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

Authors:
Goyal A, Chavez SL, Reijo Pera RA.
In:
Source: PLoS ONE
Publication Date: (2013)
Issue: 8(12): e82838
Research Area:
Stem Cells
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Platform:
96-well Shuttle™ System
4D-Nucleofector™ X-Unit
Experiment
Summary (by Lonza): In this study alternative approaches were investigated to increase pluripotency of iPSCs derived from fibroblasts. First the authors identified potential new reprogramming factors by comparing expression profiles of human ESCs, iPSCs and fibroblasts. Then they analyzed the candidates for their efficacy by reprogramming fibroblasts. They combined overexpression of different factors in combination with morpholino-based silencing. Due to the proven suitability of the Nucleofector™ Technology for transfecting morpholinos, they used either 4D-Nucleofector X Unit or the 96-well Shuttle Add-On. Their results suggest that the induction of DNMT3B, PRMT5 and AURKB expression and silencing of SETD7 expression in human fibroblasts might enhance the reprogramming of fibroblasts into iPSCs.
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.