Interesting article on the progress and challenges of  moving stem cell
research - both embryonic and adult -- from the lab to clinical
treatments.  The authors reiterate the importance of supporting research
into both types of stem cellls, however research utilizing ESC for
neurodegenerative diseases is  moving closer to clinical trials. The
authors state:

"..How long it will take for any ES cell therapies to be tested in humans
will be determined as much by politics as by the remaining scientific
questions. Well-understood and easy-to-control cell types derived from ES
cells, such as dopamine-producing neurons or the eyes' retinal pigment
epithelium cells, could be ready for human trials in less than two years.
.."
and
"As often happens in science, stem cell research has raised as many new
questions as it has answered, but the field is advancing. Early tests of
human adult stem cells in treating cardiovascular disease are encouraging
and will certainly lead to more extensive trials in the near future.
Given much promising experimental evidence in animals, therapeutic trials
of human ES cell derivatives in neurodegenerative disease are probably
imminent.

As the appropriate source of cells for both research and eventual
therapeutic applications continues to be hotly debated, restrictions on
this research are slowing progress. But we believe that generating
replacement cells and regenerating organs are feasible and realistic
goals. The remaining hurdles are difficult but not insurmountable. "

FROM: Scientific American
http://www.sciam.com/
 May 24, 2004
 The Stem Cell Challenge

What hurdles stand between the promise of human stem cell therapies and
real treatments in the clinic?

By Robert Lanza and Nadia Rosenthal

Stem cells raise the prospect of regenerating failing body parts and
curing diseases that have so far defied drug-based treatment. Patients
are buoyed by reports of the cells' near-miraculous properties, but many
of the most publicized scientific studies have subsequently been refuted,
and other data have been distorted in debates over the propriety of
deriving some of these cells from human embryos.

Provocative and conflicting claims have left the public (and most
scientists) confused as to whether stem cell treatments are even
medically feasible. If legal and funding restrictions in the U.S. and
other countries were lifted immediately, could doctors start treating
patients with stem cells the next day? Probably not. Many technical
obstacles must be overcome and unanswered questions resolved before stem
cells can safely fulfill their promise.

For instance, just identifying a true stem cell can be tricky. For
scientists to be able to share results and gauge the success of
techniques for controlling stem cell behavior, we must first know that
the cells we are studying actually possess the ability to serve as the
source, or "stem," of a variety of cell types while themselves remaining
in a generic state of potential. But for all the intensive scrutiny of
stem cells, they cannot be distinguished by appearance. They are defined
by their behavior.

Most versatile are embryonic stem (ES) cells, first isolated in mice more
than 20 years ago. ES cells come from the portion of a very early-stage
embryo that would normally go on to form three distinctive germ layers
within a later embryo and ultimately all the different tissues of the
body. ES cells retain this potential ability to produce any cell type in
the body, making them pluripotent.

Most of the existing human ES cell lines in the world were derived from
unused embryos created for couples seeking in vitro fertilization (IVF).
Researchers working with these cells have found that they usually recover
after freezing and thawing and can differentiate into assorted cell types
in a culture dish. But it is becoming clear that not all human ES cell
lines are the same.

Seeking Stemness
Some lines will differentiate into only certain cell types; others grow
sluggishly in culture. To ensure that these cells are pluripotent before
using them in research, two possible tests, already common in nonhuman ES
cell studies, have been proposed by a group of American and Canadian
biologists hoping to set standards for experimentation with human ES
cells. One would involve injecting the ES cells into an animal's body
tissue. If they form a teratoma--a distinctive tumor containing cell
types from all three embryonic layers--their pluripotency is proved.
Another way to test putative ES cells is to mark them, then inject them
into a developing animal embryo. When the animal is born, if the marked
cells turn up in all its tissues, the cell line is deemed pluripotent.
But testing human embryonic stem cells in this manner would create a
chimeric animal with human DNA throughout its body, a prospect many find
ethically troubling. What is more, passing the latter test does not
always guarantee that the cells will differentiate in the lab.

The need to find more reliable markers that distinguish truly pluripotent
ES cells is driving widespread attempts to catalogue the genes that are
turned on or off at various times in cultured ES cells. Having such a
gene expression profile would not only provide a way of identifying
pluripotent ES cells, it would also offer tremendous insight into the
properties that confer their "stemness." Unfortunately, to date, gene
expression profiles of ES cells have yielded conflicting results, and the
search for a clear ES cell signature continues.

Of course, the goal of stem cell research is to replace or regenerate
failing body parts, such as pancreatic insulin-producing cells in
diabetics or dopamine-producing neurons in people with Parkinson's
disease. But techniques for coaxing ES cells to differentiate into
desired cell types are far from perfected.

