Among the most common knee ligament injuries are those to the anterior cruciate ligament (ACL). Annually, approximately 350,000 people require surgical ACL reconstruction, accounting for more than $6 billion of health-care costs in the United States alone. An injured ACL loses its functions as it cannot heal with larger injuries and heals slowly with smaller ones. This may introduce complications, such as abnormal joint kinematics and deterioration, prior to complete rupture. Although the use of an autologous graft is the current gold standard for ACL reconstruction surgery, it is associated with donor site morbidity and a decrease in mechanical strength at the donor site. The use of allogenic grafts instead of autografts introduces the risk of disease transmission. Furthermore, integration of soft tissue grafts (e.g., hamstring tendon) to native bone is slow and risks graft pullout. To circumvent these limitations, tissue engineering seeks to fabricate suitable biomaterials that could replace the entire ACL, stimulate regeneration of the ligament tissue, and integrate with host bone tissue. Numerous efforts have led to the development of complex, multi-phased biomaterial scaffold designs that are intended to deliver an array of cell types and biological cues. Particularly, scaffolds that possess bone-regenerating biomaterials at the ends are envisioned to facilitate rapid integration with the femur and tibia. Electrospun fiber scaffolds continue to be regularly utilized for their high tensile strength, flexibility, and ability to bend. Nevertheless, fibrous scaffolds are inert and require the incorporation of trophic factors to guide tissue regeneration. Additionally, electrospun fibers are often densely packed, which can hinder cell infiltration and subsequent tissue formation. The objective of this work was to guide bone remodeling through the incorporation of trophic factors with 1) electrospun fiber scaffolds or 2) nanoparticles that could be combined with electrospun fiber scaffolds, and 3) to develop model three-dimensional fiber-hydrogel composites that support cell viability and proliferation. Two approaches were utilized to present the trophic factor bone morphogenic protein (BMP)-2 to stimulate bone formation. In the first approach, electrospun fibers were modified through the adsorption or covalent conjugation of BMP-2. These fibers exhibited increased BMP-2 concentrations with covalent conjugation over adsorption, and the incorporation of heparin into the fibers improved both adsorption and conjugation. Mesenchymal stem cells (MSCs) – that have the capacity to differentiate into osteoblastic cells – were able to attach and proliferate on all films yet appeared to do so to a greater extent on surfaces with higher heparin contents. Additionally, markers of osteoblastic differentiation were significantly higher on surfaces with covalently conjugated BMP-2 than on those with adsorbed BMP-2. In the second approach, a nanoparticle system was produced to control BMP-2 delivery and release. Importantly, this flexible system can be fabricated separately, and then combined with a scaffold for tissue regeneration. In this approach, BMP-2 was combined with chitosan nanoparticles through adsorption, encapsulation, or covalent conjugation. The particular BMP-2 incorporation technique had no significant effect on BMP-2 incorporation efficiencies, but affected particle size and BMP-2 release kinetics. Specifically, covalent conjugation method caused the aggregation of particles while adsorption method allowed the most sustainable release. MSCs cultured in the presence of the different particles survived and proliferated, but only particles with adsorbed BMP-2 stimulated osteoblastic differentiation. Finally, three-dimensional fiber-hydrogel composites of various models were fabricated to mimic the complexity of full-sized scaffolds for ACL regeneration, and to study cell infiltration, differentiation, and tissue formation. A collagen hydrogel phase was introduced to electrospun fiber scaffolds using different approaches. MSCs seeded within a thin collagen layer were able to proliferate, sense underlying substrate and spread according to fiber orientation, while those within thicker layers were not. Additionally, cells initially present in only the collagen phase infiltrated to the fiber phase. These results demonstrate that minor changes in fabrication steps to combine the two phases could significantly alter cell function during the formation of three-dimensional fiber-hydrogel composites for tissue regeneration. / Doctor of Philosophy / The anterior cruciate ligament (ACL) is one of four ligaments that connect the thigh bone to the shin bone and stabilize the knee. Injuries to the ACL often occur during high impact sports, and ruptures can necessitate surgical intervention. ACL reconstruction surgery involves drilling tunnels through the ends of leg bones, deploying