INTRODUÇÃO: O objetivo deste estudo foi de avaliar a eficácia de um centrifugado osteogênico de medula óssea para estimular a consolidação de osteotomias da fíbula em coelhos. MÉTODOS: Este estudo experimental envolveu a utilização de dez coelhos machos adultos da raça Nova Zelândia albino. Realizou-se uma osteotomia transversa médio-diafisária da fíbula direita, seguida da adição local de uma esponja de colágeno absorvível embebida em um centrifugado osteogênico, obtido pela centrifugação de aspirado de medula óssea do osso ilíaco ipsilateral. A fíbula esquerda foi utilizada como controle, sendo feita a mesma osteotomia, porém neste caso adicionando-se somente a esponja de colágeno absorvível. O centrifugado de medula óssea elaborado em laboratório foi submetido à contagem do número de células nucleadas e a teste de viabilidade celular antes de ser administrada no local das osteotomias. Após quatro semanas os animais foram sacrificados para estudo dos calos ósseos formados. Os critérios de avaliação foram a mensuração da densidade mineral utilizando-se a densitometria óssea com DEXA, do volume do calo com tomografia computadorizada multi-slice e dos tecidos formados por meio de histomorfometria. RESULTADOS: O método utilizado para a centrifugação dos aspirados de medula óssea resultou em uma concentração média de células nucleadas três vezes maior que o número destas células nos aspirados originais, sem destruição celular significativa. A utilização deste centrifugado osteogênico resultou em um aumento médio na densidade mineral óssea dos calos de 40,3% e da quantidade relativa de tecido ósseo de 9,4%, sem aumento significativo nas quantidades relativas de cartilagem ou fibrose. Não houve aumento significativo no volume dos calos ósseos. CONCLUSÃO: A administração de centrifugado osteogênico de medula óssea utilizado neste estudo favoreceu a consolidação óssea de osteotomias experimentais em coelhos, observando-se uma melhora qualitativa do calo ósseo. / INTRODUTION: The purpose of this study was to evaluate the efficacy of a centrifuged osteogenic bone-marrow aspirate to stimulate rabbit fibular osteotomies healing. METHOD: Ten white New Zeeland rabbits were used. A transverse middle-diaphysis fibular osteotomy was performed at the right fibula, where a collagen absorbable sponge embedded in the osteogenic centrifuged bone marrow aspirate was inserted. The left fibula was used as the control group, where the collagen absorbable sponge was inserted without the osteogenic centrifuged aspirate. The centrifuged bone-marrow aspirate was arranged at the laboratory and submitted to nuclear cell count and cell viability test. The rabbits were killed at four weeks after surgery to evaluate bone callus formation. The results analysis was performed with DEXA bone densitometry to evaluate callus mineral mass, multislice computer tomography to evaluate callus volume and histomorphometry to evaluate the relative rate of tissue formation. RESULTS: The bone-marrow centrifugation technique increased the number of nucleated cells by three compared with the number of that cells in the original bone-marrow aspirates, without significant nucleated cell dead. The apply of centrifuged osteogenic bone-marrow aspirate resulted in an increased callus bone mineral mass by 40,3%, and increased relative rate of bone tissue formation by 9,4%, without increase the relative rate of cartilage or fibrous tissue. There was not increased callus volume. CONCLUSION: This study shows that the centrifuged osteogenic bone-marrow aspirate was able to improve the healing of experimental fibular osteotomies in rabbits by qualitative improve of bone callus.

