Without a Trace: iPS Cell Techniques Leave No Footprints

Scientists have figured out how to induce pluripotency in differentiated cells, then erase their tracks, leaving little or no scar in the genome of the resulting iPS cells. In companion Nature papers published online March 1, laboratories from the University of Edinburgh, U.K., and Mount Sinai Hospital in Toronto, Canada, report on non-viral methods to add pluripotency genes to the DNA of adult cells, then later cut out the genes. Because they avoided viral infection and minimized the risk of transgenes altering endogenous gene expression, the scientists believe their method could ultimately be more amenable to human cell therapies. Until then, such techniques will be useful in the laboratory. In a third paper, published March 6 in Cell, scientists from the lab of Rudolf Jaenisch at the Whitehead Institute in Cambridge showed that they were able to reprogram cells from people with Parkinson disease, then excise the viral vector and convert the iPS cells into neurons for in-vitro studies. Together, the three reports illustrate what scientists had suspected, but not previously proven: that once iPS cells are made, they will retain their pluripotency even if the transgenes are then removed.

Stem cells have the potential to develop into any kind of tissue, but their use is fraught with technical and ethical concerns. The study of undifferentiated cells has taken off since the 2006 discovery that just four genes can reprogram cells to a pluripotent state (Takahashi and Yamanaka, 2006; see also ARF related news story). Differentiating pluripotent cells into specific tissues could provide useful models for disease, as well as someday offer tissue transplants. But the most common technique uses a virus to transfer the genes necessary for pluripotency, and risks activating oncogenes if the additional genes land in the wrong place. I do not think anyone would dare to do anything clinical with those cells, said Knut Woltjen of Mount Sinai Hospital, an author on both Nature papers.

In addition, adding four separate genes means a minimum of four integrations, and probably more, said Keisuke Kaji of the University of Edinburgh and also an author of both Nature papers. That multiplies the risk of disrupting endogenous gene expression. Kaji sought a way to decrease the number of integrations, and then remove them from the genome. In one paper, he and colleagues used a non-viral DNA plasmid to add the requisite genes to mouse embryonic fibroblasts. Instead of four vectors, one with each gene, Kaji linked all four genes—c-Myc, Klf4, Oct4, and Sox2—together with 2A peptide bridges. When transcribed, the construct yielded a single mRNA, but four separate proteins.

Kaji engineered the cassette such that the pluripotency genes were flanked by loxP sites. Once the cells had dedifferentiated, he transiently transfected the enzyme cre, which removed the DNA between the loxP sites. However, this excision left some of the construct behind, including a neomycin resistance gene and the promoter. In the other Nature paper, researchers in the laboratory of Andras Nagy at Mount Sinai were able to completely delete any traces of their genome modification.

Nagy, Woltjen, and colleagues took advantage of a transposon called piggyBAC, a jumping gene originally found in moths that naturally hops in and out of chromosomes. Transposons are the only system that actually allows you to get a completely traceless removal, Woltjen said. A similar approach was also used in a study, published online in Development February 18, in which scientists from the lab of Austin Smith at the Centre for Stem Cell Research in Cambridge used piggyBAC carrying Klf4 to revert partially specialized Epi-stem mouse cells back to a pluripotent state, then later used cre to delete the transgene (Guo et al., 2009).

The Nagy lab borrowed Kaji’s four-gene cassette to assemble their transposon. They transfected mouse embryonic fibroblasts with the piggyBAC vector, plus a non-integrating, transiently expressed vector containing the gene for the enzyme transposase, which transferred the transposon into the genome. Once the cells had dedifferentiated, Woltjen and colleagues again transiently transfected transposase; this time, it cut the transposon back out. Unlike the cre-lox system, piggyBAC leaves no scar; the cellular DNA sequence was exactly as it had started.

To confirm the pluripotency of the engineered cells, the authors showed that they were capable of forming teratomas that contained multiple kinds of differentiated cells, and could combine with embryos to form chimeras. The researchers were also able to reprogram human embryonic fibroblasts with the piggyBAC transposon, and in unpublished results, were able to generate human teratomas, Nagy said. The scientists have yet to slice piggyBAC out of human iPS cells, but, Woltjen said, We really foresee no reason why this excision event won’t work effectively in humans.

Human-derived iPS cells, once re-differentiated according to a scientist’s wishes, can provide valuable in-vitro models for disease. Working in Jaenisch’s lab, joint first authors Frank Soldner, Dirk Hockemeyer, and colleagues used viral vectors to generate iPS cell lines from skin cells of five people with Parkinson disease. They then used a cre-lox system to cut out the transgenes, and caused the cells to differentiate into dopaminergic neurons.

Human neurons are generally inaccessible to scientists, Soldner said. Rodent models for Parkinson disease are based on genetic mutations associated with the disease, but those genetic mutations only account for approximately 5 percent of human cases. In the study, the scientists collected skin cells from people with idiopathic disease. Using non-genetically linked disease samples opens an avenue to study diseases that we really do not have any other good models for, said Allison Ebert of the University of Wisconsin-Madison, who was not involved in the current research. It is encouraging to see that stable iPS cell lines could be generated from a wide variety of sporadic patients. Cell lines such as these could also be useful for screening drugs, Nagy noted.

They’re one more step along the process, said Mahendra Rao, vice president of research in stem cells and regenerative medicine at Life Technologies in Frederick, Maryland. Scientists are trying several methods to generate iPS cells, he noted, and it remains to be seen which will be most useful. So far, scientists have not yet generated cells Rao would feel comfortable putting in a patient. In the meantime, however, re-differentiated iPS cells can contribute plenty to the study of disease simply by growing in a dish.

Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 2009 Mar 1 [Epub ahead of print]. Abstract

Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG, Hargus G, Blak A, Cooper O, Mitalipova M, Isacson O, Jaenisch R. Parkinson’s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 2009 Mar 136:964-977.

Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hmlinen R, Cowling R, Wang W, Liu P, Gertsenstein M, Kaji K, Sung H-K, Nagy A. piggyBAC transposition reprograms fibroblasts to induced pluripotent cells. Nature 2009 Mar 1 [Epub ahead of print]. Abstract

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