DNA Methylation

<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

Nucleosome positioning changes during human embryonic stem cell differentiation

<p>Nucleosomes are the basic unit of chromatin. Nucleosome positioning (NP) plays a key role in transcriptional regulation and other biological processes. To better understand NP we used MNase-seq to investigate changes that occur as human embryonic stem cells (hESCs) transition to nascent mesoderm and then to smooth muscle cells (SMCs). Compared to differentiated cell derivatives, nucleosome occupancy at promoters and other notable genic sites, such as exon/intron junctions and adjacent regions, in hESCs shows a stronger correlation with transcript abundance and is less influenced by sequence content. Upon hESC differentiation, genes being silenced, but not genes being activated, display a substantial change in nucleosome occupancy at their promoters. Genome-wide, we detected a shift of NP to regions of higher G+C content as hESCs differentiate to SMCs. Notably, genomic regions with higher nucleosome occupancy harbor twice as many G↔C changes but fewer than half A↔T changes, compared to regions with lower nucleosome occupancy. Finally, our analysis indicates that the hESC genome is not rearranged and has a sequence mutation rate resembling normal human genomes. Our study reveals another unique feature of hESC chromatin, and sheds light on the relationship between nucleosome occupancy and sequence G+C content.</p

Persistent and productive infection of DHHs by HCVcc.

<p>(<i>A</i>) Continuous replication of HCVcc in DHHs. Day-10 DHHs were exposed to Jc1/GLuc2A for 9 h before the inoculum was removed and the cells were changed to medium E with or without cyclophilin inhibitor CsA at 1 µg/ml. Culture supernatants were collected daily for measurement of luciferase activity. The culture medium was replaced with thorough washing every 48 h, and CsA was included every time fresh medium was used. Error bars represent standard deviations from triplicate experiments. (<i>B</i>) Secretion of HCV core antigen into the culture medium by infected DHHs. Day-13 DHHs were exposed to HCVcc for 9 h before the inoculum was removed, and the cells washed and changed to medium E, then immediately collected as the 0-h samples. The infected cells were then incubated for an additional 48 h in medium E with or without IFN-α (50 units/ml) before the culture supernatants were collected as the 48-h samples. Error bars represent standard deviations from replicate experiments. (<i>C</i>) Reinfection of Huh-7.5 cells by HCV particles produced from DHHs. The 48-h media from (<i>B</i>) were used to infect Huh-7.5 cells, which were then fixed for NS3 staining four days after infection. The infectious titer of the HCVcc produced by DHHs is shown. FFU: focus-forming units.</p

Hepatic differentiation from human embryonic stems cells (hESCs).

<p>(<i>A</i>) Representative images of cell morphology and protein marker expression of hESCs (day 0), definitive endoderm (day 4), hepatic progenitor cells (days 8–10), and hepatocyte-like cells (both immature and mature, days 11–21). For day-10 cells, double-staining of AFP and CK-7 (middle panel, 40×) showed mutually exclusive expression in the cell population. (<i>B</i>) Reciprocal expression of pluripotent marker Nanog and liver-specific marker AFP during differentiation. RT: reverse transcriptase. (<i>C</i>) Expression of mRNAs of ALB and AAT during differentiation. PHH: primary human hepatocytes; (<i>D</i>) Albumin secretion by differentiated human hepatocyte-like cells (DHHs). Culture media were collected at the indicated time points during differentiation and subjected to albumin detection with an ELISA kit. Error bars represent standard deviation from replicate experiments. (<i>E</i>) Periodic acid-Schiff staining of stem cells (WA09), DHHs, and PHHs.</p

Time course of infection for determination of the transition point at which the differentiating cells became permissive for HCV.

<p>(<i>A</i>) List of growth factors in media used in the various stages of differentiation. (<i>B</i>) Time course of DHH infection. Cells were exposed for 6 h on the indicated days before the inoculum was removed. The cells were then cultured in the appropriate medium for an additional 48 h before the cell lysates were collected for detection of NS3 expression. (<i>C</i>) Secreted luciferase activities were monitored in the same experiments described in (B). Error bars represent standard deviation of triplicate experiments. (<i>D</i>) Hepatic maturation was not required for HCV infection of day-10 cells. Day-10 DHHs were infected and then either kept in medium D (hepatic specification medium) or changed to HGF-containing Medium E (hepatic maturation medium) until day 21, when all cells were collected for western blotting. The anti-NS3 antibody also recognized a nonspecific band in the mock-infected sample. (<i>E</i>) A diagram indicating the time point for transition of DHHs to HCV permissiveness on the basis of results shown in (<i>B</i>) and (<i>C</i>).</p

Cellular determinants of HCV susceptibility.

<p>(<i>A</i>) Induction of microRNA miR-122 expression by FGF-10 during hepatic specification. Equal amounts of total cellular RNA from various cells at the indicated days were subjected to a real-time RT-PCR assay for detection of miR-122 expression. (<i>B</i>) Microarray heat map of gene expression levels in day-10 versus day-7 cells. Two independent RNA samples were processed for each time point. The numbers represent the average values and standard deviations. The conventional color spectrum with green representing downregulation and red representing upregulation was adopted. Fold of changes were also listed next to the name of the gene. (<i>C</i>) Quantitative RT-PCR results of EGFR and EphA2 induction. (<i>D</i>) Upregulation of PI4KIIIα protein during the differentiation process. The levels of CyPA and DDX-3 remained unchanged in the same samples. (<i>E</i>) Quantitative RT-PCR results of IFITM1 and IFI30 expression induction.</p

Genetic modification of hESCs and HCV-resistant DHHs.

<p>(<i>A</i>) Suppression of CyPA expression by shRNA in WA09 cells and day-21 DHHs. (<i>B</i>) CyPA knockdown did not affect the expression of pluripotency marker Oct-4 in WA09 cells. (<i>C</i>) Modified DHHs were resistant to wildtype HCV infection. Infection of both the wildtype and CyPA-KD (LA) DHHs were done at day 13 and allowed to proceed for 48 h. Luciferase in the culture supernatant for monitored. Wildtype HCVcc (Jc1/GLuc2A) infected unmodified DHHs but not CyPA-KD DHHs (redlines), and the DEYN mutant infected both cell types (blue lines). Error bars represent standard deviations of replicate experiments.</p

Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS

Histone variants are known to play a central role in genome regulation and maintenance. However, many variants are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry due to their highly similar sequences. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. Here, ultra-high-resolution FT-ICR MS and MS/MS yield quantitative relative abundances of all detected HeLa H2A and H2B isobaric and isomeric variants with a label-free approach. We extend the analysis to identify and relatively quantitate 16 proteoforms from 12 sequence variants of histone H2A and 10 proteoforms of histone H2B from three other cell lines: human embryonic stem cells (WA09), U937, and a prostate cancer cell line LaZ. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biological role of many previously unstudied histone variants and post-translational modifications

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

<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.

<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