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From the marrow, or from the blood?

DNA-array analysis allows Lynn Graf to identify genes that could determine why the source of stem cells matters in transplants
Dr. Lynn Graf talks with Dr. Shelly Heimfeld.
Dr. Lynn Graf, staff scientist, discusses with Dr. Shelly Heimfeld, director of the Large-Scale Cell Processing Facility, the characterization of genes that could lead to refinements in the stem-cell transplant procedure. Photo by Clay Eals

Clinical Research Division scientists recently began studies to understand the biological basis for a curious disparity discovered last year - that patients transplanted for advanced blood cancers fare better with stem-cells harvested from blood than those harvested from bone marrow.

The new look at this topic involves tesing out the molecular differences that distinguish blood-derived from marrow-derived cells.

Dr. Lynn Graf, a staff scientist in Dr. Beverly Torok-Storb's laboratory, identified a subset of genes whose activity differs among the same cell types harvested from the two sources. She used DNA-array analysis, a technique to examine the expression of thousands of genes at once.

Future characterization of these genes could lead to refinements in the stem-cell transplant procedure.

The study was co-authored by Dr. Shelly Heimfeld, director of the Large-Scale Cell Processing Facility, and published last fall in Biology of Blood and Marrow Transplantation. The paper was honored as the best basic-science article in February at the annual meeting of the American Society for Blood and Marrow Transplantion, for which Graf received a $5,000 award.

Crucial ingredient

Regardless of their source, adult hematopoietic (blood-forming) stem cells from a tissue-matched donor are the crucial ingredient for a transplant's success. These self-renewing cells can regenerate an entire blood and immune system that replaces the cancerous bone marrow of patients with leukemia and other blood malignancies.

Bone marrow is harvested from the hip region of donors in a procedure that requires general anesthesia. In contrast, when stem cells are harvested from blood, donors are given a drug, G-CSF, that stimulates stem-cell production and circulation. Then an apheresis machine removes their blood through an intravenous line in one arm, separates out the cells of interest and returns residual blood products back to the donor through a line in the donor's other arm.

Although blood-derived stem cells provide a survival advantage over bone-marrow-derived stem cells for patients with high-risk cancers, scientists have been at a loss to explain these findings. Some scientists theorize that G-CSF-stimulated blood contains greater numbers of stem cells than the bone-marrow preparations, while others have proposed that the cells themselves are metabolically distinct from one another. It is also possible that other, undefined graft 'facilitating' or immunocompetent cells make the difference. These issues are under investigation in Transplantation Biology.

To begin to address these questions, Graf used the Large-Scale Cell Processing Core to isolate stem cells from bone marrow and G-CSF-stimulated blood. Although completely pure preparations of stem cells would be ideal, they remain elusive. Scientists, however, have identified a cell marker called CD34 that is unique to the surfaces of a few types of blood cells, including hematopoietic stem cells. Isolating cells on the basis of CD34 expression allows a stem-cell-rich preparation to be obtained.

Graf then isolated RNA, essentially the "readout" of which genes are turned on, from each of the cell populations. The RNA was mixed with glass slides coated with DNA from more than 6,000 genes to determine which RNA molecules adhered to their respective genetic blueprints - an indication of which genes were activated in the cell. The study was the first to look broadly at gene-expression differences in the two CD34 cell populations.

Qualitative differences

Graf used stringent criteria to identify genes that were significantly and reproducibly expressed at higher or lower levels in the peripheral blood CD34 cells, compared to those derived from bone marrow. Her results indicated that qualitative differences exist between the two cell populations, meaning that their functional differences with respect to transplant outcome are unlikely to be due solely to their quantity.

"It was easiest to classify the genes that were consistently expressed at lower levels in the peripheral blood cells," she said. "We found 38 genes whose expression decreased more than threefold in the G-CSF-stimulated cells, and we were able to classify these into several broad categories."

Graf found that some of these genes specify known components of the cell-division cycle, the complex circuitry that enables one cell to become two. Based on what is known about the biology of stem cells, Graf said this was not surprising.

"Others have found that when the stem cells leave the bone marrow and are circulating in the peripheral blood, they are not cycling," she said. "Functionally, it's been shown that these non-cycling cells offer an advantage during transplantation. But even though they are quiescent, they can be activated quickly to divide and differentiate into the multiple cell types that arise from these stem cells."

Graf said although further studies must be done, her results increase scientists' understanding of these blood cells and could eventually lead to refinements in the stem-cell transplant procedure.

Recently, Graf has repeated her analysis and has found additional genes whose expression differs between the two cell populations.

"Most interesting would be to identify genes that help to explain why the cells aren't cycling," she said. "These might be genes that actually regulate the cell cycle."


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