<b>August 14 2003</b> – ANN ARBOR, MI &#8211; 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 &#8211; 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 &#8211; 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 &#8211; 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 &#8211; 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 &#8211; 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 &#8211; "a factor of 20 reduction" &#8211; 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.
