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1.
Braz. j. med. biol. res ; 53(4): e9282, 2020. graf
Article in English | LILACS | ID: biblio-1089351

ABSTRACT

Vitiligo is an acquired pigmentary disorder resulting from selective destruction of melanocytes. Emerging studies have suggested that T helper cell 17 (Th17) is potentially implicated in vitiligo development and progression. It was recently discovered that metabotropic glutamate receptor 4 (mGluR4) can modulate Th17-mediated adaptive immunity. However, the influence of mGluR4 on melanogenesis of melanocytes has yet to be elucidated. In the present study, we primarily cultured mouse bone marrow-derived dendritic cells (BMDC) and then knocked down and over-expressed mGluR4 using transfection. Transduced BMDC were co-cultured with CD4+ T cells and the expression of Th17-related cytokines were measured. The morphology and melanogenesis of B16 cells were observed after being treated with co-culture medium of CD4+ T cells and transduced BMDC. We found that mGluR4 knockdown did not affect the co-stimulatory CD80 and CD86 upregulation after lipopolysaccharide stimulation but did increase the expression of Th17-related cytokines, and further down-regulated the expression of microphthalmia-associated transcription factor (MITF) and the downstream genes, decreased melanin production, and destroyed the morphology of B16 cells. Conversely, over-expression of mGluR4 reduced the expression of CD80 and CD86, suppressed the production of Th17-related cytokines, increased the expression of MITF, and did not destroy the morphology of B16 cells. Our study confirmed that mGluR4 modulated the Th17 cell polarization and resulted in the alteration of melanogenesis and morphology of B16 cells. Collectively, these findings suggest mGluR4 might be a potent target involved in the immune pathogenesis of vitiligo.


Subject(s)
Animals , Male , Vitiligo/immunology , Dendritic Cells/cytology , Bone Marrow Cells/cytology , Cell Differentiation/physiology , Receptors, Metabotropic Glutamate/physiology , Th17 Cells/immunology , Vitiligo/genetics , RNA, Small Interfering/immunology , Th17 Cells/cytology , Flow Cytometry , Melanins/biosynthesis , Melanocytes/cytology , Mice, Inbred C57BL
2.
Rev. Assoc. Med. Bras. (1992) ; 65(5): 637-646, May 2019. graf
Article in English | LILACS | ID: biblio-1012952

ABSTRACT

SUMMARY OBJECTIVE: Aplastic anemia (AA) is an immune-mediated disease that destroys hematopoietic cells through activated T lymphocytes. B lymphocyte-mediated humoral immunity also plays an important role in the pathogenesis of AA. Regulatory B cell (Breg) subpopulation, which is defined as "B10", secretes interleukin 10 (IL-10). The objective of our experiment was to investigate whether the scale-down proportion of B10 cells in AA patients may play a key role in the pathogenesis. METHODS: A total of 38 AA patients (14 SAA patients and 24 NSAA patients) and 20 healthy control subjects were included. All subjects did not suffer from autoimmune diseases or any other diseases affecting the immune system, such as infectious diseases. Bone marrow mononuclear cells (PBMCs) were isolated and analyzed by Flow cytometry (FCM) and Immunofluorescence double-labeling assay. The relationship between the relative proportions of B10 and ProB10 and their associations to AA, as well as disease severity, were assessed by common clinical indicators and then examined. RESULTS: Our analyses revealed AA patients had significantly lower proportions of peripheral B10 and B10pro compared to healthy controls. SAA patients had a substantially lower percentage of B10 cells and B10pro cells compared to NSAA patients. In addition, B10 cells and B10pro cells were negatively correlated with absolute neutrophil counts, hemoglobin levels and platelet, and absolute reticulocyte counts in AA patients. CONCLUSIONS: The present study attempted to elucidate the potential role of the scale-down proportion of B10 cells in the pathogenesis of AA.


RESUMO OBJETIVO: A anemia aplástica (AA) é uma doença imunomediada que destrói células hematopoiéticas por meio dos linfócitos T ativados. A imunidade humoral mediada por linfócitos B também desempenha um papel importante na patogênese da AA. A subpopulação de células B reguladoras (Breg), que é definida como "B10", secreta interleucina 10 (IL-10). No experimento, investigou-se se a proporção reduzida de células B10 nos pacientes de AA pode desempenhar um papel-chave na patogênese. MÉTODOS: Um total de 38 pacientes de AA (14 pacientes de anemia aplástica grave e 24 pacientes de anemia aplástica não grave) e 20 indivíduos de controle saudáveis foram incluídos. Todos os indivíduos não sofriam de doenças autoimunes ou de quaisquer outras doenças que afetam o sistema imunológico, tais como doenças contagiosas. As células mononucleares da medula óssea (PBMCs) eram isoladas e analisadas por citometria de fluxo (FCM) e ensaio de dupla marcação por imunofluorescência. A relação entre as proporções relativas de células B10 e as células ProB10 e as suas associações à AA, assim como a gravidade da doença avaliada por indicadores clínicos comuns, foram examinadas. RESULTADOS: Nossas análises revelaram que os pacientes de AA têm proporções significativamente menores de células B10 e células ProB10 periféricas em comparação com indivíduos de controle saudáveis. Os pacientes de anemia aplástica grave tiveram uma percentagem substancialmente menor de células B10 e células B10pro em comparação com pacientes de anemia aplástica não grave. Além disso, as células B10 e B10pro foram negativamente correlacionadas com contagens absolutas de neutrófilos, níveis de hemoglobina e plaquetas e contagem de reticulócitos absolutos nos pacientes de AA. CONCLUSÕES: Além disso, o estudo presente tentou elucidar o papel imunorregulatório potencial das células B10 na patogênese da AA e fornecer uma nova estratégia para a aplicação de imunoterapia baseada na célula B para tratar a AA no futuro.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Aged , Young Adult , B-Lymphocytes, Regulatory/pathology , Anemia, Aplastic/pathology , Reference Values , Severity of Illness Index , Bone Marrow Cells/cytology , Case-Control Studies , Cells, Cultured , Fluorescent Antibody Technique , Interleukin-10/analysis , Interleukin-10/metabolism , Reticulocyte Count , Antigens, CD19/analysis , Antigens, CD19/metabolism , Flow Cytometry , Anemia, Aplastic/blood , Leukocyte Count , Middle Aged , Neutrophils
3.
J. appl. oral sci ; 27: e20180150, 2019. graf
Article in English | LILACS, BBO | ID: biblio-975883

