Analysis of telomeres in input, pluripotent, and re-differentiated cell lines.

<p>(A) Blue-stained nuclear DNA with punctate red fluorescence indicating telomeres hybridized to the PNA FISH telomere probe for input fibroblasts, IPSCs, or TER cells from lines A and B as labeled. mag. 400X. (B) Quantification of TRFs from cell lines as labeled. Yellow shading indicates significance compared to line FIBA and red shading significance compared to line FIBB. TRF indicates mean length in kilobase pairs (Kb). * = P<0.05, ** = P<0.01, *** = P<.001. Error bars indicate SEM. (C) Example of changes in TRF size in cell lines as labeled. Numbers at left are fragment lengths in kilobases. (D) Mean TRF lengths for all cell lines combined by type. Labeling as in part B. Significance calculated relative to FIB.</p

Phase-contrast images of FIB and IPSC lines used in this report (as labeled).

<p>FIBA and FIBB displayed a flat stellate cytoplasm with irregular edges characteristic of fibroblast cell types (200X). IPSC lines displayed round colonies with regular edges evident in low magnification 40X images that were composed of tightly packed cells with prominent nucleoli (200X, lower panels of IPSC lines) that appeared morphologically homogenous within the center of the colony and flattened toward the edges where they were bounded by the MEF feeder layer. IPSCB1 and IPSB2 are shown only at 40X magnification.</p

Cell lines used in this report.

<p><i>Report name</i>: name used to reference the cell lines in this report. <i>Cell Type</i>: the general cell phenotype.</p><p><i>Pass:</i> passage of cells at time of telomere analysis. <i>Parental Line</i>: name of the input cell line used to produce the corresponding cell line.</p><p><i>Vec/Factors</i>: Reprogramming factor combination used to produce cell line (if applicable). Individual letters represent each of the 6 reprogramming factors as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008124#pone-0008124-g001" target="_blank">Figure 1</a>. Parentheses indicate coupling of factors into bicistronic pairs in the vector.</p><p><i>Line Ref Name:</i> Official name of each cell line used in the report.</p>*<p>Lines lost to contamination.</p>#<p>--also passage number used for production of IPSCs.</p

Indicators of pluripotency and differentiation in IPSC lines.

<p>(A) Representative sections from teratomas generated from IPSC lines as labeled. Arrows in ectodermal (ECTO) tissue indicate neural rosettes, in mesodermal tissue (MESO) indicate cartilage, and in endodermal tissue (ENDO) indicate glandular columnar epithelium. mag. 100X. (B) Morphology of FIB lines and TER lines, as labeled, in phase-contrast images (top row) and stained for fibronectin (red, bottom row). mag. 400X. Blue is DNA stain. (C) Genome-wide methylation heat map and cluster analysis of representative input fibroblasts, IPSCs, TER, and hESC lines indicating that the overall pattern of methylation of IPSCs closely matches HES lines while differentiated TER lines cluster with input fibroblasts. Value indicates the level of methylation ranked from 0 (hypomethylated) to 1 (hypermethylated).</p

Analysis of global DNA methylation levels post-HCT.

<p>(A) Diagram of the experimental design. The differences of the global methylation levels between donors, pre-HCT recipients and post-HCT recipients were assessed by a pyrosequencing based methylation assay of repetitive DNA elements (LINE1 and NBL2) in whole blood. (B) NBL2 ΔMet values between donors, pre-HCT recipients, and 1 month post-HCT recipients. (C) NBL2 ΔMet mean values between donors and recipients up to 12 months post-transplant. The dotted line marks the ΔMet mean value between donors and 1 month post-HCT recipients (ΔMet = 5.8450). During the follow up of the transplant, the mean values barely deviated from the initial post-HCT ΔMet. (D) LINE1 ΔMet values between donors, pre-HCT recipients and 1 month post-HCT recipients. (E) LINE1 ΔMet mean values between donors and recipients up to 12 months post-transplant. The dotted line marked the ΔMet mean value between donors and 1 month post-HCT recipients (ΔMet = 5.383).</p

Patient characteristics.

<p>Patient characteristics.</p

Changes in NBL2 methylation levels are associated to HCT outcomes.

<p>(A) NBL2 ΔMet between donors and 1 month post-transplant recipient with complete and mixed chimerism. (B) ROC curve for patients with complete and mixed chimerism (AUC = 0.911). (C) NBL2 ΔMet between donors and 1 month post-transplant recipients according to severity of aGVHD. (D) ROC curve for patients with severe aGVHD versus non-aGVHD and moderate aGVHD (AUC = 0.678).</p

Association of Locus-specific DNA methylation to aGVHD.

<p>(A) IFNγ methylation up to 12 months post-transplant is shown in the left panel. The black line marked the mean value in the cohort and the dotted line marked the mean value 1 month post-HCT. In the central panel are represented IFNγ methylation values according to severity of aGVHD 1 month post-HCT. IFNγ ROC curve for patients with severe aGVHD versus non-aGVHD and moderate aGVHD (AUC = 0.782) is shown in the right panel. (B) FASL methylation up to 12 months post-transplant and methylation values according to severity of aGVHD 1 month post-HCT. FASL ROC curve for patients with severe aGVHD versus non-aGVHD and moderate aGVHD (AUC = 0.769) is shown in the right panel. (C) IL-10 methylation up to 12 months post-transplant and methylation values according to severity of aGVHD 1 month post-HCT. IL-10 ROC curve for patient with and without aGVHD (AUC = 0.764) is shown in the right panel. (D) PRF1 methylation up to 12 months post-transplant and methylation values according to severity of aGVHD 1 month post-HCT.</p

Promoter DNA methylation profiling using bead arrays and differential methylation analysis after HCT.

<p>Scatter plots showing DNA methylation in donors versus post-transplant recipients 1 month and 6 months post-HCT. Red dots represent CpG sites with methylation values altered more than 20% relative to donor values.</p