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Umbilical cord blood is a rich source of hematopoietic stem cells that has been used for transplantation for over 30 years, especially when there is no compatible hematopoietic stem cell donor available. Its use has decreased more recently, since the development of methods to improve haploidentical transplants has allowed the use of mobilized peripheral blood as a source of hematopoietic stem cells. Public cord blood banks collect, process and store cord blood samples from voluntary donations. In addition, many public banks are involved in research to enhance hematopoietic stem cell therapies and develop new treatments for haematological and genetic diseases. The COVID-19 pandemic, which emerged in 2019, has had a profound and wide-ranging impact on human health and treatment. The area of hematopoietic stem cell transplantation was deeply affected by reductions in bone marrow, peripheral blood and cord blood donations; logistical challenges; exposure of healthcare workers and other challenges. The present study reviews the impact of the COVID-19 pandemic on cord blood banking and transportation around the world with a special focus on Brazil.
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Bancos de Sangue , COVID-19 , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Sangue Fetal , Pandemias , SARS-CoV-2 , Humanos , COVID-19/epidemiologia , Sangue Fetal/citologia , SARS-CoV-2/isolamento & purificação , Brasil/epidemiologia , Doadores de SangueRESUMO
Cell therapy and tissue engineering have been intensively researched for repair of articular cartilage. In this study, we investigated the chondrogenic potential of canine adipose-derived mesenchymal stromal cells (ASCs) combined to high molecular weight hyaluronic acid (HA) in vitro, and their therapeutic effect in dogs with chronic osteoarthritis (OA) associated with bilateral hip dysplasia. Canine ASCs were characterized after conventional 2D culture or 3D culture in HA, showing adequate immunophenotype, proliferation and trilineage differentiation, as well as chondrogenesis after cultivation in HA. ASC/HA constructs were used to treat 12 dogs with OA, sequentially assigned to control, ASC and ASC/HA groups. Animals were examined for clinical, orthopedic and radiological parameters. Lameness at walk and pain on manipulation were reduced in the ASC group and mainly in the ASC/HA group. Range of motion and detection of crepitus on hip rotation and abduction improved similarly in all groups. For articular edema, muscle atrophy, Norberg angle values and radiographic analyses, there were no variations throughout the period. These results indicate that ASC/HA constructs are safe and may be an effective therapeutic tool in treating canine chronic osteoarthritis, which should be confirmed with larger studies and additional clinical parameters.
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Mesenchymal stromal cells (MSCs) have attracted great attention for therapeutic applications. Since cells derived from different tissues have different properties, using the right tissue source may impact their efficiency in regenerative medicine. This study describes for the first time the isolation and characterization of MSCs derived from the equine coronary corium, which may be useful for treating diseases such as laminitis. Seven coronary corium samples were used for isolation of cells (ccMSCs). Adherent cells were characterized for morphology, immunophenotype, proliferation and differentiation potential, in vitro migration and colony-forming capacity. The cells displayed the characteristic fibroblastoid morphology, with population doubling time increasing until passage 7 and reaching a plateau in passage 10. Cells were negative for CD14 and CD45, and positive for CD73 and CD90. ccMSCs showed chondrogenic and osteogenic, but not adipogenic differentiation, and migrated with nearly total closing of the empty area in 48 h, in the scratch assay. The clonogenic potential was in average 18% to 23%. This study describes for the first time the establishment of mesenchymal stromal cell cultures from the equine coronary corium. The results are similar to MSCs isolated from many other equine tissues, except for restricted differentiation potential. As coronary corium stem cell regulation may contribute to the pathogenesis of equine chronic laminitis, the use of ccMSCs in cell therapy for this significantly debilitating disease should be further investigated.
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Derme/citologia , Cavalos , Células-Tronco Mesenquimais/citologia , Animais , Células Cultivadas , Medicina Regenerativa , Dermatopatias/terapiaRESUMO
Cirrhosis, a late form of liver disease, is characterized by extensive scarring due to exacerbated secretion of extracellular matrix proteins by myofibroblasts that develop during this process. These myofibroblasts arise mainly from hepatic stellate cells (HSCs), liver-specific pericytes that become activated at the onset of liver injury. Consequently, HSCs tend to be viewed mainly as myofibroblast precursors in a fibrotic process driven by inflammation. Here, the molecular interactions between liver pericytes and inflammatory cells such as macrophages and neutrophils at the first moments after injury and during the healing process are brought into focus. Data on HSCs and pericytes from other tissues indicate that these cells are able to sense pathogen- and damage-associated molecular patterns and have an important proinflammatory role in the initial stages of liver injury. On the other hand, further data suggest that as the healing process evolves, activated HSCs play a role in skewing the initial proinflammatory (M1) macrophage polarization by contributing to the emergence of alternatively activated, pro-regenerative (M2-like) macrophages. Finally, data suggesting that some HSCs activated during liver injury could behave as hepatic progenitor or stem cells will be discussed.
