piggyBac transposition reprograms induced pluripotent stem cells in a safer way

Why this methodology advancing is so exciting?

1. “First, PB transposition permits technical simplification and improved accessibility of reprogramming methodology by making use of effortless plasmid DNA preparation and commercial transfection products for delivery. This eliminates the need for specialized biohazard containment facilities or the production of high-titre, limited-lifetime viral stocks26.”

2. “Second, the range of somatic cell types that could be used for reprogramming is not limited by a decreased susceptibility to viral infection26.”

3. “Third, PB-mediated delivery will allow the option of xeno-free production of human iPS cells contrary to current viral production protocols that use xenobiotic conditions.”

4. “Finally, accurate transgene removal through transposase expression has been demonstrated in various cell lines8, 11, 12, 27, 28, 29. We have harnessed this potential and show here that the reprogramming factors can be removed without a trace from iPS cells once exogenous expression becomes dispensable.”

These are points at the end of the paper. My summary is that using transient expression of transposase to achieve single-round DNA transfection for reprogramming and to clean the genome after reprogramming – very cool and “safe”.

It is from the Nagy’s group in Canada. I listened to his lectures and he is really a technical geek in mouse developmental field. Still deeply impressed by his aggregation method to produce chimeras!

Nature. 2009 Mar 1.

piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells.
Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hämäläinen R, Cowling R, Wang W, Liu P, Gertsenstein M, Kaji K, Sung HK, Nagy A.

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.

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.

PMID: 19252478

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Actually, there is another very similar paper on Nature back-to-back

Nature. 2009 Mar 1.

Virus-free induction of pluripotency and subsequent excision of reprogramming factors.

Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K.

MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh, Edinburgh EH9 3JQ, UK.

Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors. However, such iPS cells contain a large number of viral vector integrations, any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming, complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia. Two transient transfection-reprogramming methods have been published to address this issue. However, the efficiency of both approaches is extremely low, and neither has been applied successfully to human cells so far. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. When the single-vector reprogramming system was combined with a piggyBac transposon, we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models.

PMID: 19252477

Update 2009-4-7:
I just noticed that this paper actually used two methods: 1. Cre out the viral sequence, which was used by Dr. Jaenisch’s group as well (ref); 2. piggyBac system …
Admire~~

F1000 review

Yi Eve Sun with Wiehong Ge
University of California Los Angeles Medical School, United States of America
Neuroscience

This paper demonstrates a virus-free, tightly controlled transgene delivery system to derive induced pluripotent stem (iPS) cells, followed by excision of reprogramming factors. It used doxycycline-inducible transcription factors, delivered by the piggyBac transposon/transposase system, to reprogram human embryonic fibroblasts, and pCAG-driven single plasmid cassette carrying a loxP site to deliver four factors for mouse iPS cell derivation. Both systems allow elimination of extrogenous reprogramming factors and minimize the genome modification.

The usage of integrating viral-mediated iPS cell technology had presented potential issues with violating genomic integrity. This paper, together with that from Nagy’s group, published back-to back-in the same issue of Nature {1}, provides virus-free and factor-free iPS derivation systems, which represent the most current advancement in the iPS cell field. Moreover, these studies provide valuable tools to gain information during the reprogramming process, a gradual and stochastic progress that is difficult to elucidate with current methodology. By adjusting the doxycycline dose and period, they can easily find the suitable transgene expression level and the minimum transgene expression time. Moreover, by comparison with transgene-free iPS cells, iPS cells with residue transgenes show the deficit of their differentiation potential. This result is confirmed by Rudolf Jaenisch’s current published work in Cell {2}, where they found partially silenced transgenes perturb the transcriptional profile of iPS cells.

References: {1} Woltjen et al. Nature. 2009 Mar 1. [PMID: 19252478]. {2} Soldner et al. Cell 2009, 136:964-77 [PMID: 19269371].