papel estabelecido durante a diferenciação osteogênica, sugerindo um mecanismo de regulação dependente da quantidade de RNAm do seu gene alvo, o COL1A1. / Osteogenesis imperfecta (OI) is characterized as a genetic disorder in which a generalized osteopenia leads to short stature, bone fragility and serious skeletal deformities. Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that can differentiate into osteoblasts, adipocytes and myoblasts that have been given great importance as a source cell therapy. The aim of this study was to analyze the gene expression profile during osteogenic differentiation from mesenchymal stem cells from bone marrow taken from patients diagnosed with Osteogenesis Imperfecta and control subjects. Samples were collected from three normal individuals and five samples from patients with Osteogenesis Imperfecta. Mononuclear cells (MON) were isolated to obtain mesenchymal cells that were expanded until third passage when the stimulus for osteogenic differentiation was induced. Analyses were also conducted to count the CFU-F and for four of the five samples from patients with OI, the number of CFU-F observed was lower than generally found for normal samples. Cells were collected for analysis of cell immunophenotyping by flow cytometry and RNA was extracted from the resulting sample called T0. Remaining cells were stimulated for osteogenic differentiation. After a day in culture with stimulation, cells from another bottle had their RNA extracted (T1), and the same procedure was performed on days 2 (T2), 7 (T7), 12 (T12), 17 (T17) and 21 (T21). All samples have shown potential of in vitro differentiation into osteoblasts and adipocytes. Immunophenotyping of mesenchymal cells was performed and samples of all patients had immunophenotypic profile consistent with previous works. We identified mutations in COL1A1 and / or COL1A2 responsible for developing the disease for four of five patients. For the patient with Osteogenesis Imperfecta and Bruck Syndrome, coding region of the gene PLOD2 was also sequenced, but no mutations were found. The gene expression analysis was performed by microarray and identified several genes with differential expression. Some genes of fundamental importance in osteoblast differentiation showed lower expression in samples from patients with OI, suggesting a minor involvement of MSCs of patients with osteogenic lineage. Other genes also confirmed their differential expression by Real Time PCR. We observed an increased expression of genes related to adipocytes, suggesting an increased adipogenic differentiation at the expense of osteogenic differentiation. The expression of PLOD2 gene variants proved to be different between normal samples, OI and the patient with Bruck Syndrome. There was also evidence of differential expression of 29b microRNA, with established role during osteogenic differentiation, suggesting a mechanism dependent regulation of mRNA abundance of its gene target, COL1A1.

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≤0,05). Foi observado que CTMs têm efeito repressivo sobre a proliferação e as expressões fenotípicas e genotípicas de OBs (P≤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≤0.05). It was observed that MSCs repressed proliferation, phenotypic and genotypic expressions of OBs (P≤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.

Orientador: Ângela Cristina Malheiros Luzo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T17:27:32Z (GMT). No. of bitstreams: 1 Manzini_BrunaMaria_M.pdf: 3170870 bytes, checksum: b4d5fa2d17ceaa12512b21b7a473e3bd (MD5) Previous issue date: 2014 / Resumo: Introdução: Cirrose e hepatite fulminante ocasionam diminuição severa da função hepática. Recentes avanços em terapia celular podem ser uma alternativa de tratamento. Objetivo desse estudo é analisar se MSCs obtidas de tecido adiposo (TA), sangue de cordão umbilical (SCU) e medula óssea (MO) se diferenciam em hepatócitos funcionais. Materiais e Métodos TA foi obtido de lipoaspiração, SCU do Banco Público de Cordão Umbilical e MO da Unidade de Transplante de Medula Óssea. Termo de Consentimento Livre e Esclarecido foi obtido de todos. TA foi submetido à digestão por colagenase, SCU e MO ao gradiente de Ficoll. As células obtidas foram cultivadas (DMEM meio baixa glicose, SFB) por 3 dias. As células aderentes foram tratadas com tripsina e cultivadas com meio acima e na quarta passagem, caracterizadas por citometria de fluxo, microscopia confocal e diferenciadas para linhagem mesoderrmal para confirmação da diferenciação em MSCs. Atividade da enzima Telomerase (TEA) e cariótipo foram realizados. Diferenciação para hepatócitos: 1,0 x103 MSCs foram semeadas em DMEM, HGF, bFGF, nicotinamida (7 dias), quando receberam meio de maturação (oncostatina, dexametasona e ITS) por 36 dias. A hepatogênese foi analisada por morfologia, funcionalidade (detecção da reserva de glicogênio, marcação PAS), absorção de Indocianina Verde e expressão gênica (PCR-TR dos genes albumina (Alb), Alpha- fetoproteina (AFP), tirosina amino-transferase (TAT) e glutamina sintetase (GS) nos dias 9, 18, 25 e 36). Transplantes: MSCs indiferenciadas e diferenciadas em hepatócitos derivadas de TA e MO infundidas em animais, sacrificados no 7° e 15° dias após transplante, para confirmação e avaliação da regeneração hepática por células humanas através de histopatológico. Resultados: MSCs foram obtidas das três fontes. Análises de telomerase e de cariótipo não demonstraram potencial tumorigênico. MSCs adquiriram morfologia cuboide semelhantes a hepatócitos. Análise funcional efetiva, demonstrando armazenamento de glicogênio e absorção de ICG. Expressão de albumina subiu progressivamente (3.7 no dia 9; 10 no dia 25). AFP inicialmente alta (4.5 dia 9; 5.0 no dia 18), diminuindo depois do dia 18 (3.0 dia 25 e 1.5 dia 36). GS aumentou 3 vezes mais durante processo de diferenciação. TAT foi maior que 6.6 (dia 25 e 36). Anatomopatológico dos transplantes demonstrou que apesar de todas as células conseguirem reverter a lesão, MSCs indiferenciadas foram mais efetivas. Conclusões:. Embora MSCs se diferenciaram em hepatócitos funcionais, devido demora em obter MSCs-SC relativa à diferenciação e expansão, talvez medula óssea e tecido adiposo sejam mais efetivos no processo de regeneração hepática utilizando terapia celular com MSCs / Abstract: Introduction: Liver cirrhosis and fulminant hepatites greatly diminishes liver function. Cell therapy could be a new tool for treatment. This study aimed analyze whether mesenchymal stem cells (MSCs) obtained from adipose tissue (AT), umbilical cord blood (UBC) and bone marrow (BM) could differentiate into functional hepatocytes. Material and Method: Adipocyte tissue (AT) was obtained from lipoaspiration, umbilical cord blood from Umbilical Cord Blood Bank and bone marrow (BM) from BM Transplantation Unit donors. All patients and donors signed free informed consent. AT was submitted to collagenase digestion, UCB and BM to Ficoll gradient. Cells were cultured (DMEM low glucose medium, SFB) for 3 days. Detached adherent cells at passage four were characterized as MSCs by flow cytometry, mesodermal lineages differentiation. Telomerase enzyme activity, karyotype analyses discharged tumorigenicity. Hepatocyte differentiation protocol was: DMEM, HGF, bFGF, nicotinamide for 7 days; addition of maturation medium (oncostatin, dexamethasone and ITS) during 36 days. Hepatogenesis was analyzed by morphology and gene expression (RT-PCR) of albumin, AF, TAT and GS genes on day 9, 18, 25 and 36. Functionality confirmed by glycogen storage detection, indocyanine green( ICG) absorption and animal experiment performed with transplantation of undifferentiated AT and BM MSCs and hepatocyte-like cells derived from AT and BM-MSCs into BALB/NOD mice submitted to fulminant hepatitis by CCl4 intraperitoneal injection. Animals were sacrificed at 7 and 15 days post transplantation to perform anatomohistopathological analyses. Results: All MSCs acquired cuboid form as hepatocyte-like cells, stored glycogen and absorbed ICG demonstrating functionality. Albumin expression gradually increased (3.7 on day 9 , 10 on 25 ). AFP expression was high at the beginning (4.5 day 9 ; 5.0 on day 18 ) , decreasing after day 18 ( 3.0 and 1.5 on day 25 day 36 ). GS expression increased 3 times during the differentiation process. TAT expression was greater than 6.6 on day 25 and 36. Animal experiments showed that undifferentiated MSCs (U-MSCs) and derivate hepatocyte ¿like cells regenerate the injured liver, but in different degrees. U-MSCs promoted better liver regeneration. Conclusion: MSCs differentiated into funcional hepatocytes, U-MSCs promoted better regeneration at animal experiments, and might be a useful tool for regenerative medicine in liver injuries improving cell therapy in this field / Mestrado / Fisiopatologia Cirúrgica / Mestra em Ciências

