This present thesis comprise two major parts both experimental and numerical study which have been conducted in four distinct steps as following: (1) Design, construction, and evaluation of control and hydrodynamic of a bioreactor system. (2) Visualization of fluid flow perfusion in the hollow fibre membrane bioreactor (HFMB) using a biomedical noninvasive imaging technique, i.e. positron emission tomography (PET). (3) Development of a mathematical model for analyzing a hybrid hollow fibre membrane bioreactor (hHFMB) and (4) Development of a dynamic and two-porous media model for analyzing the HFMB with the aid of computational fluid dynamics (CFD), specifically for bone tissue engineering application. The experimental part includes the steps 1 and 2. In the step 1, the flow perfusion bioreactor system has been designed and constructed. The experimental evaluations of hydrodynamic, and control were performed. In this system, mean pressure, mean flow rate, frequency and waveform of the pulsatile pressure and flow rate can be modulated and controlled over the time to simulate both physiological and non-physiological conditions. The temperature, dissolved oxygen, and pH can be controlled.This bioreactor system can be applied to a variety of scaffold configurations, geometries, and sizes as the cell/tissue culture chamber is adjustable in length.This system is autoclavable, and compatible with noninvasive medical imaging techniques. Designing of the inlet and outlet manifold of the bioreactor were performed according to data obtained from CFD simulation of the flow distribution to achieve high efficiencies in the uniformity of flow perfusion. In the second step, PET was proposed for the very first time and a small animal PET system was used to obtain new information about steady and pulsatile flow patterns in the HFMB for tissue engineering applications. The non-homogeneous tracer distribution, as found with PET imaging, implies the occurrence of non-efficient regions with respect to mass transfer. In steady inlet flow condition, a non-uniform distribution of radioactive tracer was obtained. In contrast, the pulsatile inlet flow generated more uniform perfusion than that of steady flow. Further, it was found that in the case of pulsatile flow, the accumulation of the tracer within the bioreactor was efficiently less than that of steady inlet flow at the same condition. Therefore, in one hand these findings have the potential to improve bioreactor design and in the other hand can explore a very important rout to employ PET in developing bioreactors for tissue engineering applications. The numerical part includes the step 3 and 4 in which the numerical study has been performed for 3-D bone tissue growth in HFMB as a case study for large-scale tissue culture. In the step 3, the feasibility of utilizing newly proposed hHFMB for the growth of mesenchymal stem cells (MSCs) to form bone tissue was investigated using numerical simulations. To this aim, a mathematical model using a CFD code was developed to optimize the design and operation parameters of hHFMB for the growth of MSCs. The volume averaging method was used to formulate mass balance for the nutrients and the cells in the porous extracapillary space (ECS) of the hHFMB. The cell-scaffold construct in the ECS of the hollow fibres and membrane wall were treated as porous medium. Cell volume fraction dependent porosity, permeability, and diffusivity of mass were used in the model. The simulations allowed the simultaneous prediction of nutrient distribution and nutrient-dependent cell volume fraction. In addition, this model was used to study the effects of the operating and design parameters on the nutrient distribution and cell growth within the bioreactor. The modeling results demonstrated that the fluid dynamics within the ECS and transport properties and uptake rates in hHFMB were sufficient to support MSCs required for clinical-scale bone tissue growth in vitro and enabled to solve nutrition difficulties because of high cell density and scaffold size. In the step 4, the new dynamic and two-porous media model has been used for analyzing the nutrient-dependent MSCs growth in order to form the bone tissue in the HFMB. In the present model, hollow fibre scaffold within the bioreactor was treated as a porous domain. The domain consists of the porous lumen region available for fluid flow and the porous ECS region, filled with collagen gel containing cells, for growing tissue mass. Furthermore, the contributions of several design and process parameters, which enhance the performance of the bioreactor, were studied. In addition, the dynamic evaluation of cell growth, oxygen and glucose distributions were quantitatively analyzed. The obtained information can be used for better designing of the bioreactor, determining of suitable operational conditions and scale up of the bioreactor for engineering of clinical-scale bone tissue.--Résumé abrégé par UMI.

Les tendinites sont des lésions communes chez le cheval athlète, ayant un impact financier et sportif considérable. Les cellules souches mésenchymateuses (CSMs) de moelle osseuse (MO) sont empiriquement utilisées en clinique pour améliorer la guérison des affections myoarthrosquelettiques. Cependant, il est nécessaire de standardiser les protocoles d’isolement des CSMs équines et d’analyser leurs effets sur la guérison tendineuse pour ajuster leur dose. Les objectifs de cette étude étaient de comparer 3 méthodes d’isolement des CSMs équines et d’établir un modèle de guérison tendineuse minimal invasif pour analyser l’effet des CSMs sur cette guérison. Des CSMs de MO du sternum de juments étaient isolées par 3 protocoles couramment utilisés (adhérence au pétri (Classique) et 2 méthodes par gradient de densité (Percoll et Ficoll)). La viabilité des cellules après isolement, le rendement d’isolement, le nombre de CSMs obtenues après 14 jours de culture et leurs caractéristiques fonctionnelles (renouvellement et différentiation) étaient comparés entre les 3 protocoles. Les résultats suggéraient que le Percoll était le meilleur protocole en termes de rendement et de capacité de renouvellement des cellules. La différence n’était pas significative pour leur viabilité et leur capacité de différentiation. Un modèle de guérison tendineuse, consistant en une ténectomie du tendon extenseur latéral du doigt fut ensuite développé. Cependant, la grande variabilité interindividuelle de qualité de guérison dans le groupe pilote implique une ré-évaluation du modèle. Des études futures, avec des CSMs isolées par le Percoll dans de nouveaux modèles de guérison tendineuse devraient permettre de déterminer la dose adéquate de CSMs. / In equine athletes, tendinitis lesions are common and lead to substantial financial losses. Bone marrow (BM) mesenchymal stem cells (MSCs) are employed clinically empirically to enhance healing of musculoskeletal injuries. However, there is a need to standardize equine MSC isolation protocols, to analyze the effects of MSCs on tendon healing and to optimize dosage. The objectives of the study were to compare 3 methods of equine MSC isolation and develop a minimally invasive model of tendon healing to analyze the effects of MSCs on tendon healing. BM MSCs from the sternum of mares were isolated by 3 protocols (adherence to a plastic culture dish (Classic) and two gradient density separation protocols (Percoll and Ficoll)) to compare for cell viability, MSC yield, number of MSCs attained after 14 days of culture and functional characteristics (self-renewal and multilineage differentiation) of the MSCs. The results suggested that the Percoll protocol was the best of those assessed in terms of MSC yield and self-renewal potential and that MSCs retrieved with the Ficoll protocol had the lowest self-renewal. There were no significant differences in terms of cell viability and differentiation capacity. A tendon healing model was then developed and consisted of a 0.5 cm tenectomy of the lateral digital extensor tendon. However, interanimal variation of healing quality was so high within the pilot group that the model should be re-evaluated. Further studies using MSCs isolated with Percoll in other novel models of tendon healing would allow determination of the adequate dosage of MSCs.

