Basic Medical Research Award
Ernest McCulloch and James Till
For ingenious experiments that first identified a stem cell the blood-forming stem cell which set the stage for all current research on adult and embryonic stem cells.
The 2005 Albert Lasker Award for Basic Medical Research honors two scientists who first identified a stem cell, which set the stage for all current research on adult and embryonic stem cells. By the turn of the 20th century, scientists were postulating the existence of self-renewing cells that could specialize for a wide variety of purposes. In a series of ingenious and elegant experiments 60 years later, Ernest McCulloch and James Till demonstrated that such a type of cell in the blood-formingor hematopoieticsystem existed. They established the properties of stem cells, which still hold true today. Furthermore, they lay the foundation for the isolation of stem cells and for the detection of proteins that help these precursor cells develop and mature. Till and McCulloch's discoveries explained the basis of bone marrow transplantation, which prolongs the lives of patients with leukemia and other cancers of the blood. Moreover, the team set a new standard of rigor for the field of hematology, transforming it from an observational science to a quantitative experimental discipline.
In the late 1950s, McCulloch and Till, newly appointed scientists at the Ontario Cancer Institute in Toronto, began to explore how ionizing radiation affects mammalian cells. This enterprise held great importance for several reasons. Scientists were trying to understand why and under what circumstances radiation therapy defeated cancer. Furthermore, the Cold War was in full swing, so people wanted to devise strategies to save military personnel who might sustain whole-body irradiation from nuclear weapons. Finally, the technique of bone marrow transplantation was in its infancy; investigators knew that this treatment replenished the essential cells of the blood system and were eager to define the source of these cells.
Till and McCulloch worked out a system for measuring the radiation sensitivity of bone marrow cells. The researchers accomplished this feat by zapping mice with a dose that would kill the animals within 30 days if they did not receive a bone marrow transplant of fresh, undamaged cells. To obtain the donor material, the team divided bone marrow from unirradiated animals into portions, and exposed each to a different amount of radiation. The largest dose killed enough donor cells to obliterate their ability to rescue the mice; the smallest dose left much of it intact. The investigators knew how many unirradiated cells were needed to save the animals, so by counting the mouse survivors, they could infer the number of cells that had withstood a given amount of radiation.Clumps or Clones?
The scientists subsequently repeated the experiment but performed autopsies on the animals 10 days after transplantation. They noticed spleen nodules that contained dividing cells, some of which were specializingor differentiatinginto the three main types of blood cells: red cells, white cells, and platelets. The number of nodules was directly proportional to the number of live marrow cells the irradiated animals had received. The crucial entity was rare: About 10,000 marrow cells had to be injected for each nodule observed.
Aspects of the experiment and its results reminded the researchers of the test for live bacteria, which depends on the ability to reproduce. Scientists disperse bacteria on a Petri dish and each bacterium multiplies to form a colony. Counting colonies reveals the number of viable cells that were in the original sample. McCulloch and Till's experiment, however, didn't distinguish whether the spleen nodules originated from single cells that reproduced and differentiated, or came from clumps of multiple kinds of cells that then simply divided. The researchers wanted to find out whether all of the cells in a noduleor colony, adopting the language of bacteriologydescended from a single cell (and thus represented a clone) or from multiple cells.
To accomplish this task, they needed cells that carried unique inheritable markers. They realized that irradiating cells would produceat low frequencyexactly such markers in the form of visibly abnormal chromosomes. By dissecting spleen nodules into their constituent cells, Till and McCulloch could determine whether each cell from a given nodule contained the same rare chromosome. If so, the "colony-forming unit" must have been a single cell; if not, it must have been composed of multiple cells.
Andrew Becker, a graduate student working with McCulloch and Till, examined hundreds of cells from 42 nodules obtained from 36 animals. Most contained only normal cells but four contained cells with distinctive chromosomes. Almost all of the dividing cells in each of these nodules carried a unique chromosomal alteration. The colonies thus arose from a single cell.
