The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells.

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2'-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

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.

Establishing an islet transplantation program in a developing country.

Diabetes is an emerging epidemic throughout the world. In our city alone, there are approximately 25,000 known diabetics (5% to 10% type 1) among a total population of 1.7 million inhabitants, and the incidence is increasing among all age groups. Islet transplantation is a potential treatment for type 1 diabetes mellitus. For this reason, we intended to establish an islet transplantation program. This required competent and well-trained professionals, a specially planned facility adhering to rigid regulations regarding safety and sterility, and a detailed study of the ethical laws and rules involving transplantation. In this article, we describe the process including any difficulties or barriers encountered due to limited resources in a developing country. We also describe all stages of personnel training and the necessary equipment and work area of a similar specialized center following the guidelines of the Brazilian National Agency for Health Care. Finally, we discuss our expectations for the initial phase of our islet transplantation program.

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.