ABSTRACT

Abstract Objectives This investigation aimed to assess the differentiation inhibitory effects of ProRoot MTA® (PMTA) and Biodentine® (BIOD) on osteoclasts originated from murine bone marrow macrophages (BMMs) and compare these effects with those of alendronate (ALD). Materials and Methods Mouse BMMs were cultured to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of NF-κB (RANKL), treated with lipopolysaccharide. After application with PMTA, BIOD, or ALD, cell toxicities were examined using WST-1 assay kit, and RANKL-induced osteoclast differentiation and activities were determined by resorption pit formation assay and tartrate-resistant acid phosphate (TRAP) staining. The mRNA levels of osteoclast activity-related genes were detected with quantitative real time polymerase chain reaction. Expressions of molecular signaling pathways were assessed by western blot. All data were statistically analyzed with one-way ANOVA and Tukey's post-hoc test (p<0.05). Results Mouse BMMs applied with PMTA, BIOD, or ALD showed highly reduced levels of TRAP-positive osteoclasts. The BIOD treated specimens suppressed mRNA expressions of cathepsin K, TRAP, and c-Fos. Nonetheless, it showed a lower effect than PMTA or ALD applications. Compared with ALD, PMTA and BIOD decreased RANKL-mediated phosphorylation of ERK1/2 and IκBα. Conclusions PMTA and BIOD showed the inhibitory effect on osteoclast differentiation and activities similar to that of ALD through IκB phosphorylation and suppression of ERK signaling pathways.


Subject(s)
Animals , Mice , Osteoclasts/drug effects , Root Canal Filling Materials/pharmacology , Bone Marrow Cells/drug effects , Cell Differentiation/drug effects , Silicates/pharmacology , Calcium Compounds/pharmacology , Alendronate/pharmacology , Bone Density Conservation Agents/pharmacology , Osteoclasts/physiology , Osteogenesis/drug effects , Phosphorylation/drug effects , Root Resorption/prevention & control , Time Factors , Bone Marrow Cells/cytology , Cell Survival/drug effects , Cells, Cultured , Blotting, Western , Reproducibility of Results , MAP Kinase Signaling System/drug effects , I-kappa B Proteins/drug effects , RANK Ligand/analysis , RANK Ligand/drug effects , Real-Time Polymerase Chain Reaction , Tartrate-Resistant Acid Phosphatase
4.
Braz. j. biol ; 78(2): 306-310, May-Aug. 2018. tab
Article in English | LILACS | ID: biblio-888877

ABSTRACT

Abstract The goal of this study was to analyze cytotoxicity, genotoxicity and mutagenicity to bone marrow cells of mice of nature identical synthetic flavorings, passion fruit and strawberry, and artificial synthetic flavorings, vanilla, chocolate, tutti-frutti and cookie, at doses 0.5; 1.0; 2.0; 5.0 and 10.0 mL/kg. The additives were given to the animals by gavage in a single daily application for seven days. Data were subjected to analysis of variance (ANOVA) followed by post Tukey's post hoc test, p <0.05. Animals treated with 2.0; 5.0 and 10.0 mL/Kg of flavorings chocolate, strawberry and cookie, and 5.0 and 10.0 mL/Kg of flavorings vanilla and passion fruit died on the fifth and sixth day of the experiment, respectively. The doses 0.5 and 1.0 mL/Kg of the six additives significantly reduced erythropoiesis in the examined tissue. Also, treatments 0.5 and 1.0 mL/Kg of chocolate, and 1.0 mL/Kg of strawberry and biscuit induced the formation of micronuclei in the bone marrow erythrocytes, at a significant frequency. Therefore, under the study conditions, the six microingredients analyzed were cytotoxic and genotoxic, and additives strawberry, chocolate and cookie were also mutagenic in at least one of the evaluated doses.


