Scientists at the University of Michigan Medical School have discovered the biological equivalent of a grocery store bar code on the surface of primitive, blood-forming stem cells in mice. Called hematopoietic stem cells, they give rise to all the different types of specialized cells found in blood.
By reading the bar code, scientists can separate stem cells from their more advanced descendants – progenitor cells that already are committed to becoming one type of blood cell. The secret, according to U-M scientists, is to look for the presence or absence of cell surface receptor proteins expressed by a family of genes called SLAM.
Scientists knew that the 10 or 11 genes in the SLAM family helped regulate the development and activation of white blood cells called lymphocytes, but no one knew they also were associated with hematopoietic stem cells.
"SLAM is the first family of receptors whose patterns of gene expression can be used to precisely distinguish hematopoietic stem cells from progenitor cells, and to identify stem cells in tissue sections," says Sean Morrison, Ph.D., an associate professor of internal medicine and of cell and development biology in the U-M Medical School and a Howard Hughes Medical Institute investigator.
Results of the research study will be published in the July 1 issue of Cell.
The discovery will be valuable to scientists working in the rapidly advancing field of stem cell science. Currently, scientists must search for many different markers and use complex procedures to separate rare hematopoietic stem cells, or HSCs, from other cells in a blood sample. Using SLAM markers will streamline the process considerably, says Mark J. Kiel, a student in the U-M Medical School's M.D./Ph.D. program who is co-first author of the study.
"The classical markers work, but they are complex and it takes a high level of skill to obtain a pure sample of HSCs," Kiel says. "We showed that using just two SLAM markers gives you similar or better purity than 10 to 12 of the best classical markers combined. It reduces the skill and time required to pick out a hematopoietic stem cell and makes the process simpler and less labor intensive."
"Many of the current markers we use to purify HSCs are expressed as a gradient from high to low across the hematopoietic cell hierarchy," says Omer H. Yilmaz, co-first author and a student in the Medical School's M.D./Ph.D. program. "What's really exciting about this finding is that now we have markers that turn on and off abruptly as stem cells differentiate."
Kiel and Yilmaz used microarray analysis to measure levels of gene activity in three types of cells – hematopoietic stem cells, multipotent progenitor cells and fully developed bone marrow cells. They selected genes that were expressed at higher levels in the HSC population, as opposed to the other two types of cells. CD150, the founding member of the SLAM family of cell surface receptors, was near the top of the list.
To see if CD150 was expressed on hematopoietic stem cells, Kiel and Yilmaz transplanted CD150+ and CD150- cells into laboratory mice whose bone marrow had been destroyed by lethal doses of radiation. Healthy bone marrow was restored in six of six mice receiving CD150+ cells, but reconstitution was successful in only one of nine mice receiving CD150- cells.
When they followed the same procedure to test another SLAM marker, CD244, the results were completely opposite. Cells with CD244 receptors on their surface membranes were unable to reconstitute bone marrow in irradiated mice for more than a few weeks, while cells without CD244 receptors were able to reconstitute indefinitely. This indicated that CD244+ cells were a marker for multipotent progenitor cells (MPPs), but not for hematopoietic stem cells.
In a third step, the team tested another SLAM marker called CD48, and found it to be expressed only on more advanced cells. It was not present on either HSCs or progenitor cells.
"Each marker is expressed at a different stage of the hematopoiesis hierarchy," Morrison says. "Selecting for cells with the combination of CD150+, CD48- and CD244- markers produced a sample that was highly enriched for HSCs. These cells were extremely rare – fewer than one out of 10,000 whole bone marrow cells had this combination of surface markers."
In the Cell paper, U-M scientists describe how the new markers helped them see hematopoietic stem cells in stained tissue sections from mouse bone marrow and spleen, something that was not possible with existing markers. Knowing where primitive hematopoietic stem cells hang out in blood-forming organs will give scientists important clues to how they work, according to Morrison. For this reason, the identification of markers that allow the localization of HSCs in tissues has been a long-sought goal in the field of hematopoiesis.
"Until this study, we didn't know exactly where to look for HSCs within bone marrow and the spleen," Morrison explains. "Most scientists believed they stayed in close contact with cells called osteoblasts, which line the inside of hollow cavities in bone that contain bone marrow. Using SLAM markers, we discovered hematopoietic stem cells also congregate in the sinusoidal endothelium – porous cells lining the inside of blood vessels that carry oxygen throughout bone marrow and the spleen.
"The presence of HSCs in sinusoidal endothelium could explain how stem cells are able to enter the bloodstream so quickly," Morrison adds. "It also suggests that endothelial cells produce substances that regulate and nurture the growth of these stem cells. If we can identify these growth factors, it could help us learn how to manipulate and grow colonies of HSCs outside the body, which could lead to new medical treatments for sickle cell anemia, leukemia and other types of cancer."
As with most discoveries in stem cell science, U-M scientists say their work raises new questions for future research. So far they have identified three SLAM markers on hematopoietic stem cells, but they have no idea what the other eight genes in the family do. SLAM gene activity creates receptors on the surface of blood-forming stem cells, but what do these receptors do? Are SLAM receptors present on other types of stem cells?
Perhaps the most important unanswered question for future applications in medicine is whether these same markers are expressed on human hematopoietic stem cells. Kiel and Yilmaz confirmed the existence of SLAM markers on HSCs in several strains of laboratory mice, but whether they will be found on human cells remains "a big maybe," according to Kiel.
"If SLAM family receptors are expressed on human HSCs, these new markers could dramatically enhance the purification of such cells, potentially making bone marrow transplants safer and more effective," says Morrison, who adds that Kiel and Yilmaz have already started tests to look for them.
Toshide Iwashita, M.D., Ph.D., a former research fellow in Morrison's laboratory and co-first author on the study, was responsible for the tissue section analysis. Cox Terhorst, Ph.D, from the Beth Israel Deaconess Medical Center and Harvard Medical School also collaborated in the research.
Morrison's research is supported by the Howard Hughes Medical Institute, the National Institutes of Health and the U.S. Army Research Office. Kiel is supported by a U-M Medical Scientist Training Program Fellowship and Yilmaz is funded by a predoctoral fellowship from the U-M's Institute of Gerontology.