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Inflamação/metabolismo , Hepatopatias/metabolismo , Fígado/metabolismo , Miofibroblastos/metabolismo , Pericitos/metabolismo , Animais , Humanos , Inflamação/patologia , Fígado/patologia , Hepatopatias/patologia , Miofibroblastos/patologia , Pericitos/patologiaRESUMO
Cell therapy has shown impressive effects in experimental cardiomyopathy models. To a lesser extent, gene therapy has also been studied. In both cases, translation to clinical therapy has been disappointing. This paper is intended to describe the experience and achievements of a multicenter working group located in Porto Alegre, southern Brazil, in experimental and translational research projects for cell-based and gene therapy methods in the treatment of dilated and ischemic cardiomyopathies. The results of preclinical and clinical studies showed that bone marrow mononuclear stem cells indeed have an effect in improving myocardial perfusion and contractile function, but the overall results are poorly translated to the clinical level. Gene therapy studies with direct myocardial injections of naked VEGF 165 plasmid showed improvement in myocardial perfusion and function in animal models. A randomized clinical trial found that this method is safe and improved myocardial perfusion, but the benefits disappeared after 1 year. An animal experiment associating VEGF 165 with angiopoietin was undertaken in mini pigs to extend the durability of that therapy. In conclusion, our efforts to better understand the mechanisms and functions of gene and cell-based therapies in cardiology resulted in significant findings and propose a future look at cell-free therapeutic approaches.
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Cardiomiopatias/terapia , Cardiomiopatia Dilatada/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Angina Pectoris/terapia , Animais , Transplante de Medula Óssea/métodos , Brasil , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Genética/métodos , Insuficiência Cardíaca/terapia , Humanos , Células-Tronco Mesenquimais/metabolismo , Isquemia Miocárdica/terapia , Miocárdio/metabolismo , Transplante Autólogo , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Osteoarthritis associated with hip dysplasia is one of the most common orthopedic abnormalities in dogs, with an incidence of up to 40% in some breeds. Tissue therapy of cartilage has received great attention, with use of mesenchymal stromal cells and different types of biomaterials. The present study aimed to evaluate the effect of platelet lysate (PL) on the proliferation and differentiation of canine adipose tissue-derived mesenchymal stromal cells (ASCs), in liquid culture or hydrogels. PL was prepared from blood collected from healthy dogs and submitted to freezing-thawing cycles, and hydrogel was formed with canine thrombin. The effect of PL on the proliferation and differentiation of canine ASCs was evaluated in liquid and hydrogel systems, with microscopy, quantification of dsDNA, histology and quantification of glycosaminoglycans. The addition of 5% or 10% PL to the culture medium induced a greater proliferation rate than the presence of 10% fetal bovine serum. The cultivation of ASCs in PL gel, with normal or chondrogenic medium, resulted in maintenance of proliferation level similar to the conventional 2D cultivation, and induction of chondrogenic differentiation, especially in the presence of the chondrogenesis induction medium.
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Tecido Adiposo/fisiologia , Condrogênese/fisiologia , Liases/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Animais , Plaquetas/enzimologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Liases/administração & dosagem , Células-Tronco Mesenquimais/fisiologiaRESUMO
Mesenchymal stem/stromal cells (MSCs) are multipotent cells distributed in all tissues and characterized by adherence, morphology, immunophenotype and trilineage differentiation potential. The present study aimed to isolate and characterize adherent MSC-like populations from different tissues of Ctenomys minutus, a threatened wildlife rodent popularly known as tuco-tuco. Adherent cells were isolated from bone marrow, brain, liver, pancreas and adipose tissue of three adult animals collect in southern Brazil. Cultures showed typical morphology and proliferation potential. Adipose-derived MSCs showed trilineage potential. Cultures derived from adipose tissue, bone marrow and brain were immunophenotyped with negative results for CD31, CD44, CD45, CD106, and MHC class II, as well as strong positive results for CD29. Low fluorescence levels were seen for CD49d, CD90.2 and CD117. Cultures were negative for CD49e, except for brain-derived cultures that were weakly positive. CD11b was negative in adipose-derived MSCs, but positive in brain and bone marrow-derived cultures. The scratch assay showed high migration potential for pancreas and adipose tissue-derived cells. This study represents the first report of isolation and characterization of cultures having characteristics of MSCs from Ctenomys minutus. The collection of biological information for biobanks represents an important contribution to the creation of strategies for prevention of loss of genetic diversity.