Resumo Os aromatizantes são essenciais para a indústria na confecção de alimentos industrializados. Porém, pouco se sabe sobre o potencial tóxico desses microingredientes alimentares. Dessa forma, objetivou-se neste trabalho analisar, em células de medula óssea de camundongos, a citotoxicidade, genotoxicidade e mutagenicidade de aromatizantes alimentares sintéticos idênticos ao natural, de maracujá e morango, e artificiais, de baunilha, chocolate, tutti-frutti e biscoito, nas doses 0,5; 1,0; 2,0; 5,0 e 10,0 mL/Kg. Os aditivos foram administrados aos animais via gavagem em aplicação diária única durante sete dias. Os dados obtidos foram submetidos ao procedimento estatístico ANOVA com pós teste de Tukey, com p < 0.05. Os animais tratados com 2,0; 5,0 e 10,0 mL/Kg dos aromatizantes de chocolate, morango e biscoito, e 5,0 e 10,0 mL/Kg dos aromatizantes de baunilha e maracujá vieram a óbito no quinto e sexto dia de experimento, respectivamente. As doses 0,5 e 1,0 mL/Kg dos seis aditivos reduziram significativamente a eritropoiese do tecido analisado. Ainda, os tratamentos 0,5 e 1,0 mL/kg de chocolate, e 1,0 mL/Kg de morango e biscoito induziram a formação de micronúcleos aos eritrócitos de medula em frequência significante. Portanto, nas condições de estudo estabelecidas, os seis microingredientes analisados foram citotóxico e genotóxicos, e os aditivos de morango, chocolate e biscoito também foram mutagênicos em pelo menos uma das doses avaliadas.


Subject(s)
Animals , Mice , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/pathology , Flavoring Agents/toxicity , Cytotoxins/toxicity , Mutagens/toxicity
5.
Braz. j. biol ; 78(1): 1-12, Feb. 2018. tab, graf
Article in English | LILACS | ID: biblio-888851

ABSTRACT

Abstract Handroanthus impetiginosus has long been used in traditional medicine and various studies have determined the presence of bioactive chemical compounds and potential phytotherapeutics. In this study, the genotoxicity of the lyophilized tincture of H. impetiginosus bark (THI) was evaluated in mouse bone marrow using micronucleus assays. The interaction between THI and genotoxic effects induced by the chemotherapeutic agent, doxorubicin (DXR), was also analyzed. Experimental groups were evaluated 24 to 48 h after treatment with N-nitroso-N-ethylurea (NEU; 50 mg/kg), DXR (5 mg/kg), sodium chloride (NaCl; 150 mM), and THI (0.5-2 g/kg). Antigenotoxic assays were carried out using THI (0.5 g/kg) in combination with NEU or DXR. Analysis of the micronucleated polychromatic erythrocytes (MNPCEs) indicated no significant differences between treatment doses of THI (0.5-2 g/kg) and NaCl. Polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) ratios did not indicate any statistical differences between DXR and THI or NaCl, but there were differences between THI and NaCl. A significant reduction in MNPCEs and PCE/NCE ratios was observed when THI was administered in combination with DXR. This study suggested the absence of THI genotoxicity that was dose-, time-, and gender-independent and the presence of moderate systemic toxicity that was dose-independent, but time- and gender-dependent. The combination of THI and DXR also suggested antigenotoxic effects, indicating that THI reduced genotoxic effects induced by chemotherapeutic agents.


Resumo Handroanthus impetiginosus tem sido usada durante um longo período pela medicina tradicional e vários estudos têm demonstrados a presença de compostos químicos e potencial fitoterapêutico. Esta pesquisa avaliou a genotoxicidade da tintura da casca liofilizada de H. impetiginosus (THI) usando o ensaio do micronúcleo em medula óssea de camundongos. A interação entre THI e os efeitos genotóxicos induzidos pelo quimioterápico doxorrubicina (DXR) também foram analisados. Grupos experimentais foram analisados a 24-48 h após o tratamento com N-Nitroso-N-etiluréia (NEU; 50 mg/kg), DXR (5 mg/kg), NaCl (150 mM) e THI (0,5-2 g/kg). O ensaio antigenotóxico foi conduzido utilizando THI (0,5 g/kg) em combinação com NEU ou DXR. A análise de eritrócitos policromáticos micronucleados (EPCMNs) não mostrou diferenças significativas entre as doses de tratamento (0,5-2 g/kg) e NaCl. As proporções de eritrócitos policromáticos (EPC)/eritrócitos normocromáticos (ENC) não revelaram diferenças estatísticas entre DXR e THI ou NaCl, porém houve diferenças entre THI e NaCl. Uma redução significativa em EPCMNs e na razão EPC/ENC foi observada quando THI foi administrado em combinação com DXR. Essa pesquisa sugere ausência de genotoxicidade de THI, dose-, tempo- e sexo-independente, e moderada toxicidade sistêmica dose-independente, mas tempo- e sexo-dependente. A associação do THI e DXR também sugere efeitos antigenotóxicos. Por conseguinte, THI pode reduzir os efeitos genotóxicos induzidos pelo quimioterapêutico.


