L'arthrose (OA) est une maladie dégénérative avec un impact remarquable sur la qualité de vie. Pourtant, aucune intervention pharmacologique entièrement appropriée, aucune thérapie biologique ou procédure n'entraînent la dégradation progressive de l'articulation OA. Ici, nous explorons les cellules souches mésenchymateuses (MSC) - précurseurs multi-potentiels de cellules qui peuvent être isolées à partir de différents niveaux de dégradation du cartilage. Nous émettons l'hypothèse que les cellules progénitrices mésenchymateuses (CPM) pourraient servir comme une thérapie potentielle. Le cartilage ostéoarthritique humain a été obtenu de 25 patients subissant un remplacement total du genou et classé en différents niveaux de dégradation. Les niveaux d'expression de l'ARNm de CD105, CD166, Notch 1, Sox9, Acan, Col II A1 et Col I A1 ont été mesurés au jour 0, au jour 14 (2 semaines in vitro) et au jour 35 (après chondrogénèse). Les cellules de toutes les classes d'OA ont augmenté de façon significative les marqueurs MPC de l'ARNm avec expression in vitro. Les cellules proliférées ont exprimées des marqueurs spécifiques aux MPC: CD105, CD166, CD73, CD90, Notch – 1 and Nucleostemin. La chondrogénèse induit une diminution de l'ARNm de CD105, de Notch 1 et de Sox9 seulement dans l'OA légère et modérée. Cependant, seules les pastilles modérées dérivées d 'OA ont révélé des signes de cartilage hyaline élevé - collagène II et faible expression de fibrocartilage - collagène I à la fois au niveau de l’ARNm et de la protéine. Une nouvelle conclusion émerge de nos données et confirme les différences dans les marqueurs MPC entre les différents niveaux de dégradation. Seules les cellules dérivées d 'OA modérées ont été capables de former une matrice hyaline composée de protéoglycanes et de collagène II avec le niveau faible en collagène I fibrocartilagineux. Nos résultats montrent que les CPM provenant d’un cartilage d’un niveau de dégradation modéré ont un fort potentiel d'auto-réparation. / Osteoarthritis (OA) is a degenerative disease with a remarkable impact on quality of life. Yet no fully appropriate pharmacological intervention, biologic therapy or procedure stops the progressive degradation of the OA joint. Herein, we explore mesenchymal stem cells (MSCs)—multipotent precursors of cells that can be isolated from different grades of OA cartilage. We hypothesize that mesenchymal progenitors cells (MPC), could emerge as a potential therapy. Human osteoarthritic cartilage were obtained and scored (according to the OARSI) from 25 patients undergoing total knee replacement. mRNA expression levels of CD105, CD166, Notch 1, Sox9, Acan, Col II A1 and Col I A1 were measured at day 0, day 14 (2 weeks in vitro) and day 35 (after chondrogenesis). Cells from all OA grades significantly increased MPC markers mRNA with in vitro expression. Proliferated cells expressed MPC specific antigens: CD105, CD166, CD73, CD90, Notch – 1 and Nucleostemin. The chondrogenesis induced decrease in CD105, Notch 1 and Sox9 mRNA only in mild and moderate OA. Yet, only moderate OA – derived pellets revealed high hyaline cartilage marker – collagen II and low fibrocartilage marker – Collagen I expression at both mRNA and protein level. A novel finding emerges from our data and confirms differences in MPC markers between OA grades. Only moderate – OA derived cells were able to form hyaline – like matrix composed of proteoglycans and collagen II with law level of fibrocartilaginous collagen I. Further studies that investigate the mechanistic effects of chondrogenic progenitor populations associated with aging and the progression of OA are crucial to our understanding of the clinical relevance of these cells for use in cartilage repair therapies.

