O linfonodo é um órgão linfoide secundário que apresenta uma arquitetura altamente organizada com diferentes compartimentos para tipos celulares específicos. Dentre as células estruturais que compõem este órgão, as células estromais como células fibroblásticas reticulares (FRCs) e células duplo negativas (DNCs) parecem ter papel importante na modulação da resposta imunológica e na tolerância periférica. As FRCs são caracterizadas pela expressão de podoplanina (gp38, PDPN) e localizam-se principalmente na zona de células T, enquanto as DNCs (gp38-) apresentam fenótipo, localização e função pouco descritos. Embora estas células tenham sido muito estudadas em modelos murinos os estudos sobre FRCs e DNCs humanas são escassos e, portanto nosso estudo deve contribuir para a compreensão da biologia e a função dessas células, podendo favorecer o conhecimento sobre a eficiência e as disfunções da resposta imune no linfonodo. Com esse intuito, isolamos e caracterizamos fenotípica e funcionalmente as FRCs e DNCs de linfonodos de pacientes com câncer, diverticulite e doadores de fígado. Nossos resultados mostraram a integridade e a distribuição celular no linfonodo. As células aderentes derivadas dos linfonodos estudados preecheram todos os critérios internacionais de caracterização de estroma, e, portanto, foram consideradas células estromais. Através da expressão de gp38 identificamos duas subpopulações de celulas estromais: FRCs (gp38+ e CD31-) e DNCs (gp38- e CD31-) e verificamos que as frequências destas células variam entre as amostras, sugerindo que a doença pode interferir na composição celular estromal dos linfonodos. As duas populações celulares foram estimuladas com citocinas inflamatórias como IFN-y ou TNF-alfa + IL-1beta por 24 e 48 horas e avaliadas quanto à expressão gênica e proteica. Em condições homeostáticas, genes relacionados com a indução e controle da proliferação foram diferencialmente expressos nas FRCs e DNCs, este dado foi confirmado in vitro, uma vez que as FRCs apresentaram maior potencial proliferativo em relação às DNCs. O estímulo com IFN-y induziu aumento de expressão nas DNCs e FRCs para citocinas, quimiocinas, moléculas de histocompatibilidade e moléculas envolvidas na regulação da resposta imunológica. Em resposta ao estímulo com TNF-alfa +IL-1beta, observamos aumento na expressão de moléculas comuns ao estímulo com IFN-?, entretanto, também observamos expressão de moléculas de citocinas, quimiocinas inflamatórias e moléculas de histocmpatibilidade especificamente relacionados a este sinal em ambas as populações. Em conjunto, nossos dados sugerem que DNCs e FRCs apresentam diferenças no perfil de resposta segundo os estímulos inflamatórios aos quais estão expostas, aumentando a expressão diferencial de moléculas envolvidas na regulação positiva e negativa da resposta imune / The lymph node is a secondary lymphoid organ that has a highly organized architecture with different compartments for specific cell types. Among the structural cells that comprise this organ, stromal fibroblastic reticular cells (FRCs) and double negative cells (DNCs) seems to play an important role in modulating the immune response and peripheral tolerance. FRCs are characterized by podoplanin (gp38, PDPN) expression and are located mainly in the T cell zone, while DNCs (gp38-) present phenotype, location and function not well described. Although these cells have been studied in murine models, studies on human FRCs and DNCs are limited and therefore our study should contribute to the understanding of biology and function of these cells and should promote knowledge of efficiency and disorders in the lymph node immune response. For this purpose, we have isolated and characterized phenotypic and functionally lymph nodes derived FRCs and DNCs from patients with cancer, diverticulitis and liver donors. Our results showed lymph node integrity and its cellular distribution. Adherent cells lymph nodes-derived fullfill the international criteria for stroma characterization, and therefore, they have been considered stromal cells. Using gp38 expression we were able to identify two stromal cells subpopulations: FRCs (gp38 + and CD31-) and DNCs (gp38- and CD31-) and found that this cells frequency varies among samples, suggesting that the disease may interfere with lymph nodes stromal cell composition. These two cells populations were stimulated with inflammatory cytokines such as IFN-y or TNF-alfa + IL-1beta for 24 and 48 hours and evaluated for gene and protein expression. In homeostatic conditions, genes involved in the induction and control of proliferation were differentially expressed by FRCs and DNCs, this data has been confirmed in vitro, since the FRCs showed higher proliferative potential compared to DNCs. IFN-y stimulation induced increase DNCs and FRCs expression for cytokines, chemokines, histocompatibility molecules and molecules involved in regulating the immune response.In response to TNF-alfa + IL-1beta stimulation, we observed common molecules expressed by the IFN-? stimulation, however, we also observed expression of cytokines, chemokines and histocompatibility molecules specifically related to this signal in both cells populations. Together, our data suggest that DNCs and FRCs differ in the response profile according to inflammatory stimuli to which they are exposed, increasing the differential expression of molecules involved in the positive and negative regulation of immune response

