Orientador: Alexandre Leite Rodrigues de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T08:37:52Z (GMT). No. of bitstreams: 1 Cartarozzi_LucianaPolitti_M.pdf: 5081962 bytes, checksum: 794c493e497a269465a5b035175dfbcb (MD5) Previous issue date: 2013 / Resumo: A regeneração nervosa periférica é um processo complexo dependente do rearranjo e ativação das células de Schwann. O estímulo das células de Schwann pode ser alcançado através do enxerto de células tronco exógenas. Com o intuito de entender a importância do enxerto de células tronco mesenquimais (MSC) no processo regenerativo periférico, utilizamos o modelo de tubulização do nervo isquiático. As próteses tubulares foram preparadas a partir de membranas de poli-caprolactona (PCL) e preenchidas com selante de fibrina (FG), utilizado, neste caso, como substrato para as MSC. A técnica de tubulização foi feita em ratas fêmeas Lewis adultas, divididas em 4 grupos (n = 5 por grupo): normal, PCL (tubo vazio); FG (tubo preenchido com selante de fibrina) e FG+MSC (tubo preenchido com selante de fibrina e enxertado com MSC). Sessenta dias após lesão, os nervos regenerados foram processados para imunoistoquímica e microscopia de luz. A presença de MSC GFP-positivas foi detectada nos nervos dos animais que receberam enxerto de MSC, indicando que a sobrevivência, a longo prazo, das células tronco no tecido. A regeneração axonal, analisada por imunoistoquímica, revelou expressão de elementos básicos do nervo periférico, ou seja, componentes dos axônios e da lâmina basal tiveram a expressão equivalente em todos os grupos experimentais. A organização axonal foi observada através da marcação anti-neurofilamento. A presença das células de Schwann foi analisada através da marcação anti-S100 e o anticorpo anti-colágeno IV foi utilizado para detecção da lâmina basal. A imunomarcação anti-p75NTR, o receptor de baixa afinidade para neurotrofinas, foi utilizada para investigar a reatividade das células de Schwann. A marcação basal deste, em nervos não lesionados, foi aumentada pelo processo regenerativo, sendo estatisticamente maior no grupo FG+MSC (77% em relação ao nervo contralateral; p<0.001). Além disso, houve colocalização de MSC GFP-positivas e imunomarcação anti-BDNF, evidenciando uma possível via de atuação das células sobre o comportamento das células de Schwann. A partir da análise das secções semi-finas dos nervos pudemos avaliar que a área dos nervos regenerados no interior das próteses tubulares foi estatisticamente igual nos diferentes grupos experimentais. Quando quantificamos o número de axônios mielinizados por uma área fixa, o grupo FG+MSC apresentou maior densidade de axônios em relação ao grupo controle (25%, p<0,05). Da mesma maneira, quando analisamos os parâmetros morfométricos nos diferentes grupos experimentais, o grupo FG+MSC apresentou uma tendência a apresentar axônios de maior calibre e bainha de mielina mais espessa, em relação aos demais grupos, sendo que, a EBM, no intervalo de 1,46 a 2,25?m, foi significantemente maior em relação aos grupos PCL e FG (p<0,05). Como consequência, os animais do grupo FG+MSC mostraram recuperação motora significativamente maior na sétima e oitava semana de análise do índice funcional do nervo fibular. Os achados deste estudo mostram que as MSC enxertadas conjuntamente com selante de fibrina influenciam positivamente o processo regenerativo, modulando a reatividade das células de Schwann / Abstract: Peripheral nerve regeneration is a complex process that is dependent on the rearrangement and activation of Schwann Cells (SC). Such stimulation of SCs may be achieved by the use of exogenous stem cells. In order to better understand the importance of mesenchymal stem cell (MSC) grafting in the peripheral regeneration process we have used the model of sciatic nerve tubulization. Tubular prostheses were prepared from polycaprolactone (PCL) membranes and filled with fibrin sealant (FS), which was used as a substrate for the MSC. The technique of tubulization was applied in adult Lewis female rats that were divided into four groups (n = 5 per group): normal, PCL (empty tube), FS (tube filled with fibrin sealant) and FS + MSC (tube filled with fibrin sealant and grafted with MSC). Sixty days after injury, the regenerated nerves were processed