Public release date: 14-Aug-2003

Contact: Sally Pobojewski

pobo@umich.edu
734-615-6912
University of Michigan Health System

Stem cell defects are key to Hirschsprung's disease

Serious genetic disorder affects one in 5,000 infants

ANN ARBOR, MI  Scientists at the University of Michigan Medical School have identified defective stem
cells as the key to a serious, sometimes life-threatening, intestinal disorder called Hirschsprung's disease,
which affects one in 5,000 newborn infants.

Babies born with this disease don't develop ganglion cells  specialized nerve cells in the large intestine,
which trigger contractions to push feces through the colon and out the rectum. The result is chronic
constipation and intestinal obstructions requiring surgery, sometimes immediately after birth.

Scientists have known for years that Hirschsprung's is a genetic disease, and some of the mutations
associated with the disorder have been identified. But no one knew exactly how these mutations impaired
the development of the intestinal nervous system.

Based on new research data, U-M scientists now say the basic problem is that neural crest stem cells, which
give rise to nerves in the embryonic digestive system, never reach the lower part of the developing gut.

Details of the U-M study will be published in the Aug. 15 issue of Science. It is the first published paper
linking Hirschsprung's disease to functional defects in neural crest stem cells, or NCSCs.

"Other scientists found that mutations associated with Hirschsprung's disease cause changes in the
proliferation, survival and migration of neural crest cells," says Sean J. Morrison, Ph.D., a Howard Hughes
Medical Institute assistant investigator and assistant professor of internal medicine and of cell and
development biology in the U-M Medical School. "But it was unclear exactly which neural crest cells were
affected or how these changes impaired the formation of the intestinal nervous system."

"We found that the mutated genes causing Hirschsprung's disease act in neural crest stem cells to impair
their ability to form a normal intestinal nervous system," Morrison adds.

Neural crest stem cells are one variety of tissue or "adult" stem cells. Unlike embryonic stem cells, which
can become nearly any cell in the body, NCSCs normally develop into the neurons and supporting cells of
the peripheral nervous system  as well as connective tissues, such as dermis, bone and vascular smooth
muscle.

NCSCs first show up in the embryo's primitive neural tube, which forms the spinal cord. When everything
goes right, neural crest stem cells then migrate through the developing gut, seeding the primitive digestive
system with stem cells capable of generating a healthy, functioning intestinal nervous system.

But in embryos with Hirschsprung's disease, some or all of these neural crest stem cells get stuck and never
migrate past the esophagus. "The stem cells can't give rise to a nervous system in the hindgut, because
they never make it to the hindgut in the first place," Morrison says.

To identify the genes expressed in neural crest stem cells, Morrison's research team first used flow
cytometry technology to isolate the small number of NCSCs  just two percent of all cells in the gut of a
14.5-day-old embryonic rat. Then they used microarray technology to analyze gene expression profiles for
the NCSCs and compare them to gene expression profiles for blended cells from the entire rat fetus. The
goal was to identify genes that were actively producing protein in gut neural crest stem cells and compare
them to active genes in other cells from the same animal.

"Of the 10 genes we found to be most up-regulated in stem cells versus fetal cells, four are known to be
involved in Hirschsprung's disease," says Toshihide Iwashita, M.D., Ph.D., co-first author on the paper and a
research fellow in Morrison's lab. "Finding that a number of genes linked to Hirschsprung's are all expressed
within NCSCs will allow us to more effectively study the ways in which these genes interact to cause the
disease."

Previously an assistant professor of pathology at Japan's Nagoya University, Iwashita has spent eight years
studying one of the major genes involved in Hirschsprung's disease  a gene called Ret. Mutations that
inactivate the function of Ret are a major cause of Hirschsprung's disease.

"Ret was expressed in neural crest stem cells at rates 100 times higher than in fetal cells generally,"
Morrison says. "Three other genes  Sox10, Grfa-1 and endothelin receptor type B also were expressed at
higher levels in NCSCs, as compared to fetal cells. Since four of our top 10 genes are linked to
Hirschsprung's, we believe it's likely that other genes up-regulated in NCSCs are involved with the disease,
as well. Identifying mutations in these genes may make it possible to find the cause of some of the currently
unexplained cases of Hirschsprung's disease."