L’apoptose endothéliale initie le processus menant au remodelage vasculaire et au développement de la néointima dans la vasculopathie du greffon. La formation de néointima résulte de l’accumulation de leucocytes, de matrice extracellulaire et de cellules positives pour l’actine musculaire lisse alpha (αSMA+) dans l’intima des artères, artérioles et capillaires du greffon. Les cellules αSMA+ dans la néointima sont des cellules musculaires lisses vasculaires (CMLV) dérivées du donneur ainsi que des cellules souches dérivées du receveur, dont des cellules souches mésenchymateuses (CSM). L’acquisition d’un phénotype anti-apoptotique chez ces cellules est déterminante pour le développement de la néointima. Le laboratoire de Dre Hébert a démontré que les cellules endothéliales (CE) apoptotiques libèrent des médiateurs induisant une résistance à l’apoptose chez les CMLV et les fibroblastes. Notamment, les CE apoptotiques relâchent la cathepsine L qui clive le perlécan et ainsi libère un fragment C-terminal correspondant au troisième motif laminine G du domaine V du perlécan (LG3). Le LG3 est anti-apoptotique pour les fibroblastes. Nous avons donc émis l’hypothèse que le LG3 est un des médiateurs clés libéré par les CE apoptotiques favorisant le développement de la néointima via l’induction d’un phénotype anti-apoptotique chez les cellules néointimales αSMA+. Nous avons démontré que les médiateurs libérés par les CE apoptotiques induisent un phénotype anti-apoptotique chez les CSM dépendant de l’activation de la voie ERK1/2. De plus, le LG3 active la voie ERK1/2 via son interaction avec les intégrines beta 1 et induit une réponse anti-apoptotique chez ces cellules. Cependant l’activation de ERK1/2 par le LG3 est plus faible en comparaison de son activation par le milieu conditionné par des CE apoptotiques. Nos résultats suggèrent que les CE apoptotiques libèrent aussi de l’EGF qui agit de façon paracrine sur les CSM en coopération avec le LG3 pour induire un phénotype anti-apoptotique chez les CSM. Nous avons poursuivi l’étude de l’effet du LG3 in vivo sur le remodelage vasculaire en transplantation. Nous avons pour cela développé un modèle murin de rejet vasculaire qui consiste en une transplantation aortique entre des souris alloincompatibles. Nous avons ensuite injecté du LG3 chez les souris receveuses en post-transplantation. Nous avons observé dans ce modèle que des niveaux augmentés de LG3 sérique augmentent la formation de néointima, favorisent l’accumulation de cellules néointimales αSMA+ et diminuent le nombre de cellules CD31+ au niveau du greffon aortique. Parallèlement nous avons vérifié que le LG3 induit aussi un phénotype anti-apoptotique chez les CMLV et nous avons démontré un nouvel effet du LG3, soit une activité pro-migratoire, qui dépend de l’activation de la voie ERK1/2 chez les CMLV. Nous avons complété cette étude par l’analyse des niveaux de LG3 sérique dans une cohorte de patients receveurs d’allogreffe rénale. Nous avons observé chez ces patients, une association entre des niveaux élevés de LG3 sérique et un rejet vasculaire. Le LG3 contribue à la formation de néointima par son activité pro-migratoire et pro-survie chez les cellules néointimales et aussi de par son activité angiostatique. Nos résultats suggèrent que le LG3 est un nouveau médiateur important dans le remodelage vasculaire en transplantation / In allogeneic transplanted organs, endothelial apoptosis is associated with vascular remodeling and neointima formation which in turn leads to allograft vasculopathy, a maladaptive form of vascular repair. In allograft vasculopathy, neointima results from the accumulation of leukocytes, extracellular matrix and alpha-smooth muscle actin positive (αSMA+) cells in the intima of allogeneic arteries, arterioles and capillaries. Neointimal αSMA+ cells comprise vascular smooth muscle cells (VSMC) derived from the donor and stem cells derived from the recipient, including mesenchymal stem cells (MSC). Acquisition of an anti-apoptotic phenotype of neointimal cells is central to the development of vascular obliterative changes. Dr Hébert’s team demonstrated that apoptotic endothelial cells release mediators which in turn induce a state of resistance to apoptosis of VMSC and fibroblasts. Apoptotic endothelial cells release cathepsin-L which cleaves perlecan therefore releasing a C-terminal fragment harbouring a laminin G motif and referred to as LG3. LG3 is anti-apoptotic for fibroblasts. We hypothesized that LG3 is a key mediator produced by endothelial apoptosis of importance in favoring neointima formation via the induction of an anti-apoptotic phenotype in αSMA+ neointimal cells We demonstrated that mediators released by endothelial apoptosis induce an ERK1/2-dependent anti-apoptotic phenotype in MSC. We identified LG3 as one of the mediators implicated in the induction of this anti-apoptotic response. Interactions between LG3 and beta 1 integrins expressed on MSC trigger ERK1/2 activation albeit to a lesser degree than medium conditioned by apoptotic endothelial cells. We showed that apoptotic endothelial cells also release EGF which cooperates with LG3 to induce an anti-apoptotic phenotype on MSC through cross-talk between EGF receptor and integrin-dependent pathways. Next, we characterized the impact of LG3 on allogeneic vascular remodeling in vivo. We developed a murine model of vascular rejection based on orthotopic transplantation of an aortic segment between two fully MHC-incompatible mice in absence of immunosuppression. Recombinant LG3 was injected intravenously post-transplantation in recipients resulting in higher circulating levels of LG3. In LG3-injected mice, accumulation of αSMA+ neointimal cells was enhanced resulting in significantly increased intima/media ratios in the allogeneic aortic graft. Aortic grafts of LG3-injected allografts also showed decreased CD31+ cells. We also demonstrated, using cell-based approaches, that LG3 exerts a pro-migratory activity on VSMC through beta 1-integrin and ERK1/2 -dependent pathways. In line with these observations we also reported augmented serum LG3 in human renal transplant patients in association with acute vascular rejection episodes. Collectively these results suggest that the pro-migratory, pro-survival and angiostatic activities of LG3 contribute to neointima formation. Our results suggest that LG3 is a novel mediator of importance in the development of obliterative vascular remodeling associated with rejection of allogeneic organs.

La vasculopathie du greffon est une pathologie caractérisée par un rétrécissement progressif et oblitérant des vaisseaux sanguins menant à une ischémie et une perte de fonction du greffon. Le rétrécissement vasculaire est dû à une accumulation de matrice extracellulaire (MEC) et de cellules mononuclées positives pour l’actine musculaire lisse alpha (alphaSMA) dont les cellules souches mésenchymateuses, le tout formant une néointima oblitérante. Cette pathologie est la cause principale de la perte des greffons rénaux et cardiaques à long terme. Le rejet vasculaire aigu est un prédicteur de la vasculopathie du greffon. L’équipe du Dr Hébert a démontré que l’apoptose endothéliale, qui joue un rôle important dans le développement du rejet vasculaire, initie la libération de LG3, un fragment du protéoglycan perlécan. Les taux sanguins et urinaires de LG3 sont augmentés chez les receveurs d’allogreffe rénale avec rejet vasculaire et vasculopathie du greffon. Les résultats obtenus en laboratoire durant ma maîtrise ont permis de mieux caractériser l’impact du LG3 sur un type cellulaire important participant à la formation de néointima : les cellules souches mésenchymateuses. Mes travaux ont démontré que le LG3 induit à la fois la migration horizontale des MSC et la transmigration des MSC. Cette migration est dépendante de la voie de signalisation d’ERK1/2, précédemment identifiée comme voie centrale dans la formation de néointima. De plus, nos résultats démontrent que la kinase Src est activée en amont de l’activation de la voie MAPK. La migration horizontale et la transmigration induites par le LG3 sont aussi dépendantes des intégrines alpha2beta1, ainsi que l’activation de la voie MAPK. Dans un modèle de transplantation murin, nous avons également démontré que l’injection sérique de LG3 recombinant favorise l’accumulation de cellules positives pour alphaSMA dans la néointima. En outre, lorsque le receveur est déficient pour l’intégrine alpha2, mais que le greffon est sauvage, la formation de néointima induite par l’injection de LG3 est diminuée dans le greffon suggérant que les cellules du receveur jouent un rôle important dans la formation de la néointima. Enfin, nous avons démontré que l’injection de LG3 augmente aussi le nombre de cellules positives pour la forme phosphorylée d’ERK1/2 (p-ERK1/2) dans la néointima du greffon et que cette accumulation est dépendante de la présence des intégrines 2 1 chez les cellules du receveur.Lorsque le receveur est sauvage, il y a une augmentation du nombre de cellules positives pour p-ERK1/2. L’investigation de ces mécanismes dans le remodelage vasculaire expose de nouvelles opportunités pour inhiber la réponse cellulaire qui mène au remodelage inadapté lors d’un dommage vasculaire chronique et ainsi prolonger la survie du greffon. / Graft vasculopathy is diseases characterized by a progressive and obliterate narrowing of the blood vessels leading to ischemia and loss of graft function. This vascular narrowing is due to an accumulation of extracellular matrix and mononuclear cells positive for alpha smooth muscle actin (alphaSMA) including mesenchymal stem cells, thus forming an occlusive neointima. This condition is the leading cause of long term loss of kidney and heart transplants. Acute vascular rejection is a predictor of graft vasculopathy. Dr. Hébert’s team has demonstrated that endothelial apoptosis, which plays an important role in the development of vascular rejection, initiates the release of LG3, a fragment of the proteoglycan perlecan. Blood and urine levels of LG3 are increased in renal allograft recipients with vascular rejection and graft vasculopathy. The results obtained in the laboratory during my Master have helped to better characterize the impact of LG3 on an important cell type involved in neointima formation: the mesenchymal stem cells. My work has shown that the LG3 induces both the horizontal migration and the transmigration of MSC. This migration is ERK1/2-dependent, previously identified as a key molecule involved in neointima formation. In addition, our results demonstrate that Src kinase is activated by upstream activation of the MAPK pathway. Horizontal migration and transmigration induced by LG3 are also dependent on alpha2beta1 integrins, and the activation of the MAPK pathway. In a murine transplantation model, we also demonstrated that intravenous injection of recombinant LG3 promotes the accumulation of alphaSMA positive cells in the neointima. In addition, when the recipient is deficient for the alpha2 integrin but the graft is wild type, LG3 fails to induce neointima formation in the graft suggesting that recipient cells play an important role in the neointima formation. Finally, we demonstrated that intravenous injection of LG3 also increases the number of positive cells for the phosphorylated form of ERK1/2 (p-ERK1/2) in the neointima. This accumulation is dependent on the presence of alpha2beta1 integrins on recipient cells: when the recipient is wild type, there is an increase in the number of cells positive for p-ERK1/2.The investigation of these mechanisms in vascular remodeling presents new opportunities to inhibit the cellular response that leads to inadequate remodeling during chronic vascular damage and prolong graft survival.

Le but de la conception d’un biomatériau est de mimer les modèles qui puissent être représentatifs de la matrice extracellulaire (MEC) existant in vivo. Cet objectif peut être atteint en associant une combinaison de cellules et des facteurs biologiques à un biomatériau sur lequel ces cellules peuvent se développer pour reconstruire le tissu natif. Dans cet étude, nous avons crée des surfaces bioactives nanostructurées en combinant la nanolithographie et la fonctionnalisation de surface, en greffant un peptide RGD ou BMP-2 (bone morphogenetic protein 2). Nous avons étudié l’effet de cette nanodistribution sur le comportement des cellules souches mésenchymateuses en analysant leur adhésion et différentiation. Nous notons que la nanodistribution des peptides induit une bioactivité qui a un impact sur l’organisation du cytosquelette, la conformation des fibres de stresse de l’actin, la maturation des adhésions focales (AFs), et le commitment des cellules souches. En particulier, l’aire, la distribution, et la conformation des AFs sont affectes par la présence des nanopatterns. En plus, le RGD et le BMP-2 changent le comportement cellulaire par des voies et des mécanismes différents en variant l’organisation des cellules souches et la maturation de leurs AFs. La nanodistribution influence de façon évidente les cellules souches en modifiant leur comportement (adhésion et différenciation) ce qui a contribué et ce qui contribuera à améliorer la compréhension des interactions des cellules avec la MEC. / The aim of biomaterials design is to create an artificial environment that mimics the in vivo extracellular matrix for optimized cell interactions. A precise synergy between the scaffolding material, bioactivity, and cell type must be maintained in an effective biomaterial. In this work, we present a technique of nanofabrication that creates chemically nanopatterned bioactive silicon surfaces for cell studies. Using nanoimprint lithography, RGD and mimetic BMP-2 peptides were covalently grafted onto silicon as nanodots of various dimensions, resulting in a nanodistribution of bioactivity. To study the effects of spatially distributed bioactivity on cell behavior, mesenchymal stem cells (MSCs) were cultured on these chemically modified surfaces, and their adhesion and differentiation were studied. MSCs are used in regenerative medicine due to their multipotent properties, and well-controlled biomaterial surface chemistries can be used to influence their fate. We observe that peptide nanodots induce differences in MSC behavior in terms of cytoskeletal organization, actin stress fiber arrangement, focal adhesion (FA) maturation, and MSC commitment in comparison with homogeneous control surfaces. In particular, FA area, distribution, and conformation were highly affected by the presence of peptide nanopatterns. Additionally, RGD and mimetic BMP-2 peptides influenced cellular behavior through different mechanisms that resulted in changes in cell spreading and FA maturation. These findings have remarkable implications that contribute to the understanding of cell-extracellular matrix interactions for clinical biomaterials applications.

Introdução. O transplante de células é possibilidade terapêutica promissora para muitas doenças neurológicas. Nos últimos anos, a possibilidade do isolamento de células-tronco dos tecidos adultos, por exemplo da medula-óssea, atrai a atenção da comunidade científica, estratégia que minimiza os problemas éticos relativos ao uso de tecido fetal para implantes visando ao tratamento de doenças neurológicas. Entretanto, a eficiência da transdiferenciação de células-tronco mesenquimais em neurônios, bem como os mecanismos envolvidos nesse processo, permanecem desconhecidos. A obtenção de neurônios maduros ocorreu somente em sistemas de co-cultura, o que induz a questão se a diferenciação representa um potencial das células per si, ou se é possível somente devido à fusão com neurônios maduros. Objetivos. No presente trabalho, pretendeu-se verificar o potencial de as células-tronco mesenquimais tornarem-se neurônios e esclarecer os possíveis mecanismos envolvidos nesse processo. Material e métodos. Células-tronco mesenquimais foram isoladas de 20 doadores voluntários normais e caracterizadas por análise de separação celular ativada por fluorescência. A multipotencialidade foi investigada ao se diferenciar as células em condrócitos e osteócitos. A capacidade de auto-renovação foi confirmada pelo ensaio de incorporação de BrdU. Ulteriormente, as células foram diferenciadas por uma semana em meio contendo AMPc, IBMX, ou combinação de ambos, e os resultados foram comparados com o cultivo em meio básico. Diferentes bloqueadores de Ca2+ ou inibidores de PKA foram usados como tentativa de se impedir a diferenciação, ocorrência que foi mensurada com imunocitoquímica para NF-200 (marcador de neurônios maduros). O registro eletrofisiológico por meio de patch clamp foi usado para se confirmar o fenótipo neuronal. As figuras foram configuradas em microscopia confocal. Para análise estatística foi utilizada ANOVA com teste post-hoc. Resultados. As células isoladas expressaram CD90, 105, 44 e 13 mas foram negativas para CD34 e 45. Isto significa que não são de origem hematopoiética; 98,74 ± 0,43% das células incorporaram BrdU em 24 horas. Após o isolamento, foi possível diferenciá-las em condrócitos ou osteócitos. Em situação controle, não foram evidenciadas células positivas para NF200. Por outro lado, ocorreu positividade em 10,75% ± 1,35 (p<0,0001) das células sob IBMX e, em 15,18% ± 1,12, sob a combinação cAMP e IBMX (p<0,0001). Foram registradas correntes de Na+ e K+ dependentes de voltagem, mas não potenciais de ação. A diferenciação foi inibida com PKAi (5,73% ± 0,42, p<0,0001), nifedipina (5,79% ± 0,98, p<0,0001), Ni2+ (7,06% ± 1,68, p<0,0001) e Cd2+ (0 ± 0, p<0,0001). Discussão. Isolou-se uma população de células-tronco estromais da medula-óssea de seres humanos que se mostrou multipotencial e auto-renovável. O aumento da concentração de AMPc no meio elevou a concentração de neurônios para 15%. A diferenciação parece depender da via PKA mas também envolve a concentração intracelular de Ca2+. Conclusão. O correto entendimento de como as células-tronco mesenquimais diferenciam-se pode contribuir para aumentar a eficácia do método e, talvez um dia, tornar possível o uso dessa ferramenta no campo clínico. / Introduction. Cell transplantation has been considered a promising therapeutic approach for many neurological diseases. The possibility of isolation of stem cells from adult tissues, i.e. bone marrow, has attracted the attention of the scientific community in the recent years. This strategy is interesting on avoiding the ethical issues regarding the use of fetal tissue for neural implants. Moreover, the efficiency of the transdifferentiation of mesenchymal stem cells (MSCs) into neurons, and the mechanisms involved in this process remain largely unknown. The obtention of mature neurons was described only in coculture systems, what raised the question if the differentiation is a potential of the cells itself, or if it is possible only due to fusion with mature neurons. Objectives. In the present investigation, we aimed to verify the potential of MSCs to differentiate into neurons, and also to clarify the possible mechanisms involved on it. Material and methods. MSCs were isolated from 20 healthy human subjects and characterized by FACS-analysis. Multipotentiality was addressed by differentiating them into chondrocytes and osteocytes. The self-renewal capacity was confirmed with BrdU-incorporation assay. Afterwards, cells were differentiated for 1 week in a medium containing cAMP, IBMX, or a combination of both, and the results were compared with cells treated in basal-medium condition. Different Ca2+-blockers and PKA-inhibitor peptide were used on an attempt to impair differentiation, which was quantified with NF-200 immunostaining (a marker of mature neurons). Patch-clamp recording was used to confirm neuronal phenotype. Pictures were taken in confocal microscope. For statistical analysis ANOVA with a post-hoc test was used. Results. The isolated cells expressed CD90, 105, 44, and 13, but were negative for CD34 and 45, meaning that they were non-hematopoiethic; 98.74 ± 0.43 % of them incorporated BrdU in 6hs. After isolation, they differentiated into chondrocytes and osteocytes. In a control situation, no NF200 positive cell was seen. On the other hand, 10.75% ± 1.35 (p<.0001) of positivity was seen under IBMX and 15.18% ± 1.12 in the combination of cAMP with IBMX (p<.0001). Na+ and K+-voltage gated currents were recorded. Differentiation was impaired with PKAi (5.73% ± 0.42, p<.0001), nifedipin (5.79% ± 0.98, p<.0001), Ni2+ (7.06% ± 1.68, p<.0001), and Cd2+ (0 ± 0, p<.0001). Discussion. We were able to isolate a population of stromal stem cells from the bone marrow of human subjects, since they were multipotential and self-renewable. Increasing the concentration of cAMP raised the percentage of neurons up to 15%. The differentiation seems to be dependent on the PKA pathway, but also involved the intracellular concentration of Ca2+. Conclusions. The complete understanding of how MSC differentiate can contribute to increase the efficiency of the method and thus make possible to use this powerful tool in the clinical practice.

As células-tronco mesenquimais são células multipotentes caracterizadas pela capacidade de autorrenovação e diferenciação. Os oxisteróis abrangem um grande grupo heterogêneo derivado do colesterol pela da oxidação enzimática e não enzimática. Os efeitos dos oxisteróis no processo de morte celular, incluindo citotoxicidade e indução de apoptose, foram descritos em diversas linhagens celulares. No entanto, os efeitos dos oxisteróis são pouco conhecidos nas células-tronco mesenquimais. O 7-cetocolesterol (7-KC), um dos mais importantes oxisteróis, foi mostrado ser citotóxico em células-tronco mesenquimais de tecido adiposo. Sendo assim, este estudo descreve os efeitos citotóxicos de curta duração (24 horas) dos oxisteróis colestane-3alfa-5beta-6alfa-triol, 3,5 colestane-7-ona, (3alfa-5beta-6alfa)-colestane-3,6-diol,7-oxocolesterol-5-en-3-beta-il acetato e 5beta-6beta epoxi-colesterol em células-tronco derivadas de tecido adiposo. Os oxisteróis 3,5 colestane-7-ona e 7-oxocolesterol-5-en-3-beta-il acetato não promoveram morte celular e nem afetaram a proliferação celular. Os outros oxisteróis promoveram apoptose, necrose e autofagia, dependendo do tipo de oxisterol e da concentração. O colestane-3alfa-5beta-6alfa-triol foi o mais efetivo. A inibição da proliferação também foi promovida pelos oxisteróis, porém não foi observada alteração no ciclo celular / Mesenchymal stem cells (MSCs) are multipotent cells characterized by self-renewal and cellular differentiation capabilities. Oxysterols comprise a very heterogeneous group derived from cholesterol through enzymatic and non-enzymatic oxidation. Potent effects in cell death processes, including cytoxicity and apoptosis induction, were described in several cell lines. Very little is known about the effects of oxysterols in MSCs. 7-ketocholesterol (7-KC), one of the most important oxysterols, was shown to be cytotoxic to human adipose tissue-derived MSCs. Here, we describe the short-term (24 h) cytotoxic effects of cholestan-3alpha-5beta-6alpha-triol, 3,5 cholestan-7-one, (3alpha-5beta-6alpha)-cholestane-3,6-diol,7-oxocholest-5-en-3-beta-yl acetate, and 5beta-6beta epoxy-cholesterol, on MSCs derived from human adipose tissue. 3,5 cholestan-7-one and 7-oxocholest-5-en-3-beta-yl acetate did not promoted cell death or affect cell proliferation. The other oxysterols led to a complex mode of cell death that could include apoptosis, necrosis and autophagy, depending on the type of oxysterol and concentration. Cholestan-3alpha-5beta-6alpha-triol was the most effective. Inhibition of proliferation was also promoted by these oxysterols, but no changes in cell cycle were observed

A desnutrição proteico-energética (DPE) provoca anemia e leucopenia decorrente da redução de precursores hematopoéticos e comprometimento da produção de mediadores indutores da hematopoese, bem como alterações estruturais e ultra-estruturais na matriz extracelular medular. A hematopoese ocorre em nichos medulares distintos - endosteal e perivascular - que modulam os processos de diferenciação, proliferação e auto-renovação da célula tronco hematopoética (CTH). As células tronco mesenquimais (CTM) tem um papel importante na formação destes nichos, através da sua diferenciação nos diversos tipos celulares que os compõe. Adicionalmente, a CTM pode modular a função de outras células, como a CTH e a célula endotelial (CE) medular, através da liberação de diversos fatores de crescimento e citocinas. As CE expressam proteínas que regulam a diferenciação e movimentação das CTH na MO. Há sinais que a CTM pode ser a precursora da CE medulares, pois in vitro a CTM pode se diferenciar em CE-like. Desta forma, a CTM é um ponto chave no estudo das alterações causadas pela DPE no nicho perivascular e sobre a regulação da hematopoese. Neste trabalho, investigamos se a DPE afeta a diferenciação in vitro da CTM medular em CE-like e avaliamos se essas células apresentam diferentes capacidades em produzir alguns mediadores regulatórios da hematopoese (CXCL-12, SCF, Ang-1, IL-11, GM-CSF e TFG-&#946;), bem como possíveis alterações no perfil de expressão gênica de marcadores de função das CTM e CE-like. Utilizamos camundongos C57BL/6 machos, divididos em grupos Controle e Desnutrido, sendo que o grupo Controle recebeu ração normoprotéica (12% caseína) e o grupo Desnutrido recebeu ração hipoprotéica (2% caseína), ambos durante 5 semanas. Após este período, os animais foram eutanasiados, foi realizada a avaliação nutricional e hematológica, caracterizando a DPE. As CTM foram isoladas, caracterizadas e diferenciadas in vitro em CE-like, o que foi evidenciado pela maior expressão gênica de NT5E, FLT1, KDR, PECAM1 e VCAM1. Avaliamos a expressão dos genes CDH5, CSPG4, LEPR, NES, CSF1, CSF2, CSF3, MCAM, PROM1, ANGPT1, CXCL12, ENG, IGF1, IL3, IL11, KITL, TGFB1, WNT3A, WNT5A, ICAM1, PDGFB1 e VWF. Encontramos alterações causadas pela DPE na expressão gênica e quantificação de CXCL-12, SCF e Ang-1, os quais mostraram que as células avaliadas do grupo Desnutrido encontram-se em um estado \"pró-proliferativo\", em um esforço para restabelecer a hematopoese na DPE. Entretanto, foi observado neste trabalho e nos demais trabalhos do grupo que há hipoplasia medular na DPE e, portanto, pode-se inferir que as alterações hematopoéticas observadas na DPE não são ocasionadas por alterações na síntese de SCF, CXCL-12 ou Ang-1. / Protein-energy malnutrition (PEM) causes anemia and leukopenia as it reduces hematopoietic precursors, impairs the production of mediators that induce hematopoiesis and alters structural and ultrastructural changes in bone marrow (BM) extracellular matrix. Hematopoiesis occurs in distinct BM niches - endosteal and perivascular - which modulate the processes of differentiation, proliferation and self-renewal of hematopoietic stem cell (HSC). Mesenchymal stem cells (MSC) play an important role in the formation of these niches through their differentiation in several cell types that compose them. Additionally, MSC can modulate the function of other cells, such as HSC and endothelial cells (EC), through the release of several growth factors and cytokines. The EC express proteins that regulate the differentiation and migration of HSC in the BM. MSC seem to be the precursor of medullary EC because in vitro MSC can differentiate into EC-like cells. Thus, MSC are a key point in the study of changes caused by DPE on the perivascular niche and on the regulation of hematopoiesis. In this study, we investigated whether PEM would affect BM-MSC in vitro differentiation into EC-like cells and evaluated whether these cells would have distinct capacities of producing some regulatory mediators of hematopoiesis (CXCL- 12, SCF, Ang-1, IL-11, GM -CSF and TFG-&#946;), as well as analyzed possible changes in the gene expression profile of MSC function and EC-like cells related markers. C57BL/6 mice were divided into Control and Malnourished groups, which received for 5 weeks, respectively, a normal protein diet (12% casein) and a low protein diet (2% casein). After this period, animals were euthanized, nutritional and hematological evaluations were performed, featuring the PEM. MSC were isolated, characterized and differentiated in vitro into EC-like cells, which were evidenced by increased gene expression of NT5E, FLT1, KDR, PECAM1 and VCAM1. The expression of CDH5, CSPG4, LEPR, NES, CSF1, CSF2, CSF3, MCAM, PROM1, ANGPT1, CXCL12, ENG, IGF1, IL3, IL11, KITL, TGFB1, Wnt3a, WNT5A, ICAM1, PDGFB1 and VWF genes was also evaluated. Changes caused by PEM on gene expression and quantification of CXCL-12, SCF and Ang-1 were found, indicating that tested cells from the Malnourished group were in a \"pro-proliferative\" state in an effort to restore hematopoiesis. However, our results are in accordance to the literature regarding bone marrow hypoplasia as a consequence of PEM. Therefore, we infer hematopoietic changes observed in this work are not related to changes in the synthesis of SCF, 12 CXCL-12 or Ang-1.

Introdução: A fibrose pulmonar idiopática (FPI) é definida como um tipo de doença fibrosante intersticial crônica de etiologia desconhecida limitada aos pulmões e que apresenta padrão histológico de pneumonia intersticial usual. A prevalência de FPI é estimada em, aproximadamente, 20/100.000 em homens e em 13/100.000 em mulheres, sendo que a idade média do diagnóstico é 67 anos e a sobrevivência média é 2 a 5 anos. Estima-se que 5 milhões de pessoas sejam afetadas em todo o mundo. O tratamento clínico atual está associado com melhora parcial e transitória, com resultados duvidosos ou insatisfatórios. Na abordagem cirúrgica da FPI, tem destaque o transplante pulmonar, cuja realização é rara, devido à escassez de doadores e à limitação das equipes capazes de realizar tais procedimentos. A terapia celular é uma alternativa terapêutica com grande potencial de aplicabilidade na fibrose pulmonar. Objetivos: Utilizar um modelo de fibrose pulmonar induzida pela bleomicina em ratos para investigar os efeitos da terapia com células-tronco mesenquimais (CTM) e o meio condicionado no remodelamento pulmonar com objetivo de elucidar o mecanismo de ação das CTM e meio condicionado, na reversão da fibrose pulmonar. Para tanto, buscamos: 1) padronizar a cultura de células-tronco mesenquimais e meio de cultura; 2) caracterizar o modelo experimental de fibrose pulmonar induzida pela bleomicina por microscopia óptica antes e após o tratamento com CTM e meio de cultura; 3) avaliar a expressão de biomarcadores sorológicos (Fibrinogênio, Fator von Willebrand e PDGF); 4) quantificar a expressão de proteínas relacionadas ao estresse oxidativo (NOS), citocinas pró-inflamatórias e pró-fibróticas (IL-17 e TGF-beta) e pró-angiogênicas (VEGF e endotelina) por imuno-histoquímica; e 5) quantificar a deposição de fibras do colágeno I e V por imunofluorescência. Materiais e Métodos: Utilizou-se um total de 44 ratos Wistar machos albinos com peso médio de 250-300g e 8 semanas de idade. Quatro grupos experimentais foram compostos de 10 animais, que participaram do experimento divididos em três momentos: D0, D10 e eutanásia (D14 ou D21). Em D0, foi realizada a instilação orotraqueal de bleomicina na dose de 1,5 U/kg; em D10, foi realizada a infusão em veia caudal de células-tronco mesenquimais na dose de 106 CTM/Kg ou 200 ?l de meio condicionado. Para o preparo das CTM, foi obtido, em média, 1,2 g de tecido adiposo, procedida a dissociação com colagenase tipo I, sendo que a contagem média de células foi de 3,05 x 106 células linfomononucleares/g de tecido adiposo. Estas células foram cultivadas durante 21 dias em meio Knockout DMEM-F12 suplementado com 10% de soro fetal bovino. Os seguintes três critérios foram utilizados para comprovar o perfil das células-tronco mesenquimais: aderência plástica, expressão de CD90 por citometria de fluxo ( > 90%) e capacidade de diferenciação em três linhagens mesodérmicas. Em D10, um pool destas células alogênicas foi infundido intravenosamente, na veia caudal, a uma concentração de 1 x 106 células/ animal num volume de 200 ul de solução salina. Em D14 ou D21, os animais foram eutanasiados e analisados quanto ao peso e conforme análises microscópico-laboratoriais. As análises histológicas foram realizadas por dois especialistas diferentes em estudo duplo-cego. Os parâmetros de ganho de peso e recuperação microscópica do tecido pulmonar foram analisados em cada grupo. Resultados: Nossos dados mostram que as células-tronco mesenquimais oriundas do tecido adiposo abdominal de ratos Wistar tiveram seu perfil fenotípico, capacidade de adesão plástica e diferenciação em 3 linhagens mesodérmicas estabelecidas inequivocamente, conforme estabelecido internacionalmente pela ISSCR. As células-tronco mesenquimais e o meio condicionado induziram: a recuperação clínica após tratamento; a reversão da inflamação e de fibrose pulmonar induzida pela bleomicina; a reversão da arteriopatia no parênquima pulmonar distal; a redução das concentrações de fibrinogênio, fator von Willebrand e PDGF (marcadores sorológicos); a diminuição da expressão de enzimas oxidantes; a diminuição da expressão de endotelina e a modulação da expressão da proteína de remodelamento (IL-17), da ativação dos fibroblastos TGF-B e da síntese de colágeno. Conclusão: Neste estudo, comprovamos que as terapias com células-tronco mesenquimais derivadas do tecido adiposo e com o meio condicionado foram eficazes na modulação dos processos inflamatórios e fibrogênicos no modelo induzido por bleomicina, agindo na ativação miofibroblástica, e, também, na