Left to their own devices in a culture dish, ES cells will spontaneously
differentiate into a hodgepodge of tissue types. With timed
administration of chemicals, we can often direct them to become one cell
type or another. But they seem to prefer to become certain
tissues--readily proliferating into patches of beating heart cells, for
example--whereas other tissues are far more difficult to derive.

Putting Cells to Work
Because we still do not understand the signals that normally instruct
these cells to choose a particular pathway during embryonic development,
many researchers are studying the natural embryonic "niche" to understand
possible environmental cues. Other scientists are trying to profile
embryonic cells' gene expression patterns as they differentiate in order
to find genes that could be turned on or off to direct the cells toward a
particular tissue type.

But deriving what appear to be cells of the desired kind is just half the
battle. ES cells will easily produce dishes full of neurons, for
instance, but these are only useful if they can be placed in a living
brain, make connections and "talk" with surrounding neurons. In 2001 stem
cell researchers thought they had a major breakthrough when Ronald McKay
of the National Institutes of Health reported having generated
insulin-producing cells--a coveted goal in stem cell research--from mouse
ES cells. Last year, though, Douglas A. Melton of Harvard University
reproduced McKay's experiment and found that the cells had absorbed
insulin from their culture medium rather than producing it themselves.
Discovering markers to identify truly functional cells is another urgent
task for the stem cell research community.

It would be ideal if we could simply inject ES cells into the part of the
body we wish to regenerate and let them take their cues from the
surrounding environment. ES cells' pluripotency, however, makes this far
too dangerous an approach for human therapy. The cells might form a
teratoma or could differentiate into an undesirable tissue type, or both.
In animal experiments, teratomas containing fully formed teeth have been
reported.

Rather than risk creating a tumor or a tooth in a patient's brain or
heart with direct ES cell injections or struggling to produce specific
functional tissues, many ES cell researchers are now striving for a
middle ground. By coaxing ES cells into a more stable, yet still
flexible, progenitor-cell stage before administering them, we can avoid
uncontrolled differentiation while still taking advantage of
environmental cues to generate the desired cell types.

Even though these progenitor cells can take to their environment and
initiate the generation of new tissue, they would still be subject to
attack by the patient's own body. ES cells and their derivatives carry
the same likelihood of immune rejection as a transplanted organ because,
like all cells, they carry the surface proteins, or antigens, by which
the immune system recognizes invaders. Hundreds of combinations of
different types of antigens are possible, meaning that hundreds of
thousands of ES cell lines might be needed to establish a bank of cells
with immune matches for most potential patients. Creating that many lines
could require millions of discarded embryos from IVF clinics.

Some researchers have speculated that such an extensive bank might not be
necessary, that patients can be desensitized to ES cell derivatives or
that the antigenic properties of the cells themselves can be reduced. But
those feats have yet to be conclusively demonstrated. At present, the
only sure way to circumvent the problem of immune rejection would be to
create an ES cell line using a patient's own genetic material through
nuclear transfer or cloning. This technique has inspired considerable
controversy and has its own practical hurdles to overcome, but it has
also produced encouraging results in animal experiments for regenerating
failing tissues.


Turning Back the Clock
Cloning can be viewed as a way to restore embryonic potential to a
patient's old cells. The human body is made of more than 200 kinds of
cells, and in mammals, once a cell is committed to a particular type,
there is normally no turning back. It is said to be "terminally
differentiated." An exception to this rule is when the nucleus containing
an unfertilized egg's genetic material is extracted and the nucleus of a
somatic (body) cell is placed into the egg instead. The egg is tricked
into behaving as though it has been fertilized and begins dividing like a
normal embryo. The ES cells derived from this embryo will contain the
donor somatic cell's DNA. But the somatic cell will have been
reprogrammed--reset to a state of stemness, capable of generating any
tissue type.

One of us (Lanza) recently showed that partially differentiated stem
cells from a cloned mouse embryo could be injected into the donor mouse's
heart, where they homed in on the site of injury from a heart attack,
replacing 38 percent of the scar with healthy heart tissue within a
month. And this year, for the first time, somatic cell nuclear transfer
(SCNT) yielded a human ES cell line. A few in the scientific community
had started to wonder whether the nuclear-transfer technique would work
with primate physiology to produce therapeutic stem cells. But Woo Suk
Hwang of Seoul National University and his colleagues proved that it
could be done. The Korean team announced this past February that they had
created a human embryo through SCNT, grew it into a blastocyst and
derived a pluripotent ES cell line. Their accomplishment represents a
major milestone. It also demonstrates how many unknowns we still face.

