Células-tronco mesenquimais de equinos: isolamento, cultivo e caracterização

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Citotoxicidade, genotoxicidade e expressão gênica em células da polpa dental humana sensibilizadas por cimentos endodônticos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise comparativa de diferentes metodologias para cultivo de células-tronco mesenquimais derivadas de tecido muscular

Pós-graduação em Biociências - FCLAS

Marcadores de superfície de Células-tronco: estudo de reação cruzada inter-espécies

Mesenchymal stem cells (MSCs) are adult multipotent cells with fibroblastoid morphology and adherent to plastic. Furthermore, they can be obtained from different sources. Besides bone marrow, these cells are taken from umbilical cord blood, umbilical vein, saphenous vein, peripheral blood, arteries, liver and fetal pancreas, placenta, dental pulp and adipose tissue. MSCs derived from adipose tissue are important because of the abundant number of cells that can be obtained from this tissue, easy access and little discomfort to the patient. This study compared two techniques for obtaining MSCs from adipose tissue: mechanical dissociation (MD) and enzymatic digestion (ED). We also analyzed the inter-species cross-reactions using commercial monoclonal antibodies directed against surface antigens of stem cells from different species: mouse, horse, rabbit, monkey and human. We found that MD technique is favorable in relation to ED within 15 days of culture, and ED is more efficient in the first days of culture. The data also showed that MD causes less damage to cellular DNA. About inter-species cross-reactions, the monoclonal antibody A69 directed against stem cells from rabbits, which can be used in veterinary medicine, particularly in research involving horses

Efeitos biológicos do laser de baixa potência sobre o cultivo de células-tronco mesenquimais provenientes do tecido adiposo e da medula óssea de cães

Pós-graduação em Medicina Veterinária - FMVZ

Caracterização de célula progenitora hepática no cultivo celular de fígado de feto canino

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise da estabilidade genética de células-tronco mesenquimais humanas

Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren´t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient

Transcriptoma diferencial entre células-tronco mesenquimais humanas jovens e senescentes

Human mesenchymal stem cells (MSC) are powerful sources for cell therapy in regenerative medicine. The long time cultivation can result in replicative senescence or can be related to the emergence of chromosomal alterations responsible for the acquisition of tumorigenesis features in vitro. In this study, for the first time, the expression profile of MSC with a paracentric chromosomal inversion (MSC/inv) was compared to normal karyotype (MSC/n) in early and late passages. Furthermore, we compared the transcriptome of each MSC in early passages with late passages. MSC used in this study were obtained from the umbilical vein of three donors, two MSC/n and one MSC/inv. After their cryopreservation, they have been expanded in vitro until reached senescence. Total RNA was extracted using the RNeasy mini kit (Qiagen) and marked with the GeneChip ® 3 IVT Express Kit (Affymetrix Inc.). Subsequently, the fragmented aRNA was hybridized on the microarranjo Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix Inc.). The statistical analysis of differential gene expression was performed between groups MSC by the Partek Genomic Suite software, version 6.4 (Partek Inc.). Was considered statistically significant differences in expression to p-value Bonferroni correction ˂.01. Only signals with fold change ˃ 3.0 were included in the list of differentially expressed. Differences in gene expression data obtained from microarrays were confirmed by Real Time RT-PCR. For the interpretation of biological expression data were used: IPA (Ingenuity Systems) for analysis enrichment functions, the STRING 9.0 for construction of network interactions; Cytoscape 2.8 to the network visualization and analysis bottlenecks with the aid of the GraphPad Prism 5.0 software. BiNGO Cytoscape pluggin was used to access overrepresentation of Gene Ontology categories in Biological Networks. The comparison between senescent and young at each group of MSC has shown that there is a difference in the expression parttern, being higher in the senescent MSC/inv group. The results also showed difference in expression profiles between the MSC/inv versus MSC/n, being greater when they are senescent. New networks were identified for genes related to the response of two of MSC over cultivation time. Were also identified genes that can coordinate functional categories over represented at networks, such as CXCL12, SFRP1, xvi EGF, SPP1, MMP1 e THBS1. The biological interpretation of these data suggests that the population of MSC/inv has different constitutional characteristics, related to their potential for differentiation, proliferation and response to stimuli, responsible for a distinct process of replicative senescence in MSC/inv compared to MSC/n. The genes identified in this study are candidates for biomarkers of cellular senescence in MSC, but their functional relevance in this process should be evaluated in additional in vitro and/or in vivo assays

