The Mother of All Blood Cells

Stem cells, capable of generating an endless supply of red cells, white cells, and platelets, have also generated a heated scientific controversy--and millions of dollars for the man who claims to have found them.

By Peter Radetsky|Wednesday, March 01, 1995
Deep in the very marrow of our bones reside the living forebears of our blood, the hematopoietic (blood forming) stem cells. From these rare and elusive pluripotent cells arise our oxygen-carrying red blood cells, the tiny platelets that facilitate coagulation, and the disease- fighting white cells of our immune system. The hematopoietic stem cells are nothing less than the springs that feed the river of life that flows through our veins. And Irving Weissman has found them.

To be precise, Weissman, a Stanford immunologist, and his collaborators at SyStemix, the Palo Alto, California, biotech company he cofounded in 1988, claim to have found a strong candidate for these remarkable cells. But they’re not fooling anyone. So confident are they, that SyStemix has patented not only the process used to find the cells but the cells themselves, in effect claiming ownership of these biological entities. So confident is the giant Swiss drug-and-chemical company Sandoz Ltd., that it has bought 60 percent of the SyStemix stock for a reported $392 million, making the 55-year-old Weissman and his stockholders instant millionaires.

The stakes are indeed high. In addition to stem cells’ importance in basic research, they may make possible a host of breakthrough medical advances. By giving patients the ability to make an entirely new blood supply essentially from scratch--and thus the ability to regenerate key components of the immune system--stem cell therapies could result in new treatments for various cancers, allow for bone marrow transplants without the need for rejection-fighting drugs, make possible powerful strategies against AIDS and other blood infections, and provide genetically engineered antidotes to a wide range of inherited diseases. No wonder Weissman has caused such a stir.

Yet none of the pioneering researchers in these fields currently employ the Weissman recipe for isolating stem cells--except, of course, those at SyStemix. Inarguably, the hunt for the stem cell preceded Weissman, and some say it would be proceeding just fine without him. While researchers agree that Weissman has brought recognition to a previously little-known field, they disagree as to the value of his scientific contribution. Some consider his work pivotal, others merely useful, others virtually irrelevant--regarding him, in Weissman’s own words, as a snake oil salesman. Despite Weissman’s controversial patent, there is continuing debate as to exactly what stem cells really are and how they might best be tracked down. Says stem cell pioneer and Weissman competitor Malcolm Moore of the Memorial Sloan-Kettering Cancer Center in New York City, No one has yet definitively isolated a stem cell. There’s been a lot of talk, but it hasn’t yet been pinned down so we can say, ‘This is the stem cell.’

The modern search for the hematopoietic stem cell began with the detonation of the atomic bombs over Japan in 1945. Researchers could easily see that the intense blasts of radiation destroyed blood cells and that people often died within weeks of exposure. But scientists mimicking that exposure in mice soon realized that these deadly effects could be prevented by transplanting bone marrow from genetically identical donors into the irradiated mice. The injected marrow revived the irradiated blood; thus, the researchers reasoned, the marrow must contain cells capable of regenerating other blood cells, something that mature blood cells are not able to do.

In the 1960s those speculations became fact. James Till and Ernest McCulloch of the Ontario Cancer Institute in Toronto found that after bone marrow cells were injected into irradiated mice, the animals developed nodules on the spleen. Each nodule was chock-full of white and red blood cells. By tracking genetic markers in the cells’ chromosomes, Till and McCulloch saw that the cells within each nodule had all derived from a single progenitor: one per nodule. Then, by simply counting nodules, they were able to estimate the number of progenitor cells in each batch of transplanted marrow. The cells turned out to be rare, about 1 in 1,000. Furthermore, Till and McCulloch found that in addition to generating a wide range of new blood cells, these progenitor cells were also able to reproduce themselves.

Based on this evidence, Till and McCulloch came up with a scenario that has been considered gospel ever since. All blood cells, they said, arise from a few hematopoietic stem cells, which are hidden away in the bone marrow. (Blood-forming stem cells are not the only ones we harbor: there are supposedly stem cells for skin, liver, and the intestines, as well as stem cells behind the generation of eggs and sperm.) These cells, as remarkable as they are rare, can both renew themselves and produce trillions upon trillions of blood cells, an inexhaustible supply for the life of their host body. When these new cells mature and die off--human red blood cells, for example, last only 120 days--the stem cells produce more to take their place. On average these cells produce an ounce of new blood-- some 260 billion new cells--each and every day.