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Cultured mesenchymal stromal cells (MSCs) are cells that can be used for tissue engineering or cell therapies owing to their multipotency and ability to secrete immunomodulatory and trophic molecules. Several studies suggest that MSCs can become pericytes when cocultured with endothelial cells (ECs) but failed to use pericyte markers not already expressed by MSCs. We hypothesized ECs could instruct MSCs to express the molecules CD271 or CD34, which are expressed by pericytes in situ but not by MSCs. CD271 is a marker of especial interest because it is associated with multipotency, a characteristic that wanes in MSCs as they are culture expanded. Consequently, surface expression of CD271 and CD34 was detected in roughly half of the MSCs cocultured with ECs as spheroids in the presence of insulin-like growth factor 1 (IGF-1). Conversely, expression of CD271 and CD34 was detected in a similar proportion of MSCs cultured under these conditions without ECs, and expression of these markers was low or absent when no IGF-1 was added. These findings indicate that specific culture conditions including IGF-1 can endow cultured MSCs with expression of CD271 and CD34, which may enhance the multipotency of these cells when they are used for therapeutic purposes.
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Clinical trials using stem cell therapy for heart diseases have not reproduced the initial positive results obtained with animal models. This might be explained by a decreased regenerative capacity of stem cells collected from the patients. This work aimed at the simultaneous investigation of endothelial stem/progenitor cells (EPCs), mesenchymal stem/progenitor cells (MSCs), and hematopoietic stem/progenitor cells (HSCs) in sternal bone marrow samples of patients with ischemic or valvular heart disease, using flow cytometry and colony assays. The study included 36 patients referred for coronary artery bypass grafting or valve replacement surgery. A decreased frequency of stem cells was observed in both groups of patients. Left ventricular dysfunction, diabetes, and intermediate risk in EuroSCORE and SYNTAX score were associated with lower EPCs frequency, and the use of aspirin and ß-blockers correlated with a higher frequency of HSCs and EPCs, respectively. Most importantly, the distribution of frequencies in the three stem cell compartments showed independent patterns. The combined investigation of the three stem cell compartments in patients with cardiovascular diseases showed that they are independently affected by the disease, suggesting the investigation of prognostic factors that may be used to determine when autologous stem cells may be used in cell therapy.
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BACKGROUND: In an attempt to increase the therapeutic potential for myocardial regeneration, there is a quest for new cell sources and types for cell therapy protocols. The pathophysiology of heart diseases may affect cellular characteristics and therapeutic results. METHODS: To study the proliferative and differentiation potential of mesenchymal stem cells (MSC), isolated from bone marrow (BM) of sternum, we made a comparative analysis between samples of patients with ischemic (IHD) or non-ischemic valvular (VHD) heart diseases. We included patients with IHD (n = 42) or VHD (n = 20), with average age of 60 years and no differences in cardiovascular risk factors. BM samples were collected (16.4 ± 6 mL) and submitted to centrifugation with Ficoll-Paque, yielding 4.5 ± 1.5 × 107 cells/mL. RESULTS: Morphology, immunophenotype and differentiation ability had proven that the cultivated sternal BM cells had MSC features. The colony forming unit-fibroblast (CFU-F) frequency was similar between groups (p = 0.510), but VHD samples showed positive correlation to plated cells vs. CFU-F number (r = 0.499, p = 0.049). The MSC culture was established in 29% of collected samples, achieved passage 9, without significant difference in expansion kinetics between groups (p > 0.05). Dyslipidemia and the use of statins was associated with culture establishment for IHD patients (p = 0.049 and p = 0.006, respectively). CONCLUSIONS: Together, these results show that the sternum bone can be used as a source for MSC isolation, and that ischemic or valvular diseases do not influence the cellular yield, culture establishment or in vitro growth kinetics.
