Published online 20 November 2007 | Nature | doi:10.1038/450462a

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Race to mimic human embryonic stem cells

'Personalized' tissues come a step closer.

The donor cells used to create cloned primate embryos came from a monkey, Semos, named after the god in the science fiction work Planet of the Apes.The donor cells used to create cloned primate embryos came from a monkey, Semos, named after the god in the science fiction work Planet of the Apes.OHSU

Two much-anticipated scientific firsts announced this week bring the dream of regenerative medicine a step closer. The production of cloned primate embryonic stem cells and the reprogramming of adult human cells both represent important milestones in the quest to produce 'pluripotent' cells, which can develop into almost any of the body's roughly 200 cell types.

Human embryonic stem cells have this property, and those used in research are usually extracted from leftover embryos created during in vitro fertilization. But researchers want to create pluripotent cells that are genetically matched to individual patients. Such cells could then be transplanted to treat disorders such as Parkinson's disease and diabetes, or be used by researchers to model disease progression.

Cloning offers one way to create these cells. This week, a team led by Shoukhrat Mitalipov of Oregon Health & Science University in Beaverton report the first creation of embryonic stem cells from cloned monkey embryos (see page 497). Until now, cloned embryonic stem cells had been created only in mice. The accomplishment in primates is "like breaking the sound barrier", says Robert Lanza, of Los Angeles-based biotech company Advanced Cell Technology.

The creation in 1996 of the first cloned animal, Dolly the sheep, led to a rapid succession of clones. But continued failure to achieve cloned human or monkey embryos resulted in pessimism.

In 2003, primate-cloning researcher Gerald Schatten said: "With current approaches, NT [nuclear transfer, a cloning technique] to produce embryonic stem cells in nonhuman primates may prove difficult — and reproductive cloning unachievable1." This was after his study involving 716 monkey eggs failed to produce a single clone. Then, in February 2004, Woo Suk Hwang, then of Seoul National University in Korea, announced that he had created cloned human embryonic stem cells2. But, in January 2006, those results were shown to have been faked, and some suspected that Schatten was right.

Mitalipov's group had been trying for almost a decade to achieve reproductive cloning in monkeys, and used some 15,000 eggs in the process. After Hwang's results turned out to be fraudulent, the researchers decided to switch from reproductive cloning to trying to establish a cloned embryonic stem-cell line. They took skin cells from Semos, a nine-year-old rhesus macaque, and inserted the cells' nuclei into eggs that had had their own genetic material removed. By January 2007, they had a cell line that retained its embryonic pluripotency — and, a couple of months later, another.

Mitalipov credits their success to a US$19,000 imaging machine, named Oosight, which allows the structures in the egg that hold the DNA to be clearly seen, enabling easy extraction — the first step in nuclear transfer. Previously, researchers used a dye named Hoechst combined with ultraviolet light to locate and remove an egg's DNA. But Mitalipov's group found that this method damaged the egg.

Mitalipov says that his group's technique should work in human cells: "There's nothing specific here. But you must use this kind of imaging system," he says.

Reproductive cloning in monkeys, though, still looks to be a long way off. In April, after creating the two cell lines, Mitalipov's team tried transferring 77 embryos created into about a dozen surrogates. All failed to create pregnancies.

In the aftermath of the Hwang fraud, Nature has taken the unusual step of having these results independently verified by a team at Monash University in Melbourne, Australia3. "The scientific community has a need to have some certainty in the outcomes of nucleartransfer experiments given the recent unfortunate experience with human somatic-cell nuclear transfer. We have total confidence in these conclusions," says Alan Trounson, a member of the Monash team.

Many scientists are hesitant to apply the technique in humans because it requires women to undergo an uncomfortable procedure that involves significant health risks. Altogether, Mitalipov's team used 304 eggs to produce the two primate embryonic stemcell lines. The researchers still have little idea of what separates the majority failures from the rare successes, so a similar number of eggs would probably be required to establish human lines.

Avoiding controversy

"The basic science of this is important," says John Gearhart, director of the Stem Cell Program at Johns Hopkins Medicine in Baltimore, Maryland. "But the low frequency of success here is troubling — particularly when considering human work."

After reprogramming, cells taken from human skin became embryonic-like stem cells.After reprogramming, cells taken from human skin became embryonic-like stem cells.

But there is another promising route to creating pluripotent cells that does not require eggs or the controversial destruction of embryos. On Tuesday, Shinya Yamanaka of the University of Kyoto in Japan reported that his team had created pluripotent cells from human skin cells4 and, on the same day, a team of researchers led by James Thomson at the University of Wisconsin, Madison, reported the same5.