Till and McCulloch next wanted to know whether the colony-forming cells could renew themselves, forming new colony-forming cells. To answer this question, they and their colleague Louis Siminovitch broke up spleen nodules into their cellular components. The scientists then injected irradiated mice such that each animal received most of the cells from a single colony. If the colony-forming cells could duplicate themselves, the second-round animals would develop nodules. They did, thus establishing that colony-forming cells can self renew.
Later, the team showed that the multiple cell types within a colony arose from a single cell. This experiment addressed a key question in hematology at the timewhether three separate types of precursor cells headed the lineages that produced red cells, white cells, and platelets, or whether a single common stem cell gave rise to all three lineages.
By the early 1970s, Till and McCulloch's experimental observations were clear-cut: They revealed that bone marrow transplantation owes its restorative powers to a single type of cell that not only can divide, but can differentiate into all three types of mature blood cellsred cells, white cells, and platelets. These features meant that the colony-forming cells represented a new class of progenitor cellsones that could proliferate enough to repopulate the bone marrow of an entire animal, self-renew, and give rise to specialized cells that have limited life spans. This definition of a stem cell still holds true today.
Randomness and Genetic Programs
A particular feature of the results struck the scientists, however. Although nearly all of the spleen nodules contained new colony-forming units, some had many and some had few or none.
They repeated the experiment with these second-round cells to find out whether the colonies would breed true: Would a nodule from a colony that had produced many nodules contain many colony-forming cells? The results they obtained indicated that the number of new colonies produced wasn't genetically programmed; instead it was random. Borrowing from the field of cosmic radiation, Till worked out a theory in which chance determined a stem cell's fatewhether it would begin to differentiate or instead divide to generate two new stem cells. Till tested this model of spleen-colony growth by computer simulation and the results agreed with the experimental observations. The theory remains strong today, more than four decades after its conception.
The researchers next homed in on molecules in the stem cells and the blood-forming environment that play crucial roles in stem-cell function. Elizabeth Russell and Seldon Bernstein, of the Jackson Laboratory in Bar Harbor, Maine, studied a particular strain of mouse that was anemic and exceptionally susceptible to radiation. The animals' anemia could be cured by injection of cells from mice that carried a regular version of the so-called "W" gene. McCulloch, Till, and Siminovitch showed that these genetically normal animals were donating colony-forming cells to their anemic siblings. Furthermore, they found that bone marrow from the anemic mice didn't form colonies when injected into genetically intact but irradiated mice. The anemic mice therefore carried a genetically encoded defect in their blood-forming stem cells.
A different strain of mousewith flaws in the "Sl" geneseemed very similar to the "W" mice, at least on the surface. These animals were also anemic and unusually radiation sensitive. So Till, McCulloch, and Siminovitch performed analogous experiments on them, expecting similar results. The results surprised them. Marrow from these mice behaved normally when injected into irradiated recipients. However, marrow from genetically normal mice didn't cure their anemia. These observations suggested that, rather than carrying defects in the stem cells themselves, the anemic "Sl" mice failed to support stem-cell growth. The results established the importance of the tissue environment in promoting normal stem cell duplication and specialization. Together, the work on "Sl" and "W" opened the door to the study of genetic regulation of stem-cell formation in mice, setting the stage for finding hematopoietic cytokinesproteins made by cells that affect the behavior of other cellsand their cellular receptors.
McCulloch and Till set a high standard for work on cell progenitors, and their findings strongly supported the hypothesis that cells with the capacity to self-renew, divide, and differentiate along many lineages existed and were available for rigorous analysis in adult animals. This finding paved the way for current attempts to physically isolate such cells, study their characteristics, and develop them for medical use. It also encouraged the pursuit of other types of stem cells, including embryonic stem cells. Like the stem cells they discovered, Till and McCulloch's work has differentiated and matured in many directions.
By Evelyn Strauss, Ph.D.