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Técnicas de Cultura de Células/métodos , Doenças das Valvas Cardíacas/patologia , Células-Tronco Mesenquimais/citologia , Isquemia Miocárdica/patologia , Esterno/citologia , Idoso , Diferenciação Celular , Proliferação de Células , Separação Celular , Forma Celular , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Humanos , Imunofenotipagem , Cinética , Masculino , Pessoa de Meia-IdadeRESUMO
Studies have shown that metabolic disorders, serum inflammatory markers and weight gain (obesity) are correlated with vitamin D deficiency. Therefore, the present study correlated the serum calcidiol (s25(OH)D3) levels in a sample of individuals from southern Brazil with variables related to metabolic disorders, obesity and lifestyle habits and assessed the cytotoxic effect of calcitriol on adipose tissue-derived mesenchymal stem cells (ADSCs). The results showed a 79.23% prevalence of hypovitaminosis D in the study population and a correlation (p<0.05) between a low serum vitamin D concentration and an elevated low-density lipoprotein cholesterol (LDL-c) level. Univariate linear regression analysis using 25(OH)D3 as a regressor showed a negative association (p<0.05) with an indoor work environment (ß=-2.305), increased body fat (ß=-0.095), age (ß=-0.065) and high-density lipoprotein cholesterol (HDL-c; ß=-0.109). An in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay performed with ADSCs using five calcitriol concentrations (15.625, 31.25, 62.5, 125 and 250nM) indicated cytotoxic potential (p<0.05) at the 62.5nM concentration at 48 and 72h and at the 125 and 250nM concentrations at 24, 48 and 72h. The results reported herein corroborate one another and suggest a key association between vitamin D deficiency and the development of obesity because ADSCs are involved in adipose tissue hyperplasia and differentiate into adipocytes that can sequester the bioavailable vitamin D necessary for homeostasis.
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Tecido Adiposo/citologia , Composição Corporal/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Vitamina D/farmacologia , Adolescente , Adulto , Brasil , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Feminino , Humanos , Concentração Inibidora 50 , Modelos Lineares , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Análise Multivariada , Adulto JovemRESUMO
OBJECTIVES: This study aimed to evaluate the potential of adipose-derived stem cells (ASCs) combined with a modified α-tricalcium phosphate (α-TCP) or gelatin sponge (GS) scaffolds for bone healing in a rat model. MATERIAL AND METHODS: Bone defects were surgically created in the femur of adult SHR rats and filled with the scaffolds, empty or combined with ASCs. The results were analyzed by histology and histomorphometry on days seven, 14, 30, and 60. RESULTS: Significantly increased bone repair was observed on days seven and 60 in animals treated with α-TCP/ASCs, and on day 14 in the group treated with GS/ASCs, when compared with the groups treated with the biomaterials alone. Intense fibroplasia was observed in the group treated with GS alone, on days 14 and 30. CONCLUSIONS: Our results showed that the use of ASCs combined with α-TCP or GS scaffolds resulted in increased bone repair. The higher efficacy of the α-TCP scaffold suggests osteoconductive property that results in a biological support to the cells, whereas the GS scaffold functions just as a carrier. These results confirm the potential of ASCs in accelerating bone repair in in vivo experimental rat models. These results suggest a new alternative for treating bone defects.
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Tecido Adiposo/citologia , Materiais Biocompatíveis/farmacologia , Regeneração Óssea/efeitos dos fármacos , Fosfatos de Cálcio/farmacologia , Esponja de Gelatina Absorvível/farmacologia , Transplante de Células-Tronco/métodos , Alicerces Teciduais , Animais , Materiais Biocompatíveis/uso terapêutico , Fosfatos de Cálcio/uso terapêutico , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fêmur/patologia , Fêmur/cirurgia , Fibroblastos/efeitos dos fármacos , Formazans , Esponja de Gelatina Absorvível/uso terapêutico , Masculino , Modelos Animais , Osteogênese/efeitos dos fármacos , Ratos Endogâmicos SHR , Reprodutibilidade dos Testes , Sais de Tetrazólio , Fatores de Tempo , Resultado do Tratamento , Cicatrização/efeitos dos fármacosRESUMO
Abstract Objectives This study aimed to evaluate the potential of adipose-derived stem cells (ASCs) combined with a modified α-tricalcium phosphate (α-TCP) or gelatin sponge (GS) scaffolds for bone healing in a rat model. Material and Methods Bone defects were surgically created in the femur of adult SHR rats and filled with the scaffolds, empty or combined with ASCs. The results were analyzed by histology and histomorphometry on days seven, 14, 30, and 60. Results Significantly increased bone repair was observed on days seven and 60 in animals treated with α-TCP/ASCs, and on day 14 in the group treated with GS/ASCs, when compared with the groups treated with the biomaterials alone. Intense fibroplasia was observed in the group treated with GS alone, on days 14 and 30. Conclusions Our results showed that the use of ASCs combined with α-TCP or GS scaffolds resulted in increased bone repair. The higher efficacy of the α-TCP scaffold suggests osteoconductive property that results in a biological support to the cells, whereas the GS scaffold functions just as a carrier. These results confirm the potential of ASCs in accelerating bone repair in in vivo experimental rat models. These results suggest a new alternative for treating bone defects.