Subject(s)
Animals , Mice , DNA Damage/drug effects , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Plant Extracts/pharmacology , Doxorubicin/toxicity , Protective Agents/pharmacology , Micronucleus Tests , Cells, Cultured , Tabebuia/chemistry
8.
Yonsei Medical Journal ; : 1522-1529, 2015.
Article in English | WPRIM | ID: wpr-177074

ABSTRACT

PURPOSE: Adipose-derived stem cells (ADSCs) are known to be potentially effective in regeneration of damaged tissue. We aimed to assess the effectiveness of intracoronary administration of ADSCs in reducing the infarction area and improving function after acute transmural myocardial infarction (MI) in a porcine model. MATERIALS AND METHODS: ADSCs were obtained from each pig's abdominal subcutaneous fat tissue by simple liposuction. After 3 passages of 14-days culture, 2 million ADSCs were injected into the coronary artery 30 min after acute transmural MI. At baseline and 4 weeks after the ADSC injection, 99mTc methoxyisobutylisonitrile-single photon emission computed tomography (MIBISPECT) was performed to evaluate the left ventricular volume, left ventricular ejection fraction (LVEF; %), and perfusion defects as well as the myocardial salvage (%) and salvage index. At 4 weeks, each pig was sacrificed, and the heart was extracted and dissected. Gross and microscopic analyses with specific immunohistochemistry staining were then performed. RESULTS: Analysis showed improvement in the perfusion defect, but not in the LVEF in the ADSC group (n=14), compared with the control group (n=14) (perfusion defect, -13.0+/-10.0 vs. -2.6+/-12.0, p=0.019; LVEF, -8.0+/-15.4 vs. -15.9+/-14.8, p=0.181). There was a tendency of reducing left ventricular volume in ADSC group. The ADSCs identified by stromal cell-derived factor-1 (SDF-1) staining were well co-localized by von Willebrand factor and Troponin T staining. CONCLUSION: Intracoronary injection of cultured ADSCs improved myocardial perfusion in this porcine acute transmural MI model.


Subject(s)
Adipose Tissue/cytology , Animals , Bone Marrow Cells/cytology , Chemokine CXCL12 , Coronary Vessels , Female , Heart/physiopathology , Heart Ventricles , Mesenchymal Stem Cells , Myocardial Infarction/physiopathology , Stem Cell Transplantation , Swine , Technetium Tc 99m Sestamibi/pharmacology , Tomography, Emission-Computed, Single-Photon/methods , Troponin T , Ventricular Function, Left
9.
Article in English | WPRIM | ID: wpr-165765

ABSTRACT

The direct differentiation of hepatocytes from bone marrow cells remains controversial. Several mechanisms, including transdifferentiation and cell fusion, have been proposed for this phenomenon, although direct visualization of the process and the underlying mechanisms have not been reported. In this study, we established an efficient in vitro culture method for differentiation of functioning hepatocytes from murine lineage-negative bone marrow cells. These cells reduced liver damage and incorporated into hepatic parenchyma in two independent hepatic injury models. Our simple and efficient in vitro protocol for endodermal precursor cell survival and expansion enabled us to identify these cells as existing in Sca1+ subpopulations of lineage-negative bone marrow cells. The endodermal precursor cells followed a sequential developmental pathway that included endodermal cells and hepatocyte precursor cells, which indicates that lineage-negative bone marrow cells contain more diverse multipotent stem cells than considered previously. The presence of equivalent endodermal precursor populations in human bone marrow would facilitate the development of these cells into an effective treatment modality for chronic liver diseases.


Subject(s)
Animals , Ataxin-1/analysis , Bone Marrow Cells/cytology , Cell Differentiation , Cell Proliferation , Cells, Cultured , Female , Hepatocytes/cytology , Mice , Mice, Inbred BALB C
10.
Article in English | WPRIM | ID: wpr-128618

ABSTRACT

BACKGROUND/AIMS: Therapies involving bone-marrow-derived mesenchymal stem cells (BM-MSCs) have considerable potential in the management of hepatic disease. BM-MSCs have been investigated in regenerative medicine due to their ability to secrete various growth factors and cytokines that regress hepatic fibrosis and enhance hepatocyte functionality. The aim of this study was to determine the antifibrosis effect of BM-MSCs on activated hepatic stellate cells (HSCs) and the mechanism underlying how BM-MSCs modulate the function of activated HSCs. METHODS: We used HSCs in both direct and indirect co-culture systems with BM-MSCs to evaluate the antifibrosis effect of BM-MSCs. The cell viability and apoptosis were evaluated by a direct co-culture system of activated HSCs with BM-MSCs. The activations of both HSCs alone and HSCs with BM-MSCs in the direct co-culture system were observed by immunocytochemistry for alpha-smooth muscle actin (alpha-SMA). The levels of growth factors and cytokines were evaluated by an indirect co-culture system of activated HSCs with BM-MSCs. RESULTS: The BM-MSCs in the direct co-culture system significantly decreased the production of alpha-SMA and the viability of activated HSCs, whereas they induced the apoptosis of activated HSCs. The BM-MSCs in the indirect co-culture system decreased the production of transforming growth factor-beta1 and interleukin (IL)-6, whereas they increased the production of hepatocyte growth factor and IL-10. These results confirmed that the juxtacrine and paracrine effects of BM-MSCs can inhibit the proliferative, fibrogenic function of activated HSCs and have the potential to reverse the fibrotic process by inhibiting the production of alpha-SMA and inducing the apoptosis of HSCs. CONCLUSIONS: These results have demonstrated that BM-MSCs may exert an antifibrosis effect by modulating the function of activated HSCs.