Because Hwang's group had 242 donated eggs to work with, they were able
to experiment with techniques, timing and conditions at every step. Even
so, from hundreds of eggs the effort yielded only a single ES cell line,
and the researchers have said that they are not certain which of their
methods was responsible for that success. Much remains to be learned
about the mysterious mechanism of reprogramming within the egg and all
that could go wrong while creating and culturing a nuclear-transfer
embryo.

Scientists are still not sure whether reprogramming itself or other
aspects of handling these embryos might introduce gene mutations that
could predispose the resulting ES cells to senescence or cancer, and more
research is needed to detect these potential problems. Inherited gene
mutations, such as those that cause hemophilia or muscular dystrophy,
would have to be corrected as well before using a patient's own cells to
create ES cells. But techniques for gene-specific modifications routinely
performed in mouse ES cells have been successfully applied to human ES
cells, providing a means of safely correcting mutations before
administering cells to patients.

The overall health of ES cells derived from clone embryos has also been
questioned because efforts to produce live animals through cloning have
met with an unusually high rate of deformities and mortality. When a
cloned ES cell line's potential is tested by injecting the cells into a
developing animal blastocyst, though, the resulting animals seem to be
perfectly normal. This outcome suggests that although reproductive
cloning is clearly too unpredictable to consider for humans, ES cells
derived by nuclear transfer, at least for therapeutic purposes, are
equivalent to regular ES cells.

Similar safety questions must also be resolved for a different technique
that produces ES cells without nuclear-transfer or IVF embryos. In a
process called parthenogenesis (from Greek for "virgin birth"), an
unfertilized egg can be chemically tricked into beginning cell division
as though it has been fertilized. These pseudo-embryos, or parthenotes,
are considerably easier to grow than nuclear-transfer embryos. In animal
studies, parthenotes have yielded ES cells able to differentiate into
multiple tissue types in culture and to pass the teratoma test, forming
cells from all three embryonic germ layers.


Unlike normal body cells, which contain a set of chromosomes from each
parent, parthenotes contain a doubled set of the egg donor's chromosomes.
This duplication gives a parthenote a full complement of genes but
prevents it from being viable if it were implanted in a woman's womb.
Having a single "parent" also means that parthenote cells carry half the
normal potential combinations of antigens, making them much easier to
match to patients. A bank of fewer than 1,000 parthenogenic ES cell lines
could probably provide immunological matches for most of the U.S.
population.

How long it will take for any ES cell therapies to be tested in humans
will be determined as much by politics as by the remaining scientific
questions. Well-understood and easy-to-control cell types derived from ES
cells, such as dopamine-producing neurons or the eyes' retinal pigment
epithelium cells, could be ready for human trials in less than two years.
In the meantime, the extraordinary regenerative potential of embryonic
stem cells has intensified the search for similar cells that may be
involved in normal healing in the adult body.

Hidden Potential?
Skin begins repairing itself immediately after being injured. The human
liver can regenerate up to 50 percent of its mass within weeks, just as a
salamander regrows a severed tail. Our red blood cells are replaced at a
rate of 350 million per minute. We know that prolific stem cells must be
at work in such rapidly regenerating tissues. But their very vigor raises
questions about why other organs, such as the brain and heart, seem
incapable of significant self-repair, especially when purported stem
cells have also recently been discovered in those tissues.

The best-known stem cells in the adult body are the hematopoietic stem
cells found in bone marrow, which are the source of more than half a
dozen kinds of blood cells. Their ability to generate a variety of cell
types, at least within a specific tissue family, is why hematopoietic
stem cells have been described as multipotent.

There is great hope that similar multipotent stem cells found in other
body tissues might be drafted into repairing damage without the need to
involve embryos--or better still, that an adult stem cell with more
versatility, approaching the pluripotency of embryonic cells, might be
discovered.

But scientists are just beginning to investigate whether natural
regeneration is somehow blocked in tissues that do not repair themselves
easily and, if so, whether unblocking their regenerative capacity will be
possible. The very source, as well as the potential of various adult stem
cells, is still disputed among researchers. We cannot say for sure
whether tissue-specific adult stem cells originate within those tissues
or are descendants of circulating hematopoietic stem cells. Nor do we
know how far these cells can be pushed to differentiate into functional
tissues outside their specific type or whether such transdifferentiation
produced in the laboratory could be reproduced in a living organism.

The idea that certain adult stem cells might have greater potential first
came from observations following human bone marrow transplants, when
donor cells were subsequently found in a wide range of recipients'
tissues. These accounts implied that under the right conditions, stem
cells from the bone marrow could contribute to virtually any part of the
body. (Similar claims have been made for the so-called fetal stem cells
found in umbilical cord blood, which resemble hematopoietic stem cells.)