Les cellules souches mésenchymateuses (CSM) de la moelle osseuse ont été intensivement étudiées pour leur capacité de régénération et leurs propriétés immunomodulatrices. Beaucoup d’études ont montré que la thérapie qui utilise les CSM améliore les fonctions de tissu ostéo-articulaire particulièrement le cartilage en vue de leur capacité de différenciation en chondrocytes. Les CSM présentent un certain nombre d'avantages pour la médecine régénérative, ces cellules peuvent être facilement isolées et multipliées en culture pour obtenir un nombre approprié pour la thérapie cellulaire. De plus, elles ont une faible immunogénicité, ce que les rende aptes à la transplantation allogénique. Depuis les années 1960, de nombreuses études ont souligné les propriétés médicinales des polysaccharides notamment les β-glucanes qui ont une place particulière du fait de leurs effets immunostimulants. L’objectif de notre travail était de mettre en évidence les capacités d’un β-glucane particulier, la laminarine, sur la prolifération et la différenciation des CSM dans la perspective d’applications dans l’arthrose. Les CSM ont été cultivés dans les milieux de croissance et de différenciation chondrocytaire. La viabilité et l'apoptose des cellules ont été explorées par le comptage, les tests MTT et la coloration à l'annexine V. En outre, l'analyse des protéines spécifiques de la prolifération a été effectuée par le western blott. De plus, l'expression des marqueurs spécifiques des CSM et des chondrocytes a été étudiée à l'aide de la RT-qPCR et de l’immunofluorescence. Nos résultats ont démontré que la stimulation des CSM à la laminarine avec la dose de 1 mg/ml soit en condition de culture de croissance basique ou en chondrogenèse a inhibé la prolifération des cellules sans induire leur apoptose. Encore, dans les conditions de culture chondrogénique, la laminarine à une dose similaire a empêché la différenciation des CSM en chondrocytes. / Mesenchymal stems cells (MSCs) are a population of multipotent cells residing in several readily available adult tissue compartments, thus allowing for their ex vivo expansion. MSCs have a reliable potential for differentiation (plasticity) into cells of the mesodermal lineage (chondrocytes, osteoblasts, adipocytes). Bone marrow-derived MSCs have been a focus of stem cell research in light of their relative ease of isolation and expansion and of their high potential for differentiation. Herein, the aim of the present PhD is to explore the potential of a β-glucan (laminarin) on Mesenchymal stem cell proliferation and differentiation for future benefit for osteoarthritis treatment. MSCs were cultured in MSC growth and chondrogenic differentiation mediums. Cells viability and apoptosis were explored by cell count, MTT assays and Annexin V staining. In addition, Analysis of the specific protein of cell proliferation was performed by western blott. Furthermore, mRNA and protein expression of specifics markers for MSCs and chondrocytes were studied using qPCR and immunofluorescence. Our results demonstrated that stimulation of MSC with laminarin at a dose of 1 mg/ml in either basic growth culture or chondrogenesis inhibited cell proliferation without inducing their apoptosis. Furthermore, under chondrogenic culture conditions, laminarin at a similar dose prevented the differentiation of MSC into chondrocytes.

Breast cancer is the most common cancer in women and unspayed female dogs. The Epithelial to Mesenchymal Transition (EMT) is a process involved in embryogenesis, carcinogenesis, and metastasis. The Transforming Growth Factor- Beta (TGF-β) pathway and its associated transcription factors are crucial for EMT induction, during which epithelial cells lose their defining characteristics and acquire mesenchymal properties. EMT has been implicated as a driver of metastasis as it allows cells to migrate and invade different organs. Recent evidence indicates that cancer stem cells are required to establish metastatic tumours at distant sites, and that EMT may promote development of cancer cells with stem-cell characteristics, thus, the EMT pathway may be an important molecular determinant of tumour metastasis. The main objective of this project was to characterise TGF-β-induced EMT in breast cancer models. EMT was induced by TGF-β in human, canine and feline breast cancer cell lines, and confirmed by morphological changes and molecular changes at the protein level by Western blot analysis. Changes at the mRNA level were confirmed in human and canine mammary carcinoma cell lines by qRT-PCR; migratory properties were assessed by invasion assays in vitro in feline and canine mammary carcinoma cells. Importantly, we observed that feline and canine mammary carcinoma cells stimulated by TGF-β acquired stem cell characteristics including sphere-forming ability, self-renewal, and resistance to apoptosis, and also enhanced migration potential. Canine cells showed resistance to chemotherapeutic drugs after TGF-β stimulation. These data suggests a link between EMT and cancer stem-cells. Moreover, global changes in microRNA expression were mapped during TGF-β-induced EMT of canine mammary carcinoma cells. This gave significant insight into the regulation of EMT in canine cancer cells and identified several potential targets, which require further investigation. During EMT cells acquire migratory properties and cancer stem-cell characteristics, suggesting that EMT and the stem-cell phenotype are closely related during cell migration and metastasis, therefore making the TGF-β pathway a potential target for the development of novel therapies against cancer and its progression.