Irv Weissman was a Stanford medical student and research associate when Till and McCulloch did their groundbreaking work. I knew those experiments, he says. I knew them cold. They were thrilling. The experiments gave him some insight into the problems of organ transplantation, something he’d been interested in since his high school years in Great Falls, Montana. I thought that the most important thing would be to understand the development of the immune system, he says. If you understood that, then when you did transplants you’d know what was going on. Till and McCulloch’s stem cell revelations gave him a path to follow. I began getting further and further into understanding white cell development, moving backward from mature cells to earlier and earlier cells.

Of course, Weissman wasn’t the only one moving backward. Researchers all over the world were beginning to look at the early, immature blood cells. Among them were the hematologists and biologists who made up the Dutch Mafia, as biophysicist Jan Visser laughingly describes himself and his compatriots. And it was the Dutch Mafia that first hit stem cell pay dirt. In 1984 Visser announced that he and his colleagues in the Netherlands had isolated stem cells in mice.

It was a startling achievement, one that had eluded even Till and McCulloch. The Ontario researchers had only been able to document the cells’ existence, not to pin them down. But in the intervening years molecular techniques had grown more sophisticated. Visser had at his disposal molecular probes designed to find their prey by homing in on any number of unique characteristics. The task of finding the theorized stem cells among all the varied cells in a sample of marrow was therefore akin to trying to pick someone out of a crowd by looking for a particular combination of hair color, weight, and nose shape. This didn’t mean the job was an easy one, however. Stem cells are comparatively primitive cells that lack the diversity of features associated with their mature progeny; they seem to be distinguished primarily by their dearth of unique characteristics.

To tease out the stem cells, then, Visser employed a three- pronged strategy. First he separated the cells by density. It had been discovered some years earlier that cells with stem cell activity--that is, cells that gave every indication of indeed being the long-sought hematopoietic cells--tend to be lower in density than other bone marrow cells, so Visser placed a batch of bone marrow cells (between 60 million and 100 million of them) in a centrifuge and culled only the ones that rose to the surface. That one step got rid of some 90 percent of all the cells.

Next he turned to a substance commonly used in laboratories to purify proteins: wheat germ agglutinin. Agglutinin fuses with certain sugars associated with proteins, and Visser had found that it also sticks to the sugars in the membranes of cells with stem cell activity. So he took the remaining 10 percent of the cells and mixed them with wheat germ agglutinin tagged with a fluorescent dye. With the help of a fluorescence- activated cell sorter, he was able to separate out only those cells that emitted a fluorescent glow, a sign that the tagged agglutinin was holding tight. In this way Visser further reduced his sample by 90 percent. He was now left with just 1 percent of his original bone marrow mix.

Finally he employed monoclonal antibodies. Antibodies are large Y-shaped molecules that are among the immune system’s prime infection fighters. They make a beeline for foreign proteins, grab them, and mark them for destruction by other immune forces. By the early 1980s these tiny guided missiles were among the favorite tools of molecular biologists, since they could be engineered to go after almost any target a scientist might choose. Visser sent them after one of the few stem cell characteristics then known: a protein called H-2K, which he had discovered in greater numbers on the surfaces of stem cells than on any other cell. Cells the antibodies ignored couldn’t have the protein and thus couldn’t be stem cells. These he cast aside.

The result was a further narrowing of the search. The antibodies reduced the number of cells by two-thirds: what remained was just three- thousandths of the original blend, only about 200,000 cells. When Visser injected these relatively few cells into irradiated mice, he found that it took no more than 200 of them to regenerate each animal’s entire blood system. In other words, there was at least 1 stem cell in every 1,000 bone marrow cells, the very proportion Till and McCulloch had come up with. Of course, Visser’s sample wasn’t pure--there were other cells in the mix--but out of an initial crowd of tens of millions, he was pretty close. Three years later, using newer sorting techniques, he whittled it down even further, using only 30 cells to save an irradiated mouse. He now estimates that stem cells represent 1 in 10,000 marrow cells.

Visser published his original results in the Journal of Experimental Medicine in 1984. Four years later Weissman announced in the journal Science that he and his colleagues had found the mouse stem cell. Whereas Visser’s work had elicited polite praise, Weissman’s made headlines. The Journal of Experimental Medicine is considered scientifically one of the best journals, Visser notes with some irony. Science is more popular.

