During the early days of an embryo’s development, the heart begins to beat. It turns out that beating heart does more than circulate the embryo’s small existing blood supply. Howard Hughes Medical Institute investigators have found that the blood’s movement through the aorta triggers the production of new blood stem cells, which will give rise to all the red and white blood cells the organism needs to survive.
The researchers have also discovered that this essential biomechanical signal can be mimicked with drugs. The findings could help clinicians expand the supply of blood stem cells needed to treat leukemia, autoimmune disorders, and other diseases.
“The biomechanical stress of early blood flow is needed for an organism to grow its initial supply of blood cells,” says George Daley, an HHMI investigator at Children’s Hospital Boston and senior author on one of the reports, published May 13, 2009, in Nature. The second report, with HHMI investigator Leonard Zon as senior author, was published May 13, 2009, in the journal Cell.
The two investigators homed in on the importance of flow for blood development from different angles.
“For a long time, I’ve had the idea that the initiation of the heartbeat in an embryo is crucial for the creation of blood stem cells,” says Daley, who hopes to grow blood stem cells from pluripotent stem cells in the laboratory so that they can be infused into patients to treat a range of diseases. He began investigating the idea with bioengineers at the Massachusetts Institute of Technology in 2001, and in early experiments Daley’s team noticed that streaming a fluid across embryonic stem cells growing in a bioreactor did spur the development of new blood cells.
Daley shelved that research for a time, but it took off again when he began collaborating with Guillermo García-Cardeña, from Brigham and Women’s Hospital in Boston. García-Cardeña invented a miniaturized cell-culturing system that can impose different degrees of fluid flow on cells. The system grows cells on a surface beneath a shallow inverted cone, which spins at different speeds to create different rates of fluid flow.
García-Cardeña’s team seeded the system with embryonic stem cells, and found that spinning fluid at a specific rate increased the production of blood stem cells. The system produced the most blood stem cells when the fluid force was equivalent to the force of blood flow in a developing mouse aorta when the heart begins beating, at about day ten and a half of embryonic development. “The cells are tuned to sense the right force,” says Daley.
Researchers had previously established that blood arises in two waves within mouse embryos. Early blood is produced in small quantities outside the embryo, in the yolk sac, while the later blood stem cells bud from the walls of the developing aorta. Daley’s work shows that when the embryo’s heart starts to beat, the frictional forces against the walls of the aorta trigger the production of blood stem cells.
Zon, also at Children’s Hospital Boston, approached the problem in a different way. Zon has been working to identify compounds that boost production of blood stem cells, with the ultimate goal of increasing the number of blood stem cells in bone marrow and umbilical cord blood, which are transfused into patients to rebuild their immune systems after cancer therapy.
To this end, Zon developed a system to quickly test thousands of drugs in zebrafish. This approach tags zebrafish embryos with a purple dye that appears only in new blood stem cells. Since zebrafish embryos are translucent, laboratory workers can watch new blood stem cells as they are generated. “You could never do this screen in any other animal, you have to do this in zebrafish,” says Zon. “We're literally looking at the aorta as blood stem cells are being born.”
In 2007, Zon and colleagues identified a compound, called prostaglandin E2, that increases the production of new blood stem cells. The drug screens also highlighted a class of compounds that increased blood flow, and showed that these compounds increased the production of blood stem cells. Until then, Zon says, “it was not known at all that blood flow is a signal that produces blood stem cells in embryos.”
Zon then worked with mutant zebrafish embryos missing a key heart protein. The hearts in these embryos never begin beating. “You never get circulation in these fish, and if you look in their aorta, you see very few blood stem cells. That confirmed to us that blood flow is truly required to make the blood stem cells,” Zon says.
With further experiments, Zon’s team found a group of drugs that enabled the fish without beating hearts to produce blood stem cells. These drugs all had something in common: they generated nitric oxide, a well-known molecule used by cells to talk to each other. Normal blood flow enhances the production of nitric oxide. “That's at least one of the critical signals that blood flow is triggering,” Zon says. But their experiments demonstrate that nitric oxide can actually supplant the need for flow.
Daley, too, found that nitric oxide is crucial for development of blood stem cells. He used a drug to block nitric oxide production in pregnant mice, and found a marked decrease in blood stem cells in the embryos they carried.
“The lesson here may be that as we try to grow blood stem cells in the laboratory, any number of drugs that produce nitric oxide may be valuable,” Daley says.