for imunohistochemistry and observed under fluorescence microscopy. The presence of GFP positive stem cells was detected in the nerves of the animals that received MSC grafts, indicating the long term survival of such cells. The axonal regeneration process was studied by immunohistochemistry and revealed the presence of the basic elements of the peripheral nerve, namely axons and basal lamina components that were equivalent in all experimental groups. The axonal organization was observed with anti-neurofilament immunostaining. The presence of SCs was analyzed with anti-S100 immunostaining and anti-type IV collagen was used to detect the basal lamina. Anti-p75NTR, the low affinity receptor for neurotrophins, was used to investigate the reactivity of the SCs. A basal positive labeling in uninjured nerves was detected, which was upregulated by the regenerative process, being statistically higher in FS + MSC group (77% relative to uninjured nerve; p<0.001). Moreover there was colocalization between GFP-positive MSC and anti-BNDF immunolabeling, showing a possible pathway that these cells induce the reactivity of SCs. From sciatic nerve semi-thin sections we were able to evaluate that the areas of regenerated nerves were statistically the same in the different experimental groups. When we quantified the number of myelinated axons in 50.000?m2, the FG+MSC group showed higher density of axons when compared with PCL group (25%, p<0,05). In the same way, the analysis of morphometric parameters showed that the FG+MSC group have a tendency to present higher caliber axons and ticker myelin sheath when compared with other groups, being that the myelin sheath thickness, in the interval between 1,46 to 2,25?m, was significantly higher in FG+MSC group when compared to PCL and FG (p<0,05). As the functional result of the findings above, the FG+MSC animals showed higher motor function recovery, analyzed by FFI, at seventh and eighth weeks after lesion. The findings herein show that MSC associated with the FS scaffold improve the regeneration process by positively modulating the reactivity of SCs / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

Master of Science in Biomedical Sciences / Department of Anatomy and Physiology / Mark L. Weiss / Mesenchymal stromal cells (MSCs) derived from the umbilical cord (UC-MSCs) have therapeutic applications and are studied to understand their potential uses and immunomodulatory properties. Research must identify good manufacturing process (GMP) compliant methods to isolate and expand UC-MSCs. In addition, MSCs metabolism characteristics in culture are unknown, warranting further investigation. Viability of MSCs decreases after cryopreservation, which is detrimental to clinical translation. Previously published methods used to isolate MSCs from the umbilical cord included open dissection steps and xenogeneic components. Here, I developed improved methods by eliminating dissection which reduces contamination risks. Instead, I used the whole umbilical cord and Miltenyi dissociator tubes to mechanically and enzymatically dissociate cells in a closed system. Xenogeneic components were decreased by using medium containing pooled human platelet lysate instead of fetal bovine serum. The cell numbers isolated from umbilical cord averaged 2.68 x 10⁵ per cm, which represents greater than 20 fold improvement over the previous method. Moreover, expansion cell numbers were increased using 10% pooled human platelet lysate supplemented media. The UC-MSCs generated here met the International Society of Cell Therapy (ISCT) definition of MSCs. Metabolism characteristics of MSCs indicated that glucose was the critical metabolite, maintaining cells longer in culture than glutamine. Cell death followed depletion of glucose, too. Finally, the average viability after thawing cryopreserved MSCs was more than 95%, higher than previous methods. The improvements I introduced to our methodology could speed clinical translation of MSCs as an allogeneic cellular therapy

