Red Fish, Green Fish

Zebrafish reveal drugs that may improve bone marrow transplant

A conceptual view of the “Dr. Seuss experiment.” Blood stem cells from zebrafish marrow were either colored green and treated with different test chemicals, or colored red and left untreated. The cells were then injected into other zebrafish so researchers could see which color grafted better. Image: Ellen van Rooijen and Vera Binder

Using large-scale zebrafish drug-screening models, researchers at Harvard Medical School and Boston Children’s Hospital have identified a potent group of chemicals that helps bone marrow transplants engraft or “take.”

The findings, published July 23 in Nature, could lead to human trials in patients with cancer and blood disorders within a year or two, said senior investigator Leonard Zon, professor of stem cell and regenerative biology in the Faculty of Arts and Sciences at Harvard University.

The compounds, known as epoxyeicosatrienoic acids, or EETs, boosted stem cell engraftment in both zebrafish and mice and could make human bone marrow transplants more efficient. Better engraftment could also allow umbilical cord blood to be used as an alternative to marrow as a source of blood stem cells, greatly increasing a patient’s chances of finding a matched donor and enhancing safety.

“Ninety percent of cord blood units can’t be used because they’re too small,” explained Zon, who is also the Grousbeck Professor of Pediatrics and director of the Stem Cell Research Program at Boston Children’s. “If you add these chemicals, you might be able to use more units. Being able to get engraftment allows you to pick a smaller cord blood sample that might be a better match.”

EETs are fats that appear to work by stimulating cell migration. They were among the top hits in a screen of 500 known compounds conducted in the Karp Aquatics Facility at Boston Children’s.

While zebrafish have previously led Zon’s team to compounds that boost blood stem cell numbers, such as prostaglandin (currently in several clinical trials under the name ProHema), the new drug screen specifically tested the stem cells’ transplantability and engraftment success.

The screen was done in a lab-created strain of zebrafish called Casper. Because Casper is see-through, Zon and colleagues could visually compare engraftment of transplanted blood stem cells chemically tagged to glow green or red—in what they’ve dubbed the “Dr. Seuss experiment.”

Led by co-first authors Pulin Li, Jamie Lahvic and Vera Binder, the researchers first used tagging to color the fishes’ marrow either red or green, then removed blood stem cells for transplantation. The green cells were incubated with various chemicals, while the red cells were left untreated.

Next, the researchers injected a mixture of green and red marrow cells into other groups of zebrafish (10 fish per test chemical). The team then visually tracked the cells’ activity in the transplant recipients and measured the green-to-red ratio.

“We call this a competitive transplant model because we can literally compete a green stem cell against a red stem cell and see what wins,” said Zon. “The expectation was that if a chemical didn’t increase engraftment, all the fish would be equal parts red and green. But if it was effective, green marrow would predominate.”

That was the case for green marrow incubated with EETs, a finding that held up over thousands of marrow transplants. “In a mouse system, this experiment would cost $3 million,” noted Zon. “In fish, it cost about $150,000.”

In a smaller-scale set of mouse experiments, the team confirmed EETs’ efficacy in promoting homing and engraftment of transplanted blood stem cells in mammals.

Although EETs are chemical cousins of prostaglandin (both are made from arachidonic acid, and both are made during inflammation), EETs work in a different way, by activating a pathway known as PI3K. EETs also enhanced PI3K activity in human blood vessel cells in a dish.

After more studies in human cells to tease out how EETs work, Zon hopes to begin clinical trials of EETs within the next two years, likely in the setting of cord blood transplant. The lab is also investigating its other top hits from the zebrafish screen.

“Every new pathway that we find has the chance of making stem cell engraftment and migration even better,” said Zon. “I think we’ll end up being able to manipulate this process.”

Supporters of the study include the Howard Hughes Medical Institute, the National Institutes of Health (1R01HL04880, Z01 ES025034, P50-NS40828, P30-HD18655, ROCA148633-01A5), the German Research Foundation (DFG) and the Care-for-Rare Foundation. Zon is a founder and stockholder of Fate, Inc., and a scientific advisor for Stemgent. Coauthor George Q. Daley is a member of the scientific advisory boards of MPM Capital, Inc., Epizyme, Inc., and iPierian, Inc.

Adapted from a Boston Children’s news release.