Fig 2: Confocal analysis of particular histone modifications in undifferentiated and differentiated hiPSCs.The expression of Histone H3-S28P (green), H4-R3me2 (red; middle panel) and H3-K4me3 (red; right panel), which are primarily mediated by AURKB, PRMT5 and SETD7, respectively, was evaluated in (A) undifferentiated and (B) Day 7 differentiated hiPSCs (HUF5 clone 2) by immunofluorescent confocal microscopy. Note that H3-K4me3 expression is increased or at least retained in hiPSCs upon differentiation. (C) Similar confocal analysis of histone modifications in Day 7 differentiated hiPSCs demonstrating that only a small population of cells still expresses H3-S28P, to suggest that they are proliferative, and decreased expression of H4-R3me2 in hiPSCs following 7 days of differentiation.

Fig 3: Gene expression analysis of candidate epigenetic reprogramming factors in hESCs and hiPSCs.The expression of DNA methyltransferases (DNMTs) was analyzed in undifferentiated (D0 for Day 0) as well as Day 7 (D7) and Day 14 (D14) differentiated (A) human embryonic stem cells (hESC; H9) and (B) human induced pluripotent stem cells (hiPSCs; clone 2) from normal adult human dermal fibroblasts (HUF5) by microfluidic Quantitative-PCR (Q-PCR). Cycle threshold (Ct) values were normalized to the two most stable housekeeping genes and graphed as shown. (C) Similar Q-PCR analysis of each histone-modifying enzyme class in H9 and (D) HUF5 clone 3. Note the high levels of DNMT3B, AURKB and PRMT5 expression in undifferentiated hESCs and hiPSCs that decreases with differentiation and the elevated expression of SETD7 in D14 differentiated H9 cells and the original HUF5 fibroblasts to suggest that induction of DNMT3B, AURKB and PRMT5 expression in conjunction with SETD7 silencing may reprogram adult fibroblasts to a more hESC-like state.

Fig 4: Expression and localization of candidate epigenetic reprogramming factors in human embryos.Human blastocysts were incubated with primary antibodies for (A) DNMT3B (green), AURKB (orange) and SETD7 (red) or (B) PRMT5 (green), AURKB (orange) and SETD7 (red), the nuclear DNA stained with DAPI (blue) and visualized by multi-channel confocal microscopy. Differential Interference Contrast (DIC) was used to distinguish the inner cell mass (ICM) from the trophoectoderm of each embryo. Note that the expression of DNMT3B, AURKB and PRMT5 primarily localized to the ICM of human blastocysts, whereas SETD7 expression was predominantly detected in the outer trophoectoderm layer of the embryos (indicated by white arrows).