Reprogrammed fibroblasts identical
to embryonic stem cells
CAMBRIDGE, Mass. (June 6, 2007) — Embryonic
stem cells are unique because they can develop into
virtually any kind of tissue type, an attribute called
pluripotency. Somatic cell nuclear transfer ("therapeutic
cloning") offers the hope of one day creating
customized embryonic stem cells with a patient's own
DNA. Here, an individual's DNA would be placed into
an egg, resulting in a blastocyst that houses a supply
of stem cells. But to access these cells, researchers
must destroy a viable embryo.
Now, scientists at Whitehead Institute have demonstrated
that embryonic stem cells can be created without eggs.
By genetically manipulating mature skin cells taken
from a mouse, the scientists have transformed these
cells back into a pluripotent state, one that appears
identical to an embryonic stem cell in every way. No
eggs were used, and no embryos destroyed.
“These reprogrammed cells, by all criteria that
we can apply, are indistinguishable from embryonic stem
cells,” says Whitehead Member and MIT professor
of biology Rudolf
Jaenisch,
senior author of the paper that appeared online June
6 in Nature.
“We are optimistic that this can one
day work in human cells,” says Marius Wernig. “We
just need to find new strategies to reach that
goal.” |
What's more, these reprogrammed skin cells can give
rise to live mice, contributing to every kind of tissue
type, and can even be transmitted via germ cells (sperm
or eggs) to succeeding generations. "Germline
transmission is the final and definitive proof that
these cells can do anything a traditionally derived
embryonic stem cell can do," adds Jaenisch.
Two additional papers report similar findings. The
first, by Shinya Yamanaka of Kyoto University, will
be published in the same issue of Nature.
The second, from Konrad Hochedlinger, formerly of the
Jaenisch lab and now at the Center for Regenerative
Medicine at Massachusetts General Hospital and Harvard
Stem Cell Institute, will appear in the inaugural issue
of the journal Cell Stem Cells. Additionally,
another paper in Nature from Kevin Eggan,
also of the Harvard Stem Cell Institute and a former
member of the Jaenisch lab, describes using mouse zygotes,
rather than eggs, for somatic cell nuclear transfer.
Jaenisch cautions that "all these results are
preliminary and proof of principle. It will be awhile
before we know what can and can't be done in humans.
Human embryonic stem cells remain the gold standard
for pluripotent cells, and it is a necessity to continue
studying embryonic stem cells through traditional means."
In August 2006, a team of researchers at Kyoto University
led by Yamanaka reported a landmark discovery that
by activating four genes in a mouse skin cell, they
could reprogram that cell into a pluripotent state
resembling an embryonic stem cell. However, the resulting
cells were limited when compared with real embryonic
stem cells, and the Kyoto team was unable to generate
live mice from these cells.
A team of researchers decided to replicate this experiment,
while refining certain technical aspects. This group
was led by Jaenisch lab postdoctoral researchers Marius
Wernig, Alexander Meissner and Tobias Brambrink; graduate
student Ruth Foreman; and Manching Ku, a research fellow
from Bradley Bernstein's lab at Massachusetts General
Hospital. Konrad Hochedlinger also contributed.
Using artificial viruses called vectors, the team
activated the same four genes in a batch of mouse skin
cells. These genes, Oct4, Sox2, c-Myc and Klf4, are
called transcription factors, meaning that they regulate
large networks of other genes. While Oct4 and Sox2
are normally active in the early stages of embryogenesis,
they typically shut down once an embryo has developed
beyond the blastocyst stage.
"We were working with tens of thousands of cells,
and we needed to devise a precise method for picking
out those rare cells in which the reprogramming actually
worked," says Wernig. "On average, it only
works in about one out of 1,000 cells."
To test for reprogramming, the team decided to zero
in on Oct4 and another transcription factor called
Nanog. These two hallmarks for embryonic stem cell
identity are only active in fully pluripotent cells.
The trick would be to figure out a way to harvest Oct4-
and Nanog-active cells from the rest of the population.
The answer came in the form of a laboratory technique
called "homologous recombination." Here,
the scientists took genetic material known to be resistant
to the toxic drug neomycin, and spliced it into the
genomes of each cell right beside Oct4 and Nanog. If
Oct4 and Nanog switched on, the drug-resistant DNA
would also spring into action. The researchers then
added neomycin to the cells. Only those fully reprogrammed
cells with an active Oct4 and Nanog survived.
Next, the team ran these cells through a battery of
tests, seeing if they could discover any substantial
differences between these cells and normal embryonic
stem cells.
"In all tests, both genetic and epigenetic, there
were no molecular markers distinguishing these two
groups," says Meissner.
But definitive proof would only come through demonstrating
that these cells could actually develop into any kind
of body tissue and cell type. The researchers approached
this question in three ways.
First, they fluorescently labeled these reprogrammed
cells and injected them into early-stage embryos, which
eventually gave rise to live mice. While these mice
consisted of both the reprogrammed cells and the natural
cells from the original embryo, the fluorescent tags
indicated that the reprogrammed cells contributed to
all tissue types in the mouse—everything from blood
to internal organs to hair color.
Next, they bred these mice and found lineages of the
reprogrammed cells in the subsequent generation, proving
that these new cells had contributed to the germ line.
Finally, the team took advantage of another lab technique
that involves creating a genetically abnormal embryo
whose cells all consist of four chromosomes, rather
than two. Because of this aberrant formation, the embryo
can only form a placenta, and cannot develop into a
full-term fetus. The researchers injected the reprogrammed
cells into this embryo, and then implanted it in a
uterus. Eventually live late-gestation fetuses could
be recovered—created exclusively from the reprogrammed
cells.
"This is the most stringent criteria anyone can
use to determine if a cell is pluripotent," says
Jaenisch.
Still, many technical hurdles remain for possibly
translating this work to human cells. For example,
the homologous recombination technique used to isolate
the pluripotent cells does not yet work in human embryonic
stem cells. Also, using cells that contain viral vectors
can pose health risks.
"We are optimistic that this can one day work
in human cells," says Wernig. "We just need
to find new strategies to reach that goal. For now,
it would simply be premature and irresponsible to claim
that we no longer need eggs for embryonic stem cell
research."
This research is supported by the National Institutes
of Health. |
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