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

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

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

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

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

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

Values are averages for at least two technical replicates.

<p>Values shown for the self-reproducibility and correlation are derived from all probes.</p>a<p>Sensitivity is calculated as the number of probes detected at p-value<0.01.</p>b<p>Probe concordance is calculated as a percentage of the number of probes with matching detected calls at p-value<0.01 between the low (50 pg or single cell) and standard (1 ng) inputs divided by the total number of probes detected in the lower input.</p>c<p>24 K WG-DASL.</p>d<p>29 K WG-DASL HT.</p

Raw signal intensity correlations between replicates of low input RNAs and whole cells.

<p>(A) 50 pg UHR and BR total RNA and (B) single HeLa and brain tumor (BT) cells; 50 cell tumorsphere (TS) and adherent cells (AC). Pair-wise scatterplots of at least two replicates for each input type are shown for all 29 K probes across the full range of raw signal intensities. Correlations are the square of Pearson's correlation coefficient.</p

Pre-amplification scheme.

<p>(1) First strand cDNA synthesis is primed with tagged oligo-dT and random 9-mer primers. The tagged oligo-dT primer contains a VN anchor followed by a T-30 stretch with a 5′ PCR tag. The tagged random 9-mer consists of a 9-mer followed by the identical 5′ PCR tag. (2) Upon reaching the 5′ terminus of the mRNAs, the reverse transcriptase, via its terminal transferase activity, adds a few nucleotides (predominantly deoxycytidine) to the 3′ end of the newly synthesized cDNAs. (3) The template-switch primer, which consists of the same 5′ PCR tag as well as a 3′ riboguanine stretch, anneals via GC complimentary base-pairing to the 3′ end of the cDNAs, thereby serving as a new template for the reverse transcriptase. (4) After cDNA synthesis, both ends of the cDNAs now contain the identical PCR tag, allowing exponential amplification of the entire cDNA population through single primer PCR (5).</p