Caracterização de células-tronco mesenquimais de camundongos normais e do modelo murino de MPS I

Existe um interesse crescente pelo controle das condições de cultivo necessárias para a expansão de células-tronco de indivíduos adultos devido ao grande potencial para o desenvolvimento de pesquisa básica e de aplicações terapêuticas apresentado pelas mesmas. Atualmente, a literatura apresenta poucos trabalhos que detalhem a biologia da célula-tronco mesenquimal (MSC) de camundongo, revelando a necessidade de estudos voltados para este tema. Quatro culturas de longa duração foram produzidas com células da medula óssea de camundongos normais e IDUA knock-out através de técnicas de cultivo relativamente simples. Estas culturas puderam ser mantidas por até 40 passagens, e demonstraram ser morfologicamente homogêneas. Células dessas culturas puderam ser induzidas a diferenciarem-se ao longo de vias de diferenciação adipogênica e osteogênica, e revelaram ser capazes de suportar o crescimento e a proliferação de células-tronco hematopoiéticas. Por apresentarem tais características funcionais, essas populações celulares foram operacionalmente definidas como MSCs. Quando o repertório de marcadores de superfície dessas células foi observado por meio de citometria de fluxo, verificou-se que elas eram positivas para Sca-1, CD29, CD44 e CD49e, e eram negativas para CD11b, CD13, CD18, CD19, CD31, CD45, CD49d e Gr-1 Este perfil de moléculas de superfície assemelha-se àquele descrito para a MSC humana, e indica ausência de contaminantes hematopoiéticos. Uma verificação preliminar da freqüência da MSC na medula óssea de camundongo foi realizada, trazendo a estimativa de que uma MSC está presente numa faixa de 11.000 – 27.000 células. Finalmente, os dados revelaram que não há diferenças imediatamente perceptíveis entre camundongos normais e do modelo murino de MPS I no tocante à MSC, o que indica que os trabalhos futuros visando à correção da deficiência de α-L-iduronidase neste modelo utilizando a MSC são viáveis. O estabelecimento da metodologia para o cultivo e expansão da MSC murina através de técnicas simples vem preencher uma lacuna existente no campo dos modelos experimentais animais, trazendo novas perspectivas para o desenvolvimento de estratégias de terapia celular/genética em modelos experimentais murinos.

Efeitos da tricostatina A sobre a acetilação de histonas, proliferação celular e diferenciação de células tronco embrionárias murinas

Background: Embryonic stem cells are cells derived from early-stage embryos that are characterized by pluripotency and self-renewal capacity. The in vitro cultured murine embryonic stem cells can indefinitely propagate in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). However, when stimulated, these cells can differentiate into cell lines derived from all three embryonic germ layers. The trichostatin A (TSA) is an epigenetic modifier agent and several studies have used the TSA to stimulate cellular differentiation. However, most of these studies only assessed one TSA concentration. Therefore, this study aimed to evaluate the effects of different TSA concentrations on histone hyperacetylation during in vitro cell differentiation of murine pluripotent embryonic stem cells, cultured with or without LIF, in the quest of to standardize their application on early cultures of embryonic stem cells.Materials, Methods & Results: Undifferentiated murine embryonic stem cells were plated in the presence of different TSA concentrations (0 nM, 15 nm, 50 nM and 100 nM) in the presence or absence of LIF. Thus, the treatments were evaluated in undifferentiated embryonic stem cells cultured in the presence of LIF (Control group: 0 nM LIF(+); Group 15 nM LIF+; Group 50 nM LIF+ and Group 100 nM LIF+), and in embryonic stem cells cultured in the absence of LIF (Control group: 0 nM LIF; Group 15 nM LIF(-); Group 50 nM LIF(-) and Group 100 nM LIF-). Treatment with TSA was performed for 24 h. After that the medium was replaced with fresh medium without TSA. Samples were collected at 0, 12, 24, 36 and 48 h after the beginning of the experiment. Three replicates were performed in each experimental group. The relative amount of Histone H3 lysine 9 acetylation was analyzed in all groups, as well as the cell proliferation in the embryonic stem cells cultured in the presence of LIF. In the control group (0 nM), the absence of LIF resulted in higher levels (P < 0.05) of H3lys9ac compared to the cultures supplemented with LIF. In the embryonic stem cells cultured in the presence of LIF, the 50 nM and 100 nM treatments resulted in higher levels (P < 0.05) of H3lys9ac when compared with 0 nM and 15 nM treatments. Evaluating the Hoechst area in the 0 nM group, it was observed that the number of cells increased (P < 0.05) according to the time of culture. Treatment with 15 nM also reflected a similar distribution, but the Hoechst area in 15 nM group was lower (P < 0.05) at 24 and 48h when compared to the observed in the control group. In the 100 nM treatment, was observed that the area of Hoechst was lower (P < 0.05) to that obtained in the control group at 12, 24 and 48h. In addition, it was observed that treatment with TSA induces greater cellular differentiation when compared to control groups in stem cells cultured in the presence of LIF as well as in the absence of LIF.Discussion: In the present study it was observed that TSA treatment increased the levels of histone acetylation in murine embryonic stem cells at a 50 nM concentration, making it possible to reduce the concentration recommended in the literature (100 nM). In addtion, it was concluded that the lower TSA concentrations utilized (15 nm and 50 nM) was less harmful to cellular proliferation than the 100 nM TSA concentration.