A síndrome de Mayer-Rokitansky-Kuster-Hauser (MRKH) caracteriza-se pela aplasia congênita dos ductos Mullerianos. Devido a algumas características peculiares, as células-tronco mesenquimais estão sendo vistas como uma nova alternativa de tratamento em pacientes acometidos pela síndrome de MRKH. Considerando que alguns biomateriais servem como suporte estrutural e interferem positivamente na regeneração tecidual, a associação da linhagem celular de mucosa vaginal HMV-II e das MSC derivadas de tecido adiposo humano com biomateriais apresenta uma nova possibilidade na criação de neovagina. Nesta perspectiva, foram cultivadas células HMV-II com diferentes biomateriais (Membracel, Biofilme, Cellprene, PLGA PI quimicamente modificado) a fim de selecionar o melhor material alternativo. Ambas as células, associadas ao biomaterial selecionado, foram submetidas à análise morfológica, coloração ácido periódico-Schiff (PAS), expressão de marcadores epiteliais específicos por imunofluorescência e microcopia eletrônica de varredura (MEV). As células que interagiram com o biomaterial apresentaram marcadores epiteliais específicos e características morfológicas epiteliais. Estes resultados indicam que a interação do biomaterial com ambas as células testadas tem potencial capacidade para uma epitelização eficiente da neovagina. O crescimento das MSC com o biomaterial selecionado para implantação subsequente em pacientes com síndrome de MRKH pode representar uma alternativa válida e promissora para a reconstrução vaginal. / The Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome is characterized by congenital aplasia of the Mullerian ducts. Because mesenchymal stem cells (MSCs) secrete paracrine factors, they have been seen as a new treatment option for several diseases. Considering that some biomaterials can be used as scaffolds and interfere positively in tissue regeneration, the association of human vaginal mucosa (HMV-II) cell line and MSCs with biomaterials appears as a new option for the creation of neovagina. In this study we cultured HMV-II cells with different biomaterials (Membracel, Biofilm, Cellprene, chemically modified PLGA PI) to select the best alternative material. For that both cells were cultured with the selected biomaterial and evaluated by morphological analysis, periodic acid-Schiff (PAS) staining, expression of epithelial markers by immunofluorescence and scanning electron microscopy (SEM). The analysis of the in vitro cell-biomaterial interactions showed specific epithelial markers and epithelial morphological features for both cells. These results indicate that the interaction of the biomaterial with the two tested cells has the potential capacity for an efficient epithelialization of the neovagina. Therefore, growth of MSCs with the selected biomaterial for subsequent implantation in patients with MRKH syndrome may represent a valid and promising alternative for vaginal reconstruction.

Hui, Fan Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 153-181). / Abstract also in Chinese. / Acknowledgements --- p.ii / Abstract --- p.iii / Abbreviations --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xv / List of Tables --- p.xv / List of Figures --- p.xvi / Major Conference Presentations --- p.xix / Publications in Preparation --- p.xxii / Chapter Chapter 1 --- Study Background --- p.1 / Chapter 1. --- Introduction --- p.2 / Chapter 1.1. --- Spinal Deformities --- p.2 / Chapter 1.1.1. --- Treatment --- p.2 / Chapter 1.2. --- Spinal fusion --- p.4 / Chapter 1.2.1. --- Gold Standard of Spinal