The Niche

Reprogramming to pluripotency without genetic engineering

Researchers make iPS cells without inserting DNA

For the first time, researchers have reprogrammed cells to pluripotency without using DNA. Ever since Shinya Yamanaka of Kyoto University in Japan showed that cultured skin cells could be made to behave like embryonic stem cells by inserting additional pluripotency genes, researchers have been trying to find ways to avoid genetic engineering as a reprogramming strategy. The additional genes make the cells less predictable, more variable and more prone to undergo unwanted proliferation. Even if DNA is not inserted into the cells, researchers worry that undetected integration could occur and could change the behaviour of those cells, limiting their use in cell therapy, drug screening and disease modelling.

An obvious alternative would be to add the proteins for the gene products instead of the genes themselves, but for that to happen, proteins would have to be made not only to enter cells but also to enter the nucleus, a particularly difficult task. Furthermore, the proteins would have to persist at high enough levels in the nucleus for the duration of the reprogramming process.

Now, researchers led by Sheng Ding of The Scripps Research Institute in La Jolla, California, have found a way to overcome this barrier in mouse fibroblasts. Besides Scripps scientists, the team included Hans Schöler of the Max Planck Institute for Molecular Biomedicine in Münster, Germany, who has published with Ding before, as well as scientists from two California-based companies — Proteomtech, in Costa Mesa, and LD Biopharma, in San Diego1.

The secret to getting the proteins across the cell and nuclear membrane turns out to be adding a sort of transportation tag to each of the four proteins (c-Myc, Klf4, Oct4 and Sox2) that is typically used to reprogram cells. The tag consisted of 11 linked copies of the amino acid arginine, a highly polar species that helped the proteins pass through membranes. In addition, the researchers added valproic acid, which has been shown to boost reprogramming rates both for induced pluripotent stem cells and in somatic cell nuclear transfer. The proteins were added 4 times over 6 days at 36-hour intervals. Researchers observed the cells over 30 passages and found that they were “morphologically indistinguishable” from embryonic stem cells and expressed similar markers. Though the researchers have not yet completed the step showing that the cells can form viable sperm and eggs, the cells did pass a related test. When the cells were mixed in with normal mouse embryos and allowed to develop in a surrogate mother, the reprogrammed cells contributed to the germ layers in 13.5-day-old embryos.

Though the work has not yet been reported in human cells, and other groups will need to replicate the results, Ding predicts that his and similar techniques will replace those requiring DNA, partially because it does not require the preparation of viruses and plasmids. “Whenever you use a genetic method, even if you claim there’s nothing left [of the added DNA], it’s still not as convenient as using chemically defined methods.”

Nonetheless, Ding and other researchers agree that further studies are essential to assessing induced pluripotent stem cells made by various methods. “At the end of the day, what you want to do is just make normal cells and reduce the risk of things like mutation,” says James Thomson of the University of Wisconsin–Madison, who recently published a technique to reprogram cells without requiring any genetic integration.2 There will eventually be many ways of making the cells, he says. “Evaluating the cells — that’s going to be the hard part.”

Nonetheless, techniques like this are essential to such evaluation. “Once the cells are vector free,” says Thomson, “they can be characterized by a lot of labs.”

Related articles

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Small molecules boost reprogramming rates

References

1. Zhou, H. et al. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell doi:10.1016/j.stem.2009.04.005 (published online 23 April 2009).

2. Yu, J. et al. Human induced pluripotent stem cells free of vector and transgene sequences. Science doi:10.1126/science.1172482 (published online 26 March 2009).

Further reading

iPS reading list

Author affiliation

Monya Baker is editor of Nature Reports Stem Cells.

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