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.

The contribution of the bone marrow microenvironment in myelodysplastic syndrome is controversial. We therefore analyzed the functional properties of primary mesenchymal stromal cells from patients with myelodysplastic syndrome in the presence or absence of lenalidomide. Compared to healthy controls, clonality and growth were reduced across all disease stages. Furthermore, differentiation defects and particular expression of adhesion and cell surface molecules (e.g. CD166, CD29, CD146) were detected. Interestingly, the levels of stromal derived factor 1-alpha in patients’ cells culture supernatants were almost 2-fold lower (P<0.01) than those in controls and this was paralleled by a reduced induction of migration of CD34+ hematopoietic cells. Co-cultures of mesenchymal stromal cells from patients with CD34+ cells from healthy donors resulted in reduced numbers of cobblestone area-forming cells and fewer colony-forming units. Exposure of stromal cells from patients and controls to lenalidomide led to a further reduction of stromal derived factor 1-alpha secretion and cobblestone area formation, respectively. Moreover, lenalidomide pretreatment of mesenchymal stromal cells from patients with low but not high-risk myelodysplastic syndrome was able to rescue impaired erythroid and myeloid colony formation of early hematopoietic progenitors. In conclusion, our analyses support the notion that the stromal microenvironment is involved in the pathophysiology of myelodysplastic syndrome thus representing a potential target for therapeutic interventions.

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.

