Fig 2: Differentially expressed genes (DEGs) between isogenic hESC and hiPSC lines(a) Venn diagram showing the number of genes consistently up- or down- regulated in 3 biological replicate hiPSC lines when compared to 3 biological replicate hESC GFP lines from the same genetic background. (FDR<0.01 and fold change <2 or <1/2, see details in the Methods section). (B) Heatmap and dendrogram for all isogenic hESC and hiPSC lines based on the 49 differentially expressed genes (DEGs) that were common between the HUES2 and HUES3 backgrounds, using hierarchical clustering based on row-scaled expression levels. hiPSC lines, red; hESC lines, blue. (C) Box plot of 6 hESC GFP lines, 6 hiPSC lines, and parental fibroblasts for the 49 DEGs. Red and blue boxes indicate the expression ranges of each gene in hiPSC and hESC GFP lines, respectively. Diamonds and crosses indicate the expression levels of each gene in parental fibroblasts derived from HUES2 and HUES3 backgrounds, respectively. Genes are ordered by Student’s t-test p-value between the 6 hiPSC and 6 hESC GFP lines. Red arrows depict genes discussed in main text. (D) RNA-seq read density of hESC GFP and hiPSC lines for OCT4, LDHA, SLC2A1, and CDX2. (E) Expression levels of OCT4, LDHA, SLC2A1, and CDX2 by qPCR in hESC GFP and hiPSC lines, normalized to ACTB (n=6). Student’s t-test *, p<0.05; **, p<0.01. Mean ± s.d. (F) Lactate production levels (left) and glucose uptake levels (right) of hESC GFP (blue) and hiPSC lines (red). Shown are data from three biological replicates for lactate assay (6 technical replicates) and from six biological replicates for glucose uptake assay (p>0.05 for both assays). Mean ± s.d. (G) Representative Western blot for LDHA levels in hESC GFP (blue) and hiPSC (red) lines.

Fig 3: Dysregulation of genes in a subset of hiPSC lines(A) Heatmap of the 8 inconsistently differentially expressed genes (iDEGs) for all isogenic hESCs and hiPSC lines (as defined in Supplementary Fig. 3C) within each of the two genetic backgrounds at FDR<0.01 and fold change <2 or <0.5. hiPSC lines, red; hESC lines, blue. (B) Genome browser images of IRX2 and DPP10 RNA-seq reads in hESC GFP and hiPSC lines. (C) Expression levels of IRX2 and DPP10 by qPCR in each hESC GFP and hiPSC line, normalized to ACTB. Brown bars indicate hiPSC lines that have undergone aberrant silencing of IRX2 and DPP10. (D) Schematic for neural induction using the combination of SB431542, an ALK inhibitor, and LDN-193189, a BMP inhibitor. (E) Fold change of the neural markers NESTIN, SOX1, PAX6, and FOXG1 by qPCR in hESC GFP and hiPSC lines relative to the hESC GFP5 line. Brown bars indicate the hiPSC lines that have undergone transcriptional silencing of IRX2 and DPP10. Results are shown from three independent experiments. Mean ± s.d. (F) Immunofluorescence staining of PAX6 (green) and SOX1 (red) indicates neural differentiation at day 6 in hESC GFP and hiPSC lines. DAPI (blue). (G) Scorecard assay of embryoid bodies (EBs) derived from isogenic hESC and hiPSC lines. Heatmap and dendrogram for these EBs based on the expression levels of the indicated developmental genes.