Yamanaka's work builds on his exciting discovery last year that introducing four transcription factors into mouse skin cells 'reprogrammed' the cells into an embryo-like state. Early this summer, Yamanaka and two other groups reported using the same four factors to create cells that seemed to be indistinguishable from embryonic stem cells6.

Because of the basic differences between human and mouse cells, Yamanaka was surprised to find that these four factors produced the same result in humans. The team generated 10 pluripotent lines from a culture of some 50,000 facial skin cells that had been subjected to the four factors. The skin sample, provided by a company in the United States, had been taken from a 36-year-old Caucasian woman. Yamanaka repeated the exercise with cells from synovial (joint) fluid from a 69-yearold man with similar results. During culture, the pluripotent cells take on the flat shape of embryonic stem cells.

Why so few cells successfully form such 'induced' pluripotent stem (iPS) cells is a mystery that Yamanaka is still trying to resolve. But because he uses such a cheap resource — cells that can be attained in their millions from a single skin biopsy — the low yield is not a problem, he says. In comparison to human embryonic stem cells — after much political wrangling and laborious effort, there are only three cell lines in Japan — his technique is prolific, Yamanaka says. "In a little dish you can get ten cell lines quickly. Practically speaking it's a very high success rate."

Like the mouse iPS cells, Yamanaka's human iPS cells passed all the basic tests for embryonic stem cells, including the ability to form tumours expressing the three primary germ layers when injected under the skin of a mouse engineered to have no immune system.

“Until now, cloned embryonic stem cells had been created only in mice.”


But are they truly pluripotent? The most stringent tests, carried out in mice, are to see whether a whole individual can be created from iPS cells or whether iPS cells mixed with an embryo's are expressed in all of the resulting chimaeric mouse's tissues. Neither test can be done with human cells. "In humans, there's no answer to the question," says Yamanaka.

But, he adds, if the cells are for use in therapy or research on a disease affecting a particular tissue, it doesn't matter. Yamanaka's cells, for example, were able to form neurons, and cardiac muscle cells that — after differentiating for 12 days — started beating. But iPS cells do have drawbacks. Introduction of the four 'Yamanaka factors' requires genetic manipulation using viral vectors that health agencies would be unlikely to approve for clinical use. And one of the factors, c-myc, is thought to be responsible for tumours in mice.

Thomson, who was the first to isolate and maintain human embryonic stem cells in culture, has gone part way towards solving these problems. He also used four factors, introduced by viral vectors, to reprogramme human foreskin cells. But only two of the four are the same, and he does not use c-myc. What is more, the discovery that a different recipe resulted in successful reprogramming suggests that scientists might have a greater degree of flexibility in finding clinically acceptable variations on Yamanaka's selection.

Onto the home strait

With researchers crowding into the field, Gearhart and others anticipate rapid advancement. "The iPS strategy is a major paradigm shift in reprogramming cells, and if proved effective and safe with human cells — most likely coming very very soon — it will diminish the role of somatic-cell nuclear transfer (SCNT) for deriving patient-specific pluripotent cells," says Gearhart.

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The case for iPS-cell research and against using cloning was highlighted this weekend when the University of Edinburgh's Iam Wilmut, one of Dolly's creators, announced that he planned to turn his back on the field he pioneered in favour of research using Yamanaka's reprogramming technique.

Mitalipov maintains that the egg is the only "perfect reprogramming machine", and is confident that cloned cells will be the first to show therapeutic value. He says he has already more than doubled the efficiency of his cloning technique. And he is preparing for the clinic. Plans are underway to create cloned embryonic cell lines from Semos and some ten other monkeys, to induce diseases such as diabetes in them, and then see if the cloned embryonic stem cells can be used to treat them. Furthermore, in a move likely to raise fresh controversy, Mitalipov this week began a collaboration with Alison Murdoch and Mary Herbert of Newcastle University, UK. Murdoch's group has a licence to work with human embryos. "I can't just keep modelling," Mitalipov says. "If we show medical progress, society will accept the technology." 

  • References

    1. Simerly, C. et al. Science 300, 297 (2003). | Article | PubMed | ISI |
    2. Hwang, W. S. et al. Science 303, 1669-1674 (2003). | Article | ChemPort |
    3. Cram, D. S. et al. Nature doi:10.1038/nature06357 (2007).
    4. Takahashi, K. et al. Cell doi:10.1016/j.cell.2007.11.019 (2007).
    5. Yu, J. et al. Science doi:10.1126/science.1151526 (2007).
    6. Cyranoski, D. Nature 447, 618-619 (2007). | Article | PubMed | ChemPort |
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