Orientador: Alexandre Leite Rodrigues de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T12:08:40Z (GMT). No. of bitstreams: 1 Spejo_AlineBarroso_M.pdf: 5889410 bytes, checksum: 655242e3ec4018faf3f968013fd8d36d (MD5) Previous issue date: 2013 / Resumo: Estudos recentes mostram resultados promissores no tratamento de lesões ao sistema nervoso (SN) através do implante de células-tronco, atribuindo essa melhora funcional à produção de fatores tróficos pelas células. Neste trabalho, o efeito neuroprotetor e neurorregenerativo de células-tronco mesenquimais (CTM) de ratos Lewis-EGFP foi investigado após o esmagamento das raízes motoras L4, L5 e L6. Cinco ratas fêmeas Lewis foram utilizadas em cada um dos seguintes grupos: G1 - esmagamento de raízes motoras; G2 - esmagamento de raízes motoras e injeção de DMEM (Meio de Eagle modificado pela Dubelco) na interface da substância branca e cinzenta (funículo lateral); G3 - esmagamento de raízes motoras e injeção de CTM (funículo lateral). Após 4 semanas, a sobrevivência neuronal foi estudada por coloração de Nissl, onde se observou, a partir da contagem neuronal, aumento da sobrevivência no grupo tratado com CTM. A técnica de imunoistoquímica foi utilizada para avaliar a expressão de sinaptofisina, sinapsina, VGLUT1 (Transportador vesicular de glutamato-1) e GAD65 (Glutamato descarboxilase 65KDa). A expressão de sinaptofisina e sinapsina na superfície dos motoneurônios lesionados mostrou uma menor redução de inputs em animais tratados com CTM, sugerindo uma possível redução no processo de eliminação sináptica. Para detectar possíveis mudanças no equilíbrio de inputs excitatórios / inibitórios em aposição ao corpo dos neurônios motores, os anticorpos VGLUT1 (marcador de terminais glutamatérgicos) e GAD 65 (marcador de terminais GABAérgicos) foram utilizados. A redução dos terminais glutamatérgicos foi semelhante em todos os grupos. Enquanto a redução de terminais GABAérgicos ocorreu em maior proporção em G1 e G2, em relação ao grupo tratado com CTM. Os dados indicam que CTM podem proteger os neurônios contra a excitotoxicidade, resultando em menor perda de células. Com sobrevida de 12 semanas após a lesão, a regeneração nervosa foi avaliada através da análise morfológica do nervo isquiático (área do nervo, número e morfometria de fibras mielínicas) e da recuperação da função motora (walking track test). O grupo tratado com DMEM apresentou nervo com menor área e menor número de fibras mielínicas do que o tratado com CTM, porém, o grupo tratado com células obteve resultados semelhantes ao grupo que sofreu apenas esmagamento. A morfometria revelou fibras com menor mielinização nos três grupos lesionados, em comparação ao nervo contralateral à lesão, porém, para os lados ipsilaterais não houve diferença entre os tratamentos. A função motora apresentou-se melhor no grupo tratado com CTM, quando comparada com o tratado com DMEM, mas não em relação ao grupo que sofreu apenas esmagamento. O efeito das CTM na função motora foi agudo, mostrando eficiência de 4 a 6 semanas após a lesão. Assim, as CTM mostraram efeito neuroprotetor e contribuíram para a regeneração, porém a forma de administração dessas células, através de injeção diretamente na medula, apesar de resultar num maior número de células enxertadas, contribuindo com a sobrevivência dos neurônios, mostrou-se um problema quando avaliada a regeneração e função motora dos animais, indicando a necessidade de desenvolvimento de outras formas de administração / Abstract: Recent studies have shown promising results in the treatment of nervous system injuries through stem cells implantation, attributing this functional improvement to the production of trophic factors by these cells. In this study, the neuroprotective and neurorregenerative effects of mesenchymal stem cells (MSC) from Lewis-EGFP mice was investigated after crushing the motor roots L4, L5 and L6. Five female rats Lewis were used in each of the following groups: G1 - motor roots crushing; G2 - motor roots crushing and DMEM (Dulbeco's Modified Eagle Medium) injection in the white/gray matter interface; G3 - motor roots crushing and MSC injection in the white/gray matter interface. At 4 weeks after injury, neuronal survival was evaluated by Nissl staining, and revealed, by the neuronal count, increased survival in the group treated with MSC. The technique of immunohistochemistry was used to evaluate the expression of synaptophysin, synapsin, VGLUT1 (Vesicular Glutamate Transporter-1) and GAD65 (Glutamate decarboxylase 65). The expression of synaptophysin and synapsin on the surface of lesioned motoneurons, showed a smaller decrease of inputs in animals treated with