Resumo: Transição epithelial-mesenquimal (EMT) é a conversão de células epiteliais polarizadas para células migratórias com fenótipo fibroblasto-símile. A EMT está envolvida na progressão e metástase em diversos cânceres nos seres humanos, porém permanece a ser mais bem explorada na literatura veterinária. O objetivo desta pesquisa foi avaliar a imunoexpressão de proteínas relacionadas à EMT nos carcinomas mamários de cadelas (CMCs). Seis proteínas foram avaliadas por meio de imunoistoquímica em 94 amostras de CMCs. Tecidos mamários não neoplásicos de 17 cadelas e amostras de 9 tumores mamários benignos de cadelas foram avaliados de modo a determinar o perfil de imunoexpressão de Snai-1. Características anatomopatológicas foram comparadas com a imunoexpressão de proteínas relacionadas à EMT nos CMCs. A perda de proteínas epiteliais e/ou a aquisição de proteínas mesenquimais foi observada principalmente em neoplasias com evidência de invasão estromal; entretanto, somente foi observada significância estatística quando comparado S100A4 e invasão vascular. Snai-1 foi observado em células luminais de neoplasias simples malignas e em células mioepiteliais de tumores benignos ou malignos de caráter complexo, sendo também significativamente relacionado à baixa de expressão de Caderina-E. Conclui-se que a perda de proteínas epiteliais e/ou a aquisição de proteínas mesenquimais está associada com EMT e pode possuir importante papel na avaliação de CMCs. O padrão único de imunoexpressão de Snai-1 pode ajudar a distinção entre um adenoma e um carcinoma não metastático e aparenta estar relacionado à conversão de células mioepiteliais a um fenótipo mesenquimal completo. A perda de Caderina-E e citoqueratina e a mudança no padrão de imunoexpressão de Snai-1, Caderina-N, S100A4 e MMP-2 indica a ocorrência de EMT em carcinomas mamários de cadelas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Epithelial-mesenchymal transition (EMT) is defined as switching of polarized epithelial cells to a migratory fibroblastoid phenotype. EMT is known to be involved in the progression and metastasis of various cancers in humans, but this specific process is still little explored in the veterinary literature. The aim of this research was to evaluate the expression of EMT-related proteins in canine mammary carcinomas (CMCs). The expression of six EMT-related proteins in CMC of 94 female dogs was evaluated by immunohistochemistry. Additionally, mammary tissues from 17 female dogs with no history of mammary tumor development and from 9 bitches with benign tumors were evaluated in order to determine Snai-1 immunoexpression patterns. Anatomopathological characteristics were compared with the expression of EMTrelated proteins in CMCs. Loss of epithelial protein and/or acquisition of the expression of mesenchymal proteins were observed, particularly in tumors with evidence of stromal invasion; however, significance was only observed between the S100A4 and vascular invasion. Snai-1 was only expressed in luminal cells of histologically malignant tumors and in myoepithelial cells of benign and malignant complex tumors and was significantly related to E-cadherin loss. In conclusion, loss of epithelial proteins and/or the acquisition of mesenchymal proteins are associated with EMT and may have an important role in the evaluation of CMC patients. The unique immunoexpression pattern of Snai-1 could help to distinguish between an adenoma and a non-metastatic carcinoma and seems to be related to conversion of myoepithelial cells to a complete mesenchymal-like phenotype. Loss of E-cadherin and cytokeratin and change of immunoexpression pattern of Snai-1, N-cadherin, S100A4 and MMP-2 indicate the occurrence of EMT in canine mammary carcinomas and should result in an en bloc resection or a close follow-up. / Orientador: Noeme Souza Rocha / Coorientador: Rafael Malagoli Rocha / Banca: Deilson Elgui de Oliveira / Banca: Geovanni Dantas Cassali / Mestre

