The evidence found in zebrafish and mice may lead to new methods to coax embryonic stem cell-like cells (known as induced pluripotent stem cells or iPS cells) into producing blood-forming stem cells for use in the clinic. Such a method could be a particular boon as an alternative source of life-giving cells for the two-thirds of people with leukemia who do not have a matched donor, typically a brother or sister, for bone marrow transplantation, said Leonard Zon of Harvard Medical School. Leukemia patients are often treated with high doses of chemotherapy, but the treatment also leaves them with a crippled immune system.
Definitive hematopoietic stem cells that are capable of self-renewal and production of all mature blood lineages arise during embryogenesis, the researchers explained. Both the timing of the blood-forming stem cells' induction and the gene programs regulating this process are well conserved across vertebrate species.
"It's easy to make an iPS cell into a red blood cell," Zon said. "The real question is whether anybody can make a [hematopoietic stem] cell that is more self-renewing" The most efficient way to do that is to go to the source and sort out all the signals the embryo uses, he added. The new findings are another step in that direction.
In the developing embryo, blood is produced in multiple waves, he noted. In humans, the first red blood cells are produced in the yolk sac. The second wave is produced in the embryonic aorta and those blood stem cells then colonize the liver, which becomes the major blood-producing organ in the fetus.
Zon's group has been on a mission in search of chemicals that could amplify the production of blood-forming stem cells in the aorta of developing zebrafish. The transparent embryos of zebrafish coupled with their sheer numbers – each female can lay 300 eggs every week – make them ideal for developmental studies.
"We were looking in real aortas in real vertebrate embryos to see the actual stem cells," Zon said. "This couldn't be done in tissue culture."
They earlier reported one important ingredient for boosting the stem cells' production, prostaglandin E2. Now, they show initiating blood flow is also key.
Embryos with a mutation known as silent heart, which lack a heartbeat and circulation showed severely reduced hematopoietic stem cells, they found. Flow-modifying compounds primarily affected the stem cells' induction after the onset of heartbeat. The only chemicals capable of boosting their numbers prior to circulation or in the silent heart mutants were nitric oxide donors, they show.
Nitric oxide (NO) is known to play a key role in blood vessels, where it controls vessel tone and the formation of new blood vessels. The findings suggest that NO signaling is also an important link between blood flow and hematopoietic stem cells' formation.
Further studies in mice showed that an NO synthesizing enzyme is active in the stem cell-forming embryonic region in mammals. Treatments that block that enzyme or mouse embryos lacking the gene also had a reduction of hematopoietic clusters and transplantable hematopoietic stem cells.
"Here, we established a conserved role for NO in the developing hematopoietic system," the researchers concluded. "NO can function in vessel formation and specification, blood flow regulation and hematopoietic cluster formation, demonstrating that it is required in the stem cell niche for hematopoietic stem cell production. Although the function of NO in the adult marrow is complex, our findings during embryogenesis indicate that modulation of blood flow or NO signaling might be therapeutically beneficial for patients undergoing stem cell transplantation."
In addition to their potential clinical application, the new findings also help to answer a biological question Zon said he has long wondered about: why embryos make their blood stem cells in the lining of the aorta.
"In the big picture, it answers why stem cells are in the aorta to begin with," he said. "It is an ideal place to sense changes in blood flow to time blood stem cells for the production of blood in the future."
The researchers include Trista E. North, Children's Hospital, HHMI, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, Beth Israel Deaconess Medical Center; Boston, MA; Wolfram Goessling, Children's Hospital, HHMI, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, Brigham and Women's Hospital, Boston, MA, Brigham and Women's Hospital and Medical Oncology, Dana-Farber Cancer Institute, Boston, MA; Marian Peeters, Erasmus Stem Cell Institute, Erasmus Medical Center, Rotterdam, The Netherlands; Pulin Li, Children's Hospital, HHMI, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA; Craig Ceol, Children's Hospital, HHMI, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA; Allegra M. Lord, Children's Hospital, HHMI, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA, Gerhard J. Weber, Children's Hospital, HHMI, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA; James Harris, Beth Israel Deaconess Medical Center; Boston, MA; Claire C. Cutting, Brigham and Women's Hospital, Boston, MA; Paul Huang, Massachusetts General Hospital, Boston, MA; Elaine Dzierzak, Erasmus Stem Cell Institute, Erasmus Medical Center, Rotterdam, The Netherlands; Leonard I. Zon, Children's Hospital, HHMI, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA.