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

Undifferentiated colonies differed with respect to imprinted genes.

<p>PCR arrays did not reveal differences in the undifferentiated state (a). Parthenote-derived lines LLC6P and LLC8P are plotted on the y-axis and conventionally-derived lines H7 and CSC14 on the x-axis. Methylation microarray revealed differences between parthenote-derived lines and conventionally-derived lines, most notably in imprinted genes. Cluster analysis (b) indicated that phESC and hESC were closely related to each other and more methylated than tissue and primary cell lines. phESC and hESC are distinguishable when complete methylation data are included (c). phESC and hESC diverge further when methylation data include only imprinted genes (d). Methylation data, normalized with tissue lines as the unmethylated baseline (c and d). In c and d, correlation is plotted on the y-axis and level of discordance between 7 phESC lines is plotted on the x axis (e.g. 4 means 3 phESC lines were allowed discordance). Red represents phESC lines, green represents hESC lines, blue represents primary cell lines and dashed purple represents tissue lines.</p

phESC produce functional RPE cells after differentiation.

<p>The LLC6P line produces pigmented cells in culture (a) and characteristic “dome-like” structures (b). Cells express RPE markers Occludin (c), CRALBP and mitf (d). Functionality of LLC6P-derived RPE is demonstrated by pHrhodo pH-sensitive fluorescent particle phagocytosis (e and d). Five minutes after administration of pHrhodo (e), RPE exhibit minimal vesicular pHrhodo content. Two hours and five minutes after administration (f), RPE exhibit marked increase in vesicular pHrhodo content. Scale bars represent 100 µm (a, e, f), 500 µm (b) and 5 µm (c, d).</p

hESC and phESC differ morphologically.

<p>Conventionally-derived (a–f) and parthenote-derived (g–l) hESCs have distinct morphology and growth patterns in the undifferentiated state. High magnification and low magnification images have been provided for each colony to illustrate the slower growth of parthenote-derived colonies, irregular colony shape and lack of stromal cells (long, thin cells between colonies indicated by arrows) in the phESC cultures. Day 1 after plating (a, d, g and j). Day 2 after plating (b, e, h and k). Day 4 after plating (c, f, i and l). Scale bars represent 20 µm (a–c and g–i) and 10 µm (d–f and j–l).</p

Immunocytochemical profile following NPC differentiation.

<p>Parthenote-derived lines displayed impaired neural maturation, but expressed immature markers normally. CSC14 (a–d), H7 (e–h), LLC6P (i–l) and LLC8P (m–p). Map2 (a, e, i and m), Olig1 (b, f, j and n), Nestin (c, g, k and o) and Pax6 (d, h, l and p). Scale bars represent 50 µm (a, b, e and f) and 10 µm (c, d, and g–p).</p

Exposure of hESC and phESC to a neural differentiation protocol yields quantitatively distinct cell populations.

<p>Conventionally-derived (a and b) and parthenote-derived (c and d) hESC have distinct growth patterns after the NPC differentiation protocol. Histograms plotting cellular yield (in millions of cells) after NPC differentiation (e) reveal that parthenote-derived lines yield fewer cells after differentiation than conventionally-derived lines. Scale bars represent 10 µm. Blue bars indicate differentiation 1, red bars indicate differentiation 2 and yellow bars indicate differentiation 3, representing the three successive differentiations.</p

phESC display altered ECM profile and do not readily form spheres in non-adherent culture.

<p>During the sphere-forming stage of the NPC protocol, hESC gave rise to clusters that readily formed spheres with minimal dissociation (a). phESC at the same stage consistently dissociated (b). PCR arrays revealed differences in extracellular matrix and adhesion molecules in NPC (c). Parthenote-derived lines LLC6P and LLC8P are plotted on the y-axis and conventionally-derived lines H7 and CSC14 on the x-axis. Details of differentially expressed genes are presented numerically (d). Western blot analysis confirmed differential expression of NCAM1 (e). Scale bars represent 20 µm (a–b).</p

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

Phenotypic assays.

<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