RESUMO
The mechanisms underlying the low efficiency of reprogramming somatic cells into induced pluripotent stem (iPS) cells are poorly understood. There is a clear need to study whether the reprogramming process itself compromises genomic integrity and, through this, the efficiency of iPS cell establishment. Using a high-resolution single nucleotide polymorphism array, we compared copy number variations (CNVs) of different passages of human iPS cells with their fibroblast cell origins and with human embryonic stem (ES) cells. Here we show that significantly more CNVs are present in early-passage human iPS cells than intermediate passage human iPS cells, fibroblasts or human ES cells. Most CNVs are formed de novo and generate genetic mosaicism in early-passage human iPS cells. Most of these novel CNVs rendered the affected cells at a selective disadvantage. Remarkably, expansion of human iPS cells in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ES cells.
Assuntos
Reprogramação Celular/genética , Variações do Número de Cópias de DNA/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Seleção Genética , Linhagem Celular , Sítios Frágeis do Cromossomo/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Haplótipos/genética , Humanos , Hibridização in Situ Fluorescente , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/patologia , Mosaicismo , Mutagênese/genética , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único/genética , Seleção Genética/genéticaRESUMO
Genomic parasites have evolved distinctive lifestyles to optimize replication in the context of the genomes they inhabit. Here, we introduced new DNA into eukaryotic cells using bacteriophage Mu DNA transposition complexes, termed 'transpososomes'. Following electroporation of transpososomes and selection for marker gene expression, efficient integration was verified in yeast, mouse and human genomes. Although Mu has evolved in prokaryotes, strong biases were seen in the target site distributions in eukaryotic genomes, and these biases differed between yeast and mammals. In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption. In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers. Naturally occurring transposons and viruses in yeast and mammals show related, but more extreme, targeting biases, suggesting that they are responding to the same pressures. These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells.
Assuntos
Bacteriófago mu/genética , Elementos de DNA Transponíveis , Técnicas de Transferência de Genes , Animais , Linhagem Celular , Mapeamento Cromossômico , Eletroporação , Células-Tronco Embrionárias/metabolismo , Marcadores Genéticos , Genoma Fúngico , Genoma Humano , Genômica , Células HeLa , Humanos , Camundongos , Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC), the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans) of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses. RESULTS: The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans. CONCLUSION: The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.
Assuntos
Células-Tronco Embrionárias/química , Células-Tronco Embrionárias/citologia , Glicômica , Polissacarídeos/química , Configuração de Carboidratos , Sequência de Carboidratos , Diferenciação Celular , Regulação para Baixo , Fucose/química , Humanos , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic. RESULTS: The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. CONCLUSION: hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state.
Assuntos
Diferenciação Celular , Linhagem Celular , Embrião de Mamíferos/citologia , Indução Embrionária/fisiologia , Células-Tronco/metabolismo , Animais , Biomarcadores/análise , Técnicas de Cultura de Células/métodos , Células Germinativas/metabolismo , Células Germinativas/ultraestrutura , Humanos , Cariotipagem , Masculino , Camundongos , Células-Tronco/ultraestrutura , Teratoma/patologiaRESUMO
Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.
Assuntos
Antígenos Heterófilos/farmacologia , Células-Tronco Embrionárias/fisiologia , Células-Tronco Mesenquimais/fisiologia , Ácidos Neuramínicos/imunologia , Ácidos Neuramínicos/farmacologia , Anticorpos/farmacologia , Especificidade de Anticorpos , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Citometria de Fluxo , Homeostase , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Fenótipo , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Manganese peroxidase (MnP) production in the white-rot basidiomycete Physisporinus rivulosus T241i was studied. Separate MnP isoforms were produced in carbon-limited liquid media supplemented with Mn(2+), veratryl alcohol, or sawdust. The isoforms had different pH ranges for the oxidation of Mn(2+) and 2,6-dimethoxyphenol. Although lignin degradation by white-rot fungi is often triggered by nitrogen depletion, MnPs of P. rivulosus were efficiently produced also in the presence of high-nutrient nitrogen, especially in cultures supplemented with veratryl alcohol. Two MnP encoding genes, mnpA and mnpB, were identified, and their corresponding cDNAs were characterized. Structurally, the genes showed marked dissimilarity, and the expression of the two genes implicated quantitative variation and differential regulation in response to manganese, veratryl alcohol, or sawdust. The variability in regulation and properties of the isoforms may widen the operating range for efficient lignin degradation by P. rivulosus.
Assuntos
Regulação Fúngica da Expressão Gênica , Peroxidases/biossíntese , Peroxidases/genética , Polyporales/enzimologia , Sequência de Aminoácidos , Álcoois Benzílicos/metabolismo , Clonagem Molecular , Meios de Cultura , DNA Complementar , DNA Fúngico/química , DNA Fúngico/genética , Ativadores de Enzimas/metabolismo , Ativadores de Enzimas/farmacologia , Estabilidade Enzimática , Proteínas Fúngicas/genética , Concentração de Íons de Hidrogênio , Lignina/metabolismo , Manganês/metabolismo , Manganês/farmacologia , Dados de Sequência Molecular , Oxirredução , Polyporales/genética , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Pirogalol/análogos & derivados , Pirogalol/metabolismo , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Alinhamento de Sequência , Madeira/metabolismoRESUMO
Identification of molecular components that define a pluripotent human embryonic stem cell (hESC) provides the basis for understanding the molecular mechanisms regulating the maintenance of pluripotency and induction of differentiation. We compared the gene expression profiles of seven genetically independent hESC lines with those of nonlineage-differentiated cells derived from each line. A total of 8,464 transcripts were expressed in all hESC lines. More than 45% of them have no yet-known biological function, which indicates that a high number of unknown factors contribute to hESC pluripotency. Among these 8,464 transcripts, 280 genes were specific for hESCs and 219 genes were more than twofold differentially expressed in all hESC lines compared with nonlineage-differentiated cells. They represent genes implicated in the maintenance of pluripotency and those involved in early differentiation. The chromosomal distribution of these hESC-enriched genes showed over-representation in chromosome 19 and under-representation in chromosome 18. Although the overall gene expression profiles of the seven hESC lines were markedly similar, each line also had a subset of differentially expressed genes reflecting their genetic variation and possibly preferential differentiation potential. Limited overlap between gene expression profiles illustrates the importance of cross-validation of results between different ESC lines.