The grand challenge
What we don't know about human embryonic stem
cells could fill labs all around the world.
First came the egg-in this case, an ordinary mouse
egg.
Scientists removed the egg's nucleus and replaced it
with the nucleus from a skin cell of a mouse suffering
a genetic immune deficiency. Next they manipulated the
egg to develop into a blastocyst, a hollow ball holding
the embryonic stem cells with the potential to become
any cell in the body. The researchers then plucked out
the stem cells, corrected the genetic defect, and used
the cells to treat the immune deficiency. And the mouse
was partially cured.
Announced three years ago by the labs of Whitehead
Member Rudolf
Jaenisch and then Whitehead Fellow George Daley,
this was the first successful "proof of principle" that
somatic cell nuclear transfer actually could help to
cure disease.
But this fall as Jaenisch opens the Whitehead Human
Embryonic Stem Cell Facility, he won't be working directly
toward replicating this achievement in humans. Instead,
the first order of business is to study the cells' basic
biology. Deep and tough problems must be solved long
before embryonic stem cells can become useful clinically,
Jaenisch says.
Growing pains
One obstacle is simply in learning how to work with
these enigmatic cells.
In 1998, James Thomson at the University of Wisconsin-Madison
launched the field by deriving the first human embryonic
stem cells using embryos from in vitro fertilization
clinics. Despite seven years of experience and the creation
of more than 100 stem cell lines worldwide, scientists
still do not know the best methods for deriving and
growing them.
Biologists have encountered severe problems growing
the earliest stem cell lines, including most of the
lines eligible for federal research funding under President
Bush's 2001 decree. "Those lines are very hard to grow
and very hard to keep pristine," says Irving Weissman,
director of the Stanford Institute for Stem Cell Biology
and Regenerative Medicine. He notes that many of the
first lines derived now show genetic abnormalities that
limit their utility.
"We are far from knowing what the optimal conditions
are for culturing human embryonic stem cells," agrees
Kevin Eggan, assistant professor of molecular and cellular
biology at Harvard University. "So when we derive them
and culture them, we are almost certainly doing things
that mess them up."
| "Embryonic stem cells hold enormous potential,"
says Jaenisch. "We have to be sure that we can realize
that potential." |
What makes a stem cell?
Even as they struggle to grow human embryonic stem
cells, biologists also face basic questions about how
they work. The most fundamental of these is "stemness"-what
makes a stem cell a stem cell.
Scientists are just beginning to work out the internal
programs and external cues that give the cells their
unique ability to become any other type of cell, that
maintain them in this state, and that allow them to
self-renew, almost indefinitely.
Whitehead Member Richard
Young, collaborating with Douglas Melton, co-director
of the Harvard Stem Cell Institute, is mapping the internal
mechanisms and gene/protein interactions involved in
stemness.
To uncover the proteins that control gene expression
in stem cells, the team is employing techniques that
Young helped develop to study the regulatory pathways
of baker's yeast. "There is a perception that human
embryonic stem cells may be useful for regenerative
medicine," Young says. "But before we get there, many
of us believe that we have to understand these pathways
a little better."
Like Young, Princeton's Ihor Lemischka is tapping systems
biology techniques, such as gene chips that represent
the total genome, to identify all the genes that are
active in embryonic stem cells but not in more mature
cells. Austin Smith, director of the Institute for Stem
Cell Research at the University of Edinburgh, has taken
more classical genetic and biochemical approaches to
working out the molecular pathways for self-renewal,
creating mutations in specific genes or exposing cells
to different substances to test their effects.
Getting with the program
Understanding these pathways will help researchers
with another quest of stem cell biology: deciphering
how transferring the nucleus of an adult cell into an
egg effectively reprograms that nucleus, resetting its
genes to the beginning of embryonic development.
Scientists have employed this technique to clone animals,
starting with Dolly the sheep in 1997. In 2002, Hwang
Woo Suk and colleagues at Seoul National University
used it to derive a stem cell line that matched a specific
patient. "Understanding reprogramming at a basic level
could have a major impact on the whole field," says
Leonard Zon, professor of pediatrics at Harvard Medical
School and past president of the International Society
for Stem Cell Research.
Knowing how the process works could reward stem cell
scientists doubly. It would give them a way to derive
stem cells without using embryos (thus avoiding that
ethical controversy). It also would provide a means
of cloning stem cells customized to treat individual
patients without the need for more egg cells.
"For everyone's idealized vision of personal cell replacement
therapy to come true, we will need something like this,"
says Eggan.
Eggan gained great proficiency with nuclear transplantation
technology as a graduate student in Jaenisch's lab.
In his Harvard lab, he investigates whether embryonic
stem cells can accomplish the same sort of reprogramming
that an egg does.
As a first step to test the possibility, Eggan's group
fused a human embryonic stem cell to a skin cell and
watched to see whether the hybrid cell would act like
a stem cell; it did.
The hybrids have limited usefulness, since they contain
double the usual amount of DNA. But the experiment did
demonstrate that embryonic stem cells might
contain the same unknown substances that stimulate reprogramming
in egg cells.