Regenerative treatment of focal hyaline cartilage defects could prevent or delay the development of secondary osteoarthritis. Current surgical techniques result partly in i) the formation of mechanically inferior fibrous cartilage or ii) present the disadvantage of the donor site morbidity from harvesting cartilage biopsy as well as iii) the dedifferentiation of chondrocytes due to in vitro expansion and iv) the reduced re-differentiation potential of in vitro expanded chondrocytes. The self-healing capacities of injured and degenerated articular cartilage revealed a promising target cell population for a regenerative, autologous treatment of these defects using mesenchymal stromal cells (MSCs). Several case studies, randomized and controlled clinical studies showed the general ability of autologous, bone marrow-derived, expanded MSC transplantation to regenerate articular cartilage lesions [1]. However, these two-stage approaches are based on time- and cost-consuming expansion of MSCs under good manufacturing practice (GMP) conditions and hold a risk of contamination during this process. In 2010, CD271, the low-affinity nerve growth factor receptor, was described as a suitable surface marker to enrich MSCs from human bone marrow aspirate intraoperatively [2]. The aim of the present dissertation was to investigate the feasibility of generating cartilage grafts from either ovine (study no. 1) and human (study no. 2) non-expanded CD271+ bone marrow cells in a collagen type I hydrogel. Study no. 1 (“Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells - An in vitro proof-of-concept study”) investigated several surface marker candidates for the prospective MSC separation and examined their potential of resulting colony-forming units, respective their yield of potent MSCs [3]. This study was conducted with ovine bone marrow samples. CD271 was the most effective surface marker to isolate the target cell population. Subsequently, CD271+, CD271- and unseparated mononuclear cells (MNCs), containing the MSCs, were used to generate cartilage grafts without an expansion of these cells in monolayer culture. It could be proven, that ovine CD271+ cells were able to generate a potent hyaline cartilage graft. Study no. 2 (“Single-stage preparation of human cartilage grafts generated from bone marrow-derived CD271+ mononuclear cells”) was performed as the final translational step from animal-derived bone marrow to human donor material and is therefore strengthening the therapeutically focus of the entire work [4]. Briefly, eight bone marrow aspirates were used for MNC isolation and subsequent magnetic cell separation (MACS). The resulting CD271+ and CD271- MNCs were compared to unseparated MNCs. Subsequently, they were seeded in a clinically approved collagen type I hydrogel and cultivated for up to 5 weeks to investigate the progression of the chondrogenic differentiation processes. Graft analysis included cell viability visualization by live/dead staining, determination of the DNA and the secreted sulphated glycosaminoglycan (sGAG) content as well as the immunohistochemical staining for typical chondrogenic differentiation markers and the extracellular matrix molecules aggrecan and collagen type II. A proliferation of cells in the generated grafts was shown of CD271+ and unsep, but not CD271- MNCs. Hence, the cell number was 2.8-fold higher after 35 days compared to the first day for CD271+ MNCs grafts, while CD271- MNCs did not proliferate in the grafts and unsep MNCs showed only a slight increase in cell number. The chondrogenic potential was measured by quantification of freshly produced sGAGs and the expression of chondrogenic markers. In grafts with CD271+ MNCs, sGAG production increased over time and reached its maximum at day 35, whereas grafts with CD271- MNCs showed no measurable sGAG deposition. The amount of sGAG in unsep MNC grafts increased only slightly over the whole cultivation period. Aggrecan and collagen type II staining varied considerably between the MNCs donors. Collagen type II positive staining was observed in CD271+ MNC grafts (5/8 donors) and unsep MNC (2/8) grafts. In comparison to macroscopically healthy cartilage, three-dimensional grafts of the CD271+ group yielded a proceeding extracellular matrix production. In summary, CD271+ MNCs showed the highest proliferation rate, cell viability, sGAG deposition and cartilage marker expression compared to the CD271- or unseparated MNC fractions in in vitro generated three-dimensional cartilage grafts. Therefore, the presented work demonstrated the feasibility of generating a cartilage graft from CD271+ bone marrow-derived MNCs in a clinically approved collagen type I hydrogel without a previous monolayer expansion of these cells. This will enable the intraoperative purification of CD271+ MNCs, which contain the majority of colony-forming MSCs, by MACS technology. The clinical application will be possible with currently available and clinical approved cell separation devices. Providing a cartilage graft with non-expanded CD271+ MNCs by a fast and simple intraoperative therapeutic approach fulfils the need for a “single-step, tissue-engineered solution to focal cartilage defects, and elimination of the morbidity of the donor defect” as requested by the editors of the journal Arthroscopy [5]. References of the summary 1. Filardo G, et al. (2016). Stem cells in articular cartilage regeneration. J Orthop Surg Res 11:42. 2. Jones E, et al. (2010). Large-scale extraction and characterization of CD271+ multipotential stromal cells from trabecular bone in health and osteoarthritis: implications for bone regeneration strategies based on uncultured or minimally cultured multipotential stromal cells. Arthritis Rheum. 62:1944–1954. 3. Petters O, et al. (2018). Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells-An in vitro proof-of-concept study. J Tissue Eng Regen Med. 4. Petters O, et al. (2018). Single-Stage Preparation of Human Cartilage Grafts Generated from Bone Marrow-Derived CD271+ Mononuclear Cells. Stem Cells Dev 27:545–555. 5. Lubowitz JH and GG Poehling. (2009). Saving our cells: Advances in tissue engineering for focal cartilage defects. Arthroscopy: the journal of arthroscopic & related surgery: official publication of the Arthroscopy Association of North America and the International Arthroscopy Association 25:115–116.:1 Introduction 1 1.1 Articular cartilage 1 1.2 Cartilage lesions 2 1.3 Self-healing capability of articular cartilage 3 1.4 Treatment option for cartilage lesions 4 1.5 Mesenchymal stromal cells in cartilage regeneration 6 2 Rationale 8 3 Publication manuscripts 9 Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells - An in vitro proof-of-concept study 9 Single-stage preparation of human cartilage grafts generated from bone marrow-derived CD271+ mononuclear cells 21 4 Summary 33 5 References 35 6 Appendix 42 7 Declaration of Authorship 47 8 Curriculum vitae 48 9 Publications 49 10 Acknowledgements 49

