WHEN he has time to look out of the window, developmental biologist Sanjay Nigam likes looking at the graceful Torrey pine trees surrounding his office at the University of California, San Diego. They remind him of kidneys. Kidneys?
He explains: kidneys grow in a similar way to trees. “You start with a bud then it goes through various rounds of branching.” The finished organ contains about one million “twigs”, or tubules, which do the job of filtering toxins from blood.
Nigam’s team studies how kidneys develop in the embryo in the hope of learning how to persuade damaged adult kidneys to repair themselves. They believe that if they can pin down the chemical signals that initiate and orchestrate kidney development, they could trigger regrowth on demand simply by giving patients the compounds as drugs. And it’s not just kidneys. Researchers elsewhere are also trying to discover how to persuade the body to regrow tissues such as heart muscle, or even whole organs such as hearts and lungs.
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These are exciting times for regenerative medicine. With the imminent arrival of stem cell therapy, tissue engineering and perhaps even xenotransplants, the prospects of replacing damaged and worn-out body parts are better than ever (see “Good as new”). But these technologies are not the only game in town. Techniques aimed at coaxing the body into regrowing its own organs and tissues are quietly making enormous progress. Indeed, human trials of one regenerative therapy have already begun.
For decades, scientists have admired many animals’ impressive capacity for regeneration. Flatworms, sea squirts, fish and amphibians can regrow internal organs and even entire limbs to varying extents. But at some point in evolution the ancestors of birds and mammals lost most of their regenerative abilities. Humans can fully regenerate only a very few tissues and organs – the liver (as long as about a quarter of it remains intact), the blood, the outermost skin layer, and, as children, fingertips from the base of the nail upwards.
The key to understanding mammals’ limited regenerative capacity lies in the fact that most of their cells are irreversibly specialised or “differentiated”. In early embryos, cells are undifferentiated – they are blank slates, lacking the characteristics of any particular tissue but with the potential to develop into all of them. But as the fetus develops, more and more cells differentiate. Only a small number of undifferentiated “stem cells” persist in some tissues.
Amphibians and fish are not so constrained. If a newt or salamander, say, loses its leg, differentiated cells in the layer next to the injury dedifferentiate to a stem-cell-like state. The cells then replay the developmental sequence that generated the limb in the embryo, dividing, multiplying and eventually differentiating into bone, muscle and skin.
So why have mammals ditched such an obviously useful skill? There are several possible explanations. Some scientists think it could have been a trade-off for a lower risk of cancer, or a better immune system. Another idea is that for mammals it became more important for wounds to scar over quickly. It certainly seems that scarring, rather than regeneration, is the default mammalian response to damage. In most injured tissues, specialist cells called fibroblasts migrate to the wound where they multiply and secrete a fibrous protein called collagen, the main component of scars. Notably, many tissues in animals with good regeneration skills, such as newts and fish, seem incapable of scarring.
So is there any chance of reawakening the lost regenerative pathways in mammals? The answer is yes, to some extent. The prospect of emulating amphibian limb regeneration in human amputees remains a distant dream. But many researchers believe that regeneration on a smaller scale – of tissues, or portions of damaged organs – is a real and immediate possibility. “It’s not too much of a stretch to believe that an appropriate brew of factors will allow us to engineer organs,” says Nigam.
One organ where there are already signs of success is the kidney. Kidney failure is a major cause of death and disability and the only treatment short of a transplant is dialysis. The disease has several causes, the most common being diabetes and high blood pressure. As a result of such stresses the cells lining the tubules undergo a dedifferentiation process called epithelial-to-mesenchymal transition (EMT). The exact cause and mechanism of EMT is unknown, but eventually scar tissue forms and the tubules degenerate.
Several teams are probing mammalian kidney development with a view to reversing EMT. Nigam’s research suggests that a relatively small number of signalling molecules, or growth factors, control kidney development. Along with other groups, his team has found about half a dozen growth factors that promote tubule development and they believe there are many more still to discover. The plan is to administer these factors to patients with failing kidneys to see whether they prompt the organ to regenerate.
One growth factor is already showing promise in inducing regeneration. Called bone morphogenic protein-7 (BMP-7), it was originally discovered as a bone growth factor and is sold in a putty formulation to help difficult bone fractures heal. But researchers recently showed that it also plays a role in embryonic kidney development, promoting tubule cell differentiation into epithelial cells.
