Published online 7 March 2008 | Nature 452, 132- (2008) | doi:10.1038/452132a

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Carbon nanotubes used to reprogramme adult human cells?

Nanotubes (above) were used to introduce a complex of proteins into testicular cells (stained, below). UNIDYM

A Californian biotech company claims that it has used carbon nanotubes to ‘reprogramme’ adult human cells to an embryonic-like state — a breakthrough that removes the elevated risk of cancer that blights other techniques. But uncertainties about the cells, which have yet to be reported in a peer-reviewed journal, have left many sceptical.

PRIMEGEN

Last year, researchers led by Shinya Yamanaka of Kyoto University demonstrated that by using just four genes it was possible to reprogramme adult human skin cells to a stem-cell-like pluripotent state — meaning that they could develop into any of the body’s cell types. These ‘induced pluripotent stem’ (iPS) cells hold tremendous therapeutic potential. But to insert the genes into the cells, researchers have had to use viral vectors, which can turn the cells cancerous.

PrimeGen, based in Irvine, claims to have got around this problem by using single-walled carbon nanotubes — cylinders of carbon molecules only a few nanometers in diameter — to introduce a complex of around a dozen proteins, including the ones coded for by the four genes used by Yamanaka, plus a fifth called Nanog. The researchers used the nanotube delivery system to introduce genes into human testicular and retinal cells, and PrimeGen reports that they were quickly taken up by an impressive 80% of the cells.

The method is based on work by Hongjie Dai of Stanford University in Palo Alto, California. Dai’s group has demonstrated that nanotubes can deliver cancer-targeting drugs1, proteins and DNA2 into cells. According to Dai, the nanotubes with their cargo are absorbed by the cells through endocytosis, a process by which the cell membrane envelopes molecules outside the cell, but there is uncertainty over how this happens.

PrimeGen has now announced an alliance with Unidym, based in Menlo Park, California, which makes the nanotubes. Robert Bismuth, vice president of marketing at Unidym, says that the researchers used nanotubes because they “are quite good at getting through a cell membrane” and because their long tubular structure enables each to carry several molecules.

But on the bigger claim that they can reprogramme cells, PrimeGen president John Sundsmo admits a few problems. He says that from around day three the cells start acting like embryonic stem cells and expressing proteins characterizing pluripotency. But after day 14 “they almost all stop”. “It’s like a switch is turned on and then turned off,” Sundsmo says. True embryonic stem cells and iPS cells exhibit pluripotent markers and proliferate quickly and endlessly.

“They look really good. But what I’d really like to see is the paper published.”



Konrad Hochedlinger, a stem-cell researcher at Harvard University who has done some of the most rigorous tests on iPS cells, suggests that the introduced factors produced a momentary blip of activity before being diluted. “These findings are less interesting as they may only reflect transient transcriptional changes but not stable cellular changes,” he says.

However, Sundsmo says that the cells show a stable “intermediate” stage of reprogramming that will be useful for therapy and basic research. He admits however the word “intermediate” is “loose and somewhat arbitrary”. “There is a disconnect between pluripotency and proliferation,” he says. “We haven’t figured it out yet.” Sundsmo says that they await results of a teratoma test — where cells are allowed to develop in vivo — that could prove whether the cells actually are pluripotent.

Frank Edenhofer, a stem-cell expert at University of Bonn in Germany, calls the technique “attractive and promising”. But his own experiments have shown nanoparticles to be “toxic and display low efficiency”. Other groups have also found that nanotubes can kill cells and have difficulty releasing their cargo. Matt Becker of the National Institute of Standards and Technology in Gaithersburg, Maryland, says that the ability of nanotubes to transfect cells without doing harm “depends critically on how the materials were prepared, dispersed in solution, and specific mechanism of entry into the cell interior”.

Few scientists have seen PrimeGen’s cells. One, Peter Donovan, a stem-cell expert at the University of California in Irvine and a member of PrimeGen’s advisory board, says: “They look really good. But what I’d really like to see is the paper published.”

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Most scientists contacted by Nature have expressed scepticism because PrimeGen announced its findings on 26 February at an investors meeting rather than in a scientific publication. Another PrimeGen scientific advisory board member, stem-cell pioneer Rudolf

Jaenisch of the Massachusetts Institute of Technology in Cambridge, told Nature, without giving a reason, that he quit the board the following day. PrimeGen seemed unaware of Jaenisch’s move when contacted by Nature. Sundsmo says that they decided to announce the results ahead of publication because the company has already filed patent applications. He says: “This is conceptually a very simple observation that is easily repeated.”

Two years ago, PrimeGen similarly publicized experimental results describing the derivation of pluripotent stem cells from sperm precursor cells (see Nature 440 , 586–587 ; 2006). At the time PrimeGen told Nature they would publish a paper, but haven’t. Sundsmo says that the science behind that paper, which has been at the core of the company’s technology, had to be reformulated because of internal disagreement over what the experiment showed. Sundsmo says that half the company was let go and that the paper has been accepted for publication.