Fusion --- p.4 / Chapter 1.2.2. --- Decortication in Spinal Fusion --- p.4 / Chapter 1.2.3. --- Autograft in Spinal Fusion --- p.4 / Chapter 1.2.4. --- Local Factors Influencing Spinal Fusion --- p.5 / Chapter 1.2.5. --- Ultimate Goals of Spinal Fusion --- p.7 / Chapter 1.2.6. --- Limitations of Spinal fusion --- p.7 / Chapter 1.3. --- Alternatives of Different Components for Enhancing Spinal Fusion / Chapter 1.3.1. --- Bone Graft Substitute --- p.9 / Chapter 1.3.2. --- Bioactive Factors --- p.15 / Chapter 1.4. --- Limitations of the Alternative Methods in Spinal Fusion Enhancement --- p.19 / Chapter 1.4.1. --- BMPs --- p.19 / Chapter 1.4.2. --- Gene Therapy --- p.20 / Chapter 1.4.3. --- Biophysical Stimulation --- p.20 / Chapter 1.5. --- Recent Methods in Enhancing Spinal Fusion --- p.21 / Chapter 1.5.1. --- Low Intensity Pulsed Ultrasound --- p.21 / Chapter 1.5.2. --- Mesenchymal Stem Cells in Spinal Fusion --- p.24 / Chapter 1.6. --- Conclusion --- p.26 / Chapter Chapter 2 --- "Hypothesis, Objectives and Plan of Study" --- p.29 / Chapter 2. --- "Hypothesis, Objectives and Plan of Study" --- p.30 / Chapter 2.1 --- Study Hypothesis --- p.31 / Chapter 2.2 --- Study Objectives --- p.31 / Chapter 2.3 --- Plan of Study --- p.32 / Chapter 2.3.1 --- For First Objective --- p.32 / Chapter 2.3.2 --- For Second Objective --- p.32 / Chapter 2.3.3 --- For Third Objective --- p.33 / Chapter Chapter 3 --- In vitro Study of Effect of Low Intensity Pulsed Ultrasound on Mesenchymal Stem Cells --- p.34 / Chapter 3.1. --- Introduction --- p.35 / Chapter 3.2. --- Materials and Methods --- p.36 / Chapter 3.2.1. --- Experimental Animal --- p.36 / Chapter 3.2.2. --- Materials and Reagents --- p.36 / Chapter 3.2.2.1. --- Dulbecco，s Modified Eagle Medium (DMEM) --- p.36 / Chapter 3.2.2.2. --- Phosphate Buffered Saline (PBS) --- p.37 / Chapter 3.2.2.3. --- Osteogenic Medium (OS) --- p.37 / Chapter 3.2.2.4. --- Alkaline Phosphatase (ALP) Buffer --- p.37 / Chapter 3.2.2.5. --- ALP Substrate Buffer --- p.38 / Chapter 3.2.2.6. --- MTT Stock Solution --- p.38 / Chapter 3.2.2.7. --- MTT Working Solution --- p.38 / Chapter 3.2.2.8. --- Lysis buffer --- p.38 / Chapter 3.2.2.9. --- Alkaline Phosphatase (ALP) Working Reagents --- p.39 / Chapter 3.2.3. --- Isolation of Bone Marrow Derived Mesenchymal Stem Cells (BM derived MSCs) --- p.39 / Chapter 3.2.4. --- In vitro Low Intensity Pulsed Ultrasound Treatment --- p.40 / Chapter 3.2.4.1. --- In vitro LIPUS Devices --- p.40 / Chapter 3.2.4.2. --- Treatment Procedure and Experimantal Groupings --- p.40 / Chapter 3.2.5. --- Effect of LIPUS on Cell Viability and Osteogenesis in bone marrow derived-MSCs --- p.41 / Chapter 3.2.5.1. --- Cell Viability Assay --- p.41 / Chapter 3.2.5.2. --- Alkaline Phosphatase (ALP) Enzyme Activity --- p.42 / Chapter 3.2.5.3. --- Cell Morphology and Alkaline Phosphatase Cytochemistry --- p.42 / Chapter 3.2.6. --- Statistical Analysis --- p.43 / Chapter 3.3. --- Results --- p.43 / Chapter 3.3.1. --- Morphology --- p.43 / Chapter 3.3.2. --- Total Number of Viable Cells --- p.44 / Chapter 3.3.3. --- ALP Activity Absorbance --- p.44 / Chapter 3.3.4. --- ALP staining --- p.45 / Chapter 3.3.5. --- Qualitative Analysis --- p.45 / Chapter 3.3.6. --- Quantitative Analysis --- p.46 / Chapter 3.4. --- Discussion --- p.46 / Chapter 3.4.1. --- LIPUS have No Enhancing Effect on Proliferation of MSCs in Basal Medium Nor Osteogenic Medium --- p.47 / Chapter 3.4.2. --- LIPUS Stimulate Proliferation of MSCs in Early Period --- p.49 / Chapter 3.4.3. --- LIPUS Further Enhanced Osteogenesis of