The DNA methylation status of <i>Aid</i>^−/− iPS cells.

<p>(A–D) The DNA methylation status of the <i>Nanog</i> promoter (A), <i>Oct3/4</i> promoter (B), B1 (C) and LINE1 (D) detected by pyrosequencing. The iPS cell clones analyzed were the same as those examined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094735#pone-0094735-g002" target="_blank">Fig. 2C</a>. The data are represented as the averages ± SD of the clones. (E) The results of the comprehensive DNA methylation analysis with MBD-sequencing. Pie charts show the comparison of the detected methylated regions between <i>Aid</i>^+/+ iPS cells and <i>Aid</i>^+/+ MEFs (left), <i>Aid</i>^+/+ iPS cells and ES cells (middle), and <i>Aid</i>^+/+ iPS cells and <i>Aid</i>^−/− iPS cells (right). DMRs; Differentially methylated regions, CMRs; Commonly methylated regions.</p

Generation of iPS cells from <i>Aid</i>^−/− mice.

<p>(A) The relative expression of <i>Aid</i> and <i>Gapdh</i>. Total RNA was isolated from three ES cell clones (RF8, B6ES and MG1.19), three <i>Aid</i>^+/+ iPS cell clones (967B2, 967C1 and 979B1), three <i>Aid</i>^−/− iPS cell clones (957F1, 979F1 and 979E1), three parental <i>Aid</i>^+/+ and <i>Aid</i>^−/− MEF clones and primary B cells (pB cells), and was used for the quantitative RT-PCR analysis. The data are shown as the average ± SD. (B) The morphology of <i>Aid</i>^+/+ and <i>Aid</i>^−/− iPS colonies 25 days after the introduction of 4 Fs into MEFs. Phase contrast (left column) and GFP fluorescence (right column) images are shown. Scale bars; 200 μm. (C, D) The number of GFP-positive colonies from <i>Aid</i>^+/+, <i>Aid</i>^+/− and <i>Aid</i>^−/− MEFs induced by 4 Fs (C) and 3 Fs (D). For each genotype, three different lots of MEFs were used in each experiment, and the experiments were repeated four times. Colonies were counted 25 (4 Fs) and 30 (3 Fs) days after the induction. (E) The number of total colonies from <i>Aid</i>^+/+, <i>Aid</i>^+/− and <i>Aid</i>^−/− MEFs subjected to transduction of the 3 Fs with or without Aid. (F) The proportion of GFP-positive colonies out of the total colonies from <i>Aid</i>^+/+, <i>Aid</i>^+/− and <i>Aid</i>^−/− MEFs induced by 3 Fs with or without Aid. (G) The number of GFP-positive colonies from <i>Aid</i>^+/+ and <i>Aid</i>^−/− primary B cells induced with 4 Fs. Experiments were repeated five times.</p

Genetically Matched Human iPS Cells Reveal that Propensity for Cartilage and Bone Differentiation Differs with Clones, not Cell Type of Origin

<div><h3>Background</h3><p>For regenerative therapy using induced pluripotent stem cell (iPSC) technology, cell type of origin to be reprogrammed should be chosen based on accessibility and reprogramming efficiency. Some studies report that iPSCs exhibited a preference for differentiation into their original cell lineages, while others did not. Therefore, the type of cell which is most appropriate as a source for iPSCs needs to be clarified.</p> <h3>Methodology/Principal Findings</h3><p>Genetically matched human iPSCs from different origins were generated using bone marrow stromal cells (BMSCs) and dermal fibroblasts (DFs) of the same donor, and global gene expression profile, DNA methylation status, and differentiation properties into the chondrogenic and osteogenic lineage of each clone were analyzed. Although genome-wide profiling of DNA methylation suggested tissue memory in iPSCs, genes expressed differentially in BMSCs and DFs were equally silenced in our bona fide iPSCs. After cell-autonomous and induced differentiation, each iPSC clone exhibited various differentiation properties, which did not correlate with cell-of-origin.</p> <h3>Conclusions/Significance</h3><p>The reprogramming process may remove the difference between DFs and BMSCs at least for chondrogenic and osteogenic differentiation. Qualified and genetically matched human iPSC clone sets established in this study are valuable resources for further basic study of clonal differences.</p> </div

Characterization of iPSCs.