La grande originalité de mon projet réside dans la génération de cellules stromales mésenchymateuses (MSCs) à partir de cellules souches pluripotentes induites humaines (iPS). Je rappellerai les propriétés phénotypiques, de multipotence et immunosuppressives des MSCs et m’attarderai sur leurs différents mécanismes immunomodulateurs. Cependant, leur nombre limité et leur isolation difficile limitent leur utilisation thérapeutique nécessitant une autre source de cellules.Mon travail a donc été de générer et de caractériser des MSCs issues d’iPS (huiPS-MSCs). L'avantage des huiPS-MSCs réside dans leur plus grande disponibilité et la possibilité d'en avoir à volonté. Encore faut-il valider l’intérêt thérapeutique potentiel de ces huiPS-MSCs. Premièrement, mes résultats in vitro montrent que les huiPS-MSCs présentent une activité immunosuppressive sur les lymphocytes T (LT) activés conduisant à une induction de LT régulateurs FoxP3+ fonctionnels. Deuxièmement, dans une approche plus axée sur la thérapie, j’ai analysé in vivo l’activité́ immunosuppressive des huiPS-MSCs dans un modèle de réaction xénogénique de greffon contre l'hôte (souris immunodéficientes NSG injectées avec des LT humains). Je montre clairement, après traitement avec les huiPS-MSCs, une réduction de la proportion de LT humains producteurs de cytokines inflammatoires (IFNγ et TNFα) typiques de la pathologie et l’apparition concomitante de LT présentant un phénotype régulateur (production d’IL10 et expression de FoxP3). La fin de mon travail a été de caractériser moléculairement la régulation de l’expression de PD-L1, une molécule immuno-régulatrice puissante, entre les MSCs issues de la moelle osseuse (BM-MSCs) de donneurs sains et nos huiPS- MSCs. Les huiPS-MSCs ont une expression constitutive de PD-L1, qui est absente sur les BM-MSCs. J’ai analysé les microARNs susceptibles de limiter l’expression de PD-L1, j’ai pu en identifier plusieurs. En mesurant leur expression dans les différentes MSCs à notre disposition, je montre que cette expression est inverse par rapport à celle de PD-L1. J’ai ainsi pu démontrer l’activité immunosuppressive de nos huiPS-MSCs in vitro et in vivo avec une perspective d’induction de tolérance immune, et caractériser la régulation de l’expression de PD-L1, molécule immunosuppressive exprimée par les huiPS-MSCs. / The mesenchymal stromal cells (MSCs) present many features that render attractive as therapeutic cells. Their phenotype, multipotency and immunosuppressive properties are well described. Nevertheless, major restriction for their clinical use is due to the limited in vitro expansion and low quantity of cells that can be collected from adult tissues. The originality of my project consisted in the generation of mesenchymal stromal cells (MSCs) from human induced pluripotent stem cells (iPS). These huiPS-MSCs could fulfill some of the specification required to improve MSCs use in therapeutic approaches: welldefined and unlimited number of cells with reproducible functional characteristics. In a first approach, I characterized the huiPS-MSCs generated in the laboratory. My results highlight the immunosuppressive activity in vitro of the huiPS-MSCs on T-cell stimulation that induces a switch in T-cell cytokine polarization toward the generation of Treg cells. Secondly, in a more therapy-oriented approach, I analyzed in vivo immunosuppressive activity of huiPS-MSCs in a xenogeneic graft versus host model (NSG immunodeficient mice injected with human T lymphocytes). My data showed significantly reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines (IFNγ and TNFα). By contrast, T cells producing IL-10 and FoxP3+ Treg cells, absent in nontreated animals, were detected in huiPS-MSCs treated mice, confirming the in vitro results of a tolerizing process. The end of my work was to characterize the molecular regulation of the expression of PDL1, an immunoregulatory molecule expressed by the MSCs. Comparing bone marrow MSCs (BM-MSCs) from healthy donors and our huiPS-MSCs, I showed that the huiPSMSCs have a constitutive expression of PD-L1, which is absent on BM-MSCs. Analysing microRNAs that could limit the expression of PD-L1, I could identify several microRNAs which expression is inverse to the expression of PD-L1. For the first time, my results highlight the immunosuppressive activity of huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo and characterize the regulation of PD-L1 expression, an immunosuppressive molecule expressed by the MSCs. They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance.