MSC, suggesting a possible reduction in synaptic elimination process. To detect possible changes in the balance of excitatory / inhibitory inputs reaching the cell body of the motoneurons, antibodies for VGLUT1 (marker of glutamatergic terminals) and GAD 65 (marker of GABAergic terminals) were used. The reduction of glutamatergic terminals was similar in all groups. While the reduction of GABAergic terminals was in greater extent in G1 and G2 with respect to the group treated with MSC. The data indicate that MSC can protect neurons against excitotoxicity, resulting in decreased cell loss. With survival of 12 weeks after the injury, nerve regeneration was assessed by morphological analysis of the sciatic nerve (nerve size, number and morphology of myelinated fibers) and motor function recovery (walking track test). The group treated with DMEM showed nerve with smaller area and fewer myelinated fibers than treated with MSC, however, the group treated with cells was not better than the group that only suffered crushing. Morphometry revealed fibers with less myelination in the three injured groups, compared to the contralateral side, but there was no difference between treatments. Motor function appeared better in MSC-treated group in comparison to the DMEM-treated, but not in relation to the group which only suffered crushing. The effect of MSC in motor function was acute, demonstrating efficiency between 4 to 6 weeks after injury. Thus, the MSC shown to be neuroprotective and contributed to regeneration, but the form of administration of such cells, by direct injection into spinal cord, has to be improved or substituted by another method / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

O osteossarcoma é um tumor ósseo maligno, de maior ocorrência em cães, possui rápido crescimento e alto potencial metastático. Assim, o cão é um modelo útil para o estudo da doença em humanos, tendo em vista as semelhanças clínicas e histopatológicas que ocorrem em ambas às espécies. Atualmente, os estudos a respeito de células-tronco são promissores considerando seu alto potencial terapêutico. Entretanto, ainda prevalecem muitas dúvidas referentes ao tratamento de tumores utilizando a terapia celular. Este tema é pouco conhecido e estudado, por isso, o objetivo deste trabalho foi avaliar a interação entre células-tronco obtidas da polpa dentária canina com as células derivadas osteossarcoma canino. Foram realizados cocultivos celulares das células derivadas de polpa dentária canina, osteossarcoma canino e derivadas de osso normal canino. Analisou-se os aspectos morfológicos das células cocultivadas e controle, assim como a atividade proliferativa, a morte celular, o potencial elétrico mitocondrial e a expressão gênica. A partir dos resultados obtidos, concluiu-se que a interação entre a célula-tronco da polpa dentária canina imatura e as células de osteosarcoma canino não apresentam alterações morfológicas. Entretanto, as células-tronco derivadas da polpa dentária canina e de osso fetal canino sadio parecem servir de suporte para o crescimento tumoral. Além disso, a cocultura celular, em todos os grupos testados, promove alterações na expressão gênica e proteica. / Osteosarcoma is a malignant bone tumor most frequent in dogs. It has fast growth and high metastatic potential. Thus, the dog is an useful model for the study of human disease, due to the clinical and histological similarities found in both species. Currently, studies about stem cells are promising considering its high therapeutic potential. However, many doubts still exist regarding the treatment of tumors using cell therapy. This theme is little known and studied. Therefore, the aim of this study was to evaluate the interaction between stem cells obtained from canine immature dental pulpstem cells with osteosarcoma cells derived from dogs. Cellular coculture were performed using cells derived from canine dental pulp, canine osteosarcoma and canine normal bone. The morphological aspects of cocultured cells and control were analyzed, as well as proliferative activity, cell death, the mitochondrial membrane electric potential and gene expression. In summary, it was concluded that the interaction between stem cells from canine immature dental pulp and canine osteosarcoma cells did not show morphological changes. However, stem cells derived from canine dental pulp and healthy canine fetal bone serve to support tumor growth. Furthermore, the cell coculture in all groups tested, causes changes in gene and protein expression.