Subject(s)
Apoptosis , Bone Marrow Cells/cytology , Cell Differentiation , Coculture Techniques , Hepatic Stellate Cells/cytology , Hepatocyte Growth Factor/metabolism , Humans , Immunophenotyping , Interleukin-10/metabolism , Interleukin-6/metabolism , Liver Cirrhosis , Mesenchymal Stem Cells/cytology , Transforming Growth Factor beta1/metabolism
11.
Article in English | WPRIM | ID: wpr-215496

ABSTRACT

Proangiogenic cells (PACs) display surface markers and secrete angiogenic factors similar to those used by myelomonocytic cells, but, unlike myelomonocytic cells, PACs enhance neovascularization activity in experimental ischemic diseases. This study was performed to reveal the differential neovascularization activities of PACs compared with those of myelomonocytic cells. We cultured PACs and CD14+-derived macrophages (Macs) for 7 days. Most of the surface markers and cytokines in the two cell types were alike; the exceptions were KDR, beta8 integrin, interleukin-8 and monocyte chemotactic protein-1. Unlike Macs, PACs significantly enhanced mesenchymal stem cell (MSC) transmigration. PACs and Macs increased neovascularization activity in an in vitro co-culture of human umbilical vein endothelial cells and MSCs and in an in vivo cotransplantation in Matrigel. However, the use of Macs resulted in inappropriately dilated and leaky vessels, whereas the use of PACs did not. We induced critical hindlimb ischemia in nude mice, and then transplanted PACs, Macs or vehicle into the mice. We obtained laser Doppler perfusion images weekly. At 2 weeks, mice treated with PACs showed significantly enhanced perfusion recovery in contrast to those treated with Macs. After day 7, when cells were depleted using a suicidal gene, viral thymidine kinase, to induce apoptosis of the cells in vivo by ganciclovir administration, we found that the improved perfusion was significantly abrogated in the PAC-treated group, whereas perfusion was not changed in the Mac-treated group. PACs caused an increase in healthy new vessels in in vitro and in vivo models of angiogenesis and enhanced long-term functional neovascularization activity in the hindlimb ischemia model, whereas Macs did not. Nevertheless, the angiogenic potential and long-term functional results for a specific cell type should be validated to confirm effectiveness and safety of the cell type for use in therapeutic angiogenesis procedures.


Subject(s)
Animals , Bone Marrow Cells/cytology , Cells, Cultured , Cytokines/analysis , Human Umbilical Vein Endothelial Cells , Humans , Ischemia/pathology , Macrophages/cytology , Male , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred BALB C , Mice, Nude , Neovascularization, Pathologic/pathology , Neovascularization, Physiologic
12.
Acta cir. bras ; 29(8): 478-484, 08/2014. graf
Article in English | LILACS | ID: lil-719180

ABSTRACT

PURPOSE: To characterize bone marrow progenitors cells grown in vitro, using native goats from northeastern Brazil as animal model. METHODS: Ten northeastern Brazil native goats of both genders were used from the Piauí Federal University Agricultural Science Center's (UFPI) - Goat Farming Sector. Bone marrow aspirates where taken from the tibial ridge and seeded on culture plates for isolation, expansion and Flow Cytometry (expression markers - Oct-3/4, PCNA, Ck-Pan, Vimentina, Nanog). RESULTS: Progenitor cells showed colonies characterized by the presence of cell pellets with fibroblastoid morphology. Cell confluence was taken after 14 days culture and the non-adherent mononuclear cell progressive reduction. After the first passage, 94.36% cell viability was observed, starting from 4.6 x 106 cell/mL initially seeded. Cells that went through flow cytometry showed positive expression for Oct-3/4, PCNA, Ck-Pan, Vimentina, and Nanog. CONCLUSIONS: Bone marrow progenitor isolated of native goats from northeastern Brazil showed expression markers also seen in embryonic stem cells (Oct-3/4, Nanog), markers of cell proliferation (PCNA) and markers for mesenchymal cells (Vimentina and Ck-pan), which associated to morphological and culture growth features, suggest the existence of a mesenchymal stem cell (MSC) population in the goat bone marrow stromal cells studied. .