Introduction : L’efficacité de la greffe d’îlots n’est plus à démontrer mais elle reste l’objet de recherches pour améliorer la qualité et la survie des îlots greffés souvent fragilisés par la destruction enzymatique de leur microenvironnement lors de la procédure d’isolement. Dans ce contexte, les cellules stromales mésenchymateuses (CSM) d’origine pancréatique représentent un outil intéressant par leurs propriétés d’immunomodulation et par leur capacité de sécrétion de facteurs du microenvironnement. L’objectif de ce travail est d’évaluer l’effet des cellules stromales pancréatiques humaines sur les cellules β humaines.Méthodes : Des îlots humains purifiés ont été maintenus en culture pendant plusieurs jours. Les cellules adhérentes se formant en périphérie de l’îlot ont été sélectionnées et immortalisées. Ces nouvelles cellules « human islet-derived stromal cells » (hISC) ont ensuite été caractérisées pour déterminer leur profil mésenchymateux Nous avons ensemencé des cellules β humaines (lignée EndoC-βH1 ou cellules primaires) sur du milieu conditionné de hISC (hISC-CM) utilisé comme support de culture. L’adhérence, la survie, la prolifération, l’insulinosécrétion des cellules β cultivées sur le hISC-CM ont été mesurées et comparées à un support contrôle : la poly-L-lysine.Résultats : Les hISC présentent un profil phénotypique et transcriptomique très proche des CSM issues de la moelle osseuse. D’un point de vue fonctionnel, les hISC présentent une capacité d’immunomodulation. Elles expriment et sécrètent des protéines matricielles connues pour être présentes autour et à l’intérieur des îlots humains tels que les collagènes de type I, IV et VI, la laminine et la fibronectine. Au contact du hISC-CM les cellules EndoC-βH sur adhèrent st s’étalent. Le hISC-CM augmente l’expression du marqueur de prolifération PCNA et améliore la survie et la fonction des cellules EndoC-βH1. D’un point de vue mécanistique, l’interaction cellules β/hISC-CM active la phosphorylation de FAK (focal adhesion kinase) et ERK (extracellular signal-regulated kinases). A l’interface de cette interaction, la sous-unité β1 de l’intégrine est impliquée dans les effets observés du hISC-CM sur l’adhérence et la fonction des cellules β.Conclusion : Nos travaux démontrent l’intérêt prometteur des hISC en tant que cellules de soutien des cellules β humaines par la sécrétion de protéines matricielles pancréatiques. Ces résultats ouvrent de nouvelles perspectives pour le maintien des îlots en culture et leur conditionnement dans un microenvironnement plus physiologique permettant ainsi de préserver leur qualité fonctionnelle lors de la greffe. / Introduction : The efficacy of islet transplantation is well established. However, the procedure still needs improvements in the quality of grafted islets, often weakened by the loss of their surrounding tissue during the isolation process. In this respect, mesenchymal stromal cells (MSC) represent an interesting tool as they have immunomodulatory and anti-inflammatory properties and are known to secrete proteins involved in creating a favorable microenvironment. This work aims to investigate the effect of human pancreatic stromal cells on human β-cells.Methods : We characterized the mesenchymal profile of cells, previously immortalized in our lab from human islets of Langerhans adherent cells, hereafter named hISC (human islet-derived stromal cells). We seeded human β-cells (EndoC-βH1 cell line or primary β-cells) on hISC-conditioned medium (hISC-CM) used as coating of Petri dishes. We assessed spreading, survival, proliferation and glucose-induced insulin secretion of β-cells cultured on hISC-CM as compared to poly-L-lysine coating.Results : Phenotypic and transcriptomic profiles of hISC are close to bone-marrow MSC. The hISC have an immunomodulation capacity. They express and secrete extracellular matrix proteins known to be present around and within human islet such as types I, IV and VI collagens, laminin and fibronectin. EndoC-βH1 seeded on hISC-CM adhere and spread on cell culture surface. We show that hISC-CM has positive effects on EndocC-βH1 proliferation, survival and glucose-induced insulin secretion, as compared to poly-L-lysine. From mechanistic point of view, hISC-CM induces FAK (focal adhesion kinase) and ERK (extracellular signal-regulated kinases) phosphorylations. The β1-integrin subunit is involved in both adhesion and increased insulin secretion of β cells induced through hISC-CM.Conclusion : Our work demonstrates a promising interest of hISC as support cells for human β-cells by scaffolding factors secretion. It opens new perspectives for conditioning human β-cells in a more physiological microenvironment to preserve their functional quality before and after transplantation.

