Researchers have for the first time reprogrammed human skin cells to a pluripotent state without using viruses, according to twin studies published online today in Nature.

The approach "is truly epigenetic," Richard Young, a geneticist at the Whitehead Institute in Cambridge, Mass., who was not involved in the research, told The Scientist. "You introduce a set of master regulators, they're expressed, they reprogram the cell, and then you successfully remove them from the cell for all time."

"It opens up a gate to increased safety for the future use of the cells in cell-based therapies," said Andras Nagy, a stem cell researcher at the Mount Sinai Hospital in Toronto and one of the study's lead authors.

Human iPS cells Image: Kyoto University / AP

In the past few years, researchers have used retroviruses, lentiviruses, adenoviruses, and plasmids to insert reprogramming factors into differentiated cells to create induced pluripotent stem (iPS) cells. Although adenovirus and plasmid transfections are reversible -- which avoids the hazardous pitfalls of permanent genetic modification -- both techniques had very low efficiencies and neither was shown to work in human cells. Now, researchers in the UK and Canada have used two virus-free approaches to generate factor-removable human iPS cells.

In the first paper, a team led by the University of Edinburgh's Keisuke Kaji reprogrammed mouse and human embryonic fibroblasts using a virus-free transfection method to introduce a single vector containing the four "Yamanaka" factors -- c-Myc, Klf4, Oct4, and Sox2. In January, the Whitehead's Rudolf Jaenisch also successfully delivered the same four genes using only a single vector. But "in [Jaenisch's] iPS cells the exogenous factors remained in the genome," said Kaji. His team, however, excised the construct following reprogramming by using an enzyme that cuts out the vector and leaves only a small DNA footprint.

Kaji's team also reversibly inserted the same four factors into human cells using a DNA transposon called piggyBac from the cabbage looper moth. Unlike the vector and the enzyme method, the piggyBac transposon could be completely removed from the genome without any changes to the original DNA. "It's a cleaner system," said M. William Lensch of Children's Hospital Boston and Harvard Medical School, who was not involved in the studies. "It doesn't leave anything that's transgenic in the cell."

In the second paper, Nagy, together with Kaji, took the piggyBac work a step further. They used a slightly modified piggyBac gene-delivery system to produce reprogrammed mouse cells that could contribute to chimeric development and form all three germ layers. The same technique also produced human iPS cells that expressed several pluripotency markers and could differentiate into various specialized cell types.

"Our method provides a seamless removal of the genes from the stem cells after they did the job [of reprogramming]," Nagy told The Scientist. "The cells go back to the original, normal [DNA sequence] without leaving any trace that anything happened."

Lensch noted, however, that the researchers "need to do more work" to definitively show that the reprogrammed cells, especially the human ones, are in fact pluripotent. "The level of characterization that they've used for their human iPS cells is very low," he said. "It's one thing to show that you can push these fibroblasts back to cells that look like embryonic stem cells, but it takes more than that to be an iPS cell. What they show falls short of convincing me that they've made human iPS cells."

Nagy said he has unpublished data showing that the transposon-mediated human iPS cells can contribute to teratoma formation and give rise to all three germ layers -- the hallmarks of pluripotency. Next, the group plans to use the piggyBac method to reprogram adult fibroblasts, in addition to the embryonic fibroblasts used in the study. "We're certain that there won't be much of a difference."

Austin Smith and his colleagues at the University of Cambridge also recently exploited the piggyBac method to obtain mouse iPS cells. Publishing online last month (Feb. 18) in the journal Development, Smith's team used the piggyBac transposon to introduce a single reprogramming factor, Klf4, into partially-differentiated mouse cells called epistem cells to produce chimera-competent iPS cells. "It's another demonstration of the future popularity of this method of introducing DNA into mammalian cells transiently," said Young.

"Right now this [piggyBac method] would look to be the best method that we have" for reprogramming, Smith said. Therapeutic applications of iPS cells are still "years away," he noted. So, "actually the real power of this [method] is that it enables the basic biology of reprogramming to be studied in a much safer and cleaner way" than virus-based approaches.


Related stories:

Single-factor stem cells
[5th February 2009]

Safer iPS cells
[25th September 2008]

Epithelial cells made pluripotent
[14th February 2008]