Weissman and his team had taken a much more narrow approach to ferreting out their prey: they relied solely on a variety of monoclonal antibodies, each designed to pick out a different stem cell surface protein--or proteins on other cells that they had found to be absent on stem cells. For instance, one group of monoclonal antibodies targeted proteins found only on the surface of mature bone marrow cells, thus allowing the researchers to get rid of almost everything that was not a stem cell. Weissman dubbed the remaining cells Lin- (lineage minus) because the antibodies had subtracted all other cell lineages. We used those antibodies to get rid of 90 percent of the bone marrow, he says. The 10 percent that was left had stem cell activity.

To pinpoint the source of that activity more precisely, they used two other monoclonal antibodies targeted to two surface proteins they knew appeared on stem cells. One--Thy1--is found in low concentrations (designated lo); the other--Sca1--is far more common (designated +). By throwing out the cells that did not display Thy1 or Sca1, Weissman brought the number of remaining cells down to just .05 percent of the whole. He dubbed the cells left behind Thy1loLin-Sca1+; like Visser, he found that only 30 of them were needed to reconstitute the full range of blood cells in an irradiated mouse. What he had was a virtually pure batch of stem cells.

Reaction was strong and swift. Science accompanied Weissman’s paper with a news story entitled Blood-Forming Stem Cells Purified, in which Weissman, without so much as a word acknowledging earlier work toward the same goal, was quoted as saying, This is the end of the particular road that was the search for the stem cell. He contended that his methodology--based on identifying the actual look of the cells--was so efficient that it might be used to go after human stem cells. Neither Visser--who had focused more on the cells’ density and their propensity to bind to agglutinin--nor anyone else had made a claim like that.

Scientists Close in on a Vital Blood Cell, proclaimed the Wall Street Journal. Thrilled to the marrow: biologists finally corner the rare forebear of all blood cells, announced Scientific American. Weissman spread the word over television and radio, and the news was covered worldwide by the Voice of America. But despite the public applause, not all the response from the scientific community was complimentary. An editorial in Immunology Today, for example, while acknowledging that the researchers had indeed isolated a cell population with stem cell activity, asked, But does this represent any advance on previously published data? The editorial pointed out that Visser’s work had generated populations with similar characteristics and only moderately less purity, and concluded, guardedly, We have not yet reached the end of the road; perhaps just one of the side streets.

The suspicion spread that Weissman had done little that was new but had nevertheless claimed credit for a monumental discovery. In the process he had slighted the achievements of earlier pioneers, Visser’s in particular. Whether Weissman’s greater recognition was the result of his being published in the right place at the right time, or his being American rather than Dutch, or his simply knowing his way around a press conference better than Visser, many scientists felt that Weissman was receiving attention out of proportion to his accomplishment.

Did the Weissman group make a real contribution in the mouse? No, not at all, declares Malcolm Moore. All of the work had already been done elsewhere. Visser had done it a long time before. So there were some people very, very angry that he had taken credit for discovering the stem cell when he hadn’t done any of the primary work.

It’s unfortunate that it worked out that way, says stem cell researcher Ihor Lemischka of Princeton. I think Weissman would be the first to admit that his getting all the credit was unfair. It caused a lot of bad feeling. Now there are two camps in the field. There’s the Dutch axis and the Weissman crowd, and everybody else is in between.

Weissman has since explicitly acknowledged Visser’s prior contribution. He didn’t do so at the time, he says, because he simply wasn’t aware of Visser’s work. I didn’t even know about it until we finished our work, Weissman claims. Early in my career a prominent scientist told me that when he started something in earnest, he quit reading the literature. He didn’t want to be distracted by what others were doing, or spoil the fun of discovery. I use that as an excuse. It’s a bad thing to admit this, I know.

Whatever its cause, Weissman’s omission had unfortunate consequences. Says Moore, Weissman was an immunologist who suddenly hit the interface with experimental hematology without appreciating that people had been working for many, many years and knew about all these things. But somehow he obtained the credit for discovering stem cells. He trod on many toes.

Ten of those toes belonged to Visser, today the head of the recently formed stem cell laboratory at the New York Blood Center in Manhattan. Yet Visser is far less strident in his assessment of Weissman’s tactics than are some of his colleagues. In fact, he seems almost to admire Weissman’s flair. Did I feel that Weissman stole my thunder? he muses. At first I did. I felt sort of sorry about it. But that soon went away. He raised attention to this field that I could never have raised. In his jet stream, in all the noise he makes, I was drawn with him. We both traveled to meetings around the world to explain our differences. No, after all it was no problem. It was good for me. It gave me a lot of attention.