As células mesenquimais estromais multipotentes (CMM) são na atualidade uma fonte atrativa para aplicações na engenharia de tecidos e na terapia celular. Devido à baixa disponibilidade nos tecidos (0,01%-0,0005%) e às elevadas doses necessárias para uma infusão (aproximadamente 106 células/Kg paciente) tornou-se necessário o desenvolvimento de tecnologias de expansão in vitro, eficientes e de custo reduzido, que permitam a obtenção de CMM com manutenção das características funcionais (diferenciação e inibição da proliferação de linfócitos), imunofenotípicas e citogenéticas. As CMM são células aderentes, ou seja, necessitam de um substrato sólido para se aderir e proliferar. O procedimento convencional de expansão em garrafas estáticas, geralmente envolve um processo laborioso em que não há correto controle e monitoramento dos parâmetros de cultivo e possui uma maior susceptibilidade à contaminação devido à excessiva manipulação para atingir o número ideal de células. Além disso, este tipo de cultivo não permite uma produção em larga escala. Em função disso, o presente trabalho foi proposto com o objetivo de desenvolver um bioprocesso escalonável, economicamente viável e eficiente para expansão de CMM derivadas da medula óssea em microcarregadores. Para isso, as células foram cultivadas em microcarregador Cyotdex 3, em frasco spinner com o meio -MEM suplementado com 15% de SFB. Foram avaliadas neste trabalho, a adesão celular aos microcarregadores, crescimento, metabolismo, recuperação celular final e avaliação das propriedades funcionais e imunofenotípicas pré e pós cultivo, comparando ao cultivo já estabelecido em garrafas estáticas. De maneira geral, os resultados obtidos mostraram que foi possível expandir CMM utilizando a tecnologia de microcarregadores. A análise do metabolismo celular mostrou que não houve exaustão de nutrientes importantes como glicose e glutamina durante o cultivo, tampouco formação dos subprodutos lactato e amônia em concentrações inibitórias. As células recuperadas após a expansão mantiveram as características imunofenotípicas e funcionais. A produção média (n=10) foi de aproximadamente 4,9x105 cel/mL. Como o sistema utilizado permite o escalonamento, se utilizássemos um biorreator de 1L, seria possível a produção de aproximadamente 5x108 células que seriam suficientes para tratar mais de 3 pacientes de até 70Kg na dose de 2x106 células/Kg. Para expansão da mesma quantidade de células na forma tradicional seriam necessárias 135 garrafas de 175 cm2 com um custo total de expansão duas vezes superior à estimativa do custo de expansão utilizando microcarregadores. / Multipotent mesenchymal stromal cells are currently an attractive source for applications in tissue engineering and cell therapy. Due to the low availability in tissues (0,01%-0,0005%) and the high doses necessary for an infusion (about 106 cells/Kg patient), it has become necessary the development of effective and low cost technologies for in vitro expansion that enable to obtain MSC with maintenance of functional (differentiation and inhibition of lymphocytes proliferation), immunophenotypic and cytogenetics characteristics. MSC are adherent cells, i.e., they need a solid substrate to adhere and proliferate. The conventional procedure for expansion in static flasks normally involves a laborious process in which there is no suitable control and monitoring of the cultivation parameters besides presenting a higher susceptibility to contamination due to excessive manipulation to reach the ideal amount of cells. Moreover, this kind of cultivation does not allow a large scale production. For this reason, this work was proposed with the objective to develop a low cost, effective and scalable bioprocess for expansion of bone marrow-derived MSC in microcarriers. Cells grew on microcarriers Cyotdex 3, in spinner flasks with the -MEM medium supplemented with 15% FBS. We evaluated the cell adhesion to microcarriers, growth, metabolism, final cell recovery, and the functional and immunophenotypic properties before and after cultivation, comparing them with the cultivation already established in static flasks. In general, the results obtained showed that it was possible to expand MSC using microcarriers technology. The analysis of the cell metabolism showed that there was no depletion of important nutrients such as glucose and glutamine during cultivation, neither formation of lactate and ammonia subproducts in inhibitory concentrations. The cells recovered after the expansion kept the immunophenotypic and functional characteristics. The mean production (n=10) was about 4,9x105 cel/mL. As the system used allows the scale-up, if we had used a bioreactor of 1L it would had been possible to produce approximately 5x108 cells that would be enough to treat more than three patients of up to 70kg with a dose of 2x106 cells/kg. For the expansion of the same amount of cells in the traditional way, it would be necessary 135 T-flasks of 175 cm2 with total cost twice higher than the estimate cost of expansion using microcarriers.