Decoding the genetic disease
Even in its current state, nuclear transfer technology
offers biologists a unique tool for studying genetic
disease. Most dramatically, in June Hwang and his colleagues
used it to create embryonic stem cell lines from patients
with type 1 diabetes and a genetic immune deficiency.
The technique, Weissman says, will enable researchers
to figure out exactly how such diseases develop-discoveries
they may not be able to make any other way. "That's
why it's such a big platform technology," he says. "You
can make a cell line with a genetic disease, you can
study it in a test tube, you can send it around to everybody
who's interested, and you can also put it into an animal
model where there's a chance that the disease will happen."
Weissman hopes to eventually produce such stem cell
lines at Stanford with backing from California's Institute
for Regenerative Medicine.
Eggan has requested permission from Harvard to derive
human embryonic stem cell lines from patients with Parkinson's
and Lou Gehrig's diseases using private funding.
Training issues
Biologists have been trying to create particular cells
and tissues from human embryonic stem cells since their
discovery. Without an understanding of underlying developmental
pathways, progress has been slow.
"This question of how to make the embryonic stem cells
into tissues is a basic-science question," says Zon,
who is working at Children's Hospital in Boston with
George Daley to develop methods for directing embryonic
stem cells to become blood-forming cells. Such projects
obviously play into therapeutic hopes for embryonic
stem cells. Zon and Daley, for example, want to cure
diseases such as sickle cell anemia and immunodeficiency
disorders.
But existing techniques for differentiating embryonic
stem cells into specific cell types have proved inefficient,
leading to mixtures of cells at different stages of
development.
"If you read the papers, you'll see people say it looks
like three or four percent of the cells can develop
into [heart cells], or 80 percent seem to be motor neurons,"
says John Gearhart, director of stem cell biology at
the Johns Hopkins Institute for Cell Engineering. "We
see these numbers all over the place, and invariably
this tells you how inefficient the system is."
Crossing presidential lines
As they work to answer these key questions, each of
these researchers either uses or plans to use non-presidential
embryonic stem cell lines.
While the presidential lines have already helped biologists
with some basic research, all of them were derived using
mouse cells or components of animal blood to feed the
embryonic stem cells.
This not only makes the lines unsuitable for clinical
use but also complicates basic science. Researchers
have recently begun deriving human embryonic stem cell
lines without using any animal tissue, lines that Gearhart
thinks will give a big boost to the field. "We would
certainly benefit enormously by new lines that don't
require mouse feeder layers," he says. "I can't express
how much of a pain they are."
Without mouse feeder cells, it should be easier for
scientists to investigate the mechanisms that keep stem
cells undifferentiated and control their development,
he adds. And when biologists ultimately succeed in producing
specific cell types such as neurons or muscle cells
from such animal-free embryonic stem cell lines, those
cells might be moved more directly into clinical trials.
The presidential lines "aren't sufficient," Jaenisch
declares flatly. "They are not behaving the way they
should be anymore."
So at least for now, Whitehead's new lab must be funded
entirely with private money. But private funding cannot
completely fill the gap for U.S. researchers. "The National
Institutes of Health are the key funding source," says
Jaenisch. "If they're not there, it is a major, major
problem."
And even when researchers manage to obtain private
backing, the federal funding ban has made it much more
difficult for those wishing to work with non-approved
lines.
As they wait for public opinion and political will
to match their need, U.S. researchers work with what
state and private money they can raise. As the field
races ahead worldwide, Whitehead scientists aim to stay
in the vanguard. "Embryonic stem cells hold enormous
potential," says Jaenisch. "We have to be sure that
we can realize that potential."
****************
Four big questions
Here's a sampling of the human embryonic stem cell issues being tackled by Whitehead scientists:
1. What makes an embryonic stem cell a stem
cell? Using microarray technology developed
at the Institute, Whitehead Member Richard Young, collaborating
with Rudolf Jaenisch and others, has discovered the
first layer of circuitry that enables such cells to
be pluripotent, suppressing entire networks of genes
essential for later development.
2. How can we manipulate embryonic stem cells?
Jaenisch and Young are exploring ways to influence key
genes and proteins, as well as tampering with their
regulatory circuitry. Understanding these processes
is a prerequisite for systematically coaxing embryonic
stem cells into forming particular cell types.
3. How does an egg reprogram the genome in
somatic cell nuclear transfer? The Jaenisch
lab is investigating the exact biochemical processes
that the egg uses to reactivate the donor nucleus during
somatic cell nuclear transfer. The long-term goal is
to turn a mature cell into an embryonic stem cell without
requiring an egg.
4. How can we make adult stem cells out of
embryonic stem cells? In this area, embryonic
stem cell researchers and adult stem cell researchers
need to work together. The Lodish lab is working to
create adult blood cells out of embryonic stem cells,
drawing on the expertise of Jaenisch, Young and Whitehead
Fellow Fernando Camargo.
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