Whether he was the first to find stem cells, it is undeniable that Weissman devised a very useful approach. It was precisely because he was an outsider that he was able to pick and choose among existing techniques and combine them in a fresh way. Thy1loLin-Sca1+ was an original recipe for a stem cell.

The real value of the Weissman purification is that it describes cell-surface differences, says Lemischka. Thy1, Sca1, and lineage markers--these are tangible molecules. So in terms of saying, ‘A stem cell looks like this, it has on its surface this and that, but not these,’ Weissman defined the stem cell for the first time. Weissman’s accomplishment is more than just a purification like Visser’s; it is really informative.

McCulloch is even stronger in his praise. Weissman’s remains the most extensive purification that’s been achieved, he says. I consider his work to be seminal. The application to man was just a step beyond what he did in the mouse.

All the same, it was quite a momentous step. By the winter of 1991 Weissman and his colleagues at SyStemix had adapted the mouse stem cell recipe and announced that they had found the human stem cell. As with the mice, they selected against the lineage markers, and for the Thy1 marker. Sca1 wasn’t a practical marker to use in human cells--in humans the gene that encodes the protein has many similar-looking relatives--so the researchers substituted a protein called CD34, which had been discovered some years earlier. They dubbed the human stem cell Thy1+Lin-CD34+. Once again Weissman’s individual ingredients weren’t new, but his combination was. Yet once again the scientific community wasn’t overawed--other researchers, using many of the same markers, were already finding similar cells.

On the other hand, none of those other researchers announced their achievement the way Weissman did--with a patent. In a dramatic departure from normal scientific procedure, Weissman and his colleagues patented their method for finding their purported human stem cell and the stem cell itself some five months before their work appeared in the April 1992 issue of the Proceedings of the National Academy of Sciences. Since a patent confers exclusive rights to the thing patented, this meant that Weissman and his colleagues were the proud owners of a living human cell.

It was an audacious stroke, if not an entirely unprecedented one. You know, people patent body components all the time, says Weissman. He’s right. Genes, growth factors, blood proteins--all have received patents. Even CD34 is patented--SyStemix had to pay to use the protein. But an entire living cell?

Next thing you know, someone will patent a zygote, and you won’t be able to have a baby without a license, quipped Sloan-Kettering hematologist David Golde at the time of the announcement.

If somebody wants to commercialize having babies, who knows? replies Weissman. As with any patent, SyStemix’s patent is specifically designed to forestall the commercial exploitation of stem cells by anyone other than itself. And the company intends to guard its privilege aggressively. It doesn’t matter what process people use, they will always be infringing, declared SyStemix’s then president, Linda Sonntag.

Such talk infuriates Moore and others in the field. It seems to me an absurd patent, Moore says. I predict it won’t withstand any challenge. If they think that anybody who tries to separate and grow stem cells is going to have to pay a licensing fee to SyStemix, they’ve got another think coming. Because people can separate stem cells using different criteria from the ones outlined by the SyStemix patent. For example, in January, Harvard Medical School researchers announced that they had developed an entirely novel and relatively simple method of isolating stem cells based not on antibodies but on the principle that stem cells, which spend most of their time in a quiescent, nondividing deep sleep, are relatively unresponsive to proteins known as growth factors. The stem cells’ progeny, on the other hand, do respond to growth factors. So the Harvard team, led by hematologist David Scadden, turned on the stem cells’ offspring by applying those factors and then promptly killed them by smothering the same cells with an anticancer drug called 5-FU, which attacks metabolically active cells. Left behind were stem cells.

Jan Visser is much more philosophical than Moore. Visser sees the move as a practical necessity. Weissman needed the patent for his company.

It’s an analysis with which Weissman agrees. Money people won’t invest in something that’s not patented, he says. And if you don’t get money, you can’t do big-time research.

Weissman can do that research. Almost immediately after the patent announcement, Sandoz bought controlling interest in SyStemix, ballooning Weissman’s net worth, at the time, to an estimated $24 million. It hasn’t changed my life, he says, then chuckles. Well, I drink better wines than I did.

I would have liked to be as rich as he is, says Visser wistfully. It’s my own mistake. He did the right thing--from a family point of view. He laughs. My children are complaining.