MSCs in Osteogenic Medium --- p.49 / Chapter 3.4.4. --- 10 mins LIPUS treatment for 7 days can positively enhance osteogenic differentiation --- p.50 / Chapter 3.4.5. --- Optimum Conditions of LIPUS was Cell Type Dependent --- p.51 / Chapter 3.4.6. --- LIPUS Promoted Osteogenesis in MSCs through Accelerated Mineralization --- p.52 / Chapter Chapter 4 --- Enhancement of Posterior Spinal Fusion The Effect of Tissue-Engineered MSC and Calcium Phosphate Ceramic composite treated with LIPUS in Vivo --- p.68 / Chapter 4.1. --- Introduction --- p.69 / Chapter 4.1.1. --- TCP Biomaterials --- p.70 / Chapter 4.2. --- Materials and Methods --- p.71 / Chapter 4.2.1. --- Materials and Reagents --- p.71 / Chapter 4.2.2. --- Preparation of MSC Derived Osteogenic Cells-tricalcium Phosphate Ceramics Composite --- p.73 / Chapter 4.2.3. --- Posterior Spinal Fusion Surgery --- p.74 / Chapter 4.2.4. --- In vivo LIPUS treatment --- p.75 / Chapter 4.2.5. --- Assessment of Fusion Mass --- p.76 / Chapter 4.2.6. --- Histology --- p.77 / Chapter 4.2.7. --- Statistical Analysis --- p.79 / Chapter 4.3. --- Results --- p.79 / Chapter 4.3.1. --- Fusion by Manual Palpation --- p.79 / Chapter 4.3.2. --- pQCT Analysis --- p.80 / Chapter 4.3.3. --- Histological Analysis --- p.81 / Chapter 4.4. --- Discussion --- p.85 / Chapter 4.4.1. --- Summary of the Findings from Different Assessment Methods --- p.85 / Chapter 4.4.2. --- Addition of MSCs to TCP ceramic in Spinal Fusion --- p.87 / Chapter 4.4.3. --- The Needs of Differentiated MSC in Spinal Fusion --- p.89 / Chapter 4.4.4. --- bFGF Masked the Effect of OS in MSC --- p.91 / Chapter 4.4.5. --- LIPUS Enhanced Bone Formation --- p.95 / Chapter 4.4.6. --- LIPUS Enhanced Bone Formation through Mineralization --- p.96 / Chapter 4.4.7. --- LIPUS Enhanced Spinal Fusion through Bone Remodeling-induced Fusion Mass --- p.97 / Chapter 4.4.8. --- LIPUS Enhanced Bone Formation through Endochondral Ossification --- p.99 / Chapter Chapter 5 --- In Vivo Monitoring of Spinal Fusion in Animal Model with High-resolution Peripheral Quantitative Computed Tomography-A New Pilot Study --- p.122 / Chapter 5.1. --- Introduction --- p.123 / Chapter 5.2. --- Materials and Methods --- p.124 / Chapter 5.2.1. --- Animal Groupings --- p.124 / Chapter 5.2.2. --- Preparation of MSC Derived Osteogenic Cells-tricalcium Phosphate Ceramics Composite --- p.124 / Chapter 5.2.3. --- Posterior Spinal Fusion Operation Procedures --- p.125 / Chapter 5.2.4. --- LIPUS treatment --- p.125 / Chapter 5.2.5. --- High-resolution Peripheral Quantitative Computed Tomography …- --- p.125 / Chapter 5.2.6. --- Analysis with HR-pQCT --- p.126 / Chapter 5.3. --- Result --- p.128 / Chapter 5.3.1. --- Qualitative Observations from HR-pQCT Images --- p.128 / Chapter 5.3.2. --- Quantitative Analysis --- p.129 / Chapter 5.4. --- Discussion --- p.130 / Chapter Chapter 6 --- "Overall Summary, Discussion and Conclusion" --- p.140 / Chapter 6.1. --- Overall Summary and Discussion --- p.141 / Chapter 6.2. --- Limitations and Further Studies --- p.145 / Chapter 6.3. --- Conclusions --- p.147 / Chapter 6.4. --- Summary Flowchart of the whole thesis --- p.148 / References --- p.153

