Abstract
Previous studies have shown that induced pluripotent stem cells (iPSCs) can be derived from fibroblasts by ectopic expression of four transcription factors, OCT4, SOX2, KLF4 and c-MYC using various methods. More recent studies have focused on identifying alternative approaches and factors that can be used to increase reprogramming efficiency of fibroblasts to pluripotency. Here, we use nucleofection, morpholino technologies and novel epigenetic factors, which were chosen based on their expression profile in human embryos, fibroblasts and undifferentiated/differentiated human embryonic stem cells (hESCs) and conventionally generated iPSCs, to reprogram human fibroblasts into iPSCs. By over expressing DNMT3B, AURKB, PRMT5 and/or silencing SETD7 in human fibroblasts with and without NANOG, hTERT and/or SV40 overexpression, we observed the formation of colonies resembling iPSCs that were positive for certain pluripotency markers, but exhibited minimal proliferation. More importantly, we also demonstrate that these partially-reprogrammed colonies express high levels of early to mid germ cell-specific genes regardless of the transfection approach, which suggests conversion to a germ cell-like identity is associated with early reprogramming. These findings may provide an additional means to evaluate human germ cell differentiation in vitro, particularly in the context of pluripotent stem cell-derived germ cell development, and contribute to our understanding of the epigenetic requirements of the reprogramming process.
Highlights
Human embryonic stem cells are derived from the inner cell mass (ICM) of blastocysts and are characterized as pluripotent due to their ability to self-renew and give rise to all types of cells in the body
When we examined the expression of the different histone-modifying enzyme classes in adult human dermal fibroblasts (AHDF), Human embryonic stem cells (hESCs), and the human induced pluripotent stem cells (hiPSCs) clones we observed elevated expression of AURKB and PRMT5, enzymes that mediate the phosphorylation of serine residues and methylation of arginine residues, respectively, and their expression decreased upon differentiation (Figure 2C, 2D and Figure S2)
High expression of the histone lysine methylation enzyme, SETD7, was detected in AHDFs, but not in undifferentiated hESCs and hiPSCS and increased with differentiation between days 7, 14 and/or 21 (Figure 2C, 2D and Figure S2). This suggested that the induction of DNMT3B, AURKB and PRMT5 expression in conjunction with the silencing of SETD7 expression might assist in the reprogramming of fibroblasts to a more hESC-like state
Summary
Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of blastocysts and are characterized as pluripotent due to their ability to self-renew and give rise to all types of cells in the body. Yamanaka and colleagues reprogrammed fibroblasts by using four transcription factors (OCT4, SOX2, KLF4 and cMYC) in viral vectors [3,8] This method has several drawbacks and recent studies have focused on eliminating the use of c-MYC and utilizing alternative methods of reprogramming, including excisable constructs, nonintegrating plasmids adenovirus, episomal and piggybac transposon vectors to circumvent the genomic integration of viral transduction and increase reprogramming efficiency [9,10]. Two different approaches, the introduction of novel factors or the addition of cell permeable chemical compounds, either alone or in conjunction with one another have been successfully used to increase the reprogramming efficiency of iPSCs. For the first approach, factors typically utilized for cell immortalization such as hTERT and the SV40 large T antigen have been transfected together with the four Yamanaka factors into human fetal, neonatal and adult dermal fibroblasts [16]. IPSCs have been derived using small molecule compounds such as Valproic Acid (VPA) and 5Aza-2 ́-deoxycytidine (AZA), which may substitute for c-MYC during transfection and are thought to act by inducing epigenetic remodeling [19]
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