The stem cell hope, p.4
The Stem Cell Hope, page 4
Briggs and King thought they had the answer. While the blastula cells resulted in viable new frog embryos when transplanted into a nucleus-free egg, cells even one day older did not—no viable embryos, let alone tadpoles. They concluded, reasonably, that some change occurs just after the blastula is formed that wipes away the very early cells’ unique pluripotency. Something, they decided, was permanently altering differentiated cells to prevent them from ever turning back and becoming pluripotent again.
Fischberg was interested enough by the theory to encourage his new charge to replicate the work, but with a different species of frog, Xenopus laevis, which he happened to have in good supply in his laboratory at the time. With the right hormonal encouragement, Xenopus had the added advantage of shedding eggs year-round, giving Gurdon the opportunity to run his experiments in generation after generation, whereas the leopard frogs ovulated only once a year.
Rather than focus on the features of the transplanted cell and its ability to revert back to an embryonic state, Gurdon, still young and inquisitive, and perhaps a bit naive, turned his attention to the egg. At its heart, he realized, nuclear transplantation was about the egg’s ability to reprogram cells and establish a biological blank slate. After all, when sperm fertilizes an egg, the egg flips the ultimate identity switch by erasing the sperm’s genetic profile and remaking it into one inextricably fused and integrated with its own. But how universal was this process? Briggs and King had proven that it was not unique to sperm, but Gurdon wasn’t quite ready to accept that the process would not work for any cell, no matter how mature. Given the right conditions, he thought, the egg might be capable of exerting its genetic morphing powers on any specialized cell.
“Remember that the main point was to test the proposition that [differentiated] cells do or do not have the same genes,” he says. “In the 1950s, one didn’t know that.” To find out, he methodically transferred increasingly differentiated cells, from late blastula and even further advanced gastrula stages, which represent the next phases of embryonic development, into recipient eggs. Much to his surprise, he says, “It started working in about a year. And within two years, it was already clear that it was working pretty well.”
Just six years after Briggs and King had sounded the death knell for transplanting the nuclei of more mature donor cells, Gurdon published a one-column description of his results in the journal Nature in February 1958, contradicting their claim that only early embryonic cells could generate new frogs. He noted that transplanting even older cells resulted in viable embryos that “appear normal at various stages of development up to the feeding tadpole.”
By 1966, Gurdon had extended the work to the most mature cells transplanted at the time—intestinal cells from tadpoles. The once inept science student achieved remarkable success in generating tubfuls of tadpoles. In Gurdon’s hands, even fully differentiated cells already specialized to function in a frog’s intestinal lining were fair game for the egg to reprogram.
“My results of course did not agree with Briggs and King, and as I was a graduate student, and they were well established scientists and much respected, it was obvious that the graduate student was likely the one who got it wrong,” says Gurdon with his characteristic self-deprecating humor.
In the first hint of the careful and thorough scientist he was becoming, however, Gurdon had integrated proof of his work into the experiment. Exploiting newly developed ways of marking the nuclei of cells that Fischberg had just developed, he was able to prove that the nuclei of the donor intestinal cell had indeed given rise to new tadpoles.
The implications were enormous. It meant that rather than shutting off or losing genes as they matured, cells retained their entire complement of genes, selectively silencing or activating those that it needed to perform its specialized function. Every cell, then, could theoretically be made young, or in biological terms, embryonic, again. “The main conclusion from our studies was that every cell has a complete set of genes,” he says. “And if that is true, then in principle, you can derive an embryo or stem cells from any cell of the body. You couldn’t do that if genes are lost or permanently inactivated, because you could never take adult cells and make them then go backwards to being embryo cells—that could not work.”
Making cells go backward—that was what Wilmut, pondering his dilemma of improving the efficiency of his genetic targeting nearly three decades later, decided might provide a solution to his intractable problem of inefficiency with gene targeting. Yet while it seemed possible that cells could be reprogrammed in amphibians, there was no evidence to suggest that the same would be true in mammals such as sheep.
Although the word never appears in Gurdon’s papers describing his work, what he actually did was produce the world’s first animal clones. When I ask him about why he never referred to his experiments as cloning, he is genuinely perplexed. “I didn’t really think of it that way,” he says. “I just happened to prefer nuclear transfer because that’s what it was, so that’s the term I used.”
His scientific exactitude notwithstanding, the rest of the world had quickly caught on to the implications of Gurdon’s experiments. Walter Cronkite made the trip across the Atlantic to interview the young graduate student, and asked the inevitable question, using the C word: When did he think the same cloning process would move from frogs to mammals and eventually to people? Gurdon’s cheeky answer? “No idea, but somewhere between ten years and a hundred years.”
As it turned out, it would take three decades for Wilmut to consider attempting the feat in his sheep. Others had tried, using mouse cells and embryos, and one of Gurdon’s own students attempted to clone a rabbit, but their failures suggested the process would not work in mammals.