<p>A) Relative expression of retroviral transgenes in DF90 (left panel) and BM90 (right panel)-iPSC clones analyzed by RT-qPCR. The value of each transgene 6 days after infection of DF90 (DF90 4F day 6) and 7 days after infection of BM90 (BM90 4F day 7) was set to 1, and relative values of four transgenes in each clone are shown on a log scale. We selected four clones of which transgene expression was silenced less than 1/1000 compared to the control. B) Expression of ESC-marker genes. Primers used for OCT3/4 and SOX2 detect transcripts from endogenous genes, but not retroviral transgenes. All clones express ESC-marker genes similar to hESCs (KhES3) and hiPSCs (201B7). C) Karyotype analyses. PN, passage number. D) Histological analyses of teratomas derived from iPSCs (DF90-iPSC B3) by hematoxylin and eosin staining. Typical tissue features of each of the three germ layers were found. Scale bar, 200 µm. E) Comparison of global gene-expression patterns between each iPSC clone and hESCs (H9). The two green lines above and below the diagonal green lines indicate the boundary of 2-fold changes between the two samples.</p

DNA methylation profiles of BM- and DF-iPSCs were similar in general, but had distinct features.

<p>Hierarchical clustering using Euclidian distance with probe sets of differentially methylated regions between DFs and BMSCs.</p

Generation of iPSCs from BMSCs and DFs of the same donor.

<p>A) Schematic representation of the generation of iPSCs from dermal fibroblasts (DFs) and bone marrow stromal cells (BMSCs) of the same healthy donor. B) Donor information and established clones’ names. %, iPSC derivation efficiency. -, not established as validated clones. C) Time course of iPSC generation.</p

Global gene expression profiles do not differ between BM- and DF-derived iPSCs.

<p>A) Correlation coefficients between each cell were calculated using gene sets differentially expressed in DFs and BMSCs. Differentially expressed genes were defined as genes of which expression varied 2-fold in all pairs, between each BMs and each DFs (9 pairs). Passage number (PN) of cells were as below; BM90 (PN 1), BM 91 (PN 3), BM 94 (PN 2), DF90 (PN 5), DF 91 (PN 5), DF 94 (PN 5), DF90-iPSC B3 (PN 9), F2 (PN 4), BM90-iPSC a3 (PN 4), a12 (PN 4), a16 (PN 4), b6 (PN 4), DF91-iPSC A1 (PN 5), A5 (PN 6), A11 (PN 5), A18 (PN 7), BM91-iPSC a15 (PN 4), a18 (PN 4), b14 (PN 4), b17 (PN 4). B) Hierarchical clustering analysis in iPSC clones and hESC lines (H9) using differentially expressed gene sets in DFs and BMSCs.</p

The propensity for EB-mediated cell-autonomous differentiation in iPSC clones differs regardless of developmental origin.

<p>Gene expression of EB outgrowth cells (A) and reseeded cells (PN1; B). RNA was extracted from cells that reached semi-confluency. RT-PCR was performed with genes related to chondrogenesis (SOX9 and ACAN), osteogenesis (RUNX2 and OSTERIX), or adipogenesis (PPARγ). The capacity to differentiate into bone and cartilage lineages was similar between DF- and BM-iPSC clones. The expression of genes representative of each of the three germ layers was also analyzed, which showed no difference among clones.</p

Induction of osteogenic differentiation in iPSCs.

<p>A) Time course of osteogenic differentiation. EB, embryoid body. OG, outgrowth. B-E) Comparison of the relative expression of osteogenesis-related genes (RUNX2, COL1A1, OCN and OSX) by RT-qPCR. The value of DF-iPSC B13 was set to 1 in each experiment.</p

Reprogramming extinguishes lineage-specific gene expression.

<p>We presented the top six genes that were BMSC-specific (A) or DF-specific (B). Expression in BM90 (A) or DF90 (B) was set to 1 in each graph.</p