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

Human esophageal squamous cell carcinoma (hESCC) is a very aggressive form of cancer due to its ability to easily metastasize into proximal lymph nodes and adjacent organs. The role of the extracellular matrix (ECM) and its stromal cells in metastasis remains unclear. To better understand the effect of the ECM and fibroblast cells on esophagus cancer cell migration and invasion, we propose a biomimetic human esophagus model cultured with hESCC and human primary fibroblast cells (fibroblast). To mimic the extracellular matrix of human esophagus we use decellularized porcine esophagus matrix (DEM) to culture with hESCC and fibroblasts in static conditions. This DEM can recapitulate the human esophagus tumor microenvironment with relevant cues. This model will provide valuable information regarding esophagus cancer cell migration with respect to the heterogeneous extracellular matrix and stromal fibroblast cells. We expect to discover the mechanisms by which extracellular matrix and stromal cells affect cancer migration and invasion in vitro. Characterizing this process will provide vital insight towards the effects of fibroblasts cells on facilitating migration and invasion of esophageal cancer cells. This esophagus cancer model also provides promising potential to study drug screening and develop new strategies against esophagus metastasis. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

Bone is an important organ influenced by mechanical load, hormones, nutrition and disease. During bone remodelling, osteoclasts resorb bone and osteoblasts form new bone. Osteoblasts are derived from mesenchymal stem cells (MSCs) such as adipose-derived stromal cells (ASCs). The mitogen-activated protein kinase (MAPK) pathway has been shown to interfere with osteoblast differentiation from an early stage. Runt related transcription factor 2 (RUNX2) exerts an effect downstream from p38 MAPK. RUNX2 phosphorylation by p38 MAPK may increase osteoblast differentiation markers such as alkaline phosphatase (ALP), osteoprotegerin (OPG) and receptor activator of nuclear factor kB ligand (RANKL). Palmitoleic acid (PLA), an omega-7 monounsaturated fatty acid (MUFA), promotes anti-osteoclastogenic effects, however, the effects of PLA on osteoblasts has not been reported. Osteoporosis is a condition which has debilitating effects in the elderly. Unsaturated fatty acids (UFA) have been studied for their beneficial effects on human health for a number of years. Polyunsaturated fatty acids (PUFA) have been studied as a potential therapeutic agent to prevent and assist in managing the condition. Few studies have been conducted on the effects of MUFA on bone therefore this study aimed to investigate the effects of PLA on osteoblast differentiation using ASCs and MG-63 osteosarcoma cells as an osteoblast model. ASCs and MG-63 osteosarcoma cell lines were exposed to PLA (20-100 μM) in osteogenic media (OM). The effects of PLA on cell viability was evaluated on undifferentiated cells. Thereafter, cells were exposed to PLA for 7, 14 or 21 days. Subsequently ALP activity, calcium mineralisation, gene expression, protein expression and adipogenesis were assessed. In this study, PLA had no significant effects on cell viability in undifferentiated cells. Furthermore, PLA had no significant effects on ALP activity, calcium mineralisation or phosphorylated extracellular signal-regulated kinase (pERK)/extracellular signal-regulated kinase (ERK) expression in differentiating cells, however, ALP activity increased at 7 day in ASCs and 21 days in MG-63 cells. Alizarin Red S staining increased at 21 days in both cell lines with a significant increase in the ASCs, however, calcium nodules were not visible. In the ASCs, PLA significantly increased the gene expression of ALP at 7 and 14 days compared to control (p<0.01 and p<0.05) while RANKL was significantly decreased at 7 days compared to the control (p<0.05). In the MG-63 cells, RUNX2 and OCN were significantly reduced at 7 days compared to control (p<0.05) and ALP, RUNX2, Osx and RANKL were significantly reduced at 14 days compared to control (p<0.001 and p<0.05). In the ASCs, lipid accumulation was not present after 21 days while in MG-63 cells, there was a significant increase in lipid accumulation at a high concentration of PLA after 21 days compared to control (p<0.05). This is the first study to explore the effects of PLA on osteoblast formation using ASCs and MG-63 osteosarcoma cells. Results suggest that PLA exerted changes in the ASCs and MG-63 cells during osteoblast differentiation, however, these changes were not significant. To conclude, PLA showed some significant effects on osteoblast-specific gene expression, however, most of the osteoblast-specific gene expression was downregulated, particularly in the MG-63 cells, after PLA treatment. / Dissertation (MSc)--University of Pretoria, 2020. / Physiology / MSc / Unrestricted

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

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17978号 / 医博第3842号 / 新制||医||1001(附属図書館) / 80822 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長澤 丘司, 教授 河本 宏, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM