McCulloch, Ernest

Ernest A. McCulloch

University of Toronto Ontario Cancer Institute

Till, James

James Till

University of Toronto Ontario Cancer Institute

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-forming — or hematopoietic — system 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 specializing — or differentiating — into 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 nodule — or colony, adopting the language of bacteriology — descended 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 produce — at low frequency — exactly 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 time — whether 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 cells — red cells, white cells, and platelets. These features meant that the colony-forming cells represented a new class of progenitor cells — ones 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.

diagram of hematopoiesis

From stem to stern. This simplified diagram of hematopoiesis illustrates the two main properties of stem cells — self-renewal and differentiation — defined by Till and McCulloch. Each stem cell can either replace itself or can begin the path toward specialization, becoming a committed precursor cell. Precursor cells are converted to differentiated cells through the action of cytokines, some of which are shown. Some details of this scheme are still controversial.

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 fate — whether 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 mouse — with flaws in the “Sl” gene — seemed 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 cytokines — proteins made by cells that affect the behavior of other cells — and 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

Key publications of Ernest McCulloch and James Till

McCulloch, E. and Till, J.E. (1960). The radiation sensitivity of normal mouse bone marrow cells, determined by quantitative marrow transplantation into irradiated mice. Rad. Res. 13, 115–125.

Till, J.E. and McCulloch, E.A. (1961). A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Rad. Res. 14, 213–222.

Becker, A.J., McCulloch, E.A., and Till, J.E. (1963). Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature. 197, 452.

Siminovitch, L., McCulloch, E.A., and Till, J.E. (1963). The distribution of colony-forming cells among spleen colonies. J. Cell. Comp. Physio. 62, 327.

Till, J.E., McCulloch, E.A., and Siminovitch, L. (1964). A stochastic model of stem cell proliferation, based on the growth of spleen colony-forming cells. Proc. Natl. Acad. Sci. USA. 51, 29–36.

McCulloch, E.A., Siminovitch, L., and Till, J.E. (1964). Spleen colony formation in anemic mice of genotype W/Wv. Science. 144, 844–846.

McCulloch, E.A., Siminovitch, L., Till, J.E., Russell, E.S., and Bernstein, S.E. (1965). The cellular basis of the genetically determined hemopoietic defect in anemic mice of genotype Sl/Sld. Blood. 26, 399–410.

Till, J.E. and McCulloch E.A. (1980). Hemopoietic stem cell differentiation. Biochim. Biophys. Acta. 605, 431–459.

Award presentation by Thomas Stossel

To begin to explain what today's Basic Science Lasker Award winners, Ernest McCullough and James Till, accomplished, let me point out that the reason we are all enjoying this luncheon is that we have blood cells — red blood cells that carry oxygen around, white blood cells that protect us from germs and platelets that keep us from bleeding. We constantly produce these cells, and we make trillions of them every day.

Since the late 19th century we could recognize these different blood cells but we had no understanding as to how they were produced. To be sure, we knew that they came from inside of the bones — in the bone marrow — and we could identify immature forms of them. But how they really were made was a mystery.

Thanks to McCullough and Till, we now know that these cells originate from bone marrow cells that we cannot readily see, called blood-forming (or ‘hematopoietic’) stem cells. Groping about how to impart what this means, I was struck that no really appropriate analogies exist in our common experience — oak seeds make oak trees; carrot seeds turn into carrots; and when the seeds do their work, they’re gone. The best I could do was to propose that stem cells are like the chefs in the kitchen who prepared the appetizer, the main course and the dessert for our lunch from their respective ingredients. I need to stretch the analogy by saying we aren’t able to visualize the chefs in the kitchen, so all we see are appetizers, main courses and desserts in various stages of preparation, just as when we look in the bone marrow we see blood cells in development but not the stem cells that produced them. The absence of a more suitable comparison underscores the magnitude of McCullough’s and Till’s conceptual achievement.

A driving force behind McCullough’s and Till’s research was the realization that radiation from nuclear bombs and X-ray machines can kill blood-cell production. Researchers had shown that blood cell production in mice exposed to such radiation could be rescued by injections of bone marrow from healthy mice — it’s as if we radiated the Pierre Hotel kitchen and killed the chefs, yet still managed to have our full lunch by importing cooks from the Plaza Hotel. But what is doing the job of making blood cells in the bone marrow or the lunch in the kitchen?

Starting in the late 1950s, McCullough and Till systematically measured how many bone marrow cells were needed to restore blood cell production in radiated mice. These measurements enabled them to calculate that the number of cells in the donor bone marrow carrying out this rescue was very small — just as in the kitchen, one chef can prepare a lot of meals. Even more importantly, they observed that the spleens of the radiated mice receiving replacement bone marrow contained small clumps of blood cells, which they called “colonies.” Each colony contained red blood cells, white blood cells, and platelets. This finding meant that something capable of making all blood cell types, a blood-forming stem cell, was trapped in the spleen. It’s as if when we imported the Plaza kitchen to the Pierre, most of the chefs we brought in would work in the kitchen. But a few chefs got trapped in the Pierre Hotel elevators and went to work making appetizers, main courses, and desserts there.

Now, if each chef in the elevator had a signature, like the food-coloring curlicues that adorn plates in gourmet restaurants, and we saw such distinct signatures on collections of appetizers, main courses, and desserts, we could conclude that each set of lunches in the elevator was made by a particular chef. In a truly elegant experiment, McCullough and Till imparted distinct genetic signatures to blood-forming stem cells in donor bone marrow and documented that the blood cells in each spleen colony bore a particular genetic imprint, proving that diverse blood cells come from individual stem cells.

If, like carrot seeds, the chefs in the kitchen simply transformed themselves into our lunches, ceasing to be chefs, no lunches could be served here tomorrow. In the same fashion, if stem cells merely morphed into mature blood cells, blood cell production would cease. To sustain the output of blood cells, stem cells have to be capable of self-replication. McCullough and Till demonstrated this self-renewal potential of stem cells by repeatedly taking individual spleen colonies from one set of mice and injecting them into irradiated mice to generate new colonies.

With these elegant experiments and others performed over a decade, McCullough and Till established the reality of blood-forming stem cells, even though, like our invisible chefs, no one actually knew what they looked like. In recent years, the blood-forming stem cells have indeed been isolated. As anyone who reads newspapers knows, embryonic stem cells may allow us to regenerate damaged body parts. McCullough’s and Till’s work became the foundation of the most active field of hematology research, spawning like stem cells themselves, a vast effort that has defined how and in response to what stem cells divide and mature into specific blood cells. It has made into a quantitative science the technology of blood stem cell transplantation, a major medical procedure that sustains life when blood cell production fails. As a hematologist who treats patients with blood diseases, I am especially pleased to participate in the awarding of the Lasker Award to Ernest McCullough and James Till.

Acceptance remarks

Interview with Ernest McCulloch and James Till