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Embryonic Stem Cells Teach Lessons about Fanconi Anemia

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Bone marrow failure may begin earlier than previously thought

March 2, 2010

Boston, Mass. -- Children with Fanconi anemia, a genetic disease that can be fatal in the absence of a bone marrow transplant, typically do not experience low blood cell counts until about age 7. However, by modeling the disease in the laboratory using embryonic stem cells, researchers at Children's Hospital Boston reveal that the events leading to bone marrow failure (inadequate production of blood cells) may actually begin before birth, casting a whole new light on the disease.

Researchers led by Asmin Tulpule, PhD, doctoral student in Children's Division of Pediatric Hematology/Oncology, and stem cell researcher George Daley, MD, PhD, Director of the Stem Cell Transplantation Program at Children's, generated blood cells from human embryonic stem cells (ESCs) to replicate the earliest stages of blood formation. As reported in the journal Blood online on January 20, they then knocked down, or suppressed, two genes associated with Fanconi anemia, and demonstrated that the disease compromises the formation of blood cells from the earliest stages of human development.

"When we knock down these genes, you see a profound deficit in blood cell formation, suggesting that these genes have an important role in early blood development," says Tulpule, first author of the paper. These results suggest that children with Fanconi anemia are born with what is already a depleted pool of blood stem cells.

Previous research had failed to create a suitable laboratory model for Fanconi anemia. Mouse models showed that the disease involves an inability to repair DNA damage, but the mice did not display the characteristic skeletal abnormalities, increased risk for leukemia or, most significantly, the trademark bone marrow failure seen in humans. Human ESCs proved to be a suitable model for observing the development of Fanconi anemia.

The researchers turned to existing lines of human ESCs to create their model because past research has indicated that it is difficult to generate Fanconi anemia models from induced pluripotent stem (iPS) cells, which are made from the skin cells of patients. Furthermore, scientists cannot create a disease model using a patient's blood stem cells, either.

"Blood stem cells are not easy to get from patients," Tulpule says. Because Fanconi anemia depletes a child of blood stem cells, extracting them for testing is not practical. "Human ESCs allow us to do all kinds of different studies that were unthinkable."

Fanconi anemia is caused by a deletion of any of 13 genes. In healthy cells, some of these genes manufacture a core complex of eight proteins responsible for DNA repair, and others manufacture proteins downstream of this core complex. The researchers created a model using two of the genes.

After establishing human ESC cultures in the lab, they used RNA-interfering viruses to suppress the activity of FANCA, one of the genes in the DNA repair core complex and responsible for 65 percent of Fanconi anemia cases, or FANCD2, a gene acting downstream of the core complex and responsible for up to 6 percent of cases. After gene knockdown, the ESCs were coaxed with a special brew of growth factors to become blood cells.

Compared to ESCs that did not undergo gene knockdown, the cell lines with either a FANCA or FANCD2 knockdown were less able to repair DNA damage and less prone to differentiate into blood stem cells, providing strong evidence that blood formation is hindered during the earliest stages of human development. Cell lines with a FANCD2 knockdown generated the fewest blood stem cells, consistent with the greater severity of disease in patients with mutations downstream of the core complex.

When the researchers reinserted the two genes back into the cell cultures, the ESCs were again able to generate blood stem cells.

Based on their lab model, the researchers now hypothesize that a child with Fanconi anemia is born with far fewer blood stem cells than usual. These limited stem cells are able to produce enough blood cells to make cell counts appear normal for years, but eventually, the inability to repair DNA damage takes its toll. These blood stem cells begin to die, making a bone marrow transplant the only recourse for a child.

The next step in this research is to figure out why FANCA, FANCD2 and related genes are so essential to blood formation. Preliminary experiments suggest that these genes interact with another group of genes known as HOX genes, which play a role in the development of both blood cells and limb patterning in the embryo.

The researchers' relative ease with their lab model shows how promising human ESCs are to understanding genetic diseases such as Fanconi anemia, Tulpule says.

"Using these cells allows us to have a better understanding of the disease and how to treat it," Tulpule says. In the future, human ESCs could be used for drug screening, helping researchers find agents that prevent marrow loss and augment the blood's ability to carry oxygen. Such treatments won't cure Fanconi anemia, but could keep a child alive long enough to wait for a bone marrow transplant, Tulpule says.

The study was funded by grants from the National Institutes of Health, the Burroughs Wellcome Fund, the Leukemia and Lymphoma Society, and the Harvard Stem Cell Institute. Daley is an Investigator of the Howard Hughes Medical Institute.

Asmin Tulpule, M. William Lensch, Justine D. Miller, Karyn Austin, Alan D'Andrea, Thorsten M. Schlaeger, Akiko Shimamura, George Q. Daley. Knockdown of Fanconi anemia genes in human embryonic stem cells reveals early developmental defects in the hematopoietic lineage. Blood Jan. 20, 2010 (online).

Elizabeth Andrews

Children's Hospital Boston is home to the world's largest research enterprise based at a pediatric medical center, where its discoveries have benefited both children and adults since 1869. More than 500 scientists, including eight members of the National Academy of Sciences, 11 members of the Institute of Medicine and 13 members of the Howard Hughes Medical Institute comprise Children's research community. Founded as a 20-bed hospital for children, Children's Hospital Boston today is a 397-bed comprehensive center for pediatric and adolescent health care grounded in the values of excellence in patient care and sensitivity to the complex needs and diversity of children and families. Children's also is the primary pediatric teaching affiliate of Harvard Medical School. For more information about the hospital and its research visit:

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