Embryonic Stem Cells 2.0
Bruce Goldman1
Nature Reports Stem Cells
Published online: 1 May 2008 | doi:10.1038/
Scientists' enthusiasm grows for induced pluripotent cells
When Shinya Yamanaka of Kyoto University reported his transformation
of cultured mouse skin cells into a state approximating that of
embryonic stem cells1, he was met with plenty of scepticism. Other
scientists hadn't anticipated that such a feat was possible. "Nobody
else was even close to doing the same experiment," says Richard Young
of the Whitehead Institute in Cambridge, Massachusetts. "That was a
very special breakthrough.
By inserting just four genes — Oct4, Sox2, Klf4 and Myc — into
fibroblasts (cultured skin cells), Yamanaka's group had achieved the
biological equivalent of making water flow uphill. The resultant
induced pluripotent stem (iPS) cells proliferate indefinitely in
culture and differentiate into all the tissues necessary to generate
a live mouse2, 3.
From mouse to human
Scientists raced to understand the process in mice and to make it
happen in human cells. Last November, less than 16 months after
Yamanaka's initial publication, his group4 and another led by James
Thomson5 at the University of Wisconsin–Madison simultaneously
reported the generation of iPS cells from human fibroblasts. (In
contrast, it took 17 years from the first publications describing the
production of embryonic stem (ES) cells from a mouse embryo6, 7 for
the equivalent research in human cells8 to be published.) Thomson's
reprogramming mix differs slightly from Yamanaka's cocktail: it
includes Oct4, Sox2 and Nanog, which encode three well-established
pluripotency-
Lin28, that encodes an RNA-binding protein. That at least two
combinations of factors work suggests some flexibility in the
process. At least two other groups had papers submitted before
Yamanaka's and Thomson's papers were published, showing that the
techniques work in multiple hands9, 10.
The ten-year head start human ES cells got on human iPS cells has
effectively shrunk to zero, according to James Thomson.
Jessica Kolman
Thomson says he hired Junying Yu (first author on their November 2007
Science paper) as a postdoc in 2003 to attempt reprogramming,
thinking it would be a 20-year project. "We never just took those
three genes and tried them in the first place, which would have saved
us five years of work," he says. "We couldn't believe it would be
that easy."
The fact that making iPS cells does not pose the technical and
ethical challenges of working with eggs or embryos is drawing large
numbers of researchers into the field and speeding up reprogramming
research. "This is definitely the hot thing right now," says Melina
Fan, executive director of Addgene, the Cambridge, Massachusetts–
based nonprofit repository that distributes both Thomson's and
Yamanaka's viral vectors for the cell-reprogramming genes. As of 17
April, she says, there have been 704 requests from 178 labs at 142
institutions for Thomson's vectors; 514 requests from 131 labs at 113
institutions for Yamanaka's human iPS cell vectors; and over 1,500
requests from 232 labs at 215 institutions for Yamanaka's murine iPS
cell vectors. The statistics speak for themselves. Although the
Thomson and Yamanaka stem cell plasmids make up only 0.2% of
Addgene's total collection, they've accounted for over 10% of
Addgene's total plasmid requests since the beginning of 2008, Fan
says.
One thing researchers want to know is whether only certain cell types
can be reprogrammed. Liver and stomach cells have now been converted
to pluripotency11. In April, the Whitehead Institute's Rudolf
Jaenisch and colleagues reported their success in generating iPS
cells from mature B lymphocytes, each of which makes a unique
antibody because of an ability to shuffle its DNA. The genomes of
fully differentiated cells have been uniquely, characteristically and
irreversibly rearranged and thus allow investigators to show
unambiguously that iPS cells need not arise from rare resident stem
cells that might be more amenable to reprogramming12.
"Biologically there's no difference" between murine iPS and ES cells,
says Jaenisch. Both can generate all the tissues in a mouse. Human
iPS cells have not been as rigorously demonstrated to be quasi-
equivalent to ES cells, and they won't be, because doing so would
require generating human babies or foetuses. Such experimentation is
irrelevant anyway, says Douglas Melton, director of the Harvard Stem
Cell Institute in Cambridge, Massachusetts, who has derived multiple
human ES cell lines. "Nobody's trying to make people."
Thomson — who, as the first to derive human ES cells, should know —
emphasizes that ES cells themselves are an artefact of
culture. "Should we spend a lot of time arguing about whether this
new tissue-culture artefact is identical to an old tissue-culture
artefact?" he asks. What matters is what you can do with the cells.
Ken Chien, a physician-scientist at Massachusetts General Hospital,
in Boston, and at the Harvard Stem Cell Institute, is excited by the
possibility of being able to simultaneously study iPS cells derived
from a particular person and monitor the course of disease in that
person. That could reveal powerful new biomarkers and predictors. But
like ES cells, he warns, iPS cells present a fair amount of
variability. He and Melton recently showed that certain ES cell lines
are much more likely to differentiate into, say, cardiac cells than
are others13.
ES cells have been around longer and are much better characterized,
and the iPS cells still warrant further tinkering to counter a
proclivity toward tumourigenicity. Moreover, somatic cell nuclear
transfer, also called therapeutic cloning, generates an embryo, so it
can address questions about early development that direct
reprogramming cannot. Nonetheless, the enthusiasm with which the
highest-tier ES cell scientists have turned to reprogramming speaks
volumes.
From doing to using
Three applications for iPS cells are in the cards. First, the
relatively easily generated iPS cells are ready to be used right now,
as ES cells are, for studying cells' differentiation and comparing
differences between normal and diseased cells. Second, they should
soon be available for drug screening, so that assays once possible
only in animals can be performed routinely in human cells.