Análise citogenética de células-tronco derivadas do subendotélio da veia umbilical humana

Mesenchymal stem cells (MSCs) are known as a population of multi-potential cells able to proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat and stroma. Several applications of the study of EC can be emphasized the therapeutic techniques such as guided bone regeneration by implantation of EC in the affected site, without the need for bone grafts, using titanium as a vehicle. The process of cryopreservation is essential for the maintenance of cell cultures, since the cell line is frozen, it can be maintained in liquid nitrogen for an indefinite period and then thawed for therapeutic or experimental purposes. The aim of this study was to isolate a population of MSCs derived from the subendothelium of the umbilical vein human (MSCs-SUVH) to assess cytogenetic analysis by the possibility of appearance of chromosomal changes in two different situations: MSCs-SUVH regarding the process of cryopreservation and MSCs-SUVH grown on the surface of titanium. Flow cytometry analysis revealed that, this cell population was positive for the markers CD29, CD73 and CD90, but there was no expression of hematopoietic lineage markers, such as CD14, CD34 and CD45 and demonstrated capacity for osteogenic differentiation. The chromosomes obtained from the primary culture of MSCs-SUVH were analyzed by GTW banding technique, and results are described as guidelines to ISCN 2005. There was not the emergence of clonal chromosomal changes in the MSCs-SUVH in different situations analyzed. However one of the strings presented a balanced paracentric inversion, probably a cytogenetic constitutional alterations, which was present before and after the experimental situations that the MSCs-SUVH was submitted

Plasticidade de células tronco mesenquimais de medula óssea de ratos na presença de meio condicionado do nervo facial e do fator de crescimento fibroblástico 2

A number of evidences show the influence of the growth of injured nerve fibers in Peripheral Nervous System (PNS) as well as potential implant stem cells (SCs) to make it more suitable for nerve regeneration medium. In this perspective, this study aimed to evaluate the plasticity of mesenchymal stem cells from bone marrow of mice in the presence of culture medium conditioned with facial nerve explants (D-10) and fibroblast growth factor-2 (FGF-2). In this perspective, the cells were cultivated only with DMEM (group 1), only with D-10(group 2), only with FGF-2(group 3) or with D-10 and FGF-2(group 4). The growth and morphology were assessed over 72 hours. Quantitative phenotypic analysis was taken from the immunocytochemistry for GFAP, OX-42, MAP-2, β-tubulin III, NeuN and NF-200 on the fourth day of cultivation. Cells cultured with conditioned medium alone or combined with FGF-2 showed distinct morphological features similar apparent at certain times with neurons and glial cells and a significant proliferative activity in groups 2 and 4 throughout the days. Cells cultived only with conditioned medium acquired a glial phenotype. Cells cultured with FGF-2 and conditioned medium expressed GFAP, OX-42, MAP-2, β-tubulin III, NeuN and NF-200. On average, area and perimeter fo the group of cells positive for GFAP and the área of the cells immunostained for OX-42 were higher than those of the group 4. This study enabled the plasticity of mesenchymal cells (MCs) in neuronal and glial nineage and opened prospects for the search with cell therapy and transdifferentiation

Estruturação ex-vivo de vasos sanguíneos a partir da diferenciação de células tronco de coelhos

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB

Contribuição da terapia celular com células tronco em fibrose em ratos

Pós-graduação em Pesquisa e Desenvolvimento (Biotecnologia Médica) - FMB