A research team from the Massachusetts General Hospital (MGH) has identified a key protein that appears to control the development and proliferation of hematopoietic stem cells. These cells, which are found in the bone marrow and blood, are capable of developing into any kind of blood cell. The discovery of this molecular switch, a protein called p21, may solve a major limitation to the use of these stem cells - the fact that naturally they occur in very small numbers and rarely reproduce.
"Hematopoietic stem cells tend to be very rare and quiescent [inactive]," says David Scadden, MD, of the MGH Cancer Center and the MGH AIDS Research Center, who is the senior author of the study appearing in the March 10 issue of Science. "Stem cell transplants - as well as gene therapies that may someday be developed using these cells - need a lot of of cells to ensure they will engraft in a recipient's marrow. If we could induce stem cells to proliferate without differentiating into specific types of blood cells, we could get the numbers of stem cells we need from a single tube of blood instead of having to extract a large amount of bone marrow.
"Perhaps even more exciting are recent reports that some hematopoietic stem cells have the potential to develop into cells of many different types - into brain, muscle or cartilage cells in addition to blood cells," Scadden continues. "If we could identify and expand the population of those cells, the possibilities for treating conditions from injuries to cancer would be enormous."
Previous attempts to increase the proliferation of hematopoietic stem cells used cellular growth factors called cytokines, which usually ended up inducing the stem cells to differentiate into specialized blood cells. Even when cytokine treatment succeeded in causing stem cells to proliferate, the cells would "burn out" and stop reproducing after a few generations. "We wanted to try another strategy," Scadden explains. "Instead of pushing on the accelerator to speed up stem cell reproduction, we wanted to release the brake that was suppressing natural proliferation."
The growth, development and reproduction of cells involve an ordered series of steps called the cell cycle. Cells that do not normally proliferate or that proliferate rarely are suspended at a stage called G0. Previous research had identified a protein called p21 that appears in some cells to play a role in controlling whether the cell remains at G0 or whether it enters the cell cycle to proliferate, but its role in stem cells had been unknown. When the MGH investigators studied what appeared to be quiescent stem cells from human bone marrow, they found high levels of p21.
To better define the role of p21, Tao Cheng, MD, of Scadden's lab, the paper's first author, conducted a number of tests using mice lacking the gene for p21. Analysis of stem cells from these p21 "knockout mice" showed they had substantially more stem cells than did normal mice and that fewer stem cells were in a quiescent state, supporting the theory that p21 suppresses stem cell proliferation. When the mice were injected with a chemotherapy drug that targets proliferating bone marrow cells, significantly fewer of the knockout mice survived than did the normal mice, suggesting that, by maintaining a population of quiescent cells, p21 helps protect the hematopoietic system from a variety of toxic effects.
To investigate mechanisms behind the previously observed cytokine-associated stem cell burnout, Cheng and his colleagues conducted a series of bone marrow transplants on the knockout and normal mice. Mice that had received bone marrow transplants became donors for a subsequent group of mice, and the process was repeated - essentially transferring an initial group of stem cells and its descendants from one animal to another. Each time the transplanted marrow repopulated an animal's hematopoietic system, the cells were exposed to cytokines.
By the third series of transplants, some of the mice lacking p21 began to die; after the fifth transplant, none of the knockout mice survived, but half of the mice with normal p21 genes survived. "In the mice without p21," Scadden says, "it looks like cytokine stimulation is pushing all the bone marrow cells to differentiate, leaving no stem cells by the time of the third or fourth transplant. But it appears that some of the animals with p21 are capable of maintaining a limited population of stem cells. Now we need to find out whether an environment without the differentiation-pushing cytokines would allow p21-knockout cells to proliferate at higher levels."
Scadden adds that the ability to grow large populations of stem cells has potential even beyond broadening the applicability of current stem cell transplant techniques. "One existing underused resource is the supply of umbilical cord blood in special banks around the country. The amounts taken from each cord currently would not be sufficient to treat an adult needing a stem cell transplant. If we could expand the number of cells available from donated cord blood, that would be a huge resource, particularly for minority patients for whom it often is difficult to find matched donors."
The study's co-authors along with Cheng and Scadden are Neil Rodrigues, MS, Hongmei Shen, PhD, and David Dombkowski of the experimental hematology group of the MGH AIDS Research Center, and Yong-guang Yang, MD, PhD, and Megan Sykes, MD, of the MGH Transplantation Biology Research Center. The research was supported by grants from the Defense Advanced Research Projects Agency of the Department of Defense, the Richard Saltonstall Charitable Foundation and the National Institutes of Health.
Journal
Science