Effects of mesenchymal stromal cells play a role the oxidant/antioxidant balance in a murine model of asthma.

Asthma is a heterogeneous disease characterised by chronic airway inflammation. One of the most devastating consequences of this inflammatory process is the generation of reactive oxygen and nitrogen species responsible for oxidative stress. The aim of this study is to analyse the efficiency of treatment with human bone marrow-derived mesenchymal stromal cells (hMSC) in maintaining the oxidative balance in a murine model of allergic asthma by quantifying nitrotyrosine in lung tissues. After confirmation of asthma in the experimental model, samples of lung parenchyma were submitted to immunohistochemical assessment. Intravenous administration of hMSC reduced the levels of nitrotyrosine in the ASTHMA-hMSC group compared to those in the ASTHMA-SAL group. In conclusion, therapeutic administration of hMSC had a beneficial effect on oxidative stress, reducing the levels of nitrotyrosine in lung tissues in a model of allergic asthma.

Isolamento e caracterização de células mesenquimais do tecido adiposo de cães / Isolation and characterization of canine adipose-derived mesenchymal stem cells

As células-tronco mesenquimais (CTMs) diferenciam-se em várias linhagens e têm potencial de utilização na medicina regenerativa. As CTMs podem ser isoladas de vários tecidos de animais adultos. O objetivo deste estudo foi o isolamento das CTMs do tecido adiposo de cães, seu cultivo e diferenciação. Foram coletadas amostras de tecido adiposo subcutâneo de cinco cães. As CTMs foram isoladas, obtendo-se 146.803 (±49.533) células/g, cultivadas e diferenciadas em osteoblastos, adipócitos e condrócitos. Avaliaram-se a cinética do crescimento, a morfologia e a viabilidade celular. A caracterização citoquímica comprovou a natureza mesenquimal das células isoladas. O cultivo foi iniciado com 20.000 células/mL, verificando-se crescimento rápido até 72 horas (220.000 células/mL), fase exponencial entre 72 e 192 horas (455.000 células/mL), seguida de platô por saturação da densidade com 240 horas (355.000 células/mL). A viabilidade celular variou entre 96 e 100%. As CTMs em cultivo são fibroblásticas, fusiformes, com citoplasma basofílico e núcleo esférico. O comprimento médio das células variou entre 80,85 e 98,36µm, a largura média entre 17,40 e 28,79µm e o diâmetro médio do núcleo entre 15,46 e 17,74µm.

The applications of mesenchymal stem cells (MSCs) are becoming increasingly more promising for regenerative medicine and tissue engineering fields. MSCs can be isolated from adult animals from a variety of tissues, such as the adipose. This study focused on the isolation, culture and differentiation of MSCs from canine adipose tissue. Samples of subcutaneous adipose tissue from five dogs were collected. These cells were isolated, cultured and differentiated into osteoblasts, adipocytes and chondrocytes. We obtained 146,803 (±49,533) cells/g. Growth kinetics and viability studies were conducted during cell culture and the evaluation of cell differentiation was successfully performed by cytochemistry. The cell cultures were initiated with 20,000 MSCs/ml. Rapid growth was observed at 72 hours (220,000 cells/ml), the exponential phase between 72 and 192 hours (455,000 cells/ml) and saturation at 240 hours (355,000 cells/ml). The cellular viability ranged from 96 to 100%. MSCs in culture are fibroblastic cells, fusiform with basophilic cytoplasm and spherical nucleus. The length and width means of the cells and nuclear diameter ranged from 80.85-98.36µm, 17.40-28.79µm and 15.46-17.74µm respectively.

Expression of pancreatic endocrine markers by mesenchymal stem cells from human adipose tissue.

Mesenchymal stem cells (MSCs) from human adipose tissue have a great potential for use in cell therapy due to their ease of isolation, expansion, and differentiation, besides the relative acceptance from the ethical point of view. Our intention was to isolate and promote in vitro expansion and differentiation of MSCs from human adipose tissue into cells with a pancreatic endocrine phenotype. Human adipose tissue obtained from patients undergoing abdominal dermolipectomy was digested with type I collagenase. MSCs isolated by plastic adherence and characterized by cytochemistry and FACS were expanded in vitro. MSC differentiation into an endocrine phenotype was induced over 2 to 4 months with high glucose (25 mmol/L) media containing nicotinamide, exendin-4, and 2-mercaptoethanol. Insulin and glucagon expressions were analyzed by immunofluorescence. Cells isolated from human adipose tissue and expanded in vitro expressed MSC markers as confirmed by FACS and cytochemistry. Insulin but not glucagon production by differentiated cells was demonstrated by immunofluorescence. MSCs isolated from human adipose tissue were induced to differentiate in vitro into an endocrine phenotype that expressed insulin.

Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue.

Mesenchymal stem cells (MSCs) have been investigated as promising candidates for use in new cell-based therapeutic strategies such as mesenchyme-derived tissue repair. MSCs are easily isolated from adult tissues and are not ethically restricted. MSC-related literature, however, is conflicting in relation to MSC differentiation potential and molecular markers. Here we compared MSCs isolated from bone marrow (BM), umbilical cord blood (UCB), and adipose tissue (AT). The isolation efficiency for both BM and AT was 100%, but that from UCB was only 30%. MSCs from these tissues are morphologically and immunophenotypically similar although their differentiation diverges. Differentiation to osteoblasts and chondroblasts was similar among MSCs from all sources, as analyzed by cytochemistry. Adipogenic differentiation showed that UCB-derived MSCs produced few and small lipid vacuoles in contrast to those of BM-derived MSCs and AT-derived stem cells (ADSCs) (arbitrary differentiation values of 245.57 +/- 943 and 243.89 +/- 145.52 mum(2) per nucleus, respectively). The mean area occupied by individual lipid droplets was 7.37 mum(2) for BM-derived MSCs and 2.36 mum(2) for ADSCs, a finding indicating more mature adipocytes in BM-derived MSCs than in treated cultures of ADSCs. We analyzed FAPB4, ALP, and type II collagen gene expression by quantitative polymerase chain reaction to confirm adipogenic, osteogenic, and chondrogenic differentiation, respectively. Results showed that all three sources presented a similar capacity for chondrogenic and osteogenic differentiation and they differed in their adipogenic potential. Therefore, it may be crucial to predetermine the most appropriate MSC source for future clinical applications.

Simultaneous autologous transplantation of cocultured mesenchymal stem cells and skeletal myoblasts improves ventricular function in a murine model of Chagas disease.

BACKGROUND: Cellular transplantation is emerging as a promising strategy for the treatment of postinfarction ventricular dysfunction. Whether its beneficial effects can be extended to other cardiomyopathies remains an unexplored question. We evaluated the histological and functional effects of simultaneous autologous transplantation of co-cultured stem cells and skeletal myoblasts in an experimental model of dilated cardiomyopathy caused by Chagas disease, characterized by diffuse fibrosis and impairment of microcirculation. METHODS AND RESULTS: Wistar rats weighing 200 grams were infected intraperitoneally with 15 x 10(4) trypomastigotes. After 8 months, 2-dimensional echocardiographic study was performed for baseline assessment of left ventricle (LV) ejection fraction (EF) (%), left ventricle end-diastolic volume (LVEDV) (mL), and left ventricle end-systolic volume (LVESV) (mL). Animals with LV dysfunction (EF <37%) were selected for the study. Autologous skeletal myoblasts were isolated from muscle biopsy and mesenchymal stem cells from bone marrow aspirates were co-cultured in vitro for 14 days, yielding a cell viability of >90%. Eleven animals received autologous transplant of 5.4 x 10(6)+/-8.0 x 10(6) cells (300 microL) into the LV wall. The control group (n=10) received culture medium (300 microL). Cell types were identified with vimentin and fast myosin. After 4 weeks, ventricular function was reassessed by echo. For histological analysis, heart tissue was stained with hematoxylin and eosin and immunostained for fast myosin. After 4 weeks, cell transplantation significantly improved EF and reduced LVEDV and LVESV. No change was observed in the control group. CONCLUSIONS: The co-transplant of stem cells and skeletal myoblasts is functionally effective in the Chagas disease ventricular dysfunction.

Cell transplantation after the coculture of skeletal myoblasts and mesenchymal stem cells in the regeneration of the myocardium scar: an experimental study in rats.

UNLABELLED: In myocardial infarction and Chagas's disease, some physiopathological aspects are common: cardiomyocyte loss due to ischemia leads to a reduction of contractility and heart function. Different cells have been proposed for cellular cardiomioplasty. OBJECTIVE: Our goal was to evaluate the method of co-culture of skeletal muscle (SM) and mesenchymal stem cells (MSC) for cell therapy of heart failure in Chagas's disease (CD) and myocardium postinfarction (MI). METHODS: For MI, 39 rats completed the study at 1 month. Seventeen rats received cell therapy into the scar and 22 rats only medium. For CD, 15 rats completed the study at 1 month including 7 that received cell therapy and eight followed the natural evolution. All animals underwent ecocardiographic analysis at baseline and 1 month. Left ventricular, ejection fraction, end systolic, and end dyastolic volume were registered and analyzed by ANOVA. The co-culture method of SM and MSC was performed at 14 days (DMEM, with 15% FCS, 1% antibiotic, IGF-I, dexamethasone). Standard stain analysis was performed. RESULTS: For MI ejection fraction in the animals that received the co-cultured cells increased from 23.52+/-8.67 to 31.45+/-8.87 (P=.006) versus the results in the control group: 26.68+/-6.92 to 22.32+/-6.94 (P=.004). For CD, ejection fraction in animals that received the co-cultured cells increased from 31.10+/-5.78 to 53.37+/-5.84 (P<.001) versus the control group values of 36.21+/-3.70 to 38.19+/-7.03 (P=0.426). Histopathological analysis of the animals receiving co-cultured cells demonstrated the presence of myogenesis and angiogenesis. CONCLUSION: The results validated the product of SM and MSC co-cultures for treatment of diseases.