No one doubts that iPS cells will eventually be generated from the
cells of individuals with known medical history. That was the main
advantage claimed for somatic cell nuclear transfer, a technically
and ethically challenging procedure that has yet to be achieved in
humans. For generating person-matched cells, iPS cells may be not
only easier to use but perhaps superior, as they would share both
nuclear and mitochondrial DNA with the original patient, whereas
cells derived by somatic cell nuclear transfer carry only the same
nuclear DNA.
The third application, which is more discussed but farther in the
future, is regenerative medicine: the production of partially or
fully differentiated, immune-compatible (and perhaps gene-repaired)
tissues to put back into patients. Last year Jaenisch's group
successfully treated transgenic mice carrying the human gene for
sickle-cell anemia by giving them haematopoietic stem cells derived
from those mice's gene-repaired iPS cells14. In a collaboration with
Jaenisch15, a team led by Harvard neuroscientist Ole Isacson recently
created dopaminergic neurons from iPS cells and put them into the
brains of rats suffering from an induced version of Parkinson's
disease. The rats' physical performance improved and, later, their
brains showed successful engraftment of the dopaminergic neurons.
Losing the virus
Before iPS cells can be used for regenerative medicine in humans, a
couple of major issues have to be resolved. The most immediate one
concerns the retroviral vectors currently used to introduce the
pluripotency genes into the cell's genome. Retroviruses integrate
randomly into chromosomes, sometimes in copy numbers as high as 15 or
20 per genome. The insertion is permanent even though the retrovirus
and its cargo gene are needed only temporarily. As reprogramming
proceeds, the cell's own silenced pluripotency program whirls into
action and the added genes are silenced.
The risk is that the pluripotency genes carried by the virus may
reactivate when the cells begin traveling down their differentiation
pathways. Jaenisch, who started his career studying viral silencing,
says the enzyme that silences retroviral genes in the pluripotent
state is different from the enzyme that maintains silence during
differentiation, and the latter is a bit more error prone.
Make one mistake with Myc, a gene with a powerful cancer-causing
potential, and there's a problem. Up to a third of the mice produced
from Myc-containing iPS cells have had tumours associated with the
gene's reactivation. Several groups have shown that iPS cells can be
generated without Myc, albeit at lower frequencies16, 17, 18. But
with so many cells readily available, that's not a showstopper, says
Thomson. "You get enough."
Another problem with retroviral vectors is insertional mutagenesis.
Random insertion of retroviruses into the genome carries the risk of
accidentally turning on or off some key gene inappropriately, perhaps
later in development.
Can virus-free iPS cells be made? "It's going to happen sooner than
people think," answers Young. Working with Jaenisch, Young found that
Oct4, Sox2 and Nanog — three of the four proteins encoded by the
retroviral vectors Thomson used — cluster together to coordinately
turn on their own genes, which are normally locked down in
differentiated cells17. Once expression of the endogenous genes for
these key transcription factors reaches a tipping point, the external
genes are no longer required, Young says.
In mice, this tipping point occurs roughly 10–12 days after the
vectors are introduced, as demonstrated by Jaenisch's team19 and
another team led by Harvard's Konrad Hochedlinger20. Both groups used
inducible viral vectors that served as combined starter guns and
stopwatches, allowing the precise timing of sequences of molecular
events involved in pushing the cells being reprogrammed past the
point of no return.
Hochedlinger'
start the expression of endogenous pluripotency genes by transiently
delivering the gene products to target cells. One method, he says,
might employ nonintegrating adenoviruses whose gene payload, after
inducing pluripotency and proliferation, would be diluted.
Another approach might be to replace viral vectors by directly
introducing the required proteins with certain amino acid signaling
sequences attached to them (such as HIV's Tat epitope) that would
permit the proteins to penetrate membranes. Hochedlinger is also
collaborating with Sheng Ding, from the Scripps Institute in La
Jolla, California, in a systematic search for small nonprotein
molecules that mimic the activity of the critical transcription
factors.
Using potential starter cells that require fewer pluripotency
transcription factors might help, Hochedlinger says. For example,
some neurons, although they are not particularly easy to get or
maintain in culture, express a bit of Sox2 naturally.
Like a new computer operating system, iPS cells are muscling into the
field as their radically fewer barriers to entry, compared with those
for ES cells, accelerate the pace of research. The ten-year head
start human ES cells got on human iPS cells has effectively shrunk to
zero, says Thomson, because so much of the legacy of ES cells —
reagents, culture media, hands-on expertise and experimental history —
is transferable to iPS cells. Tissue culture is simpler than
embryology. Skin is cheap. Creating iPS cells that are innately
immunologically compatible with the patient from whose cells they
were derived presents an attractive alternative to obtaining human
eggs, executing difficult nuclear-transfer protocols and destroying
embryos. With the ethical clouds hanging over those procedures
lifting, anxieties about funding are receding. A cadre of talented
young investigators trained on ES cells and ready to surpass their
mentors is chafing at the bit. As a result of this ferment, the
convergent view of numerous leaders in the field is that the
retroviral delivery problem will be solved within a year or so.
Then it will be on to the second challenge — perhaps the greater one.
The goal of all this work, says Melton, is not just to walk a cell up
to the pinnacle of pluripotency. The goal is to then get it to roll
down a specific lineage ridge to the desired differentiation valley.
Top of pageReferences [Note: LINKS LIVE FROM WEBSITE - BELOW}
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Author affiliation
Bruce Goldman is a freelance writer based in San Francisco.
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