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Animais , Masculino , Materiais Biocompatíveis/farmacologia , Regeneração Óssea/efeitos dos fármacos , Fosfatos de Cálcio/farmacologia , Tecido Adiposo/citologia , Transplante de Células-Tronco/métodos , Alicerces Teciduais , Esponja de Gelatina Absorvível/farmacologia , Osteogênese/efeitos dos fármacos , Ratos Endogâmicos SHR , Sais de Tetrazólio , Fatores de Tempo , Cicatrização/efeitos dos fármacos , Materiais Biocompatíveis/uso terapêutico , Fosfatos de Cálcio/uso terapêutico , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Reprodutibilidade dos Testes , Resultado do Tratamento , Modelos Animais , Proliferação de Células/efeitos dos fármacos , Fêmur/cirurgia , Fêmur/patologia , Fibroblastos/efeitos dos fármacos , Formazans , Esponja de Gelatina Absorvível/uso terapêuticoRESUMO
Mesenchymal stem cells (MSCs) are known for their important properties involving multilineage differentiation potential., trophic factor secretion and localization along various organs and tissues. On the dark side, MSCs play a distinguished role in tumor microenvironments by differentiating into tumor-associated fibroblasts or supporting tumor growth via distinct mechanisms. Cisplatin (CIS) is a drug widely applied in the treatment of a large number of cancers and is known for its cytotoxic and genotoxic effects, both in vitro and in vivo. Here we assessed the effects of CIS on MSCs and the ovarian cancer cell line OVCAR-3, by MTT and comet assays. Our results demonstrated the resistance of MSCs to cell death and DNA damage induction by CIS, which was not observed when OVCAR-3 cells were exposed to this drug.
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Stem cells isolated from adipose tissue show great therapeutic potential in veterinary medicine, but some points such as the use of fresh or cultured cells and route of administration need better knowledge. This study aimed to evaluate the effect of autologous stromal vascular fraction (SVF, n = 4) or allogeneic cultured adipose-derived stem cells (ASCs, n = 5) injected into acupuncture points in dogs with hip dysplasia and weak response to drug therapy. Canine ASCs have proliferation and differentiation potential similar to ASCs from other species. After the first week of treatment, clinical evaluation showed marked improvement compared with baseline results in all patients treated with autologous SVF and three of the dogs treated with allogeneic ASCs. On days 15 and 30, all dogs showed improvement in range of motion, lameness at trot, and pain on manipulation of the joints, except for one ASC-treated patient. Positive results were more clearly seen in the SVF-treated group. These results show that autologous SVF or allogeneic ASCs can be safely used in acupoint injection for treating hip dysplasia in dogs and represent an important therapeutic alternative for this type of pathology. Further studies are necessary to assess a possible advantage of SVF cells in treating joint diseases.
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Adipose-derived stromal cells (ASCs) are usually isolated by digestion with collagenase. We have compared alternative methods to isolate ASCs in a more economically viable protocol. Nine protocols using red blood cells lysis buffer solution, trypsin, collagenase and centrifugation were compared; the isolation rate, cell viability, expansion rate, immunophenotype and differentiation in adipogenic and osteogenic lineages were analyzed. ASCs were isolated and successfully maintained by digestion with trypsin. Cells presented similar immunophenotypes, adipogenic differentiation and in vitro proliferation but an osteogenic differentiation capacity up to seven times higher than ASCs isolated by collagenase. This alternative protocol is thus efficient and more cost-effective than the commonly-used methods and may represent a promising protocol for obtaining ASCs for bone tissue engineering.
