Discovery may have implications for the treatment of hemophilia A and B

University of North Carolina at Chapel Hill researchers have made a discovery that may have implications for the treatment of liver-based genetic defects such as hemophilia A and B in humans.

Mouse embryonic stem cells treated in culture with a growth factor and then injected into the liver reverse a form of hemophilia in mice analogous to hemophilia B in humans, the new study shows. A report of the study appears in the journal Proceedings of the National Academy of Sciences today (Feb. 15).

The genetically altered mice lack the clotting substance factor IX, which in humans results in the hereditary bleeding disorder known as hemophilia B. This disease, much less common than hemophilia A, affects roughly one of every 35,000 people, primarily males.

Although embryonic stem, or ES, cells can differentiate into most cell types in the body, numerous problems have arisen in translating their potential into therapeutic strategies, the UNC School of Medicine study authors reported.

These problems include poor engraftment, limited function, rejection of engrafted cells by the immune system and teratomas, tumors involving a mixture of tissue not normally found at that site.

The new study used a line of mouse ES cells developed in the laboratory of senior co-author Dr. Oliver Smithies, Excellence professor of pathology and laboratory medicine at UNC.

A member of the National Academy of Sciences, Smithies has won many honors for gene targeting, a technique he pioneered. This technique allows for the development of mice with specific genetic mutations that mimic human illnesses such as hemophilia. In 2001, Smithies received the Albert Lasker Award for Basic Medical Research, often called "America’s Nobel."

In the study, ES cells were treated with fibroblast growth factor for seven days prior to injection. As expected, this resulted in ES cells differentiating into early endoderm like precursors, which the researchers named "putative endoderm precursors," or PEPs. Endoderm refers to the inner layer of early embryonic cells that develops into the digestive and respiratory systems.

"Not only do ES cells differentiate into PEPs, they also engraft, persist, differentiate further and then function following injection, resulting in the persistent production of factor IX protein that can only come from a hepatocyte (liver cell) and hemophilia reversal," said study lead author Dr. Jeffrey H. Fair, associate professor of surgery and division chief of abdominal transplant surgery.

Moreover, he said, the PEP cells robustly engraft within the liver and were not recognized by the immune system as foreign.

"Within a few weeks, PEPs became hepatocytes," Fair added. "They went from something that is a very early grandparent of the hepatocyte to becoming hepatocytes. After 115 days, nearly four months after injection, mice still produced factor IX without immune suppression. This occurred even in mice that were a complete immunologic tissue mismatch to the PEPs. In addition, the incidence of teratomas was low."

The researchers believe this study demonstrates the power of multidisciplinary collaboration, said co-lead author Dr. Bruce A. Cairns, assistant professor of surgery and director of research in the N.C. Jaycee Burn Center. "This approach may not only be beneficial, but required in order to solve complex problems such as these in medicine."

Although a number of questions need to be answered, this work has great potential for future applications, not only as a novel therapeutic possibility for hemophilia but also for other genetic or acquired diseases of the liver, said senior co-author Dr. Jeffery A. Frelinger, Kenan professor and chairman of microbiology and immunology.

"The data published in this study shows that embryonic stem cells partially differentiated, are able to remain in the liver and be functional without apparent immunological rejection. This transforms them into possible candidates for cellular transplantation into the liver."

Along with Fair, Cairns, Smithies and Frelinger, co-authors from the department of surgery are Dr. Michael A. LaPaglia, Dr. Montserrat Caballero, Dr. Anthony A. Meyer (chairman) and W. Andrew Pleasant. From the department of pathology and laboratory medicine are Drs. Seigo Hatada and Hyung-suk Kim. From the College of Arts and Sciences’ department of biology are Drs. Tong Gui and Darrel W. Stafford; and from the department of genetics, Dr. Larysa Pevny.

The research was supported by grants from the National Institutes of Health and the N.C. Jaycee Burn Center.