Generation of Human Induced Pluripotent Stem Cells Using Epigenetic Regulators Reveals a Germ Cell-Like Identity in Partially Reprogrammed Colonies
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.