Cell transplantation: differential effects of myoblasts and mesenchymal stem cells.

BACKGROUND: Cellular transplantation has emerged as a novel therapeutic option for treatment of ventricular dysfunction. Both skeletal myoblasts (SM) and mesenchymal stem cells (MSC) have been proposed as ideal cell for this aim. The aim of this study is to compare the efficacy of these cells in improving ventricular function and to evaluate the different histological findings in a rat model of severe post-infarct ventricular dysfunction. METHODS: Myocardial infarction was induced in Wistar rats by left coronary occlusion. Animals with resulting ejection fraction (EF) lower than 40% were included. Heterologous SM were obtained by lower limb muscle biopsy and MSC by bone marrow aspiration. Nine days after infarction, rats received intramyocardial injection of SM (n=8), MSC (n=8) or culture medium, as control (n=11). Echocardiographic evaluation was performed at baseline and after 1 month. Histological evaluation was performed after HE and Gomori's trichrome staining and immunostainig against desmin, fast myosin and factor VIII. RESULTS: There was no difference in baseline EF and left ventricular end diastolic (LVEDV) and systolic volume (LVESV) between all groups. After 1 month a decrease was observed in the EF in the control group (27.0+/-7.10% to 21.46+/-5.96%, p=0.005) while the EF markedly improved in SM group (22.66+/-7.29% to 29.40+/-7.01%, p=0.04) and remained unchanged in the MSC group (23.88+/-8.44% to 23.63+/-10.28%, p=0.94). Histopathology identified new muscular fibers in the group that received SM and new vessels and endothelial cells in the MSC. CONCLUSION: Skeletal myoblasts transplantation resulted in myogenesis and improvement of ventricular function. In contrast, treatment with mesenchymal stem cells resulted in neoangiogenesis and no functional effect.

Could the coculture of skeletal myoblasts and mesenchymal stem cells be a solution for postinfarction myocardial scar?

Currently two lines of research have been proposed for treatment of heart failure in an attempt to address its main cause: skeletal myoblast (SM) transplants, which increase the contractile muscular mass, and mesenchymal stem cell (MSC) transplants, which increase neoangiogenesis. The objective of this study was to establish methods whereby cocultures of SM and MSC proliferate and expand, making possible the interaction of these cell types prior to their transplantation to the myocardium. Seeking to support the survival of these cells after myocardial transplantation and achieve subsequent functional improvement, SM and MSC from 10 rats were isolated and cultivated in DMEM medium supplemented with 15% fetal calf serum, 1% ATB, and growth factors. Following plating in variable proportions of satellite cells/mononuclear cells namely 2:1, 1:1, 1:2, morphological observations were made regarding cell survival, adhesion to substrate, and confluence. After 48 hours nonadherent cells were aspirated from the flasks, leaving the adherent cells, SM, and MSC. The better level of cell proliferation was observed with the proportion 2:1 cocultivated at a concentration of 5 x 10(5)/mL for 14 days. The results were satisfactory; the cell production was up to 10(8), increasing the chances of transplant success after myocardial infarction. Transplants with this model are ongoing.

Aneural culture of rat myoblasts for myocardial transplant.

Due to the peculiar characteristics of skeletal muscle, myoblast transplants have emerged as a therapy for cardiomyopathy, particularly after myocardial infarction. The objectives of this study were to define the mean time of cultivation necessary to obtain a cellular concentration of 10(6) to expand the mass for transplant, and to identify the proliferation phase of myoblasts. Ten myoblast cultures were performed using newborn Wistar rats. The isolation method used enzymatic dissociation in culture medium (HAM-F12 and 199) supplement with basic-fibroblast growth factor (b-FGF) and insulin growth factor (IGF-I). The mean cultivation time to obtain the desired concentration of 10(6) was 7 days, with expansion of up to 10(8)/g. When b-FGF was used, the cellular yield was approximately 10(7), with IGF-I the cellular yield was approximately 10(8), independent of the medium. We concluded that IGF-I is the better option for mass cellular expansion of myoblasts for application in myocardial transplants.