Since the discovery of DNA packaging into chromatin, and McClintock's (1951) work on position-effect variegation providing evidence of non-mendelian inheritance, the principal of a genome maintaining 'on' and 'off' states has been widely adopted. However, the underlying mechanisms that regulate these dynamic chromatin states and their effect on disease are still poorly understood. DNA methylation and histone trimethylation at H3K9 and H4K20 are the core hallmarks of the heterochromatic constitutively 'off' state. Constitutive heterochromatin is predominantly comprised of repetitive satellite containing pericentromeric regions and telomeres and in mouse heterochromatin clusters into large chromocenters. These regions are cytologically more compact and generally transcriptionally silent across embryonic and differentiated mouse cell types. However, in addition to increased genomic instability, mouse tumour cells sustain increased satellite expression suggesting constitutive heterochromatin is disrupted. Therefore how constitutive heterochromatin is maintained has important implications for genome regulation and disease, and remains poorly understood. While satellite DNA sequences are not evolutionarily conserved, pericentromeric and telomeric heterochromatin occurs across species. Heterochromatin formation is therefore independent of the underlying DNA sequence, supporting the hypothesis that epigenetic components can regulate chromatin structure. DNA methylation is generally thought to be associated with transcriptional silencing and chromatin compaction. However, Gilbert et al (2007) showed that the complete loss of DNA methylation did not affect the compaction at heterochromatin or global genome compaction. The role of H3K9me3 in regulating heterochromatin has also been an area of keen interest. H3K9me3 patterns are established by suppressor of variegation 3-9 homologues and provide the binding site for heterochromatic protein 1 [HP1] which can in turn recruit Suv39h1. This Suv3-9h-HP1-H3K9 axis enables its propagation throughout heterochromatin. Peters et al (2001) demonstrated that in mice loss of suv39 homologues 1 and 2 caused a loss of H3K9me3 at constitutive heterochromatic domains. These Suv39h null mice demonstrated decreased genome stability, and an increased prevalence of oncogenesis. However cytological chromocenters are still present in the absence of H3K9me3. Therefore the function of H3K9me3 as a causative agent in heterochromatin formation is still debated. Broadly the aim was to investigate the phenotypic role of heterochromatic epigenetic components in cancer progression, and address whether H3K9me3 effects large scale chromatin structure. To identify heterochromatic gene silencing components, an inhibitor screen was performed in an artificial silenced reporter system. The reporter fluorophore was silenced by the presence of centromeric arrays from yeast/bacterial artificial chromosomes and human alpha satellite repeats enriched for H3K9me3. To address the function of the de-silencing components identified in cancer, the fitness of colon cancer cells [HCT116] was investigated before and after the development of resistance to the MEK inhibitor trametinib. The most intriguing result was that BET protein inhibition resulted in derepression of the reporter construct and trametinib resistant HCT116 cells were more sensitive to BET inhibitors, while subsequent investigation showed HP1 protein levels were altered. Analysis of publically available datasets of tumour drug resistance, showed elevated BET protein binding at HP1 promoters in resistant cell lines suggesting an indirect role in gene silencing. To investigate the consequence of H3K9me3 loss on chromatin structure, mouse embryonic stem cells that lacked both Suv39 homologues were used. Microccocal nuclease digestion and sucrose sedimentation demonstrated a global decompaction of large-scale chromatin fibres whilst re-expression of suv39h1 rescued H3K9me3 at chromocenters and global chromatin decompaction. Loss of Suv39h also increased chromatin associated RNA levels that were also rescued by Suv39h1 re-expression. This suggests that H3K9me3 has a role chromatin fibre compaction globally as well as at constitutive heterochromatin, potentially mediated by chromatin associated RNA. To conclude, multiple components were identified that are involved in transcriptional silencing. Evaluating their function in tumour progression demonstrated a possible role of BET proteins in the development of MEKi resistance that may be mediated through HP1 proteins. H3K9me3 and its binding partner HP1 affect global chromatin compaction. The global decompaction after Suv39h loss correlates with an increase in chromatin associated RNA, suggesting a possible mechanism for changes in chromatin compaction beyond H3K9me3.