the tissue graft through the knee joint and bone tunnels, and anchoring it within the bone tunnels. The most common grafts are autografts that use tendons of the patient's own body or allografts that are obtained from cadavers. The complications associated with autografts include pain at the site of tissue harvest, while allografts risk disease transmission. Additionally, directly affixing a soft tissue graft (e.g., the hamstring tendon) to bone within the bone tunnel suffers from slow tissue integration and risk of pull-out. Tissue engineering is a field that seeks to develop devices to direct the regeneration of damaged tissues and organs. In the context of ACL repair, it seeks to achieve a biomaterial device with the properties of ACL, that can both guide the regeneration of ligament tissue and facilitate integration with bone tunnels, eliminating the need for autografts and allografts and their associated risks. Toward the development of an engineered ACL, this work focuses on improving graft-to-bone integration. In the first project, fibrous materials are surface-modified with bone morphogenetic protein (BMP)-2 (a bone-forming protein), and then tested for their ability to stimulate formation of a bone-like tissue in cell culture. In the second project, the deployment of BMP-2 either on the surface of or within nanoparticle delivery vehicles is evaluated as an alternative strategy to stimulate bone-like tissue formation. The third project explores the inclusion of a hydrogel phase to facilitate cell infiltration and bone-like tissue formation within fibrous materials. Together these studies provide insights into how the architecture of the engineered tissue and the deployment of bone-forming proteins can be used to enhance ACL regeneration.

Yes / Mesenchymal stem cells (MSCs) are multipotent adult stem cells with great potential in regenerative medicine. One method for stimulating proliferation and differentiation of MSCs is via electrical stimulation (ES). A valuable approach for evaluating the response of MSCs to ES is to assess changes in gene expression, relative to one or more reference genes. In a survey of 25 publications that used ES on cells, 70% selected GAPDH as the reference gene. We conducted a study to assess the suitability of six potential reference genes on an immortalized human MSC line following direct current ES at seeding densities of 5000 and 10,000 cells/cm2 . We employed three methods to validate the most stable reference genes from qRT-PCR data. Our findings show that GAPDH and ACTB exhibit reduced stability when seeded at 5000 cell/cm2 . In contrast, we found that the most stable genes across both plating densities and stimulation regimes were PPIA and YWHAZ. Thus, in ES gene expression studies in MSCs, we support the use of PPIA and YWHAZ as an optimal reference gene pair, and discourage the use of ACTB and GAPDH at lower seeding densities. However, it is strongly recommended that similar verification studies are carried out based on cell type and different ES conditions.

Poor skeletal health results from aging and metabolic diseases such as obesity and diabetes and involves impaired homeostatic balance between marrow osteogenesis and adipogenesis. Tissue engineering provides researchers with the ability to generate improved, highly controlled and tailorable in vitro model systems to better understand mechanisms of homeostasis, disease, and healing and regeneration. Model systems that allow assembly of modules of MSCs, osteoblasts, and adipocytes in a number of configurations to engage in signaling crosstalk offer the potential to study integrative physiological aspects and complex interactions in the face of changes in local and systemic microenvironments. Thus, the overall goal of this dissertation was to examine integrative physiological aspects between MSCs, osteoblasts, and adipocytes that exist within the marrow microenvironment. To investigate the effects of intercellular signaling in different microenvironmental contexts, methods were developed to photolithographically pattern and assemble cell-laden PEG-based hydrogels with high spatial fidelity and tissue-scale thickness for long-term 3D co-culture of multiple cell types. This platform was applied to study effects of crosstalk between MSCs, osteoblasts and adipocytes on markers of differentiation in each cell type. Additionally, responses of MSCs to systemic perturbations in glucose concentration were modulated by mono-, co-, and tri-culture with these cell types in a model of diabetes-induced skeletal disease. Together, these studies provided valuable insight into unique and differential effects of intercellular signaling within the niche environment of MSCs and their terminally differentiated progeny during homeostatic and pathological states, and offer opportunities further study of integrative physiological interactions between mesenchymal lineage cells.