Smad7作為轉化生長因數-β信號通路中的負性調節因子為人所知，異常的Smad7表達通常會引發癌症及組織纖維化等疾病。而目前對於其在骨重建及其相關疾病中的作用尚未有研究。本研究利用Smad7部分敲除小鼠來探索Smad7在骨重建，骨質疏鬆以及間充質幹細胞分化等方面的作用。 / 本研究所用的Smad7部分敲除小鼠模型來源於已有報導過的Smad7ΔE1(KO)小鼠。該小鼠體內Smad7基因組外顯子I的翻譯區被替換，導致部分蛋白失及其功能破壞。研究結果表明，KO小鼠在6、12、24周齡時股骨遠端幹骺端均有不同程度下降的骨小梁數目、厚度，骨礦化率，骨密度，骨體積分數，及其上升的骨小梁間隙和破骨細胞表面。骨髓來源間充質幹細胞的多向分化實驗表明，KO組呈現出抑制性的成骨能力，表現為鈣結節形成減少，鹼性磷酸酶活性下降，早晚期成骨標記基因表達下降。該組亦表現出促進性的成脂能力，有較多及較早的脂滴形成，成脂標記基因表達上升。而對於骨髓來源巨噬細胞的體外破骨誘導實驗表明，KO組有更多且更大的破骨細胞形成，較大的骨吸收面積，以及上升的破骨標記基因表達。卵巢切除小鼠模型的研究表明，術後4、8、16周，KO组的股骨遠端幹骺端对比野生组有更大程度下降的骨形态学参数，以及明顯升高的破骨細胞融合標記蛋白的表達。體外實驗表明KO组有更多且更大的破骨細胞形成，以及更大面積的骨吸收。積雪草酸曾被證實在肝纖維化模型中誘導Smad7 基因的表達，也在本實驗中用以研究對骨質疏鬆疾病的作用。卵巢切除動物模型連續給藥8周後，骨質疏鬆的現象有明顯逆轉，表現為升高的骨形态学参数，及下降的股骨內破骨細胞融合標記蛋白的表達。 / 總結，本研究證實了Smad7在骨骼發育重建及骨疾病的病理機理等方面的研究提供了突破性的見解。部分敲除Smad7可以導致抑制性的成骨能力，促進性的破骨能力，以及損傷性的骨重建，亦會加速骨質疏鬆的進程，并可作為全新的藥物治療靶點，提示Smad7 本身對於骨重建及骨代謝的保護性作用，為代謝性骨疾病的研究及其臨床藥物開發提供了更廣泛的前景。 / Smad7 has been well documented as a negative regulator of TGF-β signaling, and its altered expression often leads to human diseases such as cancer and fibrosis. However, the role of Smad7 in regulating bone remodeling and related diseases remains unclear. We performed both in vivo and in vitro experiments as well as disease model and drug therapy studies using both wild-type (WT) and Smad7ΔE1 (KO) mice to investigate the functional role of Smad7 in bone remodeling, osteoporosis, and MSCs differentiation. / The Smad7ΔE1 mice were generated by replacing part of the exon1 of Smad7 gene as reported, which resulted in truncated protein and partial loss of Smad7 function. Mice were genotyped by PCR. The μ-CT, histological assays and bone histomorphometric assays in metaphysic region of the femurs showed lower trabecular number (TbN), trabecular thickness (TbTh), mineral apposition rate (MAR), higher trabecular separation (TbSp) and Osteoclast Surface (Oc.S/BS & Oc.N/BS) in the KO mice at 6, 12, to 24 weeks old; as well as lower bone mineral density (BMD) and bone volume fraction (BV/TV) at 24 weeks old in the KO mice. The in vitro BM-MSCs multi-lineage differentiation studies showed the suppressed osteogenic potential in the KO group with fewer mineralized nodules, lower ALP activity and expression of Col1A1, Runx2 and OCN; while the adipogenic potential was elevated with more lipid droplets formation and higher expression of Adipsin and C/EBPα. The osteoclastogenic potential of KO mice BMMs was also elevated, showing higher osteoclasts activity and larger resorptive areas, as well as elevated expression of TRAP and CTR. Both in vivo and in vitro studies of the osteoporotic models showed that the KO mice had lower BMD, TbTh, and higher TbSp compared to the WT mice at 4, 8, 16 weeks after OVX, similar results of lower BV/TV and TbN were observed at 4 weeks after OVX in the KO mice. The RANKL-induced osteoclastogenesis potential was elevated compared to WT mice, with more and bigger osteoclasts, larger resorptive areas, as well as elevated expression of TRAP and CTR. The osteoclastic cell fusion was also enhanced. Treatment of Asiatic acid (one traditional Chinese medicine that has been proved to induce the expression of Smad7 as reported) in the OVX mice reversed the osteoporotic process with increase BMD, BV/TV, TbN, TbTh, and decreased TbSp compared to the untreated group. The osteoclastic cell fusion was suppressed after AA treatment. / Partial loss of Smad7 function leads to impaired bone remodeling in vivo, reduced osteogenesis and enhanced osteoclastogenesis in vitro, and also accelerates the osteoporotic development and osteoclastic cell fusion. Asiatic acid may be a novel potential drug for prevention of osteoporosis. Our findings provide new evidences for a better understanding of the biological functions of Smad7 in bone remodeling and its therapeutic potential for metabolic bone diseases. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Nan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 131-153). / Abstracts also in Chinese.