Orientador: Rogério Martins Amorim / Coorientador: Ana Liz Garcia Alves / Banca: Rui Seabra Ferreira Junior / Banca: Fernanda da Cruz Landim / Resumo: Estudos demonstram o grande potencial do uso das células estromais mesenquimais multipotentes (MSCs) como terapia celular. Seu uso em lesões neurológicas, que usualmente apresentam difícil regeneração, tem sido estudado pelo fato das MSCs apresentarem baixa imunogenicidade efeitos imunomoduladores e neuroregenerativos. Nesse contexto, o presente estudo avaliou a viabilidade e a segurança do transplante de MSCs alogênicas provenientes do tecido adiposo, medula óssea e cordão umbilical de equinos, pela via intratecal através do espaço intervertebral C1-C2, por meio de exame neurológico seriado, análises hematológicas e determinação dos níveis séricos de proteínas de fase aguda. Foram utilizados 16 equinos saudáveis, divididos em quatro grupos: grupo SHAM (SHAM) que recebeu o transplante de salina tamponada com fosfato (PBS); grupo tecido adiposo (GTA), recebeu MSCs de origem do tecido adiposo; grupo medula óssea (GMO), recebeu MSCs de origem da medula óssea; e grupo cordão umbilical (GCU), recebeu células de origem da matriz do cordão umbilical. Foram realizadas três aplicações com intervalo de 30 dias em cada grupo, e coletou-se amostras de sangue, nos momentos que antecederam os transplantes, M0, M30 e M60 e 24 horas, após o transplante, M1, M31, M61. E por último foi realizada uma coleta 30 dias após M60, caracterizando M90. Não foram observadas alterações do exame clinico, hematológico e nas proteínas de fase aguda relacionadas aos sucessivos transplantes intratecais de MSC... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Studies demonstrate the great potential of using multipotent mesenchymal stromal cells (MSCs) as cell therapy. Its use in neurological lesions, which usually present difficult regeneration, has been studied because MSCs have low immunogenicity and immunoregulatory and neuroregenerative effects. In this context, the present study evaluated the viability and safety of transplantation of allogeneic MSCs from the adipose tissue, bone marrow and umbilical cord of horses, through the intrathecal route through the C1-C2 intervertebral space, through serial neurological examination, hematological analyzes and determination of serum levels of acute phase proteins. Sixteen healthy horses were used, divided into four groups: SHAM group (SHAM) that received phosphate buffered saline (PBS) transplantation; adipose tissue group (GTA), received adipose tissue MSCs; bone marrow group (GMO), received MSCs from bone marrow origin; and umbilical cord (UGC) group, received cells from the umbilical cord array. M0, M30 and M60 were collected at the time of the transplantation, M1, M31 and M61 and 24 hours after transplantation. And finally a collection was performed 30 days after M60, characterizing M90. There were no changes in the clinical, hematological and acute phase-related proteins related to successive intrathecal transplantations of MSCs, nor were there alterations that contra indicated the use of any of the cellular sources, demonstrating that the protocol used, as well as any of cellula... (Complete abstract click electronic access below) / Mestre

Ischemic brain injury represents a major cause of death worldwide with limited treatment options with a narrow therapeutic window. Accordingly, novel treatments that extend the treatment from the early neuroprotective stage to the late regenerative phase may accommodate a much larger number of stroke patients. To this end, stem cell-based regenerative therapies may address this unmet clinical need. Several stem cell therapies have been tested as potentially exhibiting the capacity to regenerate the stroke brain. Based on the long track record and safety profile of transplantable stem cells for hematologic diseases, bone marrow-derived mesenchymal stromal cells or mesenchymal stromal cells have been widely tested in stroke animal models and have reached clinical trials. However, despite the translational promise of MSCs, probing cell function remains to be fully elucidated. Recognizing the multi-pronged cell death and survival processes that accompany stroke, here we review the literature on MSC definition, characterization, and mechanism of action in an effort to gain a better understanding towards optimizing its applications and functional outcomes in stroke. / National Institutes of Health / Revisión por pares