Les lymphocytes Th17 forment une population de cellules T CD4+ pro-inflammatoires, impliqués non seulement dans l'élimination d'agents pathogènes, mais ayant aussi un rôle délétère dans l'induction de maladies inflammatoires chroniques. Ils expriment spécifiquement le récepteur de chimiokines CCR6, qui a pour ligand le CCL20 mais aussi les β-defensine-1, 2 et 3, peptides ayant une activité antimicrobienne. Les cellules souches mésenchymateuses (CSMs) représentent une population cellulaire hétérogène exerçant diverses propriétés immunomodulatrices.Les résultats obtenus dans ce travail de thèse montrent que l'environnement inflammatoire contribue à augmenter l'adhésion des lymphocytes Th17 aux CSMs, et qu'elle est régulée par l'interaction du CCR6 avec ses ligands ; que les CSMs exercent, en partie via la sécrétion de PGE2, des effets anti-inflammatoires en faisant acquérir un phénotype régulateur aux lymphocytes Th17 différenciés, soulignant ainsi la plasticité de ces derniers.De plus, nous avons montré que les lymphocytes Th17 activés par l'antigène produisent du CCL20 et induisent, via la production de l'IL-17 et de l'IL-22, la sécrétion d'hBD-2, mais pas celle des hBD-1 et 3, par des kératinocytes épidermiques humains et de la peau reconstituée; que le CCL20, ainsi que la hBD-2, induisent l'arrêt de ces cellules sur l'endothélium enflammé in vitro en conditions de cisaillement. Finalement, l'activation spécifique d'antigène des lymphocytes Th17 entraîne une perte de l'expression de CCR6, ce qui provoque ainsi un état transitoire de non réponse à une nouvelle stimulation de ces cellules avec les ligands de CCR6, permettant leur migration ultérieure hors du tissu enflammé. / Th17 cells form a population CD4+ T cells with strong pro-inflammatory properties that are not only involved in the clearance of pathogens, but also play a deleterious role of in the pathogenesis of inflammatory disease. Th17 cells specifically express CCR6, a chemokine receptor that binds to its unique chemokine ligand, CCL20, as well as to human β-defensin (hBD)-1, 2 and 3, peptides with anti-microbial activity. Mesenchymal stem cells (MSC) represent a heterogenous population that exert broad immunomodulatory effects.The results from the studies carried out during this thesis show that the inflammatory environment contributes to increased adhesion of Th17 cells to MSCs, which is mediated via the interaction of CCR6 with its ligands, and that MSCs exert, in part via the secretion of PGE2, anti-inflammatory effects through the induction of a T regulatory cell phenotype in fully differentiated tissue-infiltrating Th17 cells, thereby underscoring the plasticity of the latter cells.Furthermore, the results show that antigen-activated Th17 cells produce CCL20 and induce, via the production of both IL-17 and IL-22, the secretion of hBD-2, but not 1 and 3, by normal human epidermal keratinocytes and reconstituted skin, and that CCL20, as well as hBD-2, induce arrest of these cells onto inflamed endothelium in vitro under conditions of shear stress. Finally, antigen-specific activation of Th17 cells also causes a loss of CCR6 expression from their cell surface and thus results in a transitory state of non-responsiveness to further stimulation of these cells with CCR6 ligands, which is likely to permit their subsequent migration out of inflamed tissue.

Chronic obstructive pulmonary disease (COPD) including emphysema is a devastating condition, increasing in prevalence in the US and worldwide. There remains no cure for COPD, rather only symptomatic treatments. Due to unique challenges of the lung, translation of therapies for acute lung injury to target chronic lung diseases like COPD has not been successful. We have been investigating lung derived extracellular matrix (ECM) hydrogels as a novel approach for delivery of cellular therapies to the pulmonary system. During the course of this work we have developed and characterized a lug derived ECM hydrogel that exhibits “injectability,” allowing cells or dugs to be delivered in a liquid and encapsulated at body temperature. The hydrogel self assembles in <5 minutes and achieves mechanical stiffness similar to other soft tissue ECM hydrogels. The hydrogel can support 3D cell growth and encapsulated cell viability. Encapsulated hMSCs can also still be activated by simulated inflammatory environments. Naïve mouse macrophages exposed to the fully formed gel were not significantly induced to express markers for pro or anti-inflammatory polarized phenotypes, but increased expression for several secreted inflammatory mediators was observed. We also investigated a novel approach for preparing and solubilizing the isolated ECM proteins, using digestion time as a variable for controlling hydrogel density (interconnectivity), mechanical stiffness, component protein size distribution, and cell behavior on fully formed gels. The potential future impact for the presented research includes optimization for future animal studies, expansion to additional applications, and the development of new derivative materials.

