Published online 5 October 2011 | Nature | doi:10.1038/news.2011.578

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Researchers begin to identify past problems with cloning technique.

Blastocysts created by somatic cell nuclear transfer can be a source of embryonic stem cells. The New York Stem Cell Foundation

Scientists at the New York Stem Cell Foundation Laboratory have reprogrammed an adult human egg cell to an embryonic state using cloning technology and created a self-reproducing line of embryonic stem cells from the developing embryo. In so doing, they have managed a feat that has at times been thought impossible, then inevitable, then completed, then incomplete and unfeasible. Their work is published today in Nature1.

It is not the end-all experiment that scientists aiming to create embryonic stem cells have been hoping for — the embryos are not true clones, because the DNA of the stem-cell line does not match that of the patient who donated cells — but it is a step in that direction and addresses some of the problems that have flummoxed experiments.

Human error

In 1996, the birth of Dolly the sheep showed that it was possible to put DNA from an adult into an egg cell to create a clone. Since then, researchers have tried for what they hoped would be an easier goal: creating a stem-cell line from a cloned human embryo that could be used either for research or to develop tissue for therapy.

It proved difficult, and not just in humans. Despite efforts, only experiments in a few mammals, including mice and rhesus macaques produced cloned embryonic stem-cell lines. When it came to humans, researchers didn't have unfettered access to the key resource, eggs — at least not in the numbers that they needed to tweak the finicky procedure for human biology.

One group said they had done it. In 2004, Woo Suk Hwang, then a biologist at Seoul University, claimed to have produced a viable cloned human embryo. But his results had been faked: it transpired that his group had used up more than 2,000 unethically obtained eggs in misguided and fraudulent experiments. No lessons were learned, and questions over the legality and morality of using eggs in research slashed the supply.

“Human eggs do indeed have the magic we thought they did!”



The need for cloning was called into question in 2006 by the creation of induced pluripotent stem (iPS) cells, adult cells reprogrammed to have the developmental potential of an embryo by the introduction of a few genetic factors to the cell rather than the use of an entire egg. iPS cells seemed to achieve the same thing as cloned embryonic stem cells with far fewer ethical sticking points. But over the past few years, they have been found to differ from embryonic stem cells in subtle but important ways that affect their development.

Step by step

In conventional cloning techniques, researchers remove the single set of chromosomes from an unfertilized egg, inject the two sets from a patient's adult cell and try to get the introduced DNA to drive the egg towards embryonic development. But such cells usually stop developing after a few divisions.

Dieter Egli, a researcher at the New York Stem Cell Foundation Laboratory who led the latest study1, started from scratch. His group ran a series of experiments using 270 eggs from 16 donors, isolating the three key events of conventional cloning techniques to see which was causing the problem. The culprit turned out to be removing the egg's DNA. So the group left it in, the reprogramming worked and an embryo — albeit one with an extra set of chromosomes from the egg — developed to the blastocyst stage, comprising 70–100 cells, from which stem cells can be derived.

Egli says he was surprised "that it actually worked. Our result really proves the technical hurdles can be overcome."

Clone of contention

"Human eggs do indeed have the magic we thought they did!" says Robert Lanza, chief scientific officer at Advanced Cell Technology, a company based in Santa Monica, California, that develops therapies using stem-cell technology. "This study clearly shows the enormous power of this technology." But he is cautious about this particular experiment. He says the method has never been tried before "because it's of no clinical relevance"; the stem cells produced, with their extra set of chromosomes, are not compatible with the patient's tissue.

Miodrag Stojkovic, a cloning expert at the University of Kragujevac in Serbia, echoes these concerns. "These are abnormal cells and therefore are a very limited tool to understand early human development," he says, adding that cloned embryonic stem cells need a normal set of chromosomes before they can properly be compared with iPS cells.

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Still, reports about problems with iPS cells are growing, and the latest work will motivate the search for a 'more natural' method of reprogramming using cloning.

Egli's group is trying a number of different approaches to remove the egg's DNA and create a viable embryo, including the use of other cell types and methods used to increase efficiency in iPS cell derivation. He will be scouring the egg's chromosomes for "so far unknown factors required for development to the blastocyst stage" that cannot be replaced by the adult cell's chromosomes.

Lanza suspects that the real problem is that removing the chromosomes from the egg requires taking out the spindle apparatus that enables cell division. "Unlike in some other species, these are critical for an embryo to continue to divide. That's the problem, and that's why human cloning hasn't worked to date," he says.

See Editorial 'High-interest clones'