A esclerose múltipla é uma doença inflamatória crônica desencadeada por células T autorreativas contra antígenos proteicos da mielina. A encefalomielite experimental autoimune é o modelo murino mais utilizado para o estudo da EM. As tubas uterinas e o útero de camundongos fêmeas são órgãos ricos em células mesenquimais que são pouco utilizadas em estudos. Dessa forma, no presente projeto, caracterizamos a obtenção dessa população e avaliamos sua capacidade imunossupressora utilizando o modelo de EAE. Observamos que o tratamento é capaz de modular o perfil de linfócitos T CD4+ durante sua ativação nos linfonodos, induzindo o direcionamento para a subpopulação Tr1 e atenuando as Th1 e Th17. Assim, houve uma diminuição do número de células infiltrantes no SNC associado a uma menor ativação de células da microglia. Em conjunto, demostraramos que as meMSC utilizadas como tratamento são capazes de atrasar o desenvolvimento da EAE e, portanto, evidenciando o caráter imunomodulador das MSCs derivadas do endométrio, chamando a atenção para seu potencial terapêutico. / Multiple sclerosis is a chronic inflammatory disease triggered by autoreactive T cells against myelin protein. Experimental Autoimmune Encephalomyelitis is the most commonly used murine model for the study of MS. The uterine tubes and uterus of female mice are organs rich in mesenchymal cells which are rarely used. Thus, in the present work, we characterized the extraction of this population and evaluated its immunosuppressive capacity using the EAE model. We observed that the meMSC treatment is capable of modulating the CD4 T lymphocyte profile during its activation in the lymph nodes, inducing the expansion of the Tr1 subpopulation and attenuating Th1 and Th17. Consequently, there was a decrease in the number of infiltrating cells in the CNS associated with a reduction of microglial activation. Taken together, our results demonstrated that the meMSCs used as treatment are capable of delaying the development of EAE, therefore, evidencing its immunomodulatory features drawing attention to its therapeutic potential.