L'utilisation grandissante des cellules souches mésenchymateuses (CSM) en ingénierie tissulaire augmente la nécessité d'améliorer leur expansion. Ces travaux ont concerné l'étude d'un procédé performant d'expansion de CSM porcines en mode agité. Tout d'abord, un milieu de culture a été adapté aux CSM porcines multipotentes. Puis, différents modes d'expansion en conditions agitées ont été évalués avec les cellules fixées sur des microporteurs. La culture sur le microporteur Cytodex 1 a permis d'atteindre une vitesse spécifique de croissance de 0,54 j-1, supérieure à celle observée en flacon statique (0,31 j-1), avec les mêmes conditions de culture. En parallèle, une méthode de comptage innovante a été proposée pour le dénombrement automatique des cellules cultivées sur Cytodex 1, sans passer par une étape de trypsination. Enfin, les conditions opératoires du procédé d'expansion ont été étudiées. En comparaison d'une culture de CSM sur Cytodex 1 sans agitation, une agrégation des cellules et une baisse apparente de la concentration cellulaire ont été observées à 25 et 75 rpm. Par ailleurs, l'ajout de microporteurs au cours d'une culture de 300 h, réalisée dans un système de culture agité à 25 rpm et dans un volume de 200 mL, a permis de prolonger la prolifération cellulaire en évitant l'agrégation tout en maintenant la multipotence des CSM. Une concentration cellulaire de 3 x 105 cellules/mL a été obtenue, au lieu de 1,2 x 105 cellules/mL en flacons statiques avec les mêmes conditions de culture. Un procédé performant d'expansion de CSM porcines en conditions agitées a ainsi pu être proposé / The extensive use of mesenchymal stem cells (MSC) in tissue engineering increases the necessity to improve the expansion performance. This work aimed at studying an efficient expansion process for porcine MSC in agitated mode. First, a culture medium was adapted to the multipotent porcine MSC. Then, various expansion modes and agitation conditions were evaluated with the cells fixed on microcarriers. Cultures on the Cytodex 1 microcarrier enabled to reach a specific growth rate of 0.54 d-1, which was higher than the one observed in static T-flasks (0.31 d-1), with the same culture conditions. In parallel, an innovative counting method was proposed for the automatic enumeration of cells cultivated on Cytodex 1, without passing by a trypsination step. Finally, the operating conditions of the expansion process were studied. Compared to a culture of MSC on non-agitated Cytodex 1 microcarriers, cell aggregation occurred and an apparent decrease in the cell concentration was observed at an agitation rate of 25 and 75 rpm. Moreover, the addition of microcarriers during a 300 h culture, performed in an agitated culture at 25 rpm and in a volume of 200 mL enabled to prolong the cell proliferation without any aggregation, while maintaining the multipotency of the cells. A cell concentration of 3 x 105 cells/mL was obtained, instead of the 1.2 x 105 cells/mL in static flasks with the same culture conditions. An efficient expansion process for porcine MSC under agitated conditions has therefore been proposed

La thérapie cellulaire pour le traitement de l'insuffisance cardiaque post-infarctus semble prometteuse même si le bénéfice fonctionnel observé actuellement en recherche clinique reste souvent limité. Parmi les différent types cellulaires utilisables, les cellules souches mésenchymateuses (MSC) reconnues pour leur capacité d'immunomodulation, de transdifférenciation et de sécrétion paracrine représentent un outil intéressant pour la régénération myocardique.L'objectif de ce travail a été de mieux comprendre les mécanismes mis en place par les MSC pour réparer le myocarde lésé afin de développer ensuite une stratégie visant à optimiser les effets thérapeutiques de la greffe de MSCs dans le cadre expérimental de l'insuffisance cardiaque post-infarctus. Pour cette étude, nous avons réalisé des cocultures entre cardiomyocytes adultes et les MSC dérivées du tissu adipeux, les cellules hMADS (human Multipotent