Subject(s)
Animals , Female , Male , Bone Marrow Cells/cytology , Mesenchymal Stem Cells/cytology , Brazil , Cell Culture Techniques , Cell Differentiation , Cell Proliferation , Flow Cytometry/methods , Goats , Models, Animal , /isolation & purification , Proliferating Cell Nuclear Antigen/isolation & purification , Vimentin/isolation & purification
13.
J. appl. oral sci ; 22(3): 218-227, May-Jun/2014. tab, graf
Article in English | LILACS, BBO | ID: lil-711715

ABSTRACT

Stem cell-based regenerative medicine is one of the most intensively researched medical issues. Pre-clinical studies in a large-animal model, especially in swine or miniature pigs, are highly relevant to human applications. Mesenchymal stem cells (MSCs) have been isolated and expanded from different sources. Objective: This study aimed at isolating and characterizing, for the first time, bone marrow-derived MSCs (BM-MSCs) from a Brazilian minipig (BR1). Also, this aimed to validate a new large-animal model for stem cell-based tissue engineering. Material and Methods: Bone marrow (BM) was aspirated from the posterior iliac crest of twelve adult male BR1 under general anesthesia. MSCs were selected by plastic-adherence as originally described by Friedenstein. Cell morphology, surface marker expression, and cellular differentiation were examined. The immunophenotypic profile was determined by flow cytometry. The differentiation potential was assessed by cytological staining and by RT-PCR. Results: MSCs were present in all minipig BM samples. These cells showed fibroblastic morphology and were positive for the surface markers CD90 (88.6%), CD29 (89.8%), CD44 (86.9%) and negative for CD34 (1.61%), CD45 (1.83%), CD14 (1.77%) and MHC-II (2.69%). MSCs were differentiated into adipocytes, osteoblasts, and chondroblasts as demonstrated by the presence of lipidic-rich vacuoles, the mineralized extracellular matrix, and the great presence of glycosaminoglycans, respectively. The higher gene expression of adipocyte fatty-acid binding protein (AP2), alkaline phosphatase (ALP) and collagen type 2 (COLII) also confirmed the trilineage differentiation (p<0.001, p<0.001, p=0.031; respectively). Conclusions: The isolation, cultivation, and differentiation of BM-MSCs from BR1 makes this animal eligible as a useful large-animal model for stem cell-based studies in Brazil. .


Subject(s)
Animals , Male , Bone Marrow Cells/cytology , Mesenchymal Stem Cells/cytology , Models, Animal , Swine, Miniature , Tissue Engineering/methods , Antigens, CD/analysis , Brazil , Cell Culture Techniques , Cell Differentiation , Cell Survival , Cells, Cultured , Flow Cytometry , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Swine
14.
Einstein (Säo Paulo) ; 12(1): 75-81, Jan-Mar/2014. tab, graf
Article in English | LILACS | ID: lil-705800

ABSTRACT

Objective : To evaluate the effect of low-level laser irradiation on the proliferation and possible nuclear morphological changes of mouse mesenchymal stem cells. Methods : Mesenchymal stem cells derived from bone marrow and adipose tissue were submitted to two applications (T0 and T48 hours) of low-level laser irradiation (660nm; doses of 0.5 and 1.0J/cm2). The trypan blue assay was used to evaluate cell viability, and growth curves were used to analyze proliferation at zero, 24, 48, and 72 hours. Nuclear alterations were evaluated by staining with DAPI (4’-6-diamidino-2-phenylindole) at 72 hours. Results : Bone marrow-derived mesenchymal stem cells responded to laser therapy in a dose-dependent manner. Higher cell growth was observed when the cells were irradiated with a dose of 1.0J/cm2, especially after 24 hours (p<0.01). Adipose-derived mesenchymal stem cells responded better to a dose of 1.0J/cm2, but higher cell proliferation was observed after 48 hours (p<0.05) and 72 hours (p<0.01). Neither nuclear alterations nor a significant change in cell viability was detected in the studied groups. Conclusion : Low-level laser irradiation stimulated the proliferation of mouse mesenchymal stem cells without causing nuclear alterations. The biostimulation of mesenchymal stem cells using laser therapy might be an important tool for regenerative therapy and tissue engineering. .


Objetivo : Avaliar o efeito da terapia com laser de baixa intensidade sobre a proliferação e as possíveis alterações morfológicas nucleares em células-tronco mesenquimais de camundongos. Métodos : Células-tronco mesenquimais derivadas da medula óssea e do tecido adiposo foram submetidas a duas aplicações (T0 e T48 horas) de laser de baixa intensidade (660nm; doses de 0,5 e 1,0J/cm2). O ensaio de azul de tripan foi utilizado para a avaliação da viabilidade celular, e curvas de crescimento foram usadas para avaliar a proliferação das células em zero, 24, 48, e 72 horas. Alterações nucleares foram avaliadas por coloração com DAPI (4-6-diamidino-2-fenilindolo) em 72 horas. Resultados : As células-tronco mesenquimais derivadas da medula óssea responderam a terapia com laser de forma dose-dependente. Um maior crescimento celular foi observado quando as células foram irradiadas com dose de 1,0J/cm2, especialmente depois de 24 horas (p<0,01). As células-tronco mesenquimais derivadas do tecido adiposo responderam melhor à dose de 1,0J/cm2, com maior proliferação após 48 (p<0,05) e 72 horas (p<0,01). Nem alterações nucleares nem a mudança significativa na viabilidade celular foi detectada nos grupos estudados. Conclusão : Laser de baixa intensidade estimulou a proliferação de células-tronco mesenquimais sem causar alterações nucleares. A bioestimulação de células-tronco mesenquimais por laserterapia pode ser uma ferramenta importante para a terapia regenerativa e a engenharia tecidual. .


