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DURHAM, N.C. - Medical researchers have been searching for a
reliable method to separate rare and primitive stem cells from
human blood because these cells can regenerate a blood supply
and immune system damaged by disease or medical treatment.

Now researchers from the Duke Comprehensive Cancer
Center at Duke University Medical Center have developed a
new method to identify and isolate stem cells from samples of
umbilical cord blood based on an enzyme in the cells. The
enzyme, which is more abundant in stem cells than other blood
cells, changes a fluorescent tag the researchers developed to a
form that can't escape the cell. The brightest cells - the stem
cells - are then selected tomatically.

Current separation methods, primarily focused on detecting
proteins on the surface of stem cells, are complex and
expensive and are complicated by the possibility that not all
stem cells express these proteins, the researchers say.

The advance, reported in the Aug. 3 issue of the Proceedings of the National Academy of
Sciences (PNAS), has immediate implications for laboratory
research involving stem cells. There is also the potential for
clinical applications if further experiments show that the
selected stem cells can mature into needed blood cells in
humans,says the study's principal investigator, Dr. Clay Smith,
an associate professor of hematology and oncology.

In the shorter run, the technique could help researchers
isolate enough of the elusive cells to investigate many
fundamental questions regarding stem cells - from how they
maintain their primitive nature, to how they differentiate into
specific kinds of cells, to even what tissues might contain
them.

To date, several types of stem cell have been identified.
Embryonic stem cells, found in developing fetuses, can mature
into any of the body's cell types. Hematopoietic stem cells,
those isolated by Smith and his colleagues, are found in bone
marrow and blood, including umbilical cord blood, and can only
mature into blood cells. Recently, neuronal stem cells have
been discovered in the brain.

The Duke researchers say their technique might be a good way
to isolate as-yet-undiscovered stem cells from other tissues.
"Our method represents a new and different way to get a
purified population of stem cells," says study co-author Dr.
Michael Colvin, director of Duke Comprehensive Cancer
Center.

While researchers have coaxed isolated stem cells to mature
into almost every type of blood cell in the lab, experiments in
mice and humans are still required for formal proof that the
cells can solve clinical problems by repopulating a real-life
blood supply, Smith says. Even then, he says, more work will be
needed before the method can be applied to human therapies.

If the isolated cells can, in fact, regrow a person's blood
supply, the technique could possibly improve success rates and
reduce complications of stem cell transplants. Certain
diseases, such as leukemia, or medical treatments, like
high-dose chemotherapy, can harm blood cells. As a result, new
blood cells - such as white cells (the body's immune system),
red cells (the body's oxygen transport system), and platelets
(the body's clotting factor) - all need to be produced from
scratch.

But while stem cells may work wonders, mature blood cells
that can't be excluded from the transplants frequently wreak
havoc. Without a reliable method to separate stem cells from
the other cells, patients' "immunity fingerprints" must be
closely matched to their bone marrow donors. If not, either the
patient can reject the foreign cells or the mature foreign
blood cells can actually attack the patient's tissues.

While these problems are less common with an umbilical cord
blood transplant since its regular blood cells are not fully
matured, they are still a concern. If a patient's own bone
marrow or blood is used for the transplant, immune matching is
not required, but it's possible that unwanted diseased cells
could be returned to the patient. The new isolation technique
could potentially eliminate such problems by making it possible
to collect and deliver pure stem cells.

If the isolated stem cells work in living systems, the
method might also provide accurate counts of the stem cells
contained in transplants. This is an important potential
ability, since scientific studies have linked the number of
stem cells transplanted with the eventual success of the
graft.

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In designing their new isolation method, the researchers
took advantage of the fact that stem cells, more than any other
blood cell, contain a great deal of an enzyme known as aldehyde
dehydrogenase. Led by Colvin, researchers developed a
fluorescent tag that would be altered by the enzyme in a way
that would trap it inside the cell. Their fluorescing molecule
is called BAAA for BODIPY aminoacetaldehyde. (BODIPY is the
part of the molecule that absorbs light of one wavelength and
then fluoresces, releasing light of a different
wavelength.)

Aldehyde dehydrogenase changes BAAA into BAA, or BODIPY
amino acetate, which becomes "stuck" inside a cell because it
is negatively charged. The researchers proved this in
experiments with special non-stem cells. However, despite the
fact that hematopoietic stem cells have lots of the enzyme,
they quickly expelled BAAA before it could be transformed to
BAA - in the same way that multidrug resistant cancer cells
quickly pump out chemotherapy drugs.

But the researchers had a solution. They knew that a drug
called "verapamil" can stop cancer cells from pumping out
chemotherapy drugs. So, the study's first author, Robert
Storms, a research associate in the Center for Genetic and
Cellular Therapies at Duke, treated the cord blood samples with
verapamil to prevent stem cells from expelling BAAA. It was a
success. With ample time to work, the aldehyde dehydrogenase in
the stem cells changed BAAA into BAA and the fluorescent tag
built up inside the desired cells.

After being treated with both BAAA and verapamil, the cord
blood samples are processed by a computerized cell sorter,
which shines a laser on one cell at a time and monitors the
cell's fluorescence. The machine gives the brightest cells -
the cells with lots of aldehyde dehydrogenase and hence lots of
BAA trapped inside - an electric charge to separate them from
the others. Those charged cells then drip one by one into a
collection vessel while all the other cells are discarded. The
sorter can analyze 3,000 cells per second.

Aldehyde dehydrogenase's normal role in stem cells isn't
known, but it might play an important role in embryo
development and hence cell differentiation, Colvin says.
Exactly how it works awaits further investigation.

The researchers used funding from the National Institutes of
Health and a Public Health Service Grant from the National
Cancer Institute to develop the technique.