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

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

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

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

Morphological characteristics and marker expression of FIB, IPSC, and TER cells.

<p>FIBA and derivatives are used as examples. <b>A–C</b>) Phase-contrast images showing the morphology of FIB, IPSC, and TER lines as labeled. Insets in A and C show reactivity of FIBA and TERA with human-specific nuclear (HuNu) antigen antibody (green). Blue is DAPI co-stained DNA. <b>D</b>) Graphical representation of the relative expression of pluripotency-related gene mRNAs in all cell lines (IPSC value set at 100%). Pluripotency marker genes were detected at low levels in FIB and TER cells compared to IPSCs. <b>E–F</b>) Immunocytochemical analysis of IPSCs (<b>E</b>) and TER cells (<b>F</b>), indicating that TER cells had not only lost high-level of expression of pluripotency markers such as Nanog, but also gained expression of fibroblasts markers such as fibronectin (FBN) (see also Ref. 7). Magnification: 800X, insets 250X.</p

Structural comparison of mitochondria in FIB, IPS, TER, and ESC cell types.

<p><b>A–C</b>) Electron tomography of the mitochondrial configurations observed. Three primary configurations were observed: <b>A</b>) <i>top panel</i>: A central slice through a tomographic volume showing the <i>orthodox</i> configuration. <i>bottom panel</i>: Top and side views of the segmented and surface-rendered volume of the orthodox mitochondrion showing individual cristae in various colors (top view (<i>upper</i>) side view (<i>lower</i>)). <b>B</b>) As in A, for the <i>condensed</i> configuration. <b>C</b>) As in A,B for the <i>ultracondensed</i> configuration. Arrowheads point to greatly enlarged crista junctions. <b>D–K</b>) Representative TEMI of mitochondria in all cell lines. (Mag = 7500-10000X). FIB and IPSC lines displayed a mix of mitochondrial configuration, most of condensed configuration (FIBA (<b>D</b>), FIBB (<b>E</b>), IPSCA (<b>F</b>), IPSCB (<b>G</b>)). TER cells displayed a preponderance of orthodox mitochondria (TERA (<b>H</b>), TERB (<b>I</b>)), and native ESCs displayed a preponderance of condensed mitochondrial forms (ESCA (<b>J</b>), ESCB (<b>K</b>)). <b>L</b>) Bar graph of the ratio of mitochondria scored as orthodox or condensed configuration (O/C) in FIB, IPS, TER, and ESC cell types as labeled. Error bars  =  SEM. Letters above TER bar indicate statistical difference (P<0.03) from all three other cell types.</p

Functional comparison of mitochondria in FIB, IPSC, TER and ESCs.

<p><b>A</b>) Total cellular ATP (fmoles/cell) in each cell type as labeled. <b>B</b>) ADP/ATP ratio in each cell type as labeled. <b>C</b>) Relative mitochondrial mass in tested cell types. Error bars indicate SEM. Letters above individual bars indicate values that differ statistically (P<0.05) from other cell type(s). (FIB = F, IPS = I, TER = T, ESC = E).</p