Attempts to directly test this theory in living organisms, however, have
not found consistent evidence of such plasticity. In March separate
reports from Leora Balsam and her colleagues at Stanford University and
from a group led by Charles E. Murry of the University of Washington both
described using powerful tracking methods to see if hematopoietic stem
cells would incorporate into injured heart muscle, a nonhematopoietic
tissue. Neither group detected contribution of new tissue by the stem
cells.


What has increasingly been found is extensive fusion of bone marrow stem
cells to cells in the heart, liver and brain, offering an alternative
explanation for the presumed transdifferentiation. In future studies of
adult stem cell potential, it will be crucial to rule out the possibility
that stem cells are merely fusing to local cells rather than generating
new ones.

Still, tissue-specific cells have already produced encouraging results.
In the German TOPCARE-AMI study of patients with severe heart damage
following myocardial infarction, the patients' own heart progenitor cells
were infused directly into the infarcted artery. Four months later the
size of the damaged tissue swath had decreased by nearly 36 percent, and
the patients' heart function had increased by 10 percent.

The small number of stem cells that can be isolated from any adult tissue
remains the biggest technical hurdle to applying this type of research
more widely in the clinic. In mouse bone marrow, stem cells are as rare
as one in 10,000, and the ratio may be even greater in humans. In most
tissues, there is no predictable location for stem cells, and we possess
only limited tools for identifying them using surface markers or gene
expression signatures.

Once isolated, adult stem cells are also notoriously slow and
labor-intensive to grow. As is true of embryonic cells, so little is
understood about the factors that may control the adult stem cells' fate
that we do not yet know whether extensive time spent in culture could
harm their ability to restore tissues in patients.

Rather than hunting for a patient's stem cells to remove, cultivate and
then replace them, we may be able to summon the body's hidden stores.
Increasing evidence suggests that stem cells, like metastatic tumor
cells, respond to common chemical signals leading them to sites of
injury. One of us (Rosenthal) recently showed in mice that stem cells
will travel great distances to reach an injury when summoned with the
help of a protein called IGF-1.

Marshaling the body's own ability to trigger tissue regeneration by stem
cells will require a better grasp of the roles played by such chemical
signals. Rosenthal and her collaborator Antonio Musaṛ have demonstrated
that IGF-1 helps to beckon stem cells, but we suspect that this molecule
may also take part in causing some of the injured cells to revert to a
multipotent state and begin differentiating into the required tissue
types. This phenomenon, known as epimorphic regeneration, underlies the
ability of newts and zebra fish to regrow entire limbs and organs.

Regenerative medicine's ideal would be to find a means to cause such
controlled dedifferentiation of adult tissue--in essence turning a
terminally differentiated cell back into a stem cell. Many

researchers are looking for the magic molecules that can produce this
transformation, and some very preliminary successes have recently been
reported. But therapeutic regeneration through dedifferentiation is a
long way off and will most likely come from a much better understanding
of stem cells themselves--both adult and embryonic.

Which Way Forward?
As often happens in science, stem cell research has raised as many new
questions as it has answered, but the field is advancing. Early tests of
human adult stem cells in treating cardiovascular disease are encouraging
and will certainly lead to more extensive trials in the near future.
Given much promising experimental evidence in animals, therapeutic trials
of human ES cell derivatives in neurodegenerative disease are probably
imminent.

As the appropriate source of cells for both research and eventual
therapeutic applications continues to be hotly debated, restrictions on
this research are slowing progress. But we believe that generating
replacement cells and regenerating organs are feasible and realistic
goals. The remaining hurdles are difficult but not insurmountable.

-------------------------------------------------------------------------
-------
ROBERT LANZA and NADIA ROSENTHAL are leading stem cell researchers. Lanza
is medical director of Advanced Cell Technology, Inc., as well as adjunct
professor at the Institute of Regenerative Medicine at Wake Forest
University School of Medicine. Lanza's current research centers on
embryonic stem cells; he has also done groundbreaking work in cloning and
tissue engineering. Rosenthal is head of the European Molecular Biology
Laboratory in Rome. She directs the EMBL Mouse Biology program,
concentrating her research on stem cell-mediated regeneration of
neuromuscular and cardiac tissues, embryonic heart development, and
developing mouse models of human diseases. Before joining EMBL, Rosenthal
directed a laboratory at the Harvard Medical School's Cardiovascular
Research Center and was a consultant on molecular medicine for the New
England Journal of Medicine.

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