Figure 1.
Experimental design of this study aimed at identifying novel epigenetic reprogramming factors.
(A) We analyzed the expression of the DNA Methyltransferase (DNMT) family and members of the different histone-modifying enzyme classes in adult human fibroblasts, undifferentiated/differentiated human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) by microfluidic Quantitative-PCR (Q-PCR) to identify candidate factors for epigenetic reprogramming. (B) The expression of the candidate reprogramming factors and the epigenetic modifications that they mediate were analyzed at the protein level in human blastocysts and undifferentiated/differentiated hiPSCs, respectively, by confocal microscopy. (C) Human neonatal and adult fibroblasts were transfected with the candidate epigenetic reprogramming factors, which were fluorescently-labeled for visualization, and colony formation assessed via brightfield and fluorescent imaging, immunofluorescence of pluripotency-related cell surface antigens and Q-PCR of epigenetic, pluripotency and cell lineage genes.
Figure 2.
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.
Figure 3.
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).
Figure 4.
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.
Figure 5.
DNMT3B-GFP/SETD7-MO transfection induces the formation of colonies that are pluripotent, but exhibit minimal proliferation.
Human adult dermal fibroblasts (HUF5) were nucleofected with a plasmid containing DNMT3B, which was tagged with GFP, and a 3’carboxyfluorescein (3’COF)-labeled SETD7 morpholino and the transfection efficiency assessed by epifluorescence. (A) The presence of DNMT3B-GFP and SETD7-MO was detected in the nucleus and cytoplasm, respectively, in transfected cells 2-3 days following nucleofection and (B) persisted in colonies that formed for several days afterwards. A greater number of clones appeared when the cells were transfected with both factors and the colonies obtained by nucleofection with DNMT3B-GFP appeared more hESC-like over those nucleofected with SETD7-MO alone. (C) When HUF5 cells were nucleofected with both DNMT3B-GFP and SETD-MO, the formation of several small clones was observed, which proliferated until approximately day 10. (D) Despite minimal proliferation, at least a portion of the colonies obtained by nucleofection with DNMT3B-GFP and SETD7-MO were positive for the pluripotency-related cell surface markers, Stage Specific Embryonic Antigen-3 (SSEA-3) and the keratin sulfate-related antigen, TRA-1-60. (E) Microfluidic Quantitative-PCR (Q-PCR) analysis of epigenetic, pluripotency and cell lineage genes in DNMT3B-GFP transfected HUF1 and HUF5 cells with and without SETD7-MO transfection compared to the original HUF5 adult fibroblasts and conventionally generated human induced pluripotent stem cells (hiPSCs) on day 0, 7 and 14 of differentiation with and without Bone Morphogenetic Proteins (BMPs). Note the similarities and differences in the expression of certain epigenetic regulators between DNMT3B-GFP and SETD7-MO nucleofected clones, undifferentiated and differentiated hiPSCs and adult fibroblasts as well as elevated expression of several pluripotency markers and germ cell-specific genes in DNMT3B-GFP/SETD7-MO transfected colonies. Grey squares indicate no expression, whereas blue, white and red squares correspond to low, medium and high expression, respectively. A direct comparison of global gene expression ratios between the different cell types is shown in Table S2.
Figure 6.
Addition of other factors and chemical compounds for increasing epigenetic reprogramming efficiency.
(A) The experimental design for increasing reprogramming efficiency in human adult, neonatal and fetal fibroblasts with the use of (1) the additional reprogramming factors, AURKB, PRMT5, SV40, hTERT and/or NANOG, (2) the cell-permeable chemical compounds, AZA and/or VPA, to assist in chromatin remodeling for gene activation and (3) an alternative transfection approach whereby fibroblasts are first nucleofected with SETD7-MO and then transfected with DNMT3B, hTERT and SV40 via a cationic lipid reagent. (B) Brightfield imaging of colonies obtained by nucleofecting neonatal human foreskin fibroblasts (HFF-1) with DNMT3B-GFP/SETD7-MO in the presence of AZA and VPA, which represented the largest number of clones obtained. (C) Gene expression analysis of epigenetic regulators, pluripotency genes and trophoectoderm markers as well as (D) markers of the three germ layers and germ-cell specific genes in HFF-1 cells transfected with different combinations of reprogramming factors and chemicals by microfluidic Quantitative-PCR (Q-PCR) for comparison to fibroblasts similarly nucleofected three times with the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC). Note the lack or negligible expression of trophoectoderm and germ layer markers, but high levels of early and mid germ cell-specific genes in HFF-1 colonies not observed in conventionally generated hiSPCs. Grey squares indicate no expression, whereas blue, white and red squares correspond to low, medium and high expression, respectively.