Saviors

Someday the transplant you need may be growing on the hoof—or in a lab.

By Robert Pool|Friday, May 01, 1998
RELATED TAGS: BIOTECHNOLOGY
Meet Pig 23. Unlike the famous cloned sheep Dolly, this pig has no media-friendly moniker, only a number on a tag stapled to his left ear. I don’t name my animals, says Karl Ebert, chief scientific officer of Midas Biologicals in North Grafton, Massachusetts, and the biologist who created Pig 23 here at the Tufts School of Veterinary Medicine. This particular animal, a male Yorkshire white, has pale pinkish skin covered with light bristles that are softer than they look, an expressive snout, and the stereotypical curly tail—all in all, a very piggy pig, no different in appearance from his thousands of cousins that end up as pork chops and ham at the butcher counter.

But Pig 23 will serve humans in a different way. He is being grown in the hope that animals like him might one day prove suitable organ donors for humans. In each of his cells, tucked in somewhere among 19 pairs of pig chromosomes, is a bit of humanity: a single human gene. He might not look it, but Pig 23 and a few dozen others like him may harbor the solution to one of the most vexing problems of modern medicine.

Over the past 20 years, breakthroughs in transplant surgery have extended the lives—and hopes—of patients with failing organs. Today the three-year survival rate for liver recipients is over 70 percent. The numbers are even better for kidney, pancreas, and heart transplants. But despite years of public-education campaigns, there just are not enough organ donors. In 1996 some 20,000 Americans received transplants, but another 50,000 were on waiting lists; of those waiting, 4,000 died before a suitable organ became available.

To survive, most desperate patients must rely on another’s tragedy. Fatal strokes, heart attacks, traffic accidents, and homicides are their best hopes. Wealthy patients seeking transplants, however, do have other options, but they are ethically troubling. In India, for example, the shortage of organs has created a thriving, and mostly legal, trade in kidneys from living donors. Each year an estimated 2,000 poor but healthy Indians, looking for enough money to provide a dowry or build a house, sell one of their kidneys to a rich person in need. Buyers from around the world may pay $10,000 or more for both the operation and the organ—a bargain, given that the alternative is a lifetime of dialysis.

The organ trade in China is even more troubling. In February the New York Times reported that fbi agents had arrested two Chinese men in New York City on charges of conspiring to arrange organ transplants from executed Chinese prisoners. The men were said to have offered access to organs from 50 of the 200 prisoners executed each year on the island province of Hainan.

Given the grim scenarios, it is small wonder that many are eager to find an organ source that is not another human body. Using organs from genetically altered pigs is one option; growing, or culturing, human replacement organs in laboratories is another. Both strategies have been under development for several years, and both are just starting to bear fruit.

A representative of option number one is rooting around in his pen at the Tufts veterinary school, perhaps nosing after a stray morsel of food. His virtues as a potential organ donor are not obvious, but they are real. For one, a pig’s internal organs are designed and laid out much like those of a human, which is why fetal pigs are the standard choice for dissection in anatomy courses. And the size is right, too. Although pigs can grow to well over half a ton, researchers believe the transplanted organs will take growth cues from the human host and expand to fit the available space—a child could be given the liver of a young pig, for instance, and that liver would grow along with the child, stopping its growth as the child reached adulthood.

As organ donors, the only nonhuman animals that make more sense than pigs are apes, our closest relatives. But most people would question the ethics of breeding chimpanzees or gorillas or orangutans for their organs. Besides, apes reproduce so slowly that it would be nearly impossible to raise a large supply. Pigs, by contrast, multiply quickly. A sow is ready to become pregnant by six months, and she can have two litters annually, averaging about ten piglets each. And raising pigs for their flesh is a venerable, 7,000-year-old tradition. With 95.7 million pigs making that one-way trip each year to the supermarket, an extra few thousand sacrificed for a nobler cause than sausage would probably not bring out the Porcine Liberation Army.