Les glioblastomes (GBM) sont des tumeurs astrocytaires au pronostic défavorable. L’échec des thérapies actuelles (chimio et radiothérapies) est principalement lié à la résistance des cellules souches cancéreuses (CSCs). Ces cellules ont besoin de communiquer en permanence avec leur microenvironnement pour leur survie et pour maintenir une niche favorable à leur développement. Le transfert de matériel entre les CSC, les cellules tumorales et le microenvironnement contribue à l’échappement thérapeutique. Des travaux récents révèlent l’importance des récepteurs aux neurotrophines TrkB et TrkC dans la survie et la croissance des CSC de GBM. Nos travaux préliminaires dans le cancer bronchique démontrent que les récepteurs aux neurotrophines sont transférés aux cellules du microenvironnement via les exosomes afin de les contrôler. Cependant, le mécanisme de diffusion de récepteurs oncogéniques à partir de CSC n’a jamais été étudié. Notre objectif principal était donc de déterminer l’implication des récepteurs des neurotrophines dans le transfert du phénotype agressif des CSC vers les cellules du microenvironnement afin de favoriser la résistance thérapeutique du glioblastome. Nos résultats ont permis d’établir un lien entre le stade de différenciation des cellules tumorales, l’expression des neurotrophines et leur interaction avec le microenvironnement tumoral via les exosomes. Le transfert de TrkB au sein des exosomes joue un rôle clé dans la progression tumorale du GBM et dans l’agressivité cellulaire. Néanmoins, le transfert des récepteurs aux neurotrophines via les exosomes pourrait également être impliqué dans les mécanismes de radiorésistance. Des études menées sur des cellules de GBM humain irradiées et traitées par des exosomes démontrent l’implication de ces derniers dans l’échappement thérapeutique. Parmi les cellules du microenvironnement ciblées par les exosomes, les CSM sont celles qui ont été les moins étudiées bien qu’elles possèdent un tropisme spécifique pour le GBM. Nos travaux démontrent que les exosomes de GBM modifient le phénotype des CSM et augmentent leurs capacités prolifératives et migratoires. La fonction exacte du transfert des récepteurs des neurotrophines devra être analysée dans ces différents modèles afin de préciser son importance dans la physiopathologie du glioblastome et sa progression. L’expression des récepteurs aux neurotrophines dans ces exosomes permet d’envisager leur utilisation en tant que biomarqueurs diagnostiques et/ou pronostiques dans le GBM. Mots clés : Glioblastomes, cellules souches cancéreuses, neurotrophines, TrkB, radiothérapie, cellules souches mésenchymateuses, exosomes. / Glioblastoma are tumors derived from astrocytes with a dark prognosis. Current therapies fail to inhibit relapses due to radioresistant properties of cancer stem cells (CSC). Communication between CSC and their microenvironment is required for maintain “stem cells niche” and cell survival . The transfer of materials between CSC, tumor cells and microenvironment contributes to therapeutic resistance. In glioma, recent studies reveal the major role of TrkB and TrkC in survival of CSC. Our previous work, in lung cancer, have shown that neurotrophin receptors exhibits a control on microenvironment cells and angiogenesis through exosome transfer. However, similar mechanism of oncogenic receptor transfer from CSC has never been studied. Our main goal was to determine the involvement of neurotrophin receptors in the transfer of biological aggressiveness to microenvironment cells in order to promote therapeutic resistance in glioblastoma. Our findings suggest a relationship between cell differentiation status, expression of neurotrophin receptors and their interaction with the microenvironment through exosomes. TrkB-containing exosomes play a key role in the control of glioblastoma progression and cell aggressiveness. Mechanisms of radioresistance might also be dependent of the transfer of neurotrophin receptors through exosomes. Indeed, our results on irradiated human GBM cells and treated by exosomes demonstrate the involvement of exosome in radioresistance mechanisms. Although mesenchymal stem cells (MSCs) are considered as stromal components of glioblastoma, their communication with CSC, particularly through exosomes, remain largely undefined. Our results show that GBM-derived exosomes modify the phenotype of MSCs and increase their proliferative and migratory abilities. The putative function of neurotrophin receptors transfer should be analyzed in these models to determine their prime role in glioblastoma pathogenesis and progression. This finding suggest that the neurotrophin receptor expression in exosomes could be used as diagnostis and prognosis biomarkers of GBM.