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Tecido Adiposo/citologia , Separação Celular/métodos , Células-Tronco/fisiologia , Proliferação de Células , Sobrevivência Celular , Imunofenotipagem , Manejo de Espécimes/métodosRESUMO
Mucopolysaccharidosis I (MPS I) is a congenital disorder caused by the deficiency of α-l-iduronidase (IDUA), with the accumulation of glycosaminoglycans (GAGs) in the CNS. Although GAG toxicity is not fully understood, previous works suggest a GAG-induced alteration in neuronal membrane composition. This study is aimed to evaluate the levels and distribution of gangliosides and cholesterol in different brain regions (cortex, cerebellum, hippocampus and hypothalamus) in a model using IDUA knockout (KO) mice (C57BL/6). Lipids were extracted with chloroform-methanol and then total gangliosides and cholesterol were determined, followed by ganglioside profile analyses. While no changes in cholesterol content were observed, the results showed a tissue dependent ganglioside alteration in KO mice: a total ganglioside increase in cortex and cerebellum, and a selective presence of GM3, GM2 and GD3 gangliosides in the hippocampus and hypothalamus. To elucidate this, we evaluated gene expression of ganglioside synthesis (GM3, GD3 and GM2/GD2 synthases) and degradation of (Neuraminidase1) enzymes in the cerebellum and hippocampus by RT-sq-PCR. The results obtained with KO mice showed a reduced expression of GD3 and GM2/GD2 synthases and Neuraminidase1 in cerebellum; and a decrease in GM2/GD2 synthase and Neuraminidase1 in the hippocampus. These data suggest that the observed ganglioside changes result from a combined effect of GAGs on ganglioside biosynthesis and degradation.
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Córtex Cerebral/metabolismo , Gangliosídeo G(M1)/metabolismo , Expressão Gênica , Lipídeos de Membrana/metabolismo , Mucopolissacaridose I/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cerebelo/metabolismo , Córtex Cerebral/patologia , Colesterol/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipotálamo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucopolissacaridose I/patologia , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Neuraminidase/genética , Neuraminidase/metabolismo , Sialiltransferases/genética , Sialiltransferases/metabolismoRESUMO
Stem cells are known by their capacity of self-renewal and differentiation into at least one specialized cell type. Mesenchymal stem cells (MSCs) were isolated initially from bone marrow but are now known to exist in any vascularized organ or tissue in adults. MSCs have a great therapeutic potential, due to their ability to migrate to sites of tissue injury and secrete trophic factors that hasten endogenous repair. They have also been shown to present immunosuppressive properties that may be used in the treatment of autoimmune or graft-versus-host diseases. Clinical trials employing MSCs show that the therapy is safe, but the efficiency needs to be in tested in phase III and IV studies. We describe here protocols for the isolation of human MSCs from human bone marrow and adipose tissue. The safe use of these cells demand a thorough in vitro characterization, as described in protocols of immunophenotyping by flow cytometry and analysis of their capacity to differentiate into adipogenic, osteogenic, and chondrogenic lineages.
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Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Tecido Adiposo/citologia , Tecido Adiposo/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Separação Celular , Ensaios Clínicos como Assunto , Citometria de Fluxo , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/imunologiaRESUMO
Mesenchymal stem cells (MSC) are currently considered the most promising type of adult stem cells for therapeutic applications, because they can be easily isolated from the bone marrow and other tissues, and manipulated for different applications. The genetic transformation of MSC using genes that enhance their homing ability, as well as their proliferation and survival capacities when transplanted to sites of injury, is an important alternative to improve MSC function, especially for tissue regeneration. This chapter describes protocols for the transformation of MSC using plasmid vectors by lipofection and electroporation, as well as retroviral vectors representing viral transformations.
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Células-Tronco Mesenquimais/metabolismo , Transformação Genética/genética , Vetores Genéticos/genética , Humanos , Células-Tronco Mesenquimais/citologiaRESUMO
Stress studies of the broad-spectrum antiparasitic nitazoxanide were conducted in order to isolate and elucidate the major degradation product involved in thermal, acid, alkaline, oxidative and photolytic decomposition of the drug in solution and solid state. The major degradation product was identified and characterized using techniques namely LC-DAD, (1)H NMR, (13)C NMR, IR, and MS/MS. The stability of nitazoxanide raw material and nitazoxanide in tablets and in suspension powder was studied under different conditions and the results suggest the formation of the same deacetylated degradation product occur in all cases. This product was also studied in order to determine the preliminary cytotoxicity in vitro with mononuclear cells. Compared with nitazoxanide, the degradation product showed a higher cytotoxicity at a concentration of 40 µg mL(-1) after 48 h of incubation, under tested conditions. Therefore, stress studies showed that special care must be taken during the preparation, manufacture, and storage of this pharmaceutical drug.