5 research outputs found

    DNA Methylation

    Get PDF
    <p><b>A</b>. X Chromosome DNA Methylation and XIST Expression. Methylation levels of genes in the X-chromosome (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118307#pone.0118307.s009" target="_blank">S6A Table</a>) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to their location (from the beginning to the end of the chromosome). Samples that show loss of DNA methylation for the “Enz” cluster are highlighted in blue, those that show DNA methylation for the “Ecm” cluster are highlighted in pink, and for both clusters in mauve. Genes located in the regions of loss of DNA methylation are listed to the right of the heatmap. XIST expression is shown on the line graph, with the detection limit for the microarray indicated by the red line. <b>B</b>. DNA methylation at imprinted loci. Methylation levels for imprinted probes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118307#pone.0118307.s009" target="_blank">S6B Table</a>) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to chromosome location; genes are listed to the left. The inset at the right shows a detail of the NESP/GNAS complex locus, indicating the positions of the CpG sites that were hypermethylated (red triangle) vs. hypomethylated (green triangle) in the late passage samples relative to the NESP/GNAS and NESPAS exons. <b>C, D, E</b>. Heatmaps showing differential DNA methylation genes for early vs. late passage <b>(C)</b>, mechanical vs. enzymatic passage <b>(D)</b>, and Mef vs. Ecm substrate <b>(E)</b>. In heatmap <b>(C)</b>, the black boxes indicate genes for which the DNA methylation levels in the late passage MefMech (P103) samples was more similar to those in the early passage samples. Probes were selected by multivariate regression. Functional enrichments identified by GREAT analysis are shown to the right of the heatmaps, visualized using REVIGO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118307#pone.0118307.ref013" target="_blank">13</a>]. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. In the functional enrichment results, the size of the node indicated the number of contributing GO terms, and color of the nodes indicates the FDR (darker color for lower FDR), and the edge length indicates the similarity between GO terms (shorter edge for more similar terms).</p

    Increased Risk of Genetic and Epigenetic Instability in Human Embryonic Stem Cells Associated with Specific Culture Conditions

    Get PDF
    <div><p>The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.</p></div

    Detail of recurrent deletions in chromosome 17.

    No full text
    <p><b>A</b>. The short arm of the chromosome is enlarged and the regions that showed deletions in the MefEnz, EcmMech, and EcmEnz conditions are indicated by red bars. Blue lines enclose the common area among all the conditions and the genes in the common area are indicated in the lower part of the figure. Genes for which the level of expression correlated with copy number are highlighted by red squares. <b>B</b>. Graphs representing the expression levels of TP53, SENP3, and SOX15, as measured by gene expression microarray. The ideogram of the chromosome was from the U.S. Department of Energy Genome Programs.</p

    Phenotypic assays.

    No full text
    <p>Horizontal brackets indicate significant differences between the specified conditions, as calculated using ANOVA (p-value<0.05). <b>A</b>. The MTT assay measures cell proliferation, with absorbance being correlated with cell growth. <b>B</b>. Doubling time was calculated from cell counts taken over 3 days (average of 3 replicates per condition). <b>C</b>. The EdU incorporation was determined via FACS, and reveals the cell cycle distribution. The graph shows the percent of cells in S phase. <b>D</b>. Relative telomerase activity is shown. <b>E</b>. Average number of telomere repeat fragments (TRF, average of 3 replicates) for each condition. <b>F</b>. Percent of apoptotic cells, determined using TUNEL-FACS. <b>G</b>. Example of an OCT4-positive focus in a teratoma section. <b>H</b>. Bar graph showing, for each culture condition, the percent of teratoma sections containing at least one OCT4-positive focus, and the average number of foci per section. The error bar indicates the standard deviation of counts from 6 (EcmEnz), 7 (MefMech), or 8 (MefEnz and EcmMech) sections. Raw counts can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118307#pone.0118307.s007" target="_blank">S4 Table</a>).</p

    Chromosome 12 detail in MefEnz.

    No full text
    <p>Detailed view of the duplications in chromosome 12 for MefEnz. <b>A</b>. The green areas indicate the regions of duplication that were identified by CNVPartition 3.2.0. The areas that were detected manually rather than by the software are indicated in a scale from grey to black. Estimates of the percentage of the population carrying the duplication were performed using the BAF distance for heterozygous SNPs as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118307#pone.0118307.ref003" target="_blank">3</a>]. <b>B</b>. LogR Ratio (LRR) and BAF plots of replicate A of MefEnz showed changes in genetic aberrations over time. <b>C</b>. The CNV data were further validated via TaqMan Copy Number Assays. Probe 1 corresponds to Hs03295596_cn, Probe 2 to Hs04404518_cn, Probe 3 to Hs03812366_cn, Probe 4 to Hs03841181_cn, Probe 5 to Hs06947059_cn, and Probe 6 to Hs06363301_cn. <b>D</b>. Karyotyping results confirmed the CNV data.</p
    corecore