There is just one problem, but it’s a whopper. Put a pig organ into a human being, and the human body will attack it so quickly and so violently that the organ will begin to turn black and die even before the surgeon can close the incision. This so-called hyperacute rejection is a defense mechanism that the body reserves for tissues that seem particularly foreign—organs from chimpanzees or baboons don’t trigger it, for instance, while those from sheep or dogs or pigs do—and it is difficult to control. So if pigs are to stand in as organ donors, a way to shut down hyperacute rejection must be found. This is where Pig 23 and his kin come in.

Hyperacute rejection kills by suffocation. The organ is starved for oxygen because the blood starts to clot, explains Stephen Squinto, vice president for research in molecular sciences at Alexion Pharmaceuticals in New Haven, Connecticut. Put a pig’s liver into a baboon and as soon as the organ is hooked up to the baboon’s blood supply, antibodies in the blood latch onto the endothelium—the inner lining of the organ’s blood vessels—and signal the rest of the immune system to attack. The first assault comes from the complement system, a collection of proteins that infiltrate the cells of the endothelium and disrupt their workings. Gaps open between the cells, creating leaks in the blood vessel, and the once-smooth endothelium becomes rough, allowing blood cells to stick to it and clots to form. Smaller blood vessels can become entirely blocked within minutes, and without oxygen the organ dies.

In 1992 a group of scientists from Yale formed Alexion, with an interest in discovering ways to regulate immune responses. At that time the gene for human complement inhibitor had just been cloned, Squinto explains, and that discovery offered a natural approach to lengthening the time a pig organ could survive inside a human. Complement inhibitor is a protein manufactured by the body’s cells. The protein shields human cells from the complement attack, allowing the immune system to destroy an invader with a minimum of collateral damage to the body’s own cells. At Alexion, researchers decided to create genetically engineered pigs whose cells produced human complement inhibitor.

Within three years, working with William Velander at Virginia Tech in Blacksburg, the Alexion team did just that. Velander took fertilized eggs from sows and, using an extremely fine needle, injected into each egg billions of strands of dna, each containing the complement inhibitor gene and the regulatory region that turns it on and off. Occasionally, by chance, one or more copies of the gene were absorbed into an egg’s own dna during the flurry of gene activity that accompanies an egg’s development into an embryo. Velander had no way of knowing in which eggs this would happen, so he returned all of them to the sows and waited. When the piglets were born four months later, most were pure pig, but a small percentage had the human complement inhibitor gene within their dna. With a blood test, Velander picked out which pigs were producing the human protein. From these, Alexion scientists bred a small herd of pigs whose organs would, they hoped, be more acceptable to humans.

Tests on baboons showed that the complement inhibitor did work, but not well enough. Hearts from the genetically engineered pigs, says Squinto, kept beating for ten days in baboons given immunosuppressive drugs, compared with two days for hearts from normal pigs. But ten days is not nearly enough time to justify putting such a heart into a human patient.

But that does not mean the pigs’ livers are useless. Another American company, Nextran, in Princeton, has performed clinical trials in which blood from patients with failed livers is routed out of their bodies, through the extracted liver of a genetically modified pig, and back to the patient. Although the human blood will eventually kill the liver, the organ can keep a desperate patient alive until a suitable human liver is found.

Alexion is taking a different tack. By adding a second human gene to its pigs, it hopes to extend the two-week survival period to several months. Five years ago, Squinto explains, a group of Australian researchers discovered just what makes a pig’s organs so repugnant to the human immune system. The problem is that cells lining a pig’s blood vessels are studded with a sugar molecule called alpha-galactose, and the human immune system has swarms of antibodies for alpha-gal. These antibodies act as miniature bull’s-eyes, directing complement proteins and other attack modules of the immune system to zero in on the pig tissue. Without alpha-galactose, the tissue would seem much less threatening to the human immune system.

