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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
BACKGROUND: Polyploid cells can be found in a wide evolutionary spectrum of organisms. These cells are assumed to be involved in tissue regeneration and resistance to stressors. Although the appearance of large multinucleated cells (LMCs) in long-term culture of bone marrow (BM) mesenchymal cells has been reported, the presence and characteristics of such cells in native BM and their putative role in BM reconstitution following injury have not been fully investigated. METHODS: BM-derived LMCs were explored by time-lapse microscopy from the first hours post-isolation to assess their colony formation and plasticity. In addition, sub-lethally irradiated mice were killed every other day for four weeks to investigate the histopathological processes during BM regeneration. Moreover, LMCs from GFP transgenic mice were transplanted to BM-ablated recipients to evaluate their contribution to tissue reconstruction. RESULTS: BM-isolated LMCs produced mononucleated cells with characteristics of mesenchymal stromal cells. Time-series inspections of BM sections following irradiation revealed that LMCs are highly resistant to injury and originate mononucleated cells which reconstitute the tissue. The regeneration process was synchronized with a transient augmentation of adipocytes suggesting their contribution to tissue repair. Additionally, LMCs were found to be adiponectin positive linking the observations on multinucleation and adipogenesis to BM regeneration. Notably, transplantation of LMCs to myeloablated recipients could reconstitute both the hematopoietic system and BM stroma. CONCLUSIONS: A population of resistant multinucleated cells reside in the BM that serves as the common origin of stromal and hematopoietic lineages with a key role in tissue regeneration. Furthermore, this study underscores the contribution of adipocytes in BM reconstruction.
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Transplante de Medula Óssea , Medula Óssea , Camundongos , Animais , Adiponectina , Hematopoese/efeitos da radiação , Células da Medula Óssea , Camundongos Transgênicos , Camundongos Endogâmicos C57BLRESUMO
In spite of significant advancements of therapies for initial eradication of cancers, tumor relapse remains a major challenge. It is for a long time known that polyploid malignant cells are a main source of resistance against chemotherapy and irradiation. However, therapeutic approaches targeting these cells have not been appropriately pursued which could partly be due to the shortage of knowledge on the molecular biology of cell polyploidy. On the other hand, there is a rising trend to appreciate polyploid/ multinucleated cells as key players in tissue regeneration. In this review, we suggest an analogy between the functions of polyploid cells in normal and malignant tissues and discuss the idea that cell polyploidy is an evolutionary conserved source of tissue regeneration also exploited by cancers as a survival factor. In addition, polyploid cells are highlighted as a promising therapeutic target to overcome drug resistance and relapse.
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Neoplasias/genética , Poliploidia , Animais , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Evolução Molecular , Humanos , RegeneraçãoRESUMO
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
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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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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The all-trans-retinoic acid (ATRA) is the most active form of vitamin A that helps to regulate the proliferation, differentiation and apoptosis of several types of cells, mainly the adipocytes, and causes weight loss through the reduction of adipogenesis and lipogenesis. In this present study we demonstrated that ATRA concentrations of 20.75, 50 and 100 µM decreased the cell viability in vitro of human adipose-derived stem cells (ADSCs), and in ADSCs during adipogenic differentiation. The cells cycle assessment showed that ATRA increased the cell frequency in Sub-G1 at 4.02x and decreased it in G1 in 2.54x. Moreover, the membrane integrity loss increased by 4.66x and apoptosis increased by 33.56x in ATRA-treated cultures. The gene expression assay suggested that the treatment using ATRA leads to mitochondrial membrane permeabilization and to consequent release of proapoptotic BAK and BAX molecules (increased expression 5.5 and 5.4x respectively); in addition, it increased CASP3 expression (by 8.8x). These events may activate the Bcl-2 (4.1x increase), GADD45 (increase 3.14x) and PPAR-γ (16x increase) expressions, as well as, to reduce the p53 (by -1.38x) expression; therefore, these events should be further mediated by increased RARα expression (by 3.8x). The results evidenced that ATRA may be a good proposal for mesotherapy strategies in order to control the development of subcutaneous adipose tissue; as this tissue have a higher development in some specific areas and ATRA interferes not only in the ADSCs differentiation but also in the apoptosis of ADSCs, preadipocytes and adipocytes.
