Additional Supporting Information may be found in the online version of this article.

STEM_683_sm_suppfigure1.tif9091KSupplementary Figure 1. Characterization of BJ-iPSCs. (A) BJ-derived iPSC lines, iB4 and iB5, possessed a normal karyotype. (B) DNA fingerprinting analysis confirmed the somatic relatedness of iB4 and iB5 to BJ fibroblasts, and of iPS2 and iPS4 to HFF1 fibroblasts. (C) In vitro EB-based differentiation of iB4 and iB5 demonstrated that both lines were capable of generating cell types representative of the three germ layers, including ectoderm (NESTIN, PAX6, and TUJ-1), mesoderm (smooth muscle actin, SMA), and endoderm (SOX17 and alpha feto protein, AFP). Scale bars represent 20μm. (D) In vivo differentiation of iB4 and iB5 confirmed their pluripotency properties, as both lines efficiently generated teratomas containing derivatives of the three germ layers. At the bottom, a table showing the growth of BJ-iPSC teratomas over time is reported.
STEM_683_sm_suppfigure2.tif1957KSupplementary Figure 2. Global gene expression analysis and qPCR confirmation. (A) Transcriptome analysis was performed using Illumina bead chips. Hierarchical clustering of neonatal fibroblasts (BJ and HFF1), hESCs (H1 and H9), and iPSCs (iB4, iB5, iPS2, and iPS4) showed that all iPSCs acquired a hESCs-like transcriptional signature. (B) Microarray-based expression of pluripotency and differentiation-associated genes in H9, iB4, and iB5 expressed as log2 ratio relative to BJ fibroblasts. (C) qPCR-based expression of the same genes in H9, iB4, and iB5, carried out by using the Embryonic Stem Cells StellARray, confirmed the array-derived results.
STEM_683_sm_suppfigure3.tif2571KSupplementary Figure 3. Generation of an amplicon library for mtDNA sequencing. Two sets of PCR primers (Set A and Set B) were utilized, each set designed to cover the entire mitochondrial genome. The expected length of each amplicon was approximately 650 base pair. From each individual PCR reaction, a corresponding aliquot was loaded on 2% agarose gel. Gel electrophoresis confirmed successful PCR reactions with respect to product specificity and product size. The GeneRuler 100 BP ladder (Fermentas, MD, USA) served as marker.
STEM_683_sm_suppfigure4.tif4618KSupplementary Figure 4. Heatmap representation of genes involved in cellular glucose metabolism. Values represent the log2 ratio of the array average signal of the given gene divided by the average signal of BJ fibroblasts (fold change 1.5, detection p value ≤0.01, and differential p value ≤0.01). Up-regulated genes are shown in red, down-regulated genes in green.
STEM_683_sm_suppfigure5.tif2257KSupplementary Figure 5. Bioenergetic profiles of fibroblasts, hESCs, and iPSCs. (A) Representative OCR profile following mitochondrial perturbation. For every cell line, multiple replicates were seeded and divided into two groups (shown by the red and blue lines). All replicates were first exposed to 1μM oligomycin (time point A), blocking Complex V and thus inhibiting OXPHOS. Half of the replicates (blue line) were then treated with 0.3 μM FCCP (time point B), which uncouples mitochondria and leads to consumption of energy and oxygen without the generation of ATP. In the remaining half, we injected 1μM rotenone (time point B), a Complex I inhibitor which blocks the beginning of mitochondrial respiration. (B) Mitochondrial respiration (oxygen consumption rate, OCR) and glycolysis (extracellular acidification rate, ECAR) profiles in fibroblast cells BJ and HFF1. (C) OCR and ECAR profiles in hESC lines H1 and H9. (D) OCR and ECAR profiles in iPSC lines iB5 (with low mtDNA mutational load) and iPS2 (with high mtDNA mutational load). Error bars represent the standard error of the mean (SEM).
STEM_683_sm_supptable1.doc27KSupporting Table 1. List of primers used for mtDNA sequencing (Set A and Set B)
STEM_683_sm_supptable2.doc34KSupporting Table 2. List of primers used for real-time PCR analysis
STEM_683_sm_supptable3.doc32KSupporting Table 3. Sequencing Statistics
STEM_683_sm_supptable4.doc166KSupporting Table 4. Homoplasmic mtDNA mutations in iPSCs
STEM_683_sm_supptable5.doc92KSupporting Table 5. Heteroplasmic mtDNA mutations in iPSCs

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