Journal Article

piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells

Hämäläinen, Riikka, Wang, Wei, Cowling, Rebecca, Michael, Iacovos P., Gertsenstein, Marina, Sung, Hoon-Ki, Mileikovsky, Maria, Woltjen, Knut, Desai, Ridham, Kaji, Keisuke, Nagy, Andras, Liu, Pentao, Mohseni, Paria

2009

Overview

Virus-free iPS cells The discovery that non-germline adult cells can be reprogrammed to become pluripotent, able to differentiate into any cell type, opened up exciting possibilities. Reprogrammed cells — called induced pluripotent stem (iPS) cells — should have great potential in regenerative medicine, but most current methods of producing them involve viral gene delivery that could cause abnormalities in the induced cells. Two groups in this issue report on a collaboration that has succeeded in producing pluripotency in human cells without using viral vectors. Stable iPS cells were produced in both human and mouse fibroblasts using virus-derived 2A peptide sequences to create a multicistronic vector incorporating the reprogramming factors, delivered to the cell by the piggyBac transposon vector. The 2A-linked reprogramming factors, not required in the established iPS cell lines, were then removed. This paper uses the piggyBac transposon to generate stable iPS cells from human and mouse fibroblasts; the individual piggyBac insertions can then be removed from established iPS cell lines. The study also demonstrates removal of reprogramming factors joined with 2A sequences (described in an accompanying paper; doi:10.1038/nature07864) delivered by a single transposon from murine iPS lines. Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state 1 , 2 , 3 , 4 . 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 1 , lentiviral 5 , adenoviral 6 and plasmid 7 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 8 , 9 , 10 , 11 . 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 12 . Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition 13 . 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 12 , 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 14 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: Animals / Biological and medical sciences / Cell Differentiation / Cell differentiation, maturation, development, hematopoiesis / Cell Line / Cell physiology / Cells, Cultured / Cellular Reprogramming - genetics / DNA Transposable Elements / Embryonic stem cells / Fibroblasts / Fibroblasts - cytology / Fibroblasts - physiology / Fibroblasts - virology / Fundamental and applied biological sciences. Psychology / Gene Order / Gene silencing / Gene Transfer Techniques / Genetic aspects / Genetic Vectors - genetics / Humanities and Social Sciences / Humans / Kruppel-Like Factor 4 / letter / Methods / Mice / Mice, Nude / Molecular and cellular biology / multidisciplinary / Physiological aspects / Pluripotent Stem Cells - physiology / Proteins / Science / Science (multidisciplinary) / Sequence Alignment / Stem cells / Transcription Factors - genetics / Transfection / Transgenes - genetics