Effects of low-intensity electrical stimulation and adipose derived stem cells transplantation on the time-domain analysis-based electromyographic signals in dogs with SCI.

INTRODUCTION: The application of low-intensity electrical stimulation (LIES) to neural tissue increases neurochemical factors responsible for regeneration as nerve growth factor. Stem cell (SC) therapy for patients with Spinal cord injury (SCI) promote some increase functional improvement. OBJECTIVE: Investigate the electromyographic response in paraplegic dogs undergoing LIES and SC transplantation. METHODS: 27 dogs paraplegics with SCI were divided into three groups with different types of therapy. GADSC: two SC transplants (n = 9); GLIES: LIES (n = 8); GCOMB: two SC transplants and LIES (n = 10). Adipose derived mesenchymal stem cells (ADSCs) were transplanted by lumbar puncture in the amount of 1.2 × 106 cells/50 µL. Acupuncture needles positioned in the interspinous space were used for stimulation. The electrical stimulation was applied with a mean voltage ∼30 mV and four consecutive modulated frequencies (5 Hz, 10 Hz, 15 Hz and 20 Hz) within 5 min each. The patients motor performance was evaluated before (Pre) the procedure and after 30 (Post30) and 60 (Post60) days, from electromyography root mean square (EMGRMS) registered with subcutaneous electrodes in the vastus lateralis muscle, while the animals were in quadrupedal position. RESULTS: All three groups showed a significant intra-group increase of EMGRMS (Pre vs. Post30 or Pre vs. Post60). However, there were no statistically significant differences between Post30 and Post60. The inter-group test (GADSC X GLIES X GCOMB) did not present significance when compared the instants Pre (p = 0.34), Post30 (p = 0.78) and Post60 (p = 0.64). CONCLUSION: Some dogs recovered motor activity, expressed by the EMGRMS, in all groups, in pre vs. post (30 or 60 days) comparisons.

Generation of patient-specific induced pluripotent stem cell lines from one patient with Jervell and Lange-Nielsen syndrome, one with type 1 long QT syndrome and two healthy relatives.

Four human iPSC cell lines (one Jervell and Lange-Nielsen Syndrome, one Long QT Syndrome-type 1 and two healthy controls) were generated from peripheral blood obtained from donors belonging to the same family. CytoTune™-iPS 2.0 Sendai Reprogramming Kit (containing OCT3/4, KLF4, SOX2 and cMYC as reprogramming factors) was used to generate all cell lines. The four iPSCs have normal karyotype, express pluripotency markers as determined by RT-PCR and flow cytometry and differentiated spontaneously in vitro into cells of the three germ layers, confirming their pluripotent capacity.

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.

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: 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.

Disparate chondrocyte metabolism in three-dimensional fibrin cultures derived from autogenous or commercially manufactured fibrinogen.

OBJECTIVE: To compare chondrocyte proliferation and metabolism in three-dimensional fibrin cultures formed from polymerized autogenous fibrinogen with that of commercially manufactured fractionated fibrinogen. ANIMALS: Fibrinogen and chondrocytes for in vitro experimentation derived from 2 horses, ages 12 and 14 months, donated for reasons unrelated to skeletal or hematologic abnormalities. PROCEDURE: Fibrinogen was isolated from whole blood, using plasma cryoprecipitation and centrifugation, and fractionated fibrinogen was purchased. Each was mixed with 10 x 10(6) chondrocytes/0.5 ml of fibrinogen, and was polymerized by addition of 0.5 ml of calcium-activated thrombin. Thirty 1-ml fibrin-chondrocyte disks were formed from each fibrinogen source and cultured for 0 (n = 6), 7 (n = 12), or 14 (n = 12) days. Chondrocyte metabolism and cell proliferation in each fibrin type were objectively assessed by assays for total proteoglycan content, [35S]proteoglycan accumulation, proteoglycan monomer size, and total DNA. Cell morphology and cartilage-specific cell function was evaluated by routine histologic, alcian blue histochemical, type-II collagen immunohistochemical, and type-II collagen in situ hybridization methods. RESULTS: Histologic examination indicated better retention of chondrocyte morphology in autogenous composites. Autogenous fibrinogen also stimulated greater chondrocyte proliferation (DNA content increased 1.4-fold on day 14) and supported higher proteoglycan accumulation (increased 1.4-fold on day 14), compared with commercial, fractionated fibrinogen. Abundant intracellular type-II procollagen mRNA was detected in autogenous fibrin cultures by in situ hybridization, and translation was confirmed by extensive pericellular type-II collagen accumulation. CONCLUSIONS: Autogenous fibrinogen has an inherent capacity to maintain chondrocyte phenotypic metabolism that is reduced or absent in commercially prepared fibrinogen. Enhanced, differentiated cell function may be useful for in vivo applications, but represents an added variable that may confound in vitro experiments, and should be considered when designing studies of chondrocyte function.

Heart valve operations in patients with active infective endocarditis.

Sixty-two consecutive patients underwent heart valve operation for active infective endocarditis. There were 42 men and 20 women whose mean age was 49 years (range, 21 to 79 years). The infection was in the aortic valve in 37 patients, the mitral valve in 18, the aortic and mitral valves in 5, and the tricuspid valve in 2. Twenty-four patients had prosthetic valve endocarditis. Staphylococcus and Streptococcus were responsible for 86% of the infections. Annular abscess was encountered in 33 patients. Complex valve procedures involving reconstruction of the left ventricular inflow or outflow tract or both were performed in 31 patients. There were three operative deaths (4.8%). Predictors of operative mortality were prosthetic valve endocarditis, preoperative shock, and annular abscess. Patients were followed for 1 month to 130 months (mean follow-up, 43 months). Only 1 patient required reoperation for persistent infection. There were ten late deaths. Most survivors (96%) are currently in New York Heart Association class I or II. The 5-year actuarial survival was 79% +/- 7%. These data demonstrate excellent results in patients with native valve endocarditis, and support the premise that patients with prosthetic valve endocarditis should have early surgical intervention.

Surgical treatment of aortic root abscess.

A significant number of patients with infective aortic valve endocarditis develop aortic annular abscess. Although antibiotics may occasionally sterilize an aortic root abscess, most patients require surgical intervention. A review of our experience with 21 consecutive patients surgically treated for aortic root abscess disclosed that 13 patients had prosthetic valve and eight had native aortic valve endocarditis. The predominant microorganism was Staphylococcus aureus, particularly in those patients with native aortic valve endocarditis. The abscess was limited to the aortic annulus in 10 patients and was either multiple or had perforated the left ventricular outflow tract in 11 patients. Most patients were desperately ill at the time of operation. Repair was accomplished by aggressive debridement of all apparently infected tissue and reconstruction of the left ventricular outflow tract with autologous pericardium. Although postoperative complications were common, only one patient died in hospital. Operative survivors have been followed up from 3 to 68 months (mean, 29 months). One patient died of complications of repair of a thoracoabdominal aneurysm 34 months after surgery; his prosthetic aortic valve and patch were intact at autopsy. No patient has experienced recurrent infection, pericardial patch aneurysm, or prosthetic valve dehiscence.