Adipose Derived Stem cells) afin de mimer le microenvironnement cardiaque in vitro. Des travaux antérieurs à ma thèse réalisés au laboratoire avaient montré que la communication intercellulaire entre ces deux types cellulaires grâce à des structures nanotubulaires aboutissait à la reprogrammation du cardiomyocyte vers le stade progéniteur. Durant ma thèse, nous avons ensuite pu montrer in vitro, toujours grâce au système de coculture, que ce meme type de communication hetérologue via des connexions nanotubulaires constituées de f-actine et de tubuline, modifiait la sécrétion paracrine des cellules souches hMADS. Les cellules souches ainsi reprogrammées, par les échanges intercellulaires de matériel cardiaque améliorent de façon significative leur potentiel angiogénique et de chémoattraction in vitro. Le bénéfice sur les MSCs de la coculture a été confirmé dans le traitement de l'insuffisance cardiaque post-infarctus chez la souris. Dans ce modèle nous avons pu montré que les cellules souches cocultivées avaient un capacité de régénération myocardique nettement supérieures aux cellules souches naives et que l'amélioration fonctionnelle était associée à une stimulation de la vascularisation et de la mobilisation des progéniteurs cardiaques endogènes. Enfin, des résultats similaires ont été observés dans notre modèle préclinique d'ischémie-reperfusion myocardique porcin encourageant la poursuite des travaux de recherche basés sur la communication intercellulaire afin d'optimiser l'efficacité thérapeutique des cellules souches dans la reconstruction cardiaque..En conclusion, nos travaux ont mis en évidence que la communication intercellulaire entre les cardiomyocytes souffrants et les cellules souches conditionnent de façon importante les effets thérapeutiques des cellules souches et que la manipulation ex vivo de ces phénomènes pourrait constituer une approche pour optimiser la thérapie cellulaire cardiaque chez l'homme. / Cell therapies represent one of the most promising approaches to rebuild damaged heart particularly those based on mesenchymal stem cells (MSC). These cells are known for their plasticity, immune privilege and strong self-renewal ability. Intramyocardial delivery of MSC ameliorates heart function after infarction in clinical studies but mechanisms by which MSC exert their therapeutic action is far from being understood and further investigations are required for improving the modest efficiency observed.The objective of this work was to better understand mechanisms by which MSC repair damaged myocardium in order to develop strategies optimizing their therapeutic effects. To mimic in vitro the microenvironment of an injured heart, we developed a species mismatch co-culture system consisting of terminally-differentiated cardiomyocytes (CM) and MSC from adipose tissue called hMADS for human Multipotent Adipose Derived Stem cells. Previous works in the laboratory showed that cell-to-cell communication processes between CM and hMADS involving tunnelling nanotubes (TNT) reprogram adult CM toward a progenitor-like state.During my PhD, we found that crosstalk between hMADS and CM through TNT altered the secretion by hMADS of cardioprotective soluble factors and thereby maximized the capacity of stem cells to promote angiogenesis and chemotaxis of bone-marrow multipotent cells. Additionally, engraftment experiments into mouse infracted hearts revealed that in vitro preconditioning of hMADS with CM increased the cell therapy efficacy of naive stem cells. Functional improvement was associated with higher angiogenesis and homing of bone marrow progenitor cells at the infarction site. Finally, similar results were observed in our preclinical study using a porcine model of myocardial infarction.In conclusion, our findings established the relationship between the paracrine regenerative action of MSC and the nanotubular croostalk with CM and emphasize that ex vivo manipulation of theses communication processes might be of interest for optimizing current cardiac cell therapies.