Even Wilmut had no interest in generating a herd of cloned sheep simply for the sake of cloning. What he was after was a rich population of cells that could grow well in a petri dish, proliferating at a reliable and steady pace once they had been tweaked to contain specific genes for therapeutically useful proteins. “The thing you need is a cell that can grow for a long time in the lab,” he says. “There are lots of steps to genetically modifying a cell, so once you put in a gene and find the ones that have the right change, then you have to grow them, and they have to be working cells that can grow for a long time.” Wilmut figured these cells could then be cloned, using Gurdon’s technique, and presto! Problem solved. If, as Gurdon had proved, the developmental clock on cells could be turned back and they could be reprogrammed to become entirely new beings, then why not turn genetically altered sheep cells into entirely new sheep, thus guaranteeing that the animals would produce whatever desired protein was needed, rather than relying on the notoriously fickle process of injecting genes into the nuclei of embryos?
The cells Wilmut was after were embryonic stem cells. And while cloning, or nuclear transfer, seemed a logical and even brilliant way of bypassing the inefficiencies of traditional genetic engineering, executing it would require a number of firsts. No one had successfully repeated Gurdon’s experiments with mammalian cells, so there was no reason to believe that it would be possible to isolate embryonic sheep stem cells, much less genetically modify them and then clone them using nuclear transfer. (“To this day, there are no embryonic stem cells from sheep,” he says emphatically, a reminder, of course, of what shepherds already knew—that sheep are notoriously delicate animals and perhaps not the most ideal species on which to conduct rule-bending studies of development. Sheep cells, it seems, are especially finicky and don’t thrive well in a cell culture.)
Wilmut wasn’t deterred by these hurdles. In fact, his conviction that marrying nuclear transfer and embryonic stem cell techniques would provide a far more efficient way of genetically modifying animals was only reinforced as he enjoyed the warm glow of a pint and the company of colleagues in a Dublin pub one January evening in 1987.
Attending the annual meeting of the International Embryo Transfer Society in Ireland that winter, Wilmut was learning what was really going on at the lab benches around the world by raising a glass with fellow embryologists and engaging in the off-the-cuff discussions that are often the most valuable part of any scientific gathering. Over the course of the evening, he heard about a scientist in Texas who had more or less successfully cloned a cow. Steen Willadsen, originally from Denmark, had taken a blastocyst cell from a very early, week-old cow embryo, injected it into an egg whose nucleus had been removed, just as Gurdon had done with the frogs, and successfully obtained embryos that appeared to divide and develop normally—at least for a few days—before dying.
For Wilmut, it was as if a bolt of lightning had struck. If Willadsen had successfully cloned a blastocyst-stage cell, Wilmut thought, then it might be possible to take those blastocyst cells, genetically engineer them to ferry a particular protein-making human gene, and then select out only those cells in culture that exhibited a precocious tendency to express that gene. Those cells could then be cloned using nuclear transfer, as Willadsen had done, to generate a live animal guaranteed to be a highyielding producer of the protein in question. It was a way of turning the haphazard method of creating Tracy into the animal biotech equivalent of a sure thing. Such an animal would be a true clone, rather than a chimera, as Tracy was, since she was the genetically designed product of her sire’s egg and sperm and Wilmut’s introduction of the AAT gene.
The key to Willadsen’s success, it turns out, was stem cells. The remarkably morphing cells he had lured out of the blastocyst possessed a latent ability to become all of the cells in the adult animal, and when transplanted into an enucleated (nucleus-free) cow egg, they had done just that—begun to generate what might have become a new calf.
Wilmut hoped to exploit the same cells, and manipulate them to carry specific genetic changes that would produce pharmaceutically useful proteins, except in a species far more plentiful in his environs—sheep. “We hoped initially these would be embryo stem cells,” he says, “so we had a biggish project funded by a mixture of private and government money to try to both derive stem cells from sheep embryos and do nuclear transfer from them.”
At the time, however, the science of embryonic stem cells was hardly a robust field with a thick network of researchers to nurture and encourage it. The very concept of a stem cell, in fact, had only recently been born in the labs of a mouse biologist tucked away in Bar Harbor, Maine.
In 1953, about same time Gurdon was beginning his breakthrough studies on Xenopus nuclear transplantation at Oxford, Leroy Stevens, a developmental biologist, arrived at the Jackson Laboratory along the coast of Maine and was presented with what he later called a “crazy” assignment. His lab leader had received a grant from a major tobacco company to investigate the potential carcinogenic properties of cigarettes—or, more specifically, of the paper that the tobacco was rolled in, which company officials believed might be the true cancer-causing culprit. After exposing mice to the various components in cigarettes, including the tobacco, he stumbled upon a grotesquely fascinating tumor in the scrotum of a commonly used strain of lab mice, strain 129. Scientists had known that male mice in this group tended to form tumors spontaneously, although they didn’t know why. And they didn’t much care; the cancers were more of a nuisance than a cause for investigation, since the growths essentially contaminated experiments and forced the researchers to start all over again. In most labs, the mice were sacrificed.
But the sheer size of the tumors that Stevens was seeing in a few of his animals piqued his curiosity, so he performed an autopsy and sliced open the gonadal tissue. What he found was a fantastically freakish amalgam of some of the body’s various tissue and cell types: traces of muscle, bone, cartilage, skin, teeth, tubules that went nowhere, gut and nerve cells—a biological shop of horrors. “It almost looked as if you’d taken the embryo and chopped it up and rearranged it in crazy ways,” says Gail Martin, a professor of anatomy at University of California, San Francisco, who would build on Stevens’s work. Stevens also found that in addition to the wild combination of different tissue types, the tumors contained pockets of undifferentiated stem cells, the tabula rasae of the cellular world, which start out as blank slates but through the course of dividing and growing eventually give rise to all of the different cell types in the body.
While Gurdon homed in on the egg and its ability to reverse development, Stevens had tapped another promising vein of regenerative ore: tumor cells. Clearly, the biological soup of tissues in the tumors provided a powerful platform for studying how those stem cells turned themselves into differentiated tissue—or, in the case of cancers, took a molecular wrong turn in development. At the time, there was precious little in the way of developmental maps that tracked a cell’s progression from an undifferentiated to a differentiated state.
Conducting a series of experiments in which he methodically sliced off and then injected groupings of the tumor cells into mice, Stevens managed to peel away the stem cells from the junkyard of tissues in the tumor, and isolate the ones that were responsible for morphing into the aberrant array of tissues in the tumor. He called these stem cells embryonal carcinoma (EC) cells for their ability to seed cancers.
But was this biological plasticity a unique feature of the gonadal germ cells, or even of the tumor? Cancer is, after all, a disease in which the cells become stuck in a perpetual “on” position, dividing, growing, and dividing again and again with abandon. And the EC cells came from cells in the testes that had gone malignantly rogue.
Stevens’s work suggested that if the tumor he had studied arose from a stem cell that triggered an uncontrolled explosion of growth in a bunch of different tissues, then maybe a similarly undifferentiated mother cell in early development would give rise to all the tissues in a normal embryo. If normal embryos harbored undifferentiated cells, then isolating these would prove a boon for developmental biologists, giving them a cell they could now study in order to understand how the merging of egg and sperm created first one cell and eventually an entire being of millions of cells. But no one had yet answered the obvious question: Could you extract a truly undifferentiated cell directly from an embryo?
It would take Martin Evans, a geneticist at the University of Cambridge in England, and Gail Martin, in San Francisco, nearly a decade to simultaneously supply the answer. Working separately on their respective continents, Evans and Martin (Martin had recently completed her Ph.D. in Evans’s lab) managed to grow up stem cells extracted from a three–to five-and-a-half-day-old mouse embryo on a specially concocted broth of nutrients, cells, and nurturing sera.
In Martin’s case, success was a matter of serendipity. Day after day, she mated mice and flushed blastocysts from the pregnant females seventysix hours after she detected the copulation plug that indicated successful fertilization. She surgically scraped away the inner cell mass of stem cells in the days-old blastocysts. Then she painstakingly extracted the stem cells from these inner cell mass structures with a micropipette, sucking them up one by one and plating them on a culture dish. And day after day, she watched the cells die.
Finally, in frustration, she recalled a trick she had used as a graduate student. While earning her degree, Martin had become adept at working with tumors in another species—chickens—that required quite a bit of coaxing to grow in the lab. They needed a molecular cheerleading squad goading them to divide and grow. “What they needed was another cell type to provide them with factors—and I didn’t know what,” she says. Eventually, she learned that the chicken tumors simply required some reminders of home, in the form of feeder cells that mimicked their normal avian environment. Irradiated mouse fibroblast cells, which are the most common cells in connective tissue such as the skin, did the trick. The mouse cells could not divide, but they did release nutrients and other growth factors that convinced the chicken cells to divide.
“One day I thought, ‘Eh, I’ll throw the [mouse] stem cells in fibroblasts to see if they could make the stem cells grow,” says Martin. “And lo and behold, as long as I added the fibroblast feeders, I could get individual stem cells to grow and form big colonies. If I didn’t add the feeders, they just died.”
Martin published a description of the new cell lines in Proceedings of the National Academy of Sciences in December 1981. To distinguish her cells, which she grew from stem cells taken from mouse embryos, from the embryonal carcinoma cells that Stevens and others had extracted from the gonadal tumor cells, Martin called hers embryonic stem cells—and is credited with coining the term that still is associated with the very earliest stem cells isolated from animal or man.
Evans’s and Martin’s papers quickly launched a flood of new studies and laid the foundation for research into stem cells, but that field had nothing to do with curing human diseases—at least not initially. With the ability to grow an infinite number of mouse embryonic stem cells indefinitely, scientists set their more immediate sights on answering questions of embryonic development. It would take more than a decade for the technology’s potential as a source of fresh and healthy cells to replace damaged or deficient ones in disease to bubble up as a possible advantage. So while the embryonic stem cells did usher in a revolution in biology, it just wasn’t the one that we are now familiar with.