One solution would be to make a pig without the gene coding for the production of alpha-galactose, but, says Squinto, this is not yet feasible. So the Alexion researchers have taken an ingeniously indirect approach. They have created pigs that convert the alarming alpha-gal into an innocuous human counterpart. These pigs carry a second human gene, for an enzyme called H-transferase. When this enzyme is working, most of the precursor for alpha-galactose is transformed instead into the sugar molecule that appears on human type O blood cells, which all humans tolerate. To perform this act of creation, Alexion approached Ebert, one of the pioneers in genetically engineered farm animals. The result is Pig 23 and a score of otherwise normal pigs that produce the human H-transferase enzyme—and just a tiny bit of alpha-gal.

Recently Alexion has crossbred the two lines of pigs, those with the complement inhibitor and those with the H-transferase enzyme. Some of the offspring, which were born in late January, should have both bits of humanity. And this genetic patchwork, the company hopes, will provide organs that can survive long enough in humans to be valuable, at least as a bridge to keep a patient alive until a human organ can be found.

There is good reason to think Alexion will succeed, says Squinto. Alexion researchers have created mice with both human genes, and cells from these mice withstand the onslaught of the human immune system far better than cells from mice with just one of the genes. Given these results, he adds, it’s reasonable to hope that a pig organ could survive up to several months in a baboon or a human treated with immunosuppressive drugs.

The humanization of the pig will not stop here. Several groups are wrestling with the difficult problem of knocking out the pig gene for alpha-galactose and replacing it with the human gene for type O blood sugar. From there, researchers will take it step by step, identifying and replacing the various features of pig tissue that alarm the human immune system. It’s conceivable that pig tissue might one day be made nearly invisible to a human immune system.

Ultimately, Ebert says, we want to make a universal donor—a pig whose organs are accepted by the human body as completely as type O blood.

The biggest stumbling block, in fact, may not be the difficulty of humanizing pigs, but the difficulty of policing the pig-human boundary. Just as the virus responsible for aids apparently originated in monkeys and was somehow transmitted to humans, a similar virus, researchers worry, might be hiding in pigs. Some have even hypothesized that a primate’s violent rejection of nonprimate tissue hints at an ancient exposure to a lethal virus. They speculate that the primates that survived exposure were those carrying a mutation that allowed them to recognize—and ferociously reject—virus-infected cells.

Pig-to-human transplants might revive this ancient battle. If such a virus is transferred into humans whose immune systems have been suppressed by drugs, it might proliferate and mutate into a deadly killer. Last March researchers in England reported that pigs do indeed carry at least one virus in their kidney cells that can infect human cells. Because the virus is actually encoded in the pigs’ dna, it would be impossible to get rid of it without genetic engineering, and even then no one would know what other viruses might be lurking in pigs.

This has created a bit of a problem, Squinto admits, although he says it doesn’t have to stop the show. It’s possible, for instance, that pigs are distant enough genetically from people that viruses hiding in their dna will pose little risk. Feline leukemia virus has never been much of a threat to humans, he notes. Even the researchers involved in detecting the pig virus recognize that it doesn’t reproduce easily in human cells and may well not spread—or cause disease—inside the human body. And they, too, acknowledge the dire need for transplant organs.

Still, the specter of cross-species infection has prompted fear. Last year Britain imposed a moratorium on transplanting animal organs into humans until further research proved them safe. In the United States the Food and Drug Administration, which oversees experimental medical procedures, is drawing up guidelines that may increase requirements for monitoring cross-species infections and rein in experimentation on humans. Before surgeons begin putting pig organs into humans, disease specialists will need to determine just how dangerous it is. If it is judged too hazardous, the hopes of those needing new organs will rest with one other possible source of replacement parts: the tissue engineers.

Last October, London’s Sunday Times reported that a researcher at the University of Bath had created headless tadpole embryos. It wasn’t actually the first time a scientist had grown headless animals—headless embryonic mice were created in a Texas lab in 1994—but when combined with the recent breakthrough in cloning, it touched off a rash of speculation: Could scientists grow headless humans to provide organs for transplantation? Might wealthy people clone themselves, minus the head, to have spare parts in case of accident or disease? It may be possible to do it, says David Mooney, a tissue-engineering specialist at the University of Michigan, but it will be extremely difficult.