O câncer de mama é uma doença heterogênea que é caracterizada por células epiteliais de mama malignas. As células-tronco cancerígenas (CST) no câncer de mama podem aumentar o potencial de agressividade através do tumor. O objetivo deste estudo é avaliar a expansão clonal do microambiente tumoral e a diferenciação celular após o estímulo com células estaminais mesenquimais. As células MSC derivadas da geléia de Wharton foram co-cultivadas com MCF- 7 em proporções de 1%, 10%, 30%. A co-cultura de MCF-7 com MSC mostrou alteração na localização da e-caderina para o citoplasma e, de preferência, o núcleo. Para a n-caderina, a co-localização foi predominantemente na membrana após a exposição do MSC. As células MCF-7 apresentaram colocalização do citoplasma e do núcleo no biomarcador de ?-catenina. Este fenômeno é confirmado à WB com o aumento dos níveis de proteínas de ecaderina no citoplasma no MCF-7 após o estímulo do MSC. A morfologia das transições amênico-mesenquimatosas foi mostrada no ensaio 3D em algumas colônias, no entanto esta morfologia não é predominante. A co-cultura de MCF- 7 com MSCs aumenta o número de mammosferes e influencia o aumento de CD44 + / CD24-, esse fenômeno foi possível sob estimulação de 30% das células MSC. A linhagem celular de câncer de mama MCF-7 em associação com MSC pode aumentar o potencial de agressividade através do tumor. Essa interação no microambiente do tumor é determinante para a expansão clonal e diferenciação celular, que são mecanismos relevantes no processo de disseminação metastática. / Breast cancer is a heterogeneous disease that is characterized by malignant breast epithelial cells. Cancer stem cells (CST) in breast cancer can boost a potential for aggressiveness trough the tumor. The goal for this study is to evaluated the tumor microenvironment clonal expansion and cellular differentiation after stimulus with mesenchymal stem cells. MSC cells derived from Wharton\'s jelly were co-cultured with MCF-7 in proportions 1%,10%,30%. The co-culture of MCF-7 with MSC showed alteration on localization of ecadherin to the cytoplasm and preferably nucleus. For n-cadherin, the colocalization were predominantly in membrane after MSC exposition. MCF-7 cells showed cytoplasm and nucleus co-localization on ?-catenin biomarker. This phenomenon is confirmed to WB with increasing the proteins levels of ecadherin on cytoplasm in MCF-7 after MSC stimulus. Amoeboid to mesenchymal transitions morphology were showed on 3D assay in some colonies, however this morphology is not predominant. The co-culture of MCF-7 with MSCs increase in the number of mammospheres and influence the increase of CD44+/CD24-, this phenomenon was possible under stimulation of 30% of MSC cells. The breast cancer cell line MCF-7 in association with MSC can boost a potential for aggressiveness trough the tumor. This interaction on tumor microenvironment is determinant for the clonal expansion and cellular differentiation, which are relevant mechanisms in the process of metastatic dissemination.

As células-tronco mesenquimais (MSCs) da medula óssea compreendem uma população de células multipotentes com propriedades imunorreguladoras e capacidade de secreção de fatores de crescimento, desempenhando um papel fundamental na regulação da hematopoiese. À luz dessas propriedades, alguns estudos fornecem uma análise das relações estabelecidas entre células-tronco hematopoiéticas normais (HSCs) e MSCs quando expostas à cocultura. Jing et al. (Haematologica, 2010) demonstram neste tipo de arranjos de cocultura a geração de três populações distintas de células: células não aderentes (Fração A), células aderidas à superfície de MSCs (Fração B) e células abaixo das MSCs (Fração C). Além disso, dados recentes apontam para a associação da progressão da doença com a evidência de transferência de mitocôndrias funcionais (mt) e espécies reativas de oxigênio (ROS) das MSCs para as células leucêmicas. É teorizado como um mecanismo de MSCs, a fim de reduzir as espécies reativas de oxigênio (ROS). No entanto, os desempenhos diferenciais nesses processos de transferência entre MSCs normais e leucêmicas em sistemas de cocultura em cada uma dessas populações de células distintas não foram estabelecidos. As células leucêmicas (CD45+) têm um aumento de quase três vezes na proliferação em todas as três populações após a cocultura com MSCs leucêmicas, mas não após a cocultura com MSCs saudáveis. As células CD45+ da fração A têm uma baixa taxa de proliferação em cocultura com MSCs normais comparadas com as células leucêmicas. Em 5d, as MSCs leucêmicas (CD73+) aumentam 20 vezes a coloração de mitotracker em comparação com 