Orientador: Ana Liz Garcia Alves / Resumo: O transplante de células tronco mesenquimais (CTMs) em doenças articulares vem se demonstrando eficaz. O objetivo desse estudo foi avaliar a resposta imune de repetidas aplicações intra-articulares de CTMs alogênicas e a imunogenicidade destas, utilizando ensaios de microcitotoxicidade para avaliar as respostas de anticorpos citotoxicos dependentes de complemento. Quinze equinos adultos foram distribuídos aleatoriamente entre três grupos. Seis animais no grupo ALO onde articulações hígidas receberam CTMs alogênicas, seis no grupo ALO LPS em que as articulações foram previamente inflamadas, pela indução experimental da sinovite com 0,5ng de LPS e três animais no grupo AUTO que foi mantido como controle negativo e receberam CTMs autólogas. Foram realizadas 2 aplicações na mesma articulação de células tronco mesenquimais derivadas de membrana sinovial com intervalo de 4 semanas. Foram colhidas amostras seriadas nos 3 dias após cada aplicação e a cada 7 dias por 8 semanas do liquido sinovial para análise e soro sanguíneo para os ensaios de microcitotoxicidade e exame ultrassonográfico para avaliar efusão articular através da distancia entre a superfície do talus e a capsula articular. Para análise estatística foi utilizado os testes Two Way Repeated Measures ANOVA e teste Turkey's. Foi evidenciada uma reação inflamatória moderada pelo aumento da celularidade do liquido sinovial após as aplicações. Também, foi observado que após a segunda aplicação alogênica, as articulações apres... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Transplantation of mesenchymal stem cells (MSCs) in joint diseases has been shown to be effective. The objective of this study was to evaluate the immune response of repeated intra-articular applications of allogeneic MSCs and their immunogenicity using microcytotoxicity assays to evaluate complement dependent cytotoxic antibody responses. Fifteen adult horses were randomly assigned to three groups. Six animals in the ALO group where healthy joints received allogeneic MSCs, six in the ALO LPS group in which the joints were previously inflamed, by the experimental induction of synovitis with 0.5ng of LPS and three animals in the AUTO group that was maintained as negative control and received Autologous MSCs. Two applications were performed in the same articulation of mesenchymal stem cells derived from synovial membrane with interval of 4 weeks. Serial samples were taken 3 days after each application and every 7 days for 8 weeks of synovial fluid for analysis and blood serum for the microcytotoxicity and ultrasound examination to evaluate joint effusion through the distance between the surface of the talus and the joint capsule. Two-way ANOVA and Turkey's test were used for statistical analysis. A moderate inflammatory reaction was evidenced by increased synovial fluid cellularity after the applications. Also, it was observed that after the second allogeneic application, the joints presented effusion until 28 days after application when compared to the autologous applications ... (Complete abstract click electronic access below) / Mestre

Preterm birth (<37 weeks) is the world’s principal cause of death of children <5 years of age. Bronchopulmonary dysplasia (BPD) is the most common complication of preterm birth. BPD is characterized by an arrest in alveolar and vascular development within the lung. It is a multifactorial disease, caused by a combination of supplemental oxygen, mechanical ventilation, and inflammation. BPD is also an independent risk factor for abnormal neurodevelopment. The link between BPD and abnormal neurodevelopment is poorly understood and there are currently no effective cures for these complications. We hypothesized that a crucial cell population for brain development, i.e., the neural progenitor cell (NPC) is functionally impaired in BPD and that this impairment is associated with abnormal neurodevelopment. Based on our previous findings, we also predicted that human umbilical cord-mesenchymal stromal cell (UC-MSC) extracellular vesicles (EVs), could mitigate both the lung and brain injuries in experimental BPD. We utilized several animal models of BPD, across multiple species, to determine the effects of hyperoxia, mechanical ventilation, and inflammation on the developing lungs and brain. We also utilized UC- MSC therapy to mitigate these injuries. We discovered that hyperoxia exposure damages the developing lungs as well as the brain, leading to cerebrovascular and NPC impairments, as well as reduced neurogenesis. These impairments were associated with neurobehavioural deficits in adulthood. Furthermore, we found that inflammation in combination with mechanical ventilation and hyperoxia also impairs NPC function. Importantly, we demonstrated that UC-MSC EVs can reduce inflammation, improve vascular growth, restore lung growth, and mitigate impairments in NPC self-renewal. This work highlights novel mechanisms of BPD-associated abnormal neurodevelopment and offers potential regenerative medicine therapies to alleviate these outcomes for this vulnerable population.

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