Animal models are useful for testing and developing future treatments and procedures before they are tried in humans. Before bone marrow transplants were first tried clinically in the 1950’s for the treatment of radiation poisoning or leukemia, they had already been shown to work in rats, dogs, and primates. But even after the proven success of the method to replenish a patient’s hematopoietic stem cells – the precursors of all the different types of blood cells – animal models continued to be useful for improving the procedure and better understanding the system’s biology. Now, more than 50 years after those first experiments, a new animal model for transplanting marrow has been developed – under water.
Zebrafish, the tiny, striped fish often found in pet stores, lead a double life as scientific heroes. Because of their fast reproductive cycle, translucent embryos (seen above), and well-studied genome, zebrafish are an increasingly popular animal model for scientists to study embryonic development, genetics, and diseases such as cancer. The ability to easily mutate zebrafish genes and screen for interesting biological changes makes the species an ideal fit for studying the function of hematopoietic stem cells and how they can be better used in medical procedures. But there was only one problem for a team of researchers at the Harvard Stem Cell Institute: nobody had tried to do a marrow transplant in zebrafish before.
“We wanted to be able to have an assay where you could compare mutant marrow with wild type marrow and see whether the hematopoietic stem cells function differently,” said Jill de Jong, member of the Harvard research team and now assistant professor of pediatrics at the University of Chicago Medical Center. “The only way to do that was with a transplant assay. Since you’re talking about mutants in fish, it really would have to be a transplant assay in fish – and that didn’t exist.”
Translating a stem cell transplant procedure developed in mammals to fish required several modifications. For one, zebrafish do not carry their hematopoietic stem cells in bone marrow, but rather in their kidneys. In recipient fish, nobody had calibrated the amount of radiation needed to knock out the native marrow cells, or the amount of donor cells needed to successfully replenish the marrow and blood. And while it is easy to match mice for transplantation purposes – because they are inbred and immunologically identical – the fish require more precise matching of donor and recipient, just like humans. The low success rate in the first batch of zebrafish transplants reflected this difficulty.
“These fish were like random donors, they were not immunologically matched at all,” de Jong said. “In some ways, it’s kind of miraculous that it even worked at all.”
But one by one, the kinks were worked out and the procedure was standardized (and published earlier this year in the journal Blood). A number of the immune system MHC genes, which are carefully matched in human bone marrow transplants, were located on chromosome 19 of the zebrafish genome. Each fish could then be genotyped and paired with a closer match for the transplant, which raised the success rate of the procedure.
With a reliable assay in place, de Jong can now start using it to look for new genes involved in the regulation and control of hematopoietic stem cells, as well as the development of leukemia. After creating mutant zebrafish with unusual blood (using the random process of forward genetics), their marrow can be competed against normal marrow to find mutant cells that are better at taking root in the transplant recipient. The genes responsible for that improved outcome could then be identified, and used to better understand hematopoietic stem cell biology. Eventually, what de Jong calls her “zebrafish transplant factory” could produce new discoveries that lead to information on what causes hematopoietic stem cells to occasionally form leukemias and how to achieve more effective bone marrow transplants in humans.
“We hope to find a gene that we didn’t already know, because it’s really an unbiased screening mechanism,” de Jong said. “We can thus use that method to identify new genes that are important for hematopoietic stem cell engraftment and function.”
Even one of the difficulties in developing the zebrafish transplant may be a positive. Because the fish, like humans, must be immunologically matched to facilitate transplant, researchers can study the factors that control whether a matched or mismatched transplant is rejected or accepted. That information could help expand the donor pool for a patient (by allowing for less than perfectly-matched transplants) and prevent complications of bone marrow transplants, such as graft versus host disease.
“I’m excited at the idea that we could have a model for graft versus host disease in the fish,” de Jong said. “We could then utilize the really strong genetics and unbiased screening capability in zebrafish to find things that decrease GVHD in our model, and those could potentially be transferred to human transplants.”
de Jong, J., Burns, C., Chen, A., Pugach, E., Mayhall, E., Smith, A., Feldman, H., Zhou, Y., & Zon, L. (2011). Characterization of immune-matched hematopoietic transplantation in zebrafish Blood DOI: 10.1182/blood-2010-09-307488