Le myélome multiple (MM) est une hémopathie maligne de la lignée B qui se manifeste par <p>une accumulation de plasmocytes monoclonaux dans la moelle osseuse. Le MM se caractérise par <p>la nature inéluctable de ses rechutes de plus en plus réfractaires au traitement. Au sein de la moelle <p>osseuse, les cellules malignes interagissent avec leur microenvironnement notamment composé de <p>cellules stromales mésenchymateuses (CSM). Les CSM apportent un soutien aux cellules <p>plasmocytaires (PC) malignes via des interactions cellulaires directes (VLA-4, VCAM-1, CD44,…) <p>ou par la production de cytokines (IL-6, SDF1, VEGF,…). De ces interactions résulte l’apparition <p>d’altérations constitutives au sein des MM-CSM telles qu’une production réduite de dépôts de <p>calcium et une augmentation de la sécrétion de nombreux facteurs (IL-6, GDF-15, etc.). <p>L’objectif de ce travail est de mieux caractériser les anomalies constitutives (génomiques, <p>prolifératives, immunomodulatrices, etc.) des MM-CSM et d'évaluer l’impact des traitements reçus <p>par les patients sur ces anomalies. Ces analyses permettront de mieux définir le rôle des CSM dans <p>la physiopathologie et la progression de la maladie afin de mettre en évidence d’éventuelles cibles <p>thérapeutiques.<p>Compte tenu de l’altération de nombreux acteurs du cycle cellulaire mise en évidence par <p>notre analyse du profil d’expression génique des MM-CSM après comparaison avec celui des CSM <p>de donneurs sains (DS-CSM), nous avons décidé d’étudier leurs capacités prolifératives. Nos <p>observations démontrent que a) les MM-CSM sont caractérisées par une réduction de leur <p>clonogénicité et par un temps de doublement plus important par rapport aux DS-CSM b) l’entrée <p>précoce des MM-CSM en sénescence est confirmée par l'augmentation drastique du nombre de <p>cellules exprimant la « senescence-associated β-galactosidase », par l’augmentation de la taille <p>cellulaire, par l’expression spécifique de membres du « senescence-associated secretory profile » <p>(SASP) et par la surexpression de TP53 et TP21, deux facteurs importants du cycle cellulaire et de <p>la sénescence c) l’ostéoblastogenèse des MM-CSM est altérée en raison de ce processus de <p>sénescence, à savoir, une réduction de la production de calcium et une hausse précoce de l’activité<p>de la phosphatase alcaline des MM-CSM par rapport aux DS-CSM d) l’activité de support à <p>l’hématopoïèse des MM-CSM est augmentée (« increased Blast-Colony Forming Cells and LongTerm Culture Initiating Cells »). <p>Dans la seconde partie de ce travail, on s’est intéressée plus particulièrement aux capacités <p>immunomodulatrices des MM-CSM. Nous avons observé, par réaction mixte lymphocytaire et <p>stimulation mitogénique (PHA/IL-2), a) une réduction de l’inhibition de la prolifération des <p>lymphocytes T activés en présence de MM-CSM par rapport aux DS-CSM b) une inversion du ratio <p>Th17/Treg lorsque des lymphocytes T activés sont cultivés en présence de MM-CSM par rapport <p>aux DS-CSM ;les mécanismes potentiellement impliqués dans ces altérations ont été investigués c)<p>les MM-CSM présentaient une expression anormale du CD40/40L, VCAM1, ICAM-1, LFA-3 <p>HLA-DR et HLA-ABC et des taux d’IL-6 et d’IL-10 altérés ont été mesuré dans le milieu de <p>culture lorsque les lymphocytes T activés étaient cultivés en présence de MM-CSM par rapport aux <p>DS-CSM. <p>Nos résultats suggèrent que les MM-CSM entrent précocement en sénescence avec de <p>profondes altérations de leurs propriétés biologiques et notamment leur capacités <p>d'immunomodulation et de différenciation ostéogénique. Nous concluons que l’entrée en <p>sénescence des MM-CSM pourrait être à l’origine d’une altération du microenvironnement tumoral <p>qui favoriserait rechutes et résistances aux traitements. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Extracellular nucleotides act as local intercellular messengers. In response to various stimuli, nucleotides can be released from the cytosol of damaged cells, exocytosis vesicles and efflux through membrane channel. In the extracellular fluids, nucleotides are rapidely degraded by ecto-nucleotidases, such as CD39,CD39L1 and CD73. Extracellular nucleotides activate the P2 receptor family .This family of receptors can be divided into 2 groups: P2X1-7R ( ionotropic receptors) and P2YR ( G protein-coupled receptors). Nowadays, there are eight accepted human P2Y receptors: P2Y1,2,4,6,11,12,13,14.<p>To determine the physiological roles of P2Y receptor family, our laboratory generated different strains of P2Y knockout mice (P2Y4 ,6 and 13). In collaboration with A. Gartland ,I. And Arnett TR Orriss ( Sheffield and London University) ,it has been observed that the P2Y13R-/- mice exhibit an impaired bone tissue metabolism that leads to a reduction in the volume of the femoral trabecular bone and the number of trabeculae. This phenotype is correlated with the decrease in the number of osteoblasts at the endo-cortical bone surface .<p>We therefore examined whether P2Y13 R activation was involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). In the first part of our work we have shown that :<p>Induction of the MSCs differentiation is associated with the release of ATP and its conversion to ADP (agonist of the P2Y13R). ATP release probably involves the pannexine1 while the conversion of ATP into ADP is probably due to the activity of the ecto-nucleotidase CD39L1.