piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells

Abstract

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.

Mechanism and methods to induce pluripotency

“…To meet this need, several nonintegration systems and removable systems have been developed. These include episomal vectors (Yu et al, 2009), the piggyBac transposon system (Kaji et al, 2009;Woltjen et al, 2009;Yusa et al, 2009), and minicircle vectors (Jia et al, 2010).…”

“…Although iPSCs can be obtained by removable transposon or the non-integration episomal system, all of these methods still involve introducing foreign DNA into the cell so that the resulting cell lines need to be sequenced to verify that they are indeed free of genome alteration (Kaji et al, 2009;Woltjen et al, 2009;Yu et al, 2009). Thus, a reprogramming approach that does not use any DNA is highly desirable.…”

Differentiation of Stem Cells into Insulin-Producing Cells: Current Status and Challenges

“…In addition, the viral vectors after integration with the host may lead to unexpected genetic disorder (Okita et al 2007;Takahashi et al 2007;Tateishi et al 2008;Yu et al 2009). Therefore, new and improved methods of generating iPS cells, including minimal or no genetic modification are needed (Huangfu et al 2008;Kaji et al 2009;Okita et al 2008;Stadtfeld et al 2008;Woltjen et al 2009;Yu et al 2009;Zhou et al 2009). Tateishi et al (2008) reported that iPS cells derived from human skin fibroblast by retroviral expression of OCT4, SOX2, c-MYC, and KLF4 have the potential to differentiate into insulin-producing islet-like clusters (ILCs).…”

Stem cells: novel players in the treatment of erectile dysfunction

“…14 Many groups have now shown that somatic cells can be reprogrammed by overexpression of variable sets of few transcription factors in cells that share various characteristics with ESCs. [14][15][16][17] To date, several novel reprogramming protocols are available, [18][19][20][21][22] which have broadened the spectrum of cell types and species involved in iPSCs generation. Similar to ESCs, iPSCs also show the potential for differentiation into numerous cell types.…”