3d, implicando que os blastos AML estimulam MSCs a produzir mais mt, embora os MSCs normais apresentem os mesmos níveis de mitotracker em 3 / 5d. Além disso, os níveis de mtROS diminuem em 10 vezes em 5d em comparação com 3d em leucemia, mas não em MSCs normais, sugerindo uma recuperação mediada por mt em MSCs leucêmicas após a cocultura. Finalmente, o ROS total diminui 2 vezes nas células CD45+ após cocultura com MSCs leucêmicas por 5d, mas não em contrapartida normal. Em essência, esses achados sugerem diferentes mecanismos de doação mitocondrial de MSCs para blastos LMA. Além disso, o estudo fornece um passo importante nacompreensão da natureza complexa do metabolismo do tumor, não apenas na célula maligna, mas também dentro do microambiente que a suporta. / Bone marrow mesenchymal stromal cells (MSCs) comprise a population of multipotent cells with immunoregulatory properties and the capability of secreting growth factors, playing a key role in the regulation of hematopoiesis. In light of these properties, some studies provide analysis of the relations established between normal hematopoietic stem-cells (HSCs) and MSCs when exposed to coculture. Jing et al. (Haematologica, 2010) demonstrate in these kind of coculture arrangements the generation of three distinct cells populations: non-adherent cells (supernatant), phasebright cells (adhered to the surface of MSCs) and phase-dim cells (beneath the MSCs). Furthermore, recent data pointed to the association of disease progression in AML with the evidence of functional mitochondria (mt), and reactive oxygen species (ROS) transference from MSCs to the blasts cells. It is theorized as a mechanism of MSCs in order to reduce the reactive oxygen species (ROS). Nevertheless, the differential performances in these transference process among normal and leukemic-MSCs in coculture systems in each of those distinct cells populations were not established. AML cells (CD45+) have an increase of almost 2.5-fold in proliferation in all of 03 populations after coculture with leukemic-MSCs but not after coculture with a normalMSCs. The CD45+ cells in phase-bright/dim have a low proliferation rate in coculture with normal-MSCs compared with the leukemic cells. In 5d, the leukemic-MSCs (CD73+) increase 20-fold the mitotracker staining compared with 3d, implying that AML blasts stimulate MSCs to produce more mt, albeit the normal-MSCs present the same mitotracker levels in 3/5d. Additionally, the mtROS levels decrease by 10-fold in 5d compared with 3d in leukemic, but not in normal-MSCs, suggesting mt mediated recover in leukemic-MSCs after coculture. Finally, total ROS decrease 2-fold in CD45+ cells after coculture with leukemic-MSCs for 5d, but not in normal counterpart. In essence, these findings suggest different mechanisms of mitochondrial donation from MSCs to AML blats. Moreover, the study provides an important step in the understanding of the complex nature of tumor metabolism, not only in the malignant cell, but also within the microenvironment which supports it.

Diversos estudos tem demonstrado que as células-tronco mesenquimais (CTM) são imbuídas de uma forte atividade imunossupressora in vitro, no entanto, os resultados de imunoterapias utilizando CTM têm sido variáveis até o momento. Nossa hipótese para tal variação são interações que devem ocorrer entre as CTM e fragmentos de patógenos circulantes nos pacientes, resultando na modulação da atividade imunossupressora. Para avaliar a ocorrência deste fenômeno em CTM de medula óssea, inicialmente foi avaliado a presença de diversos TLR através da marcação com anticorpos e posterior quantificação por citometria de fluxo, sendo observada a presença dos TLR2, TLR3, TLR4 e TLR9. No intuito de avaliar alterações no potencial imunossupressor, linfócitos T ativados e marcados a nível intracelular foram co-cultivados com CTM estimuladas com LPS, POLY IC e oligonucleotídeos com motivos CpG: DSP30, CpG-A e CpG-B, sendo sua proliferação quantificada por citometria de fluxo. Como resultados, foi observado que a estimulação com LPS e DSP30 levaram a perda e acentuação da capacidade supressora, respectivamente, enquanto o estímulo simultâneo com LPS e DSP30 resultou em sua manutenção. Tais modulações na imunossupressão foram corroboradas ao serem avaliadas modulações na expressão gênica, tendo em vista que o estímulo por LPS e DSP30 induziram no aumento da expressão de IL1 e TGF, respectivamente. Em seguida, foi avaliado o efeito das mesmas condições experimentais na indução a proliferação das CTM, ao ser mensurada alterações na quantidade de células em um equipamento de High content Screening (HCS). Como resultados, foi