The ethical problems alone would be staggering, he notes, and the scientific hurdles look daunting, since the brain plays a major role in the body’s development and survival. Anencephalic babies, born without an upper brain, die within hours or days of leaving the womb, and life-support machines don’t prolong survival by much. But perhaps the biggest reason that headless humans aren’t in our future, Mooney says, is that we won’t need them: It will be much easier to grow individual organs.

Over the past decade, a loose coalition of surgeons, cell biologists, chemists, and materials scientists have developed a new approach to creating organs. They call it tissue engineering. The strategy is to start with a small amount of tissue—from a patient, a relative, or even a stranger—and let the cells from that tissue grow and divide. Then you use these cells as a basic building material, Mooney says. Doctors are already using simple engineered tissues, such as skin and cartilage, in clinical trials, but replacements for internal organs are still a decade or two away by even the most optimistic estimates.

Perhaps the closest to application is a replacement liver being engineered by Joseph Vacanti at Children’s Hospital in Boston. Vacanti, who is a professor of surgery at Harvard Medical School and chief of organ transplantation at Children’s, creates his livers on wads of spongelike, biodegradable polymers. He floods the sponge with a solution containing liver cells, and some cells latch onto the sponge’s webbing. There, under the right conditions, they grow and make connections with other cells, eventually creating a mass of liver tissue that can be transplanted into an animal. Over time, the sponge degrades, leaving only living tissue.

Unfortunately, Vacanti notes, the liver is more than just liver cells. It is shot through with blood vessels that bring nutrients in and ship the liver’s products—including many essential proteins—around the body. It also contains a system of bile ducts that carry toxins out of the liver. And nobody knows how to grow either a circulatory or a biliary system.

Vacanti has recently taken the first step: he has created a simple circulatory system for his tiny artificial livers. He created polymer sponges with deep channels, then seeded the channels with two types of cells—liver cells and endothelial cells, the cells that line the interior of blood vessels. Somehow the two types, even if mixed together initially, sort themselves out correctly, with the liver cells slipping into the interior of the sponge and the endothelial cells lining up along the walls of the channels through it. The result is an artificial liver with simple, nonbranching blood vessels spanning it. Larger livers, with circulatory systems modeled on the complex blood supply lacing real livers, are the next step. If those engineered livers can keep alive experimental animals whose livers have been removed, Vacanti would then move to clinical trials in humans. As with livers from pigs, the natural first candidates will be people on transplant waiting lists who are near death from liver failure.

At this stage, the artificial livers might work without a system of bile ducts, since they could still serve as an interim transplant until a suitable human liver is found, but, Vacanti says, eventually we will have to build that in too. So far he has not started work on a biliary system, but he sees no reason why it should be any harder to engineer than the blood supply.

Tissue engineers predict that besides livers, it will eventually be possible to build hearts, kidneys, and almost every other organ except the brain in a similar fashion. The problems, of course, are formidable. How, for instance, does one get four or five different cell types to arrange themselves in the correct order? But tissue engineers expect to get at least some help from the tissue itself.

In the long run, Vacanti says, the dream of tissue engineers is to create universal organs that could be put into anyone who needs them without requiring immunosuppressive drugs. Someday, he says, we will have cell banks with cells that have been genetically engineered to be invisible to the human immune system. To create a liver or kidney or heart, a tissue engineer would withdraw the correct cells from the cell bank, seed them into an organ framework, and grow the organ.

Or, if Pig 23’s descendants live up to their promise, transplant surgeons of the future may find themselves ordering their organs from large farms where thousands of pigs are raised in perfectly antiseptic conditions so that no viruses or microorganisms take up lodging in their organs. And though these pigs will undoubtedly look just as piggy as Pig 23, their flesh will be, as nearly as our immune systems will be able to tell, humanlike. It could be enough to convince people to stop eating pork.
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