As distrofias musculares progressivas (DMP) são um grupo de doenças genéticas hereditárias caracterizadas pela degeneração progressiva e irreversível da musculatura esquelética. A distrofia muscular de Duchenne (DMD) é a forma mais comum e mais grave de DMP, com prevalência de 1 a cada 3500 a 5000 meninos. Em geral, a perda da ambulação ocorre entre 9 a 12 anos e complicações respiratórias e cardíacas podem levar ao óbito a partir da segunda década. A pesquisa em terapia celular iniciou-se com o objetivo de reverter ou diminuir a progressão do processo distrófico através do repovoamento do músculo com células normais. Atualmente, acredita-se em um benefício terapêutico com base nas propriedades anti-inflamatórias, anti-fibróticas e imunomodulatórias das células tronco adultas (CTA). As CTAs mesenquimais são bastante heterogêneas quanto à sua composição celular o que ocasiona inconsistência de resultados. Por isso, a caracterização e separação de sub-populações através de marcadores específicos e o enriquecimento de culturas de CTA com um subtipo celular de interesse pode aumentar a robustez e o efeito das terapias. Uma dessas subpopulações é o pericito que, ao contrário das CTAs mesenquimais, foi bem descrito quanto à sua localização e função in vivo. Além disso, pericitos derivados de tecido adiposo humano aumentaram a sobrevida de camundongos duplo mutantes para distrofina e utrofina (dko). Dessa forma, este trabalho pretendeu comparar o potencial terapêutico de CTAs mesenquimais e pericitos de um mesmo tecido adiposo em camundongos dko. Conseguimos confirmar o resultado anterior, mostrando que os pericitos tendem a melhorar a sobrevida de animais tratados, sendo ainda melhores do que células mesenquimais, mas a melhora perdura somente durante o tratamento. A sobrevida é maior no começo do tratamento, sugerindo que o quanto antes o tratamento for iniciado, com animais mais jovens e sintomas mais leves, melhor poderá ser o resultado. Outras perguntas a serem pesquisadas na tentativa de melhorar o efeito terapêutico da terapia celular com pericitos são: número de injeções, quantidade de células a serem injetadas, tempo de tratamento e idade das células \"doadoras\" / Progressive muscular dystrophies (PMD) are inherited genetic diseases characterized by progressive muscle loss and weakness. Duchenne muscular dystrophy (DMD) is the most common and aggressive form of PMD, with incidence of 1 in every 3500-5000 boys. In general, patients with DMD are confined to wheelchairs around 9-12 years of age and death occurs due to respiratory and heart dysfunction after the second decade. Cell therapy research at first aimed to recover or slow down the dystrophic process by repopulating the patient\'s muscle with normal cells. However, nowadays it is believed also that therapeutic benefits occur by the anti-inflammatory, anti-fibrotic and immunomodulation properties of mesenchymal stem cells (MSC). MSC are constituted by an heterogeneous cell population and therefore, cell sorting of the subpopulation cell of interest is being done routinely. By doing this enrichment, the effect can be more robust and powerful. One of the cell populations of interest for research is pericyte, which are cells well defined regarding their in vivo function and location, as opposed to MSC. Besides that, pericytes derived from adipose tissue were successful in increasing survival of double knockout mice for dystrophin and utrophin (dko). The present work aimed to compare the therapeutic potential of MSC and pericytes derived from the same adipose tissue sample in the dko mouse model. We confirmed our previous results, showing that pericytes tend to improve the survival of treated mice, and are even better than MSC from the same source but the trend was statistically significant only during the treatment period. Additionally, we also observed that the survival was better in the beginning of treatment, suggesting that earlier treatment may lead to a better therapeutic effect. In an attempt to increase the therapeutic effect of these procedure other questions to be asked are: the number of injections and number of cells per injection, the duration of the treatment and the \"age\" of the donor cells

One of the leading causes of death and hospital stays in the United States, myocardial infarction (MI) occurs when coronary blockages lead to downstream ischemia in the myocardium. Following the MI, the heart activates a number of pathways to repair or remodel the infarcted zone. Endothelial cells respond to ischemia by de-differentiating to form neovasculature and myofibroblasts. The resident cardiac differentiable stem cells (CDCs) are recruited via local cytokines and chemokines to the infarct zone where they too differentiate into myofibroblasts. Mesenchymal stem cells (MSCs) of the bone marrow respond to circulating factors by immobilizing to the heart and differentiating down cardiac lineages. In regenerative medicine approaches, these processes are exploited to augment the resident supply of reparative cells. Clinical trials to transplant cardiac stem cells into MI zones have been met with mixed results. When CDCs are harvested from autologous or type-matched donors, the cells are prepared with a minimum of manipulations, but the yield is quite small. Conversely, MSCs from bone marrow are highly proliferative, but the manipulations in culture required to trigger cardiac differentiation have been found to transform the cell into a more immunogenic phenotype. In addition, there is a dearth of in vivo evidence for the fate of transplanted cells. Currently, intracardiac echocardiographs are used to assess the infarcted area and to guide delivery of stem cell transplants. However, this modality is invasive, short-term, and does not image the transplanted cells directly. In this project, I addressed these shortcomings with a regenerative medicine and bioimaging approach. Our lab has developed multimodal nanoparticles based on a core of mesoporous silica, functionalized with fluorescein or tetramethylrhodamine isothiocyanate for visibility in fluorescent microscopy, Gd2O3 for magnetic resonance imaging (MRI), and trifluoropropyl moieties for ultrasound applications. After establishing in vitro models of cardiac stem cells using CDCs and MSCs, the particles were implemented and characterized in vitro. At a concentration of 125 μg/mL in culture, the particles are highly biocompatible, and labeled cells were found to be fluorescent, echogenic, and detectable with MRI in prepared agar phantoms. Ex vivo mouse hearts, first mounted in agar phantoms, then left in situ, were implemented as a model for guided delivery using ultrasound and follow-up cell tracking with MRI. These results in this project demonstrate the feasibility of this highly novel and practical approach. Additional studies will be carried out to evaluate the biocompatibility and retention versus clearance in live animal models, prior to the carrying out of true pre-clinical models for myocardial infarction.

