[asa] Embryonic Stem Cells in Mice with No Embryos and No Cloning

http://www.nature.com/news/2007/070604/full/447618a.html

Simple switch turns cells embryonic
Technique removes need for eggs or embryos.

David Cyranoski

Research reported this week by three different groups shows that
normal skin cells can be reprogrammed to an embryonic state in mice.
The race is now on to apply the surprisingly straightforward procedure
to human cells.

If researchers succeed, it will make it relatively easy to produce
cells that seem indistinguishable from embryonic stem cells, and that
are genetically matched to individual patients. There are limits to
how useful and safe these would be for therapeutic use in the near
term, but they should quickly prove a boon in the lab.

"It would change the way we see things quite dramatically," says Alan
Trounson of Monash University in Victoria, Australia. Trounson wasn't
involved in the new work but says he plans to start using the
technique "tomorrow". "I can think of a dozen experiments right now —
and they're all good ones," he says.

In theory, embryonic stem cells can propagate themselves indefinitely
and are able to become any type of cell in the body. But so far, the
only way to obtain embryonic stem cells involves destroying an embryo,
and to get a genetic match for a patient would mean, in effect,
cloning that person — all of which raise difficult ethical questions.

As well as having potential ethical difficulties, the 'cloning'
procedure is technically difficult. It involves obtaining unfertilized
eggs, replacing their genetic material with that from an adult cell
and then forcing the cell to divide to create an early-stage embryo,
from which the stem cells can be harvested. Those barriers may have
now been broken down.

"Neither eggs nor embryos are necessary. I've never worked with
either," says Shinya Yamanaka of Kyoto University, who has pioneered
the new technique.

Last year, Yamanaka introduced a system that uses mouse fibroblasts, a
common cell type that can easily be harvested from skin, instead of
eggs. Four genes, which code for four specific proteins known as
transcription factors, are transferred into the cells using
retroviruses. The proteins trigger the expression of other genes that
lead the cells to become pluripotent, meaning that they could
potentially become any of the body's cells. Yamanaka calls them
induced pluripotent stem cells (iPS cells). "It's easy. There's no
trick, no magic," says Yamanaka.

The results were met with amazement, along with a good dose of
scepticism. Four factors seemed too simple. And although the cells had
some characteristics of embryonic cells — they formed colonies, could
propagate continuously and could form cancerous growths called
teratomas — they lacked others. Introduction of iPS cells into a
developing embryo, for example, did not produce a 'chimaera' — a mouse
carrying a mix of DNA from both the original embryo and the iPS cells
throughout its body. "I was not comfortable with the term
'pluripotent' last year," says Hans Schöler, a stem-cell specialist at
the Max Planck Institute for Molecular Biomedicine in Münster who is
not involved with any of the three articles.

This week, Yamanaka presents a second generation of iPS cells, which
pass all these tests. In addition, a group led by Rudolf Jaenisch at
the Whitehead Institute for Biomedical Research in Cambridge,
Massachusetts, and a collaborative effort between Konrad Hochedlinger
of the Harvard Stem Cell Institute and Kathrin Plath of the University
of California, Los Angeles, used the same four factors and got
strikingly similar results.

"It's a relief as some people questioned our results, especially after
the Hwang scandal," says Yamanaka, referring to the irreproducible
cloning work of Woo Suk Hwang, which turned out to be fraudulent.
Schöler agrees: "Now we can be confident that this is something worth
building on."

The improvement over last year's results was simple. The four
transcription factors used by Yamanaka reprogramme cells
inconsistently and inefficiently, so that less than 0.1% of the
million cells in a simple skin biopsy will be fully reprogrammed. The
difficulty is isolating those in which reprogramming has been
successful. Researchers do this by inserting a gene for antibiotic
resistance that is activated only when proteins characteristic of stem
cells are expressed. The cells can then be doused with antibiotics,
killing off the failures.

The protein Yamanaka used as a marker for stem cells last year was not
terribly good at identifying reprogrammed cells. This time, all three
groups used two other protein markers — Nanog and Oct — to great
effect. All three groups were able to produce chimaeric mice using iPS
cells isolated in this way; and the mice passed iPS DNA on to their
offspring.

Jaenisch also used a special embryo to produce fetuses whose cells
were derived entirely from iPS cells. "Only the best embryonic stem
cells can do this," he says.

"It's unbelievable, just amazing," says Schöler, who heard Jaenisch
present his results at a meeting on 31 May in Bavaria. "For me it's
like Dolly [the first cloned mammal]. It's that type of
accomplishment."

The method is inviting. Whereas cloning with humans was limited by the
number of available eggs and by a tricky technique that takes some six
months to master, Yamanaka's method can use the most basic cells and
can be accomplished with simple lab techniques.

But applying the method to human cells has yet to be successful. "We
are working very hard — day and night," says Yamanaka. It will
probably require more transcription factors, he adds.

If it works, researchers could produce iPS cells from patients with
conditions such as Parkinson's disease or diabetes and observe the
molecular changes in the cells as they develop. This 'disease in a
dish' would offer the chance to see how different environmental
factors contribute to the condition, and to test the ability of drugs
to check disease progression.

But the iPS cells aren't perfect, and could not be used safely to make
genetically matched cells for transplant in, for example, spinal-cord
injuries. Yamanaka found that one of the factors seems to contribute
to cancer in 20% of his chimaeric mice. He thinks this can be fixed,
but the retroviruses used may themselves also cause mutations and
cancer. "This is really dangerous. We would never transplant these
into a patient," says Jaenisch. In his view, research into embryonic
stem cells made by cloning remains "absolutely essential".

If the past year is anything to judge by, change will come quickly.
"I'm not sure if it will be us, or Jaenisch, or someone else, but I
expect some big success with humans in the next year," says Yamanaka.

Additional reporting by Heidi Ledford
For more on alternative stem-cell work, see 'Stem cells: Recycling the
abnormal'; and see http://www.nature.com/stemcells

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Received on Wed Jun 6 19:27:29 2007