possível observar que somente o tratamento com DSP30 foi capaz de aumentar significativamente a quantidade de células, fenômeno corroborado ao ser mensurada a síntese de DNA, através da utilização de um produto comercial, seguido de análise por citometria de fluxo. No intuito de avaliar possíveis modulações na via NF-B, CTM estimuladas com LPS ou DSP30 foram sujeitas ao ensaio de imunoprecipitação de cromatina, utilizando anticorpos específicos a subunidades de RelA e RelB, sendo o DNA imunoprecipitado sujeito a PCR quantitativo com primers específicos para a regiões promotoras do gene VCAM-1. Como resultados, foi observado que o estímulo com LPS aumentou a atividade do RelA, enquanto não foram observados efeitos após o estímulo com DSP30. No entanto, o estímulo simultâneo com ambos os ligantes levou ao aumento de atividade de RelA e RelB. Ao serem avaliadas estas condições em um ensaio de imunofluorescência analisado em HCS, foi possível observar maiores níveis da proteína RelB no citoplasma das células tratadas com DSP30, sugerindo um aumento da sua formação. Apesar dos mecanismos moleculares subjacentes aos resultados observados ainda necessitarem de maior elucidação, nosso trabalho indica que a estimulação das CTM com DSP30 pode trazer benefícios no sentido de potencializar a imunossupressão e proliferação celular, além de impedir a perda da imunossupressão, decorrente da interação com LPS. Tais resultados poderão servir como diretrizes para o aprimoramento de imunoterapias utilizando CTM de medula óssea, principalmente em casos de pacientes com infecções por patógenos. / Several studies have shown that mesenchymal stem cells (MSCs ) are imbued with a strong immunosuppressive activity in vitro , however , the results of immunotherapies using CTM has been mixed so far. Our hypothesis for this variation are interactions that must occur between the CTM and fragments of circulating pathogens in patients , resulting in the modulation of the immunosuppressive activity. To evaluate the occurrence of this phenomenon in bone marrow MSCs was initially evaluated the presence of various TLR by staining with antibodies and subsequent quantification by flow cytometry , the presence of TLR2 , TLR3 , TLR4 and TLR9 was observed . To assess changes in the immunosuppressive , activated T lymphocytes and labeled intracellularly potential were co-cultured with MSC stimulated with LPS , poly IC and oligonucleotides with CpG motifs : DSP30 , CpG - A and CpG - B , and their proliferation measured by flow cytometry. As a result , it was observed that stimulation with LPS and DSP30 led to loss of stress and suppressing ability , respectively, while simultaneous stimulation with LPS and DSP30 resulted in maintenance. Such modulations in immunosuppression were corroborated when assessing modulations in gene expression , given that the stimulus induced by LPS and DSP30 in increased expression of TGFb and IL1 , respectively. Then , the effect of the same experimental conditions inducing proliferation of MTC to be measured changes in the amount of cells in a High Content Screening equipment (HCS ) was measured . As a result , it was observed that only the DSP30 treatment was able to significantly increase the amount of cells, phenomenon to be measured supported DNA synthesis through the use of a commercial product followed by analysis by flow cytometry . In order to evaluate possible modulations in NF-kB pathway , CTM stimulated with LPS or DSP30 were subjected to chromatin immunoprecipitation assay , using antibodies specific to subunits RelA and RelB , and the immunoprecipitated DNA subjected to quantitative PCR with primers specific to the promoter regions of VCAM-1 gene. As a result , it was observed that stimulation with LPS increased the activity of RelA , while effects were not observed after stimulation with DSP30 . However , simultaneous stimulation with both ligands led to increased activity of RelA and RelB . When these conditions are evaluated in an assay in HCS immunofluorescence analysis , we observed higher levels of RelB protein in the cytoplasm of cells treated with DSP30 , suggesting an increase in their formation. Although the molecular mechanisms underlying the observed results still require further elucidation , our work indicates that stimulation of MSC with DSP30 can bring benefits in terms of enhancing immunosuppression and cell proliferation , and prevent loss of immunosuppression resulting from the interaction with LPS . These results can serve as guidelines for the improvement of immunotherapies using CTM bone marrow , especially in cases of patients with infections caused by pathogens.