Subject(s)
Animals , Humans , Male , Mice , Cell Proliferation/radiation effects , Low-Level Light Therapy/methods , Mesenchymal Stem Cells/radiation effects , Adipose Tissue/cytology , Bone Marrow Cells/cytology , Bone Marrow Cells/radiation effects , Cell Survival/drug effects , Cells, Cultured/radiation effects , Dose-Response Relationship, Radiation , Lasers, Semiconductor/therapeutic use , Mesenchymal Stem Cells/cytology , Radiation Dosage , Reproducibility of Results , Time Factors
15.
Article in English | WPRIM | ID: wpr-53761

ABSTRACT

Recent studies suggest that the intracoronary administration of bone marrow (BM)-derived mesenchymal stem cells (MSCs) may improve left ventricular function in patients with acute myocardial infarction (AMI). However, there is still argumentative for the safety and efficacy of MSCs in the AMI setting. We thus performed a randomized pilot study to investigate the safety and efficacy of MSCs in patients with AMI. Eighty patients with AMI after successful reperfusion therapy were randomly assigned and received an intracoronary administration of autologous BM-derived MSCs into the infarct related artery at 1 month. During follow-up period, 58 patients completed the trial. The primary endpoint was changes in left ventricular ejection fraction (LVEF) by single-photon emission computed tomography (SPECT) at 6 month. We also evaluated treatment-related adverse events. The absolute improvement in the LVEF by SPECT at 6 month was greater in the BM-derived MSCs group than in the control group (5.9%+/-8.5% vs 1.6%+/-7.0%; P=0.037). There was no treatment-related toxicity during intracoronary administration of MSCs. No significant adverse cardiovascular events occurred during follow-up. In conclusion, the intracoronary infusion of human BM-derived MSCs at 1 month is tolerable and safe with modest improvement in LVEF at 6-month follow-up by SPECT. (ClinicalTrials.gov registration number: NCT01392105)


Subject(s)
Adult , Aged , Bone Marrow Cells/cytology , Cell- and Tissue-Based Therapy/adverse effects , Echocardiography , Female , Heart/physiopathology , Humans , Male , Mesenchymal Stem Cell Transplantation/adverse effects , Mesenchymal Stem Cells/cytology , Middle Aged , Myocardial Infarction/therapy , Pilot Projects , Stroke Volume , Tomography, Emission-Computed, Single-Photon , Transplantation, Autologous , Treatment Outcome , Ventricular Function, Left , Young Adult
16.
Article in English | WPRIM | ID: wpr-191055

ABSTRACT

The purpose of this study was to devise an expanded ischemic flap model and to investigate the role of AMD-3100 (Plerixafor, chemokine receptor 4 inhibitor) in this model by confirming its effect on mobilization of stem cells from the bone marrow. Male Sprague-Dawley rats were used as an animal research model. The mobilization of stem cells from the bone marrow was confirmed in the AMD-3100-treated group. The fractions of endothelial progenitor cells (EPC) and the vascular endothelial growth factor receptor (VEGFR) 2+ cells in the peripheral blood were increased in groups treated with AMD-3100. The expression of vascular endothelial growth factor (VEGF) was increased in response to expansion or AMD injection. The expression of stromal cell derived factor (SDF)-1 and VEGFR2 were increased only in unexpanded flap treated with AMD-3100. Treatment with AMD-3100 increased both the number and area of blood vessels. However, there were no statistically significant differences in the survival area or physiologic microcirculation in rats from the other groups. This endogenous neovascularization induced by AMD-3100 may be a result of the increase in both the area and number of vessels, as well as paracrine augmentation of the expression of VEGF and EPCs. However, the presence of a tissue expander under the flap could block the neovascularization between the flap and the recipient regardless of AMD-3100 treatment and expansion.


Subject(s)
Animals , Anti-HIV Agents/pharmacology , Bone Marrow Cells/cytology , Chemokine CXCL12/biosynthesis , Endothelial Progenitor Cells/cytology , Hematopoietic Stem Cells/cytology , Heterocyclic Compounds/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Neovascularization, Physiologic , Nitric Oxide Synthase Type III/metabolism , Rats , Rats, Sprague-Dawley , Receptors, CXCR4/antagonists & inhibitors , Surgical Flaps/blood supply , Tissue Expansion/methods , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor Receptor-2/biosynthesis
18.
Article in English | WPRIM | ID: wpr-20005

ABSTRACT

The technique of stem cells or hepatocytes transplantation has recently improved in order to bridge the time before whole-organ liver transplantation. In the present study, unfractionated bone marrow stem cells (BMSCs) were harvested from the tibial and femoral marrow compartments of male mice, which were cultured in Dulbecco's modified Eagle's medium (DMEM) with and without hepatocyte growth factor (HGF), and then transplanted into Schistosoma mansoni-infected female mice on their 8th week post-infection. Mice were sacrificed monthly until the third month of bone marrow transplantation, serum was collected, and albumin concentration, ALT, AST, and alkaline phosphatase (ALP) activities were assayed. On the other hand, immunohistopathological and immunohistochemical changes of granuloma size and number, collagen content, and cells expressing OV-6 were detected for identification of liver fibrosis. BMSCs were shown to differentiate into hepatocyte-like cells. Serum ALT, AST, and ALP were markedly reduced in the group of mice treated with BMSCs than in the untreated control group. Also, granuloma showed a marked decrease in size and number as compared to the BMSCs untreated group. Collagen content showed marked decrease after the third month of treatment with BMSCs. On the other hand, the expression of OV-6 increased detecting the presence of newly formed hepatocytes after BMSCs treatment. BMSCs with or without HGF infusion significantly enhanced hepatic regeneration in S. mansoni-induced fibrotic liver model and have pathologic and immunohistopathologic therapeutic effects. Also, this new therapeutic trend could generate new hepatocytes to improve the overall liver functions.


