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Duke Scientists Grow New Stem Cells in Blood by Manipulating Molecular Pathway

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Duke Health News 919-660-1306

DURHAM, N.C. -- In a major advance for stem cell research,
scientists have grown new blood stem cells in the laboratory by
manipulating a molecular pathway called Wnt. They also have
shown that these Wnt-signaled stem cells are viable and
continue to reproduce, both as stem cells and mature blood
cells, when transplanted into mice.

Their discovery -- that the Wnt pathway is critical in
regenerating blood stem cells -- lays the groundwork for
growing large numbers of blood or "hematopoietic" stem cells
for transplantation into critically-ill patients, said
Tannishtha Reya, Ph.D., assistant professor of pharmacology and
cancer biology at the Duke Comprehensive Cancer Center.

Results of the study, initiated by Reya as a postdoctoral
fellow in the laboratory of Irving Weissman, M.D., at Stanford
University and completed in her own lab at Duke, are published
in the April 27, 2003, online issue of the journal Nature.

For 25 years, doctors have used donor bone marrow to
repopulate the blood-forming and immune systems of patients
with cancers and metabolic diseases. Yet bone marrow contains
so few stem cells -- only one per 2,000 other types of cells --
that patients have no immunity while the new stem cells
struggle to regenerate and produce blood and immune cells.

Attempts to grow hematopoietic stem cells in the lab have
met with limited success, said Reya, because scientists have
never understood the basic mechanism by which they regenerate
and give rise to specific cells.

The Duke study shows that the Wnt pathway is necessary for
normal stem cell survival and proliferation. Moreover,
activating the Wnt pathway causes hematopoietic stem cells to
regenerate in their immature state instead of differentiating
into specific, mature cells, the study found. Once stem cells
differentiate, they can no longer self-renew and produce new
stem cells. Hence, maintaining their status as stem cells is
critical to growing enough cells for use in a transplant, said
Andrew Duncan a graduate student in Reya's lab and co-author of
the study.

"The Wnt protein's pathway of activation has previously been
defined, but our work demonstrates its critical role in
regulating hematopoietic stem cell growth," said Duncan.

Specifically, the Wnt protein works by binding to the
"frizzled" receptor on the stem cell surface. There, it
initiates a cascade of events that releases beta catenin inside
the cell. Beta catenin is another protein that eventually acts
in concert with components inside the cell's nucleus to turn on
critical genes, such as Notch1 and HoxB4. These genes, in turn,
can act to regulate the stem cells' ability to self-renew.

Beta catenin is the most important known regulator of the
Wnt pathway and it also provides the strongest growth signal
for stem cells, said Reya. When beta catenin was expressed by
the hematopoietic stem cells, they grew undifferentiated for at
least eight weeks, the study found. During that time, the stem
cells underwent eight to nine population doublings to generate
at least a hundred times the number of original cells. In
contrast, stem cells in which the pathway was not activated
showed minimal growth beyond a two-week period.

Despite their undifferentiated state in the laboratory, stem
cells that expressed beta catenin gave rise to normal white
blood cells in mice whose immune systems had been destroyed by
radiation. Just 125 of these stem cells were able to completely
regenerate the animals' hematopoietic systems, the study
showed.

Taking the research one step further, Reya and Duncan
decided to test whether Wnt itself -- which activates beta
catenin -- could produce the same effect on stem cells.

Although it is less potent than beta catenin, Wnt has the
benefit of being simpler to deliver to stem cells, and it
mimics the cells' natural signaling process.

Thus, Reya and Duncan treated stem cells with purified Wnt
protein, developed by Roel Nusse, Ph.D., and colleagues at
Stanford University and described in the April 27 online issue
of Nature. Purified proteins are desirable because they are
stripped of all other growth factors or conflicting signals
that might inadvertently influence stem cell behavior, said
Reya.

"We took a pure population of stem cells and applied a pure
growth factor (Wnt), so we were able to completely control how
the stem cells grew," said Reya. "Most growth factors used so
far generate signals that will make stem cells differentiate,
but purified Wnt, together with another factor, appears to
deliver a clear self-renewal signal, so the cells do not
differentiate very much but do still proliferate.

"The hope is that in the future, one may be able to expand a
patient's or donor's hematopoietic stem cells by activating the
Wnt signaling pathway and thereby provide an increased source
of cells for transplantation," said Reya.

Moreover, the Wnt protein may have potential beyond
hematopoietic stem cells, she said. Previous studies have
implicated components of the Wnt pathway in promoting growth of
primitive cells in the skin, gut and brain, said Reya. Those
findings raise the possibility that Wnt signaling might be used
as a general signaling cue for stem cell self-renewal in a
variety of tissues. Finally, Reya said, increased self-renewal
is a property that could be dangerous, as uncontrolled or
mutated cellular growth signals are a hallmark of cancer.

"Our finding that the Wnt pathway may play a role in
hematopoietic stem cell self-renewal leads us to propose that
this pathway should be studied for a role in cancer stem cell
self-renewal."

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