Pluripotency via plasmids

Researchers are one step closer to reprogramming stem cells that are safe for use in the clinic with a new virus-free method for deriving human induced pluripotent stem (iPS) cells that doesn't rely on integrating foreign DNA, according to a linkurl:study;http://www.sciencemag.org/cgi/content/abstract/1172482 published online today in __Science__.

Skin cells induced to a pluripotent state
Image: Junying Yu / UW-Madison

Since these cells' genomes have never been genetically modified, they provide the first such reprogrammed cells that can fairly and accurately be compared with human embryonic stem cells to test if they are truly embryonic-like, the researchers say. Last year, Kyoto University's linkurl:Shinya Yamanaka;http://www.med.kyoto-u.ac.jp/E/grad_school/introduction/1517/ devised the linkurl:first virus-free route;http://www.sciencemag.org/cgi/content/full/322/5903/949 to obtaining iPS cells, but his approach involved laborious and repeated insertions with plasmid vectors, and it was only shown to work in mouse cells. Now, linkurl:James Thomson;http://stemcells.wisc.edu/faculty/thomson.html of the University of Wisconsin-Madison has extended the plasmid technique to reprogram human skin cells to an embryonic-like state using only a single plasmid transfection. This latest landmark on the path to safe, patient-specific iPS cells is also the first time that researchers have produced human iPS cells without transiently inserting DNA into the genome and then reversing the changes. Thomson and his colleagues took a plasmid derived from the Epstein-Barr virus, packed it full of seven reprogramming genes -- an amalgamation of all six factors used by linkurl:Thomson;http://www.sciencemag.org/cgi/content/abstract/318/5858/1917 and linkurl:Yamanaka;http://www.cell.com/abstract/S0092-8674(07)01471-7 in 2007 to first reprogram human skin cells, plus an extra gene, the SV40 large T gene, to counteract the toxic side effects of the oncogene c-myc -- and inserted them into newborn foreskin fibroblast cells. The vectors then reprogrammed the skin cells to yield iPS cells that passed all the standard tests of pluripotency. Thomson's team then relaxed selection for the plasmids, and the iPS cells gradually lost the introduced vectors over successive rounds of cell division, but retained their pluripotency. The vector's intermediate instability is in the "sweet spot" for deriving iPS cells, Thomson told __The Scientist__: "It's sort of stable, but not too hard to get rid of." Ultimately, the researchers obtained iPS cells completely devoid of vector and transgene sequences -- albeit at low frequencies of around five in a million. Some of these cells have now survived in continuous culture for seven months and counting. "Here you don't see any transgenic vectors in the chromosomes," linkurl:In-Hyun Park,;http://daley.med.harvard.edu/assets/In_Hyun/In_Hyun.htm a stem cell researcher at Children's Hospital Boston and Harvard Medical School who was not involved in the study, told __The Scientist__. "It's much cleaner than the previous reported methods." Requiring seven reprogramming factors was "very unsatisfying," admitted Thomson, but the important thing is that the technique works. "I suspect that over time we'll pare the number of genes down, but it's a viable approach today," he said. "I am really excited about seeing that more and more means of generating genetically unaltered iPS cell lines are going to be available," linkurl:Andras Nagy;http://www.mshri.on.ca/nagy/ of Toronto's Mount Sinai Hospital, who last month linkurl:published;http://www.nature.com/nature/journal/vaop/ncurrent/abs/nature07863.html his own virus-free method of creating human iPS cells using a linkurl:transposon-mediated delivery system,;http://www.the-scientist.com/blog/display/55486/ said in an email. "They all have their own pros and cons... At this point there is no way to see which one is going to be superior over the others. Most likely there will be no single winner of this 'race.'" Thomson agreed. "I think there will be multiple methods that will more or less get us to the same endpoint," he said. Sorting out which method is best "will come down to cost effectiveness and safety, and it'll take a couple years to figure that out." linkurl:Konrad Hochedlinger;http://www.hms.harvard.edu/dms/bbs/fac/Hochedlinger.html of the Massachusetts General Hospital and the Harvard Stem Cell Institute, who was not involved in the study, said that with these non-genetically modified human iPS cells, we can finally "ask how equivalent iPS cells are, in fact, with embryonic stem cells." If the two types of stem cells really are identical, "then we're just at the same starting point as we were with embryonic stem cells," noted Thomson.
**__Related stories:__***linkurl:Piggybacking to pluripotency;http://www.the-scientist.com/blog/display/55486/
[1st March 2009]*linkurl:Single-factor stem cells;http://www.the-scientist.com/blog/display/55399/
[5th February 2009]*linkurl:Safer iPS cells;http://www.the-scientist.com/blog/display/55054/
[25th September 2008]