Other scientists have shown that neural crest cells migrate through the gut in response to a biochemical
substance called GDNF or glial-derived neurotrophic factor. "Since NCSCs express higher levels of Ret, the
receptor for GDNF, we decided to test the sensitivity of NCSCs to GDNF's signal," says Genevieve M.
Kruger, a graduate student in the U-M Medical School's M.D./Ph.D. program and co-author on the Science
paper. "We found that over 10-fold more NCSCs migrated out of guts exposed to GDNF than guts cultured
in control gels."

The researchers then examined the intestinal tracts of a strain of embryonic mice that don't have the Ret
gene. They found that stomachs and intestines from Ret-deficient mice contained far fewer neural crest
stem cells  "a factor of 20 reduction"  compared to the number of NCSCs in the lower gut of mice with the
Ret gene.

"Lacking the ability to express Ret, stem cells never migrated past the esophagus into the lower gut,"
Morrison explains. "The inability of NCSCs to migrate past the esophagus explains the absence of enteric
nerves in the stomach and intestines of these mice."

In previous research, Morrison and his research team discovered that neural crest stem cells remain in the
gut of rats and mice throughout their adult lives. In a future experiment, Morrison hopes to transplant NCSCs
from the upper to lower gut of rats born with Hirschsprung's disease to see if it is possible to develop some
functional nerves in the hindgut after birth.

"Our study raises several intriguing new ideas, but we're still not close to implementing what we've learned
in humans," Morrison cautions. "Neural crest stem cells haven't even been characterized in humans yet. But
the research does provide critical insights into the cellular and molecular mechanisms underlying
Hirschsprung's disease."

The study was funded by the Howard Hughes Medical Institute, the National Institutes of Health, the Searle
Scholars Program and the U-M's Institute of Gerontology. Ricardo Pardal, Ph.D., a U-M research fellow, and
Mark J. Kiel, a graduate student in the U-M Medical School's M.D./Ph.D. program, were collaborators in the
study.

###

Sally Pobojewski, pobo@umich.edu, 734-615-6912 or

Kara Gavin, kegavin@umich.edu, 734-764-2220
Stem Cells: Stem Cells Found In Adult Hair Follicles May Provide Alternative To
Embryonic Stem Cells
12/12/2006

Having recently identified the molecular signature of these epidermal neural crest stem cells in the
mouse, their research resolves conflicting scientific opinions by showing that these cells are distinctly
different from other types of skin-resident stem cells/progenitors. Their work provides a valuable
resource for future mouse neural crest stem cell research.

A report on the research from Dr. Maya Sieber-Blum's laboratory, co-authored by Yao Fei Hu, Ph.D.,
and Zhi-Jian Zhang, Ph.D., researchers in cell biology, neurobiology and anatomy at the Medical
College, was published in a recent issue of Stem Cells: The International Journal of Cell Differentiation
and Proliferation.

Epidermal neural crest stem cells are found in the bulge of hair follicles and have characteristics that
combine some advantages of embryonic and adult stem cells, according to lead researcher, Maya
Sieber-Blum, Ph.D., professor of cell biology, neurobiology & anatomy. Similar to embryonic stem cells,
they have a high degree of plasticity, can be isolated at high levels of purity, and can be expanded in
culture. Similar to other types of adult stem cells, they are readily accessible through a minimally
invasive procedure and could lead to using a patient's own hair as a source for therapy without the
controversy or medical issues of embryonic stem cells.

"We see the potential for cell replacement therapy in which patients can be their own donors, which
would avoid ethical issues and reduce the possibility of tissue incompatibility," says Dr. Sieber-Blum.

The Medical College team in collaboration with Prof. Martin Schwab, director of the Brain Research
Institute of the University of Zürich, recently injected these cells in mice with spinal cord injuries.
According to the study, when grafted into the spine, the cells not only survived, but also demonstrated
several desirable characteristics that could lead to local nerve replacement and re-myelination
(restoration of nerve pathways and sheaths).