<p>ADP stimulation of P2Y13 R+/+ (but not P2Y13 R-/-) adherent bone marrow stromal cells (BMSC) increased significantly the formation of alkaline phosphatase-colony forming units (CFU-ALP), as well as the expression of osteoblastic genes such as Osterix involved in the maturation of pre-osteoblasts into osteoblasts. The number of CFU-ALP obtained from P2Y13 R-/- BMSC and the level of osteoblastic gene expression after osteogenic stimulation were also strongly reduced compared to those obtained in wild-type cell cultures. In contrast, when P2Y13 R-/- BMSCs were incubated in an adipogenic medium, the number of adipocytes generated and the level of adipogenic gene expression (PPARγ2 and Adipsin) were higher than those obtained in P2Y13 R+/+ MSC. We also observed a significant increase of the number of bone marrow adipocytes in tibia of P2Y13 R-/- mice. <p>The P2ry12 gene deletion (also activated by ADP) also affects the ability of BMSCs to differentiate into osteoblasts but stimulates adipogenic differentiation.<p>In a second part of our work ,we have shown that the pro- osteogenic action of P2Y13R is indirect. Indeed ,this receptor is not expressed by MSCs but by adherent myeloid cells present in the bone marrow cell cultures (characterized by the expression of CD11b and CD45 markers) .In addition, we observed that following receptor activation by ADP ,these myeloid cells produce BMP2 factor.<p>We therefor propose that the stimulation of MSCs differentiation induces CD45- adherent cells to release ATP that is converted into ADP, most probably by the up-regulated CD39L1 ectonucleotidase. This ADP stimulates P2Y12R and P2Y13R expressed by CD45+/CD11b+ myeloid cells leading to the release of the osteogenic factor BMP2. This cytokine favours the maturation of pre-osteoblasts into osteoblasts and concomitantly inhibits the maturation of pre-adipocytes.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

La pulpe dentaire est sujette à des lésions sévères faisant suite à une carie dentaire ou à un traumatisme. La thérapeutique conventionnelle préconisée alors est le traitement endodontique, qui consiste en l’exérèse de la totalité de la pulpe dentaire et le comblement de l’espace pulpaire par un matériau inerte. Ce traitement induit une fragilisation de la dent et une plus grande susceptibilité aux infections. Au cours de ce travail, nous avons mis au point une solution alternative, en proposant le remplacement de la pulpe dentaire lésée par une « pulpe équivalente » constituée de cellules souches mésenchymateuses de la pulpe ensemencées dans une matrice de collagène. Nous avons testé ce substitut pulpaire au travers d’un modèle de pulpotomie de la molaire chez le rat, à savoir l’exérèse de la totalité du parenchyme de la chambre pulpaire et conservation du réseau vasculaire radiculaire, où nous avons implanté des « pulpes équivalentes ». Notre objectif étant notamment de déterminer le devenir des cellules souches pulpaires implantées dans la dent grâce à l’imagerie nucléaire, dans ce contexte de développement d’une thérapie cellulaire. Les cellules ont été marquées à l’111Indium-oxine préalablement à leur implantation. Nous avons montré que le marquage n'avait pas d'incidence sur la viabilité et la prolifération des cellules pulpaires. Le suivi du signal s’est fait par tomographie d'émission monophotonique, couplée à un scanner spécifique du petit animal (NanoSPECT/CT, Bioscan), hebdomadairement pendant 3 semaines. Nous avons mis en évidence que l'intensité du signal SPECT était directement liée à l'intégrité des cellules, puisque que les matrices implantées avec des cellules marquées puis lysées par choc isotonique présentaient une diminution rapide de l’intensité du marquage. Grâce à la sensibilité de la méthode d’imagerie choisie, nous avons montré l’absence de diffusion majeure des cellules dans la circulation sanguine à partir du site d'implantation, ce qui pourrait constituer un risque de minéralisation ectopique lié à l’implantation de cellules souches mésenchymateuses. Par ailleurs, l’étude par histologie des processus de réparation et régénération de la pulpe dans les dents de rat a mis en évidence une prolifération abondante de cellules de type fibroblastique au sein des matrices, ainsi que la présence de nombreux vaisseaux et de nerfs dans la matrice cellularisée et à proximité. Ces résultats, non observés dans les matrices implantées avec des cellules lysées, suggéraient donc une fonctionnalité du tissu reconstruit et suggéraient que les cellules pulpaires implantées favorisaient une néovascularisation rapide de la pulpe équivalente, vraisemblablement en induisant un recrutement de cellules endothéliales à partir du réseau vasculaire radiculaire résiduel. / The dental pulp is prone to severe injuries following a tooth decay or trauma. Conventional recommended therapy is the endodontic treatment, which consists in the removal of all of the dental pulp and filling of the pulp space with an inert material. This treatment leads to a weakening of the tooth and a greater susceptibility to infection.In this work, we have developed an alternative solution, proposing the replacement of the injuried dental pulp by an " pulp equivalent " consisting of mesenchymal stem cells from the pulp seeded in a collagen matrix . We tested this pulp