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. 2016 Dec 1;143(23):4368–4380. doi: 10.1242/dev.138982

Fig. 4.

Fig. 4.

Transcriptional and epigenetic profiling of LIF-3i-reverted hPSCs. (A) Genome-wide cross-species hierarchical clustering. Shown is a dendrogram of expression microarrays of mESC [serum/LIF; naïve (LIF-2i)], primed mEpiSC, and isogenic hPSC samples from this study before (hPSC primed) and after 5 passages in LIF-3i (hPSC naïve). Human PSC lines (n=12) included: three hESC lines H9, H7 and ES03 (gold); six sa-MP-iPSC lines E5C3, E5C1, E17C6, LZ6+2, LZ6+10 and 6.2 (red); and three fibro-iPSC lines 7ta, C1.2 and C2 (green). (B) (Top) CpG methylation. Box plot shows beta values of genome-wide autosomal differentially methylated region (DMR) CpG probes from Infinium methylation arrays [16,282 of 473,864 autosomal probes significantly (P<0.05) differentially methylated (SD>0.15); see supplementary Materials and Methods for further details] in the same isogenic primed (−) versus LIF-3i-reverted (+) hPSC samples used for the microarrays above. Gold, hESCs (n=3); red, sa-MP-iPSCs (n=6); green, fibro-iPSCs (n=3). ***P<0.001 (paired two-way t-test). (Bottom) Global 5MC and 5hMC levels from dot blot immunoassays (relative to primed) for representative LIF-3i-reverted hPSCs. Genomic DNA samples were collected before (−) and after (+) LIF-3i reversion from H9 (gold), E5C3 (red) and C1.2 (green). (C) Activities of proximal enhancer (PE) and distal enhancer (DE) elements of the human OCT4 promoter in primed (bFGF) versus LIF-3i-reverted E5C3. Shown are relative firefly luciferase activities following normalization with Renilla luciferase and negative control basal activities ±s.d. (n=3). *P<0.05 (paired t-test). (D) Stable BAC reporter transgenic OCT4 PE/DE mutant lines. (Top) Cytometry plots of representative LIF-3i-reverted C2 hiPSC subclones (n=3) stably transfected with full-length OCT4-GFP-2A-PURO PE/DE sequences (control), mutant ΔPE-OCT4-GFP-PURO constructs, or non-transfected (no construct) controls. (Bottom) Percentage GFP+ cells among naïve cultures of individual hiPSC subclones (n=3) expressing control or mutant ΔPE sequences. (E) Pluripotency circuits in LIF-3i-reverted hPSCs. (Top) Mean beta values of core module-specific CpG DMRs in primed (−) versus LIF-3i-reverted (+) hPSC; (bottom) corresponding log2 mean subtracted normalized expression of core module genes (Table S1) of the same independent hPSC samples (identical to those used above for expression microarrays). Gold, hESCs (n=3); red, sa-MP-iPSCs (n=6); green, fibro-iPSCs (n=3). (F) Pluripotency gene-specific promoter CpG methylation. Heatmap-dendrogram clustering and box plots of mean beta values of ESC module gene-specific CpG DMRs [P<0.001 (paired two-way t-test)] of LIF-3i (+) versus primed (−) hPSCs. Samples are the same 12 hPSC lines in each category, as described above. Percentages represent reduction of median beta value following LIF-3i reversions.