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Adipócitos/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Tretinoína/farmacologia , Adipócitos/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
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
This study was designed to (i) identify stable reference genes for the analysis of gene expression during in vitro differentiation of rat adipose stromal cells (rASCs), (ii) recommend stable genes for individual treatment conditions, and (iii) validate these genes by comparison with normalization results from stable and unstable reference genes. On the basis of a literature review, eight genes were selected: Actb, B2m, Hprt1, Ppia, Rplp0, Rpl13a, Rpl5, and Ywhaz. Genes were ranked according to their stability under different culture conditions as assessed using GenNorm, NormFinder, and RefFinder algorithms. Although the employed algorithms returned different rankings, the most frequently top-ranked genes were: B2m and/or Ppia for all 28day treatments (ALL28); Ppia and Hprt1 (adipogenic differentiation; A28), B2m (chondrogenic differentiation; C28), Rpl5 (controls maintained in complete culture medium; CCM), Rplp0 (osteogenic differentiation for 3days; O3), Rpl13a and Actb (osteogenic differentiation for 7days; O7), Rplp0 and Ppia (osteogenic differentiation for 14days; O14), Hprt1 and Ppia (osteogenic differentiation for 28days; O28), as well as Actb (all osteogenesis time points combined; ALLOSTEO). The obtained results indicate that the performance of reference genes depends on the differentiation protocol and on the analysis time, thus providing valuable information for the design of RT-PCR experiments.
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Tecido Adiposo/metabolismo , Algoritmos , Antígenos de Diferenciação/biossíntese , Diferenciação Celular , Perfilação da Expressão Gênica/normas , Regulação da Expressão Gênica , Adipogenia , Tecido Adiposo/citologia , Animais , Antígenos de Diferenciação/genética , Células Cultivadas , Condrogênese , Perfilação da Expressão Gênica/métodos , Osteogênese , Ratos , Células Estromais/citologia , Células Estromais/metabolismoRESUMO
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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Our body contains cells that can be propagated in vitro and give rise to cells with mature mesenchymal phenotypes. These cells are interesting not only because of their differentiation capability, which could be used for tissue engineering, but also because they secrete molecules which have trophic, chemoattractant, and immunomodulatory properties. Along decades of study, these cells have been referred to as fibroblastic cells, stromal cells, or mesenchymal stem cells. There is evidence that pericytes, cells that wrap endothelial cells in blood vessels, behave as stem cells in the tissues, and give rise to these progenitor cells when removed from the body and expanded in culture - a process that may reflect changes that occur in vivo under injury conditions. Here, we discuss the evidence that favors this thesis, and discuss culture methods, clinical and preclinical applications of mesenchymal stem cells under this perspective.
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Células-Tronco Mesenquimais/citologia , Pericitos/citologia , Técnicas de Cultura de Células , Diferenciação Celular , Plasticidade Celular , HumanosRESUMO
Quantitative real-time PCR can detect variations in gene expression. The identification of the stable reference genes (RGs) is necessary to evaluate the expression of specific genes of interest under various conditions in many cell types, including human adipose-derived stromal cells (hASCs). In this study, we used the algorithms BestKeeper, NormFinder, geNorm, and RefFinder to investigate the stability of 15 potential RGs (B2M, eEF1A1, GAPDH, H2AFZ, HMBS, HPRT1, PGK1, PPIA, RPL5, SDHA, TBP, TKT, TRFC, TUBB, and UBC) in hASCs during control, adipo-, chondro-, and osteogenic differentiation for 28 days. RPL5, GAPDH, H2AFZ, and HPRT1 were the most stable RGs, while B2M and UBC were the least stable RGs for the majority of group analyses (tri-lineage differentiation and control analyzed combined or individually; each lineage combined with the control). These RGs were used to normalize adipo- (FABP4, LPL, and PPARG), chondro- (COMP and SOX9), and osteogenic gene expression markers (BMP4, COL1A1, and RUNX2). Each marker showed a similar expression when normalized by H2AFZ, HPRT1, or RPL5, confirming that these RGs exhibit stable expression. However, GAPDH, B2M, and UBC exhibited high standard deviation (SD), down-regulated and/or up-regulated differentiation gene expression markers when compared with stable RGs, demonstrating that these RGs are unstable.