restauração tecidual e endotelial. Além disso, o meio condicionado se mostrou tão eficiente quanto as células-tronco propriamente ditas, no efetivo remodelamento pulmonar, devendo ser considerado como uma proposta terapêutica viável e inovadora / Introduction: Idiopathic pulmonary fibrosis (IPF) is defined as a type of chronic interstitial fibrosing disease of unknown etiology limited to the lungs and presenting a histological pattern of usual interstitial pneumonia. The prevalence of IPF is estimated at approximately 20/100,000 in men and 13/100,000 in women, with the mean age of diagnosis being 67 years and the average survival is 2 to 5 years. It is estimated that 5 million people are affected worldwide. The current clinical treatment is associated with partial and transient improvement, with dubious or unsatisfactory results. In the surgical approach to IPF, pulmonary transplantation is a prominent feature, which is rare because of the scarcity of donors and the limitation of the teams capable of performing such procedures. Cell therapy is a therapeutic alternative with great potential for applicability in pulmonary fibrosis. Objectives: To use a model of bleomycin-induced lung fibrosis in rats to investigate the effects of mesenchymal stem cell (MSC) therapy and conditioned medium on lung remodeling in order to elucidate the mechanism of action of MSC and conditioned medium, in the reversion of pulmonary fibrosis. To do so, we aim to: 1) standardize the culture of mesenchymal stem cells and conditioned medium; 2) characterize the experimental model of lung fibrosis induced by bleomycin by optical microscopy before and after treatment with MSC and conditioned medium; 3) to evaluate the expression of serological biomarkers (Fibrinogen, Factor von Willebrand and PDGF); 4) to quantify the expression of proteins related to oxidative stress (NOS), pro-inflammatory and pro-fibrotic (IL-17 and TGF-beta) and pro-angiogenic cytokines (VEGF and endothelin) by immunohistochemistry; and 5) to quantify the deposition of collagen I and V fibers by immunofluorescence. Materials and methods: A total of 44 male albino Wistar rats weighing 250-300g and 8 weeks of age were used. Four experimental groups were composed of 10 animals, which participated in the experiment divided in three moments: D0, D10 and euthanasia (D14 or D21). In D0, orotracheal instillation of bleomycin at the dose of 1.5 U/ kg was performed; in D10 caudal vein infusion of mesenchymal stem cells at the dose of 106 MSC/ kg or 200 ul of conditioned medium was performed. To prepare the MSC, a mean of 1.2 g of adipose tissue was obtained, dissociated with type I collagenase and the mean cell count was 3.05 x 106 lymphomonuclear cells / g of adipose tissue. These cells were cultured for 21 days in DMEM-F12 Knockout medium supplemented with 10% fetal bovine serum. The following three criteria were used to prove the profile of mesenchymal stem cells: plastic adherence, expression of CD90 by flow cytometry ( > 90%) and differentiation capacity in three mesodermal lines. In D10, a pool of these allogeneic cells was infused intravenously into the caudal vein at a concentration of 1 x 106 cells / animal in a volume of 200 ul of saline. In D14 or D21 the animals were euthanized and analyzed for weight and microscopic-laboratory analyzes. Histological analyzes were performed by two different specialists in a double-blind study. The parameters of weight gain and microscopic recovery of lung tissue were analyzed in each group. Results: Our data show that the mesenchymal stem cells derived from the abdominal adipose tissue of Wistar rats had their phenotypic profile, plastic adhesion capacity and differentiation in 3 mesodermal lines established unequivocally, as established internationally by ISSCR. Mesenchymal stem cells and conditioned medium induced: clinical recovery after treatment; bleomycin-induced reversal of inflammation and pulmonary fibrosis; the reversal of arteriopathy in the distal pulmonary parenchyma; the reduction of fibrinogen, von Willebrand factor and PDGF concentrations (serologic markers); decreased expression of oxidizing enzymes; the reduction of endothelin expression and the modulation of the expression of the remodeling protein (IL-17), the activation of TGF-B fibroblasts and the synthesis of collagen. Conclusion: In this study we demonstrated that therapies with mesenchymal stem cells derived from adipose tissue and conditioned medium were effective in the modulation of inflammatory and fibrogenic processes in the bleomycin-induced model, acting to modulate myofibroblastic activation and also in tissue restoration and endothelial cells. In addition, the conditioned medium proved to be as efficient as the stem cells themselves, in effective pulmonary remodeling, and should be considered as a viable and innovative therapeutic proposal

As doenças cardiovasculares estão entre as principais causas de morte, principalmente aquelas decorrentes do infarto do miocárdio (IM). A terapia com células-tronco tem mostrado resultados promissores após o IM em estudos clínicos e experimentais, especialmente as células-tronco mesenquimais (MSC), por apresentarem notável potencial pró-angiogênico, anti-fibrosante e imunomodulador. Entretanto, nenhum estudo foi realizado quanto à variabilidade da frequência cardíaca (VFC), tono autonômico e sensibilidade do barorreflexo, que são considerados fatores de risco apreciáveis, e se encontram atenuados na insuficiência cardíaca (IC) induzida pelo IM. Desta forma, o objetivo do presente trabalho foi examinar o efeito do transplante de MSC de medula óssea sobre a modulação autonômica e a sensibilidade do barorreflexo em ratos com IC, induzida pelo IM. Para isso, foi realizada a ligadura da artéria coronária esquerda em ratos Wistar machos (220-360g), e após dois a três dias foi feita a avaliação da área infartada pelo SPECT. As MSCs foram injetadas, intravenosamente, sete dias pós-IM. A função cardíaca foi analisada pela ventriculografia, antes e um mês após o transplante. Após a reavaliação da ventriculografia, os animais foram submetidos a registros eletrocardiográficos, após receberem eletrodos implantados subcutaneamente no dorso, e cânula na veia jugular esquerda para