As células tronco mesenquimais (MSC) têm capacidade imunossupressora envolvendo diferentes mecanismos. No entanto, ainda não está claro o que desencadeia as diferentes vias de imunorregulação, ou mesmo o que determina a magnitude de sua capacidade supressora. A nossa hipótese é que a intensidade da atividade supressora das MSC esteja vinculada à sua capacidade de desencadear múltiplas vias moleculares, simultaneamente. Utilizamos uma estratégia experimental na qual testamos o efeito das MSC humanas do tecido adiposo (AdMSC), derivadas de diferentes indivíduos, sobre a proliferação de PBMC estimuladas, obtidas do mesmo indivíduo, visando relacionar o efeito supressor com a ocorrência simultânea de modificações imunomoleculares (expressão de mRNA e proteínas), tanto nos linfócitos T como nas AdMSC. Foram comparadas as correlações entre as modificações de expressão gênica e proteica nos ensaios com maior (> 50% inibição de proliferação de linfócitos T) ou menor (<50% de inibição) capacidade supressora. A primeira novidade do nosso trabalho é que durante a atividade supressora das AdMSC, múltiplas moléculas são acionadas, simultaneamente, nos linfócitos T (LT) e nas próprias AdMSC, indicando a importância da ação integrada de múltiplas vias moleculares. Várias dessas modificações de expressão gênica ocorreram de forma dominante (em todos os experimentos), como o aumento da expressão de IDO, ILB e MMP2 nos LT e de diversas moléculas nas AdMSC (entre elas: IDO, HLAG, IL10, PDL1, SEMAD4, MMP9, FASL e várias quimiocinas). Ademais, relatamos diversos novos mecanismos potencialmente envolvidos na atividade supressora das AdMSC humanas, entre eles, a diminuição do número de células T efetoras ativadas, CD4 e CD8 expressando ICOS e CD8 positivas expressando OX40, além do aumento do número de uma população específica de células T reguladoras (CD4+CD25hiFOXP3+) expressando a ectoenzima CD73, importante na geração de adenosina, molécula com atividade imunorreguladora. Descrevemos também outras novas moléculas possivelmente envolvidas nos mecanismos de imunossupressão das AdMSC como SEMA4D, MMP9, CCL22 e RUNX3, cuja expressão aumentou nas AdMSC após o cocultivo e GARP, CTNNB1, IL1B e MMP2, nos LT. Mostramos, pela primeira vez, um perfil imunomolecular diferencial nos ensaios de maior capacidade supressora, com correlações positivas específicas entre os aumentos da expressão gênica, nos dois tipos celulares, envolvendo moléculas como HLAG e CCR4, IL13, IL4, TLR10, IL1B, GARP, S1PR1 e CTNNB1 (-catenina), nos linfócitos T e, nas AdMSC, CCL22 com MMP9 e HLAG com FOXP3. Assim, sugerimos que a magnitude da atividade supressora das AdMSC depende da combinação de múltiplas vias moleculares mobilizadas, simultaneamente, e relacionadas com a indução de um perfil Th2, migração e sobrevida celular, bem como com a atividade imunorreguladora. Além de apontar novas moléculas ao repertório imunomolecular da atividade supressora das AdMSC humanas, trazemos uma contribuição importante, apresentando uma visão mais ampla da rede de interações imunomoleculares envolvida na atividade supressora das MSC / Mesenchymal stem cells (MSC) exhibit immunosuppressive activity operating by a variety of mechanisms. However, it is still unclear what triggers the different immunoregulatory pathways, or what determines the magnitude of their suppressive capacity. We hypothesized that the magnitude of MSC suppressive activity may be related to their capacity to activate multiple pathways, simultaneously. We used an approach in which we tested the effect of different human MSC derived from adipose tissue (AdMSC) over T cell proliferation, using PBMC obtained from a single donor. We determined the relationship between AdMSC suppressive activity and the simultaneous occurrence of immunomolecular changes (mRNA and protein expression) in both AdMSC and T cells, following AdMSC/PBMC interactions. Correlations of gene expression and protein modifications were compared in assays in which AdMSC displayed high (>50% of T cell proliferation inhibition) and low (<50% inhibition) immunosuppressive activity. The first novelty from our work is that multiple molecules are simultaneously activated, in both AdMSC and T cells, indicating the importance of an integrated action of several molecular pathways. Many of these gene expression modifications were dominantly (in all experiments) upregulated, namely: IDO, IL1B and MMP2 in T cells and several molecules in AdMSC such as: IDO, HLAG, IL10, PDL1, SEMAD4, MMP9, FASL, as well as many chemokines. We also report novel mechanisms potentially involved in human AdMSC suppressive activity, such as the decrease in the number of activated T cells, CD4 and CD8 cells expressing ICOS and CD8 positive cells expressing OX40. We also found an increase in the numbers of a