Neural crest stem cells generate a wide array of cell types and tissues and actually give rise to the
autonomic and enteric nervous systems along with endocrine cells, bone and smooth muscle cells. The
cells can be isolated from the hair follicle bulge as multipotent stem cells, and then expanded in culture
into millions of cells without losing stem cell markers.

"We grafted the cells into mice that have spinal cord injuries and were encouraged by the results. The
cells survived and integrated into the spinal cord, remaining at the site of transplantation and not
forming tumors," Dr. Sieber-Blum says.

According to Dr. Sieber-Blum, subsets of the epidermal neural crest stem cells express markers for
oligodendrocytes, the nerve-supporting cells that are essential for proper neuron function. She has
been awarded a grant from the Biomedical Technology Alliance, a Milwaukee inter-institutional research
group, to determine in collaboration with Brian Schmit, Ph.D., associate professor of biomedical
engineering at Marquette University, if the grafts lead to an improvement of spinal reflexes in the injured
spinal cord of mice.

Dr. Sieber-Blum points out that the cells may also be useful to treat Parkinson's disease, multiple
sclerosis, Hirschsprung's disease, stroke, peripheral neuropathies and ALS. Certain defects of the
heart, and bone defects (degeneration, craniofacial birth defects) could also be treated through neural
crest stem cell replacement therapy. Together, these conditions affect over 11 million people today in
 the US and are estimated to annually cost more than $170 billion.

SOURCE: Medical College of Wisconsin

Stem Cell Study Raises More Questions about Therapies

By Nancy Touchette

Posted: The seemingly

October 17, 2003 remarkable ability of stem
cells to turn into other
types of cells has fueled
research on experimental
therapies, particularly
involving stem cells
isolated from a person's Bone marrow stem cells fuse with liver cells in mice
to produce a hybrid cell (blue) that contains two
bone marrow. nuclei.
Image courtesy Nature.
A boy in Michigan
received injections of his own bone marrow stem cells in February
2003 to repair his damaged heart, and his condition reportedly
improved. Clinical trials are planned or underway in several
countries to test whether bone marrow stem cells can help patients
with heart disease.

But some researchers have challenged the claim that bone marrow
stem cells are actually becoming other cells. They say these cells
are “fusing” with other cells rather than actually changing their
genetic program, and a major study published in May 2003 reported
that bone marrow stem cells had fused with liver cells in mice.

Now a new study weighs in with similar findings, showing that

bone marrow stem cells transplanted in mice fused with liver cells,
neurons and heart cells. Fusion occurs when a bone marrow cell
somehow adheres to another cell. The membranes join and the
resulting cell has two nuclei and double
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fused cells contain an abnormal • Stem cell therapies: time to
number of chromosomes, they could step back or forge ahead?
lead to cancer or other problems.

But it's also possible that cell fusion is a naturally occurring process
that plays a role repairing tissues. At the moment, no one is sure just
what's going on.
Until researchers fully understand how bone marrow cells restore
damaged tissues, clinical applications should be put on hold, says
Arturo Alvarez-Buylla of the University of California, San
Francisco, who led the new study of bone marrow stem cells in
mice.

His team transplanted bone marrow stem cells into mice. Two
months later, the bone marrow cells had fused with cells in the
brain, the liver, and the heart. The researchers did not find evidence,
however, of fusion with other cells.

Alvarez-Buylla and his colleagues would like to know which cells

are doing the fusing—and why some cells and not others appear to
fuse with bone marrow cells. They would also like to know whether
cell fusion is normal or not.

“It's not so important to point a finger at who is right and who is

wrong, but it is important to find out what is really going on,” says
Alvarez-Buylla.

“If fusion is a mistake of cell biology and cells end up getting stuck
together, this could interfere with normal cell function,” he says.
“Until we know what's going on it's too much of a risk for the
patient.”

The confusion over whether bone marrow cells fuse with other cells
has prompted some researchers to move away from the idea of
using these cells to repair damaged tissues.

For example, Piero Anversa, of the New York Medical College in

Valhalla first discovered that bone marrow stem cells could repair
damaged heart cells in mice. But he has since abandoned this work
and focused on cardiac stem cells. His team recently discovered
stem cells in cardiac tissue in humans and rats.