substitut through a model of the molar pulpotomy in rats, ie. the removal of the entire parenchyma of the pulp chamber and preservation of the root vascular network and implantation of the pulp equivalent. Our aim was to determine the fate of pulp stem cells implanted in the tooth by nuclear imaging in the context of developing a cell therapy. The cells were labeled with 111Indium - oxine prior to their implantation. We have shown that the labelling had no effect on the viability and proliferation of pulp cells. The signal tracking was done by single photon emission tomography , coupled with a specific small animal scanner ( NanoSPECT / CT , Bioscan ) weekly for 3 weeks. We demonstrated that the intensity of SPECT signal was directly related to the integrity of the cells, since the lysed labeled cells by isotonic shock showed a rapid decrease in the intensity of labeling . Due to the sensitivity of the chosen imaging method , we have shown the absence of major diffusion cells into the bloodstream from the site of implantation, which could result in a risk of ectopic mineralization related to the implementation of mesenchymal stem cells.Furthermore, the study by histology repair processes and regeneration of the pulp in teeth rat showed abundant proliferation of fibroblast-like cells within the matrix , and the presence of numerous vessels and nerves in matrix cellularized. These results , not observed in the matrices implanted with lysed cells, thus suggesting a feature of the reconstructed tissue and suggested that the pulp cells implanted favored a rapid neovascularization equivalent pulp, presumably by inducing the recruitment of endothelial cells from the residual root vascular network.

Le sujet de ce travail de thèse a concerné l'analyse de l’immunogénicité de progéniteurs cardiaques issus de cellules souches embryonnaires humaines. Le but a été double. D’une part, comprendre les mécanismes cellulaires et moléculaires qui sous-tendent cette immunogénicité et, d’autre part, mettre en place des stratégies d’immuno-intervention permettant de la surmonter.Le travail a comporté deux volets, l’un in vitro et l’autre in vivo utilisant des modèles expérimentaux murins. Les analyses in vitro, ont utilisé une méthode de culture lymphocytaire mixte où des progéniteurs cardiaques humains ont été mis en culture avec des lymphocytes allogéniques. Les résultats ont montré que les progéniteurs cardiaques sont effectivement immunogènes et que la réponse immunitaire qu’ils suscitent peut-être modulée efficacement par des cellules mésenchymateuses dérivées du tissu adipeux. De plus, nous avons confirmé l’expression des molécules d’histocompatibilité de classe I à la surface de progéniteurs cardiaques, une expression qui semble modulée au cours de la culture.Les modèles in vivo que nous avons utilisés ont consisté en l’implantation de corps embryoïdes et des progéniteurs cardiaques de souris dans un contexte allogénique. Divers sites d’implantation ont été utilisés (myocarde, capsule rénale, muscle gastrocnemius) chez des souris immunocompétentes. Les résultats ont montré qu’à la fois les corps embryoïdes et les progéniteurs cardiaques sont rejetés chez les receveurs immunocompétents non traités, avec une cinétique différente en fonction du site d’implantation. Par ailleurs, l’utilisation d’un traitement par anticorps anti-CD3, appliqué à différents temps suivant l’implantation nous a permis de prolonger la survie des cellules implantées en induisant, en fonction de la fenêtre thérapeutique, soit une immunosuppression soit une tolérance immunitaire. / The present work concerned the analysis of the immunogenicity of cardiac progenitors derived from human embryonic stem cells. Our aim was to understand the cellular and molecular mechanisms which underlie this immunogenicity and to surmount it by setting up strategies of immune-intervention. The study consisted in two major components, one in vitro and the other in vivo using experimental mice models. The in vitro analyses were assessed by the mixed leukocyte reaction method, where human cardiac progenitors were cultured with allogeneic lymphocytes. The results showed that the cardiac progenitors are indeed immunogenic and that the immune response that they induce could be modulated by mesenchymal stromal cells derived from adipose tissue. Moreover, we confirmed the expression of class I histocompatibility molecules on the surface of cardiac progenitors, an expression which seems modulated during the culture. The in vivo models that we used consisted of the grafting of embryoïdes bodies and cardiac progenitors derived from mouse embryonic stem cells in an allogeneic context. Cells were grafted in different sites of immunocompetent mice (myocardium, renal capsule, muscle gastrocnemius). The results showed highlighted that at the same time both embryoïdes bodies and cardiac progenitors are rejected among untreated immunocompetents hosts, whereas their survival is extended by anti-CD3 treatments, In addition, anti-CD3 treatment prolongs the survival of grafted cells, either by immunosuppression or by inducing immune tolerance according to the timing when it is applied.