RESUMO
PURPOSE: Human embryonic stem (hES) cells are useful tools for regenerative medicine. Maintaining hES cells for research and clinical purposes remains a challenge. The hES cells have typically been grown on a mouse or human cell feeder layer, but these methods harbor potential health problems for the recipient. A culture system using magnetic field and iron oxide nanoparticles were previously demonstrated to sustain mouse embryonic stem cells in vitro. Now, by using the BG01v/hOG cell line, we could assess the effect of this culture system on the stemness of an embryonic stem cell of human origin. METHODS: Using a variant hES cell line, BG01V/hOG, expressing an emerald green fluorescent protein (EmGFP), we grown these cells in the presence of serum-free medium supplemented with magnetic nanoparticles functionalized with citrate. The cells were positioned over a circular magnet (4000 Gauss) and monitored daily by fluorescence microscopy. RESULTS: We discovered that hES cells can proliferate when labeled with magnetic nanoparticles and in the presence of a magnetic field without losing pluripotency. CONCLUSION: These results establish an alternative method for maintaining hES cells which would minimize health concerns as well as label cells for subsequent clinical tracking.
Assuntos
Técnicas de Cultura de Células , Células-Tronco Embrionárias/metabolismo , Nanopartículas de Magnetita/química , Células-Tronco Pluripotentes/metabolismo , Animais , Linhagem Celular , Ácido Cítrico/química , Meios de Cultura Livres de Soro , Compostos Férricos/química , Proteínas de Fluorescência Verde/genética , Humanos , Campos Magnéticos , Camundongos , Microscopia de FluorescênciaRESUMO
The aim of this work was to study the derivation of bovine embryonic stem-like (ES-like) cells from the inner cell mass (ICM) of in vitro produced blastocysts. The ICMs were mechanically isolated and six out of seventeen (35 percent) ICMs could attach to a monolayer of murine embryonic fibroblasts (MEF). Ten days after, primary outgrowths were mechanically dissected into several small clumps and transferred to a new MEF layer. Cells were further propagated and passaged by physical dissociation over a 60 days period. The pluripotency of the bovine ES-like cells was confirmed by RT-PCR of Oct-4 and STAT-3 gene markers. The colonies were weakly stained for alkaline phosphatase and the mesoderm and endoderm differentiation gene markers such as GATA-4 and Flk-1, respectively, were not expressed. Embryoid bodies were spontaneously formed at the seventh passage. Results showed that bovine ES-like cells could be obtained and passaged by mechanical procedures from the fresh in vitro produced blastocysts.
RESUMO
Nonstructural protein 4 (NSP4), encoded by group A rotavirus genome segment 10, is a multifunctional protein and the first recognized virus-encoded enterotoxin. The NSP4 gene has been sequenced, and five distinct genetic groups have been described: genotypes A-E. NSP4 genotypes A, B, and C have been detected in humans. In this study, the NSP4-encoding gene of human rotavirus strains of different G and P genotypes collected from children between 1987 and 2003 in three cities of West Central region of Brazil was characterized. NSP4 gene of 153 rotavirus-positive fecal samples was amplified by reverse transcriptase-polymerase chain reaction and then sequenced. For phylogenetic analysis, NSP4 nucleotide sequences of these samples were compared to nucleotide sequences of reference strains available in GenBank. Two distinct NSP4 genotypes could be identified: 141 (92.2%) sequences clustered with NSP4 genotype B, and 12 sequences (7.8%) clustered with NSP4 genotype A. These results reinforce that further investigations are needed to assess the validity of NSP4 as a suitable target for epidemiologic surveillance of rotavirus infections and vaccine development.
Assuntos
Glicoproteínas/genética , Infecções por Rotavirus/virologia , Rotavirus/genética , Toxinas Biológicas/genética , Proteínas não Estruturais Virais/genética , Sequência de Bases , Brasil , Criança , Pré-Escolar , Fezes/virologia , Genótipo , Humanos , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rotavirus/classificação , Análise de Sequência de RNARESUMO
Nonstructural protein 4 (NSP4), encoded by group A rotavirus genome segment 10, is a multifunctional protein and the first recognized virus-encoded enterotoxin. The NSP4 gene has been sequenced, and five distinct genetic groups have been described: genotypes A-E. NSP4 genotypes A, B, and C have been detected in humans. In this study, the NSP4-encoding gene of human rotavirus strains of different G and P genotypes collected from children between 1987 and 2003 in three cities of West Central region of Brazil was characterized. NSP4 gene of 153 rotavirus-positive fecal samples was amplified by reverse transcriptase-polymerase chain reaction and then sequenced. For phylogenetic analysis, NSP4 nucleotide sequences of these samples were compared to nucleotide sequences of reference strains available in GenBank. Two distinct NSP4 genotypes could be identified: 141 (92.2 percent) sequences clustered with NSP4 genotype B, and 12 sequences (7.8 percent) clustered with NSP4 genotype A. These results reinforce that further investigations are needed to assess the validity of NSP4 as a suitable target for epidemiologic surveillance of rotavirus infections and vaccine development.