In the clinical realm, however, the SyStemix patent has yet to become a real factor. In its own labs, SyStemix has shown that its human stem cells can indeed regenerate blood in mice that have been bred with a human immune system--the so-called SCID-hu mice. And the company has received permission to begin testing its ability to do so in humans by injecting the cells into 20 multiple myeloma patients whose immune systems have been ravaged by chemotherapy. These tests should be starting right around now; it’s taken this long to develop a technology that can use the Thy1+Lin-CD34+ recipe to isolate stem cells in large enough quantities to use in humans.

But none of the other clinical trials going on right now use the Weissman recipe. At the New York Blood Center, immunogeneticist Pablo Rubinstein is spearheading a worldwide effort to transplant stem cells by utilizing human neonatal cord blood. His approach is simplicity itself. In the fetus, stem cells continue flowing through the blood before settling into the bone marrow a few days after birth. Cord blood is therefore relatively rich in stem cells; Rubinstein and his colleagues can simply collect and transplant that whole blood and know that they are transplanting stem cells. Since the fall of 1993 the team has helped perform transplants on 13 patients--all but one of them children--suffering advanced stages of leukemia or inherited disease. The idea is to provide these people with a new source of blood to replace their own diseased cells. The results have given researchers grounds for hope. Although 3 of the patients (including the adult) died from unrelated complications, the other 10 are doing well. Their blood has been fully regenerated, their disease put into remission.

Moreover, none of these patients experienced the bane of transplantation efforts, graft-versus-host disease, in which the introduced cells recognize the host’s body as foreign and attack it. With pure stem cells, there should be no graft-versus-host disease, says Rubinstein. The immune system generated from such a cell would mature in the recipient and thus would be trained to recognize the recipient as self.

Another trial using cord blood began at Children’s Hospital in Los Angeles in the spring of 1993, when pediatric gene therapist Donald Kohn performed stem cell gene therapy on three newborns afflicted with an inherited immune system disorder called ADA deficiency, known more popularly as the bubble boy disease. Children with the disease lack the gene to produce the enzyme adenosine deaminase, or ADA. Without ADA the immune system cannot function; such children face certain early death.

ADA deficiency was the target of the very first gene therapy trials, begun in 1990 at the National Institutes of Health. Those trials involved putting corrected genes into mature white cells rather than stem cells. The corrected cells thus eventually die, which means that the therapy--while revolutionary and successful--will be able only to curtail, not cure, the disease. Stem cells, on the other hand, might provide a true cure. In his attempt to provide that cure, Kohn actually needed to isolate the stem cells in the babies’ cord blood: he did so by targeting the CD34 protein, inserting the ADA gene in the cells, and returning them to the infants. He’s hopeful that the genes have taken up residence in the stem cells and are being packaged into their progeny. For the first few months we didn’t see any cells with the gene, he says. Then we started seeing a few, maybe 1 in 10,000. Now we’re up to about 1 in 1,000. Although in the meantime the infants are receiving injected doses of ADA enzyme, Kohn’s hope is that stem-cell-generated cells containing the inserted gene will eventually produce enough enzyme to obviate the need for supplemental therapy and effectively cure the children.

These trailblazing efforts may presage a spectacular future for stem cell therapies. Malcolm Moore and his team are inserting drug- resistant genes into stem cells identified by CD34 and other markers so that they can give the cells to leukemia patients or cancer patients undergoing chemotherapy, which tends to destroy healthy white cells along with the diseased cells. A number of groups--including one at SyStemix--are looking at inserting anti-HIV genes into stem cells as a possible treatment for AIDS. Finally, though it’s a long shot, the ability to isolate stem cells may even lessen the escalating need for blood transfusions.

In the end, controversy and hard feelings aside, it seems that Weissman has sparked a revolution. Today there is interest in stem cells, and money available to fuel that interest, as never before. Because of the public relations work that Weissman did, it became easier to get grants, says Visser. Because Sandoz bought SyStemix, all the big companies started to look at stem cells.

What did Irv Weissman bring? asks stem cell researcher Norman Iscove of the Ontario Cancer Institute. He made the field live. He brought it into headlines, into newscasts.

No one appreciates the field more than Weissman. I think you’ll see the first practical large-scale use of stem cells in three years for cancers, five to seven years for other conditions, he says. The great thing is that we have developed this population of cells that are just the right thing. They’re not like drugs, which have side effects. You know that what you’re providing is the right thing because stem cells are the product of over a billion years of evolution.

And Irv Weissman owns them.
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