piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells

By K. Woltjen, Iacovos P. Michael, P. Mohseni, R. Desai, M. Mileikovsky, R. Hämäläinen, R. Cowling, Wei Wang, Pentao Liu, M. Gertsenstein and 3 more. Nature, 2009

1,888 citations recorded by the source corpus.

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral, lentiviral, adenoviral and plasmid transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision, we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/2c51a29dd7ed6e10a17a97b9a5cd5bb1a1cd25ec

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Citations in the corpus

Senescence impairs successful reprogramming to pluripotent stem cells. (2009, methodology): “In a similar way, two laboratories used piggyBac transposons that can be shuttled in and out of the host genome by transient expression of transposase (Woltjen et al., 2009; Yusa et al., 2009).”
Ethical and Safety Issues of Stem Cell-Based Therapy (2018, methodology): “Several types of non-integrating methods have been developed [use of non-integrating adenoviral vectors, repeated transfection of plasmids, Cre–loxP– mediated recombination, PiggyBac-transposition] [37-41].”
Emerging trends and new developments in regenerative medicine: a scientometric update (2000 – 2014) (2014, background): “Potentially risky viral integration of transcription factors were replaced with the use of neural progenitor cells and small molecules [18] or ‘piggyBac’ transposition [19].”
Differentiation of Effector CD4 T Cell Populations (2010, positive): “Even terminally differentiated fibroblasts can be reprogrammed to become inducible pluripotent stem cells by the transduction of the transcription factors Oct4/Klf4/Sox2/Myc (288, 289).”
Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon (2009, neutral): “While this manuscript was under review, Kaji et al. and Woltjen et al. reported similar results 31, 32.”