specific Treg subpopulation (CD4+CD25hiFOXP3+) expressing the ectoenzime CD73, important in the generation of adenosine, a molecule displaying suppressive activity. Moreover, we report other novel molecules possibly involved in AdMSC suppressive activity, once their gene expression was upregulated in AdMSC (SEMA4D, MMP9, CCL22 and RUNX) and in T cells (GARP, CTNNB1, IL1B e MMP2), following AdMSC/PBMC interactions. We also show, for the first time, a differential immunomolecular profile in the assays with high suppressive activity, showing exclusive positive correlations among changes in gene expression, involving multiple molecules, such as HLAG with CCR4, IL13, IL4, TLR10, IL1B, GARP, S1PR1 and CTNNB1 (-catenin) in T cells, and CCL22 and MMP9, with HLAG and FOXP3, in AdMSC. We, therefore, suggest that the magnitude of AdMSC suppressive activity depends on a particular combination of multiple immunoregulatory pathways simultaneously activated and related with the induction of a Th2 profile, cell migration and survival, as well as with immunoregulatory activity. Besides adding new players to the scenario of MSC suppressive activity, we believe our data bring a major contribution to the field, by providing a broader view of the interactive immunoregulatory molecular networks involved in MSC immunologic activities

As doenças genéticas degenerativas atingem milhões de crianças em todo o mundo. Dentre essas doenças, a distrofia muscular, caracterizada como uma doença monogênica poderia ser tratada na sua origem através da terapia gênica. Assim, este estudo propõe à correção da mutação no gene da distrofina, causador da distrofia muscular através de modificações genéticas específicas. A criação de novas classes de terapêuticos que podem desencadear rearranjos no DNA genômico de maneira específica representa uma nova promessa para experimentos em terapia gênica. A tecnologia usada foi o RNA interferente (RNAi) que é utilizada para regulação da expressão gênica pós-transcricional. A Ku 70 é uma das proteínas específicas para a recombinação não homóloga, o RNAi foi usado na tentativa de atenuar a Ku70, prevalecendo então a expressão da recombinação homóloga, com intuito de corrigir a mutação gênica causadora da distrofia muscular em cães. Para tal avaliação, utilizamos linhagens de células tronco (CT) mesenquimais recentemente isoladas, oriundas de populações mononucleares da médula óssea de cães jovens afetados pela distrofia muscular, apresentando bons resultados em cultivo e caracterização. Este trabalho proporciona além da criação de uma nova terapêutica específica para a correção da distrofia muscular, o aumento do conhecimento e entendimento na indução de modificações genômicas em células, no desenvolvimento de novas classes de agentes terapêuticos moleculares que representam um grande potencial em estudos e no tratamento de várias doenças genéticas e infecciosas, degenerativas ou adquiridas. O presente trabalho apresenta métodos de isolamento e caracterização de células tronco-mesenquimais bem como a utilização de RNAi visando promover a recombinação homóloga entre o DNA transfectado e o alvo no DNA genômico. / The degenerative genetic diseases affect millions of children around the world. Among these diseases, muscular dystrophy, characterized as a monogenic disease can be treated at its source through gene therapy. Thus, this study proposes the correction of the gene that causes muscular dystrophy through genetic modification specific. The creation of new classes of therapeutics that can trigger rearrangements in the genomic DNA in a specific manner represents a new promise for gene therapy experiments. The technology will be used by RNA interference (RNAi) and that used to regulate gene expression post-transcriptional. The 70 Ku is a protein specific to the non-homologous recombination, RNAi was used in an attempt to mitigate the Ku70, then the prevailing expression of homologous recombination, aiming to correct the mutation that causes muscular dystrophy in dogs. For this evaluation, we use mesenchymal stem cell lines recently isolated populations derived from bone marrow mononuclear cells of young dogs affected by muscular dystrophy, presenting good results in characterization and culture. This work also provides the creation of a new specific therapy for the correction of muscular dystrophy, increased knowledge and understanding in the induction of genomic changes in cells in the development of new classes of molecular therapeutic agents have great potential in studies and treatment of various genetic and infectious diseases, degenerative or acquired. This paper presents methods for isolation and characterization of mesenchymal stem cells and the use of RNAi to promote homologous recombination between transfected DNA and genomic target DNA.