Mesenchymal stromal cells (MSCs) are being explored for treatment of inflammatory, ischemic, autoimmune, and degenerative diseases. More and more of these diseases require MSCs to be delivered locally to the diseased site rather than systemically injected into patients. However, little is understood about whether cell cryopreservation or prelicensing will affect the efficacy of the locally injected product or how the local injection environment affects MSC expression of trophic factors and interactions with patient immune cells. Several groups have disagreed on whether cryopreservation hinders MSC potency and therefore it is important to understand the effects of cryopreservation on MSC function and in what contexts cryopreservation can be used. Therefore, a better understanding of MSC phenotype after local injection is needed so that cryopreservation and prelicensing can be optimized to modulate cell potency for more efficacious MSC products. Currently, it has been shown that in vivo there are rapid drastic shifts in gene expression by MSCs which have been locally injected. One of the most prominent gene changes is in the enzyme COX-2 which leads to the production of bioactive lipids called prostaglandins, namely PGE2. PGE2 has several functions depending on the context in which other cells encounter it. In order to model the gene changes that occur in vivo, in vitro cell aggregates termed spheroids have been utilized to study the effects of local injection of MSCs. MSC spheroids have shown more potency than their 2D counterparts in shifting macrophage polarization and rescue of cells from ischemic damage. This thesis examines how process variables like cryopreservation and prelicensing affect the efficacy of the MSC product in the context of local injection. Additionally, it shows how spheroid formation alters therapeutic factor expression and activity and how drug treatment and biomaterials can be utilized to modify potency of these cells. In Chapter 2, we demonstrate that cryopreservation in the context of an ischemia/reperfusion injury in the eye does not significantly decrease MSCs effectiveness in salvaging neuronal cells. However, IFN-γ, a commonly used prelicensing cytokine to increase MSC potency, led to a decrease in the effectiveness of MSCs in this model. Chapters 3 and 4 define the changes that occur to several of MSCs’ trophic factors including immunomodulatory and growth factors and how these alterations affect MSC interactions with macrophages and T cells. Because validation and tracking of locally injected products can be cost-prohibitive for many research groups, Chapter 5 lays out a low-cost method to track fluorescently labeled cells in local injections to skin to aid in minimization of variability in results obtained from animal wound healing models. These findings demonstrate that initial preparation of MSC therapeutics is critical to their efficacy in local injection. Therefore, careful testing of potency for large-scale MSC production pipelines should be evaluated to ensure the efficacy of the resulting product. Additionally, spheroids exhibit differences in the mechanisms of action due to alterations in their secretome which can be partly overcome with co-administration of steroids such as budesonide. Therefore, steroid co-administration with MSCs being considered for local application should be further explored for use in local delivery of MSCs for the treatment of inflammatory conditions. Finally, this research demonstrates the need to further understand the mechanisms by which spheroids alter their gene and trophic factor production to better tailor MSC therapies for disease specific localized injection.