determinação farmacológica do tono autonômico. Também, foi implantada cânula na artéria femoral para registro da pressão arterial, e análise da sensibilidade do barorreflexo. Logo após os registros, os animais foram sacrificados para coleta do coração e análise histopatológica. Para a infusão, as MSCs foram caracterizadas, segundo critérios da Sociedade Internacional de Terapia Celular. A área de lesão induzida logo após a ligadura da artéria foi semelhante entre os animais. Um mês após o tratamento, o tamanho do infarto foi reduzido no grupo tratado com MSC. Verificou-se redução da fração de ejeção do ventrículo esquerdo (FEVE) após a ligadura coronariana, e previamente ao tratamento nos animais com IC. Um mês após a terapia com MSC, ou injeção de salina, não houve melhora da FEVE em ambos os grupos com IC. Os intervalos QT e QTc foram alongados pelo IM, enquanto as MSCs não tiveram efeito nestes parâmetros. A VFC mostrou redução do desvio padrão de valores sucessivos (SDNN) do intervalo RR (iRR) e a raiz quadrada da média da soma dos quadrados das diferenças de valores sucessivos (RMSSD) do iRR pós-IM, enquanto as MSCs preveniram essa redução. Na análise espectral, os espectros do iRR, após o infarto, mostraram potências menores, em unidades absolutas, das bandas de baixa frequência, LF, e alta frequência, HF, enquanto que as MSCs promoveram um aumento destes parâmetros. Os métodos não-lineares da VFC mostraram redução na entropia, e aumento da análise de flutuação depurada de tendência (DFA) do iRR pós-IM. A terapia com MSC preveniu a alteração da entropia e, também, protegeu o DFA. A frequência cardíaca foi semelhante entre os grupos; entretanto, a frequência intrínseca de marca-passo apresentou-se reduzida no grupo IC, enquanto o tratamento com MSC preservou esta atenuação. Foi observado menor tono vagal no grupo IC não tratado, enquanto o tratamento com MSC aumentou o tono vagal. O tono simpático não apresentou diferença entre os grupos. A sensibilidade do barorreflexo à bradicardia foi reduzida na IC, enquanto que as MSC preveniram essa redução. O colágeno intersticial apresentou-se aumentado na IC, mas não no grupo tratado com MSC. A pressão arterial (PA) sistólica, a PA média e os parâmetros da variabilidade da PA: SDNN e LF encontraram-se reduzidas nos grupos com IC, tratados ou não com MSC. Em conjunto, esses dados demonstram que a terapia com MSC reduziu a extensão do infarto e a fibrose intersticial no miocárdio remanescente do ventrículo esquerdo. Além disso, melhorou a modulação autonômica colinérgica do coração, a VFC e a sensibilidade barorreflexa. / Cardiovascular diseases are among the leading causes of death, especially those resulting from myocardial infarction (MI). Stem cell therapy has shown promising results after MI in both patients and experimental animals, especially those using mesenchymal stem cells (MSC), which present potential pro-angiogenic, anti-fibrotic and immunomodulatory. However, no studies have been conducted on the heart rate variability (HRV), autonomic tone and baroreflex sensitivity, which are considered risk factors and are attenuated in heart failure (HF) induced by MI. Thus, the main of this study was to examine the effect of bone marrow MSC transplantation on autonomic modulation and baroreflex sensitivity in rats with HF, induced by MI. Therefore, it was performed ligation of the left coronary artery of male Wistar rats (220-360g) and after two to three days, assessment was made of the area infarcted by SPECT. MSC were injected intravenously seven days post-MI. Cardiac function was assessed by ventriculography before and one month after transplantation. After reassessing the ventriculography, the animals underwent electrocardiographic recordings after receiving electrodes implanted subcutaneously on the back, and cannula in the left jugular vein for drug determination of autonomic tone. Also, cannula was implanted in the femoral artery for blood pressure recording and analysis of baroreflex sensitivity. Soon after the records, the animals were sacrificed for collection of the heart and histopathological analysis. For infusion, the MSC were characterized according to the criteria of the International Society for Cellular Therapy. The lesion area induced soon after artery ligation was similar among animals. One month after treatment, infarct size was reduced in the group treated with MSC. A reduction was observed in left ventricular ejection fraction (LVEF) after coronary ligation, and prior to treatment in HF animals. One month after the MSC therapy, or saline injection, there was no improvement in LVEF in both groups with HF. The QT and corrected QT intervals were lengthened by IM, while the MSC had no effect on these parameters. HRV showed a reduction in the standard deviation of successive values (SDNN) of the RR interval (RRi), and the root mean square of the successive (RMSSD) RRi after MI, while the MSC prevented this reduction. In spectral analysis, the RRi spectra, after infarction, showed lower power, in absolute units, of the low frequency, LF, and high frequency band, HF, while MSCs promoted an increase in these parameters. Nonlinear methods of HRV showed a reduction in entropy, and increased detrended fluctuation analysis (DFA) of RRi, post-MI. The MSC therapy prevented the change of entropy and also protected DFA. Heart rate was similar in both groups; however, the intrinsic heart rate was reduced in HF group, while treatment with MSC preserved this attenuation. A lower vagal tone was observed in HF untreated group, while treatment with MSC increased vagal tone. The sympathetic tone did not differ between groups. The baroreflex sensitivity to bradycardia was reduced in HF, while the MSC prevented this reduction. The interstitial collagen was increased in HF, but not in the group treated with MSC. Systolic blood pressure (BP), the mean BP and parameters of BP variability: SDNN and LF were reduced in the groups with HF, treated or not with MSC. Together, these data demonstrate that MSC therapy reduced the infarct size and interstitial fibrosis in the remaining myocardium of the left ventricle. In addition, the cells improved cholinergic autonomic modulation of the heart, heart rate variability and baroreflex sensitivity.