Subject(s)
Alanine Transaminase/blood , Alkaline Phosphatase/blood , Animals , Antigens, Differentiation/biosynthesis , Aspartate Aminotransferases/blood , Bone Marrow Cells/cytology , Bone Marrow Transplantation , Cell Differentiation , Cell- and Tissue-Based Therapy , Cells, Cultured , Collagen/metabolism , Female , Granuloma/parasitology , Hepatocyte Growth Factor/pharmacology , Hepatocytes/cytology , Liver/parasitology , Liver Cirrhosis/parasitology , Male , Mice , Mice, Inbred BALB C , Schistosoma mansoni/pathogenicity , Schistosomiasis mansoni/mortality , Stem Cell Transplantation , Stem Cells/cytology
19.
Article in English | WPRIM | ID: wpr-228628

ABSTRACT

OBJECTIVE: To assess the feasibility of T2*-corrected fat-signal fraction (FF) map by using the three-echo volume interpolated breath-hold gradient echo (VIBE) Dixon sequence to differentiate between malignant marrow-replacing lesions and benign red marrow deposition of vertebrae. MATERIALS AND METHODS: We assessed 32 lesions from 32 patients who underwent magnetic resonance imaging after being referred for assessment of a known or possible vertebral marrow abnormality. The lesions were divided into 21 malignant marrow-replacing lesions and 11 benign red marrow depositions. Three sequences for the parameter measurements were obtained by using a 1.5-T MR imaging scanner as follows: three-echo VIBE Dixon sequence for FF; conventional T1-weighted imaging for the lesion-disc ratio (LDR); pre- and post-gadolinium enhanced fat-suppressed T1-weighted images for the contrast-enhancement ratio (CER). A region of interest was drawn for each lesion for parameter measurements. The areas under the curve (AUC) of the parameters and their sensitivities and specificities at the most ideal cutoff values from receiver operating characteristic curve analysis were obtained. AUC, sensitivity, and specificity were respectively compared between FF and CER. RESULTS: The AUCs of FF, LDR, and CER were 0.96, 0.80, and 0.72, respectively. In the comparison of diagnostic performance between the FF and CER, the FF showed a significantly larger AUC as compared to the CER (p = 0.030), although the difference of sensitivity (p = 0.157) and specificity (p = 0.157) were not significant. CONCLUSION: Fat-signal fraction measurement using T2*-corrected three-echo VIBE Dixon sequence is feasible and has a more accurate diagnostic performance, than the CER, in distinguishing benign red marrow deposition from malignant bone marrow-replacing lesions.


Subject(s)
Adult , Aged , Aged, 80 and over , Area Under Curve , Bone Marrow Cells/cytology , Bone Marrow Transplantation , Contrast Media , Diagnosis, Differential , Female , Humans , Magnetic Resonance Imaging , Male , Middle Aged , ROC Curve , Sensitivity and Specificity , Signal-To-Noise Ratio , Spinal Diseases/diagnosis
20.
Yonsei Medical Journal ; : 1130-1137, 2014.
Article in English | WPRIM | ID: wpr-207148

ABSTRACT

PURPOSE: To determine whether renal injury induced by ischemia-reperfusion (I/R) could be further improved by mesenchymal stem cells (MSCs) modified with survivin. MATERIALS AND METHODS: Lentiviral vectors were used to introduce the survivin gene into MSCs and the MSCs modified with survivin were transplanted into established mice models of renal I/R injury. Seven days later, serum creatinine (Scr) and blood urea nitrogen (BUN) were measured and the survival of MSCs was determined. Hematoxylin and eosin staining was used to assess renal pathological change. The expressions of hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) in kidney tissue were detected by western blot. RESULTS: Mice transplanted with survivin-modified MSCs demonstrated good renal function recovery with Scr and BUN decline close to normal levels and improvement of renal I/R injury repair. Additionally, the survival of transplanted MSCs modified with survivin was enhanced and the expression of HGF and bFGF in kidney tissue was increased. CONCLUSION: Our results demonstrated that MSCs engineered to over-express survivin could enhance their therapeutic effect on renal I/R injury in mice, probably via the improved survival ability of MSCs and increased production of protective cytokines in ischemic tissue.


Subject(s)
Animals , Bone Marrow Cells/cytology , Inhibitor of Apoptosis Proteins/therapeutic use , Male , Mesenchymal Stem Cell Transplantation/methods , Mice , Mice, Inbred C57BL , Reperfusion Injury/drug therapy , Repressor Proteins/therapeutic use
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