Trop souvent diagnostiqués à des stades tardifs du fait de leur quasi asymptomatie, les adénocarcinomes séreux ovariens posent un véritable problème de santé publique. Malgré les progrès récents de prise en charge chirurgicale, l’émergence d’une maladie résiduelle microscopique chimiorésistante impacte grandement le pronostic des patientes.Le microenvironnement tumoral est un acteur clé de la progression tumorale et de l’émergence de résistances aux traitements anticancéreux. Durant ces travaux de thèse, nous nous sommes intéressés à deux composants majeurs du stroma tumoral, d’une part les cellules souches mésenchymateuses, d’autre part les cellules endothéliales.Nous avons pu démontrer que les cellules souches mésenchymateuses participent à la progression tumorale et l’émergence de résistances. Enfin nous avons démontré que les cellules endothéliales, via la production de facteurs angiocrines, participent à la chimiorésistance des cellules tumorales ovariennes.Dans ce travail, nous avons pu définir de nouvelles cibles thérapeutiques mettant en jeu la relation entre les cellules tumorales ovariennes et l’hôte. / Ovarian cancers constitute a poor prognosis disease. Due to their absence of symptoms, ovarian cancers are generally diagnosed at late stages. Despite major breakthrough regarding ovarian cancer surgery, minimal residual disease-induced relapse is still a hurdle for clinicians.Tumor microenvironment is a key actor on disease progression and resistance to therapy. In this study, we have focused on two major components of the tumor stroma, on one hand, the mesenchymal stem cells, and the endothelial cells on the other hand.We were able to demonstrate that mesenchymal stem cells are critically involved in ovarian cancer progression and resistance to therapy, while the endothelium, through production of angiocrine factors, is deeply involved in resistance of ovarian cancer cells to platinum and taxane based therapy.Here, we set forth the idea that disrupting the relationship between ovarian cancer cells and their host stroma constitute a new therapeutic window.

As research and clinical use of stem cell therapies progresses, it is becoming more evident that being able to visualize the stem cell transplant in vivo is of great benefit to the researcher or clinician. As a result, researchers are working to address this need. Our lab is collaborating to develop novel, multimodal nanomaterials, one with a core of mesoporous silica, and the other with a core of gadolinium oxide. Varying modifications have been made as needs arose. Human mesenchymal stem cells (MSCs) were isolated from bone marrow aspirates and confirmed to be positive for STRO-1, a common MSC marker. These cells were labeled with 125 μg/mL of varying nanoparticle types: gadolinium oxide, doped with 0.5%, 5%, or 10% europium for magnetic resonance imaging (MRI) and luminescence microscopy, and mesoporous silica nanoparticles (MSN), loaded with fluorescein for fluorescent microscopy and capped with either iron oxide or gold for MRI and computed tomography (CT), respectively. We studied the kinetics of MSN uptake by MSCs for 10 days using fluorescent microscopy. In ex vivo studies, we used the 4.7 Tesla Varian® small animal MRI scanner to detect 5*10⁴ cells labeled with ferrite-capped MSN particles and injected into the brain, lung, and heart of a perfusion-fixed mouse. Micro-CT was used to detect 1.7*10⁶ cells labeled with gold-capped MSN and delivered to the lungs via the trachea in a perfusion-fixed mouse. The results of this research are preliminary to in vivo testing using animal models as a proof-of-concept for these potentially marketable particles.

The regenerative potential of tissue injury declines with age. Recently, a significant role for Wnt/β-catenin signaling has been shown in tissue specific stem cell aging, leading to increased tissue fibrosis. Wnt/β-catenin signaling regulates the differentiation of multipotent mesenchymal stem cells into osteoblasts during fracture repair. We investigated the potential role of dysregulated Wnt/β-catenin signaling in delayed fracture union and tissue fibrosis in the elderly. Old mice displayed increased total β-catenin protein levels at 4 and 7 days post-fracture and tissue fibrosis at 14 and 21 days post-fracture compared to young mice. Furthermore, treatment with a pharmalogical agent decreased total β-catenin protein levels in the fracture callus at 4 days post-fracture and prevented tissue fibrosis at 21 days post-fracture. Our data suggests that dysregulated Wnt/β-catenin signaling in the elderly contributes to delayed fracture repair and tissue fibrosis and offers a potential therapeutic strategy to improve fracture outcome in the elderly.

The regenerative potential of tissue injury declines with age. Recently, a significant role for Wnt/β-catenin signaling has been shown in tissue specific stem cell aging, leading to increased tissue fibrosis. Wnt/β-catenin signaling regulates the differentiation of multipotent mesenchymal stem cells into osteoblasts during fracture repair. We investigated the potential role of dysregulated Wnt/β-catenin signaling in delayed fracture union and tissue fibrosis in the elderly. Old mice displayed increased total β-catenin protein levels at 4 and 7 days post-fracture and tissue fibrosis at 14 and 21 days post-fracture compared to young mice. Furthermore, treatment with a pharmalogical agent decreased total β-catenin protein levels in the fracture callus at 4 days post-fracture and prevented tissue fibrosis at 21 days post-fracture. Our data suggests that dysregulated Wnt/β-catenin signaling in the elderly contributes to delayed fracture repair and tissue fibrosis and offers a potential therapeutic strategy to improve fracture outcome in the elderly.