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.

Isolamento, caracterização e diferenciação de células-tronco mesenquimais do líquido amniótico equino obtido em diferentes idades gestacionais

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst

Background: The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system.Results: A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human.Conclusion: Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.

Histological study of the dynamics in epidermis regeneration of the carp tail fin (Cyprinus carpio, Linnaeus, 1758)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A necessidade atual em se continuar a pesquisar com células-tronco embrionárias

Embryonic stem cells are pluripotent and able to generate all cell types of the body, being the most promising cells to the study for regenerative medicine. Following the differentiation path, there are adult stem cells, which are committed with a specific cell lineage, creating limitations on their application. On the extreme opposite of embryonic stem cells there are the induced pluripotent stem cells, originated from a somatic cell after genetic reprogramming. Induced pluripotent stem cells are a recent science discovery and may substitute the use of embryonic stem cells in future research. But with nowadays knowledge, the use of adult stem cells and induced pluripotent stem cells are limited due to high expenses and long time process demand. Moreover, the development achieved on all kinds of stem cells study are, in some part, due to the study of embryonic stem cells, what makes the study of these cell type still mandatory. © Todos os direitos reservados a.

Investigação citogenética na síndrome mielodisplásica infantil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cultivo e caracterização de celulas-tronco obtidas de blastocistos equinos frescos e refrigerados

Pós-graduação em Medicina Veterinária - FMVZ

Geração de blastocisto quimérico pela agregação de trofectoderme com a massa celular interna ou com células iPS

Pós-graduação em Ciências Biológicas (Farmacologia) - IBB

Expressão de fatores de transcrição da via de sinalização LIF/JAK/STAT no desenvolvimento embrionário inicial em bovinos

Pós-graduação em Medicina Veterinária - FMVZ

Avaliação do promotor OCT-4 de equinos em uma abordagem transgênica em células-tronco embrionárias de murinos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Derivação de células tronco de tecido ovariano na espécie canina

Pós-graduação em Medicina Veterinária - FCAV