In July, a team led by Raghu Kalluri of Harvard Medical School published a paper showing that BMP-7 reversed chronic kidney failure in mice (Nature Medicine, vol 9, p 964). BMP-7 injections made the tubule cells redifferentiate, echoing the molecule’s role in development. Biotech firm Curis of Cambridge, Massachusetts, which holds the rights to BMP-7 in kidney disease, now plans to start clinical trials of BMP-7 in patients on dialysis next year.
It’s not just with kidneys that there are signs of success. Last year a team led by Mark Keating at Harvard provoked a great deal of excitement among regeneration researchers when they showed that zebrafish, a standard lab animal, could regrow heart muscle.
Zebrafish were already known for their ability to regenerate fins, retinas and spinal cords, and have long been studied alongside amphibians by scientists interested in natural regeneration. But no-one knew if they could regrow their hearts. In fact, they are the first vertebrates confirmed to have a natural capacity for heart regeneration. “It unequivocally demonstrated that a vertebrate could regenerate heart,” Keating says.
Keating’s group cut off up to a quarter of the muscle from the bottom of the heart – as much as was possible without killing the fish outright – and two months later the muscle had regrown (Science vol 298, p 2188). Although he hasn’t worked out what the mechanism is, Keating believes cells next to the injury dedifferentiate, multiply to replace the missing tissue, and then redifferentiate as heart muscle – the same process as in amphibian limb regeneration.
The significance of the finding for human medicine is obvious. In western countries heart disease kills about a third of the population. Many of those deaths are among heart attack survivors whose hearts have been permanently weakened by scar tissue.
Keating’s next goal is to discover which growth factors are involved in heart regeneration – perhaps ones that direct nearby cells to dedifferentiate or multiply. He is hoping the compounds will have the same effect on human hearts. That may not be as great a leap as it seems, as recent evidence suggests that mammals haven’t lost the ability to regenerate, it is just that the pathways are no longer active.
One strong line of evidence comes from a peculiar strain of lab mouse called the MRL, whose remarkable properties were discovered seven years ago by researchers at the Wistar Institute in Philadelphia. Immunologist Ellen Heber-Katz was investigating an autoimmune disease called lupus that MRL mice develop spontaneously. Three weeks into the study, she was bewildered to find that the animals’ ear punch-holes – usually a permanent tag – had disappeared. Heber-Katz made fresh holes, and over the next three weeks watched them heal over perfectly with no scarring and complete regeneration of cartilage, skin and hair follicles. “It was pretty amazing,” she recalls.
Further work revealed the mice could also regenerate the last tenth of their tails, and regrow their livers four times as fast as normal mice. But the team really raised eyebrows two years ago when they showed the mice could also regenerate their heart muscle (Proceedings of the National Academy of Sciences, vol 98, p 9830). After up to 15 per cent of the heart had been destroyed by freezing, the area regenerated within two months. In normal mice the injuries merely scarred over.
What is the source of the MRL mouse’s astonishing ability? The MRL mouse is a commercial strain developed about 40 years ago and its regenerative capacity wasn’t intentional. Exactly how it happens is the subject of intensive research. But by studying the offspring of MRL mice crossed with other strains, Heber-Katz’s group and others have worked out there are at least 14 genes involved. While the identity of these genes remains a mystery, Heber-Katz’s hypothesis is that they somehow block scar formation. She speculates that suppressing the normal scarring process is enough to reactivate dormant regeneration processes left over from mammals’ evolutionary past. Mark Keating also believes that the idea of a trade-off between scarring and regeneration is well worth further study, but he warns that you cannot assume blocking scarring will be enough to activate regeneration. “It is possible that humans scar because they have reduced capacity to regenerate,” he says.
The MRL mouse and the light it might shed on scarring’s role in regeneration has provoked excitement among researchers looking for a mammalian model of regeneration. Yet there is one natural example of mammals repeatedly losing and regenerating large complex organs that suggests that scarring is only part of the story in mammals, just as it is in amphibians.
Most species of deer shed their antlers every spring and regrow a new set within two months. Antler regeneration is an impressive feat – growth rates can be up to centimetre a day in larger species such as red deer. Biologist Jo Price of the Royal Veterinary College in London says the power of sexual selection could explain why this ability evolved. “Size matters,” she says. “The bigger your antlers, the better your pulling power.” Because antlers regenerate every year, if they are damaged in fighting, the disadvantage only lasts until the next growing season.
Exactly what goes on during antler regeneration is difficult to say because deer are much harder to work with than mice. But what we do know about the process suggests that the key lies in a combination of lack of scarring, dedifferentiation and replaying development. Shed antlers leave behind a wound that does not scar over. A cluster of stem cells in the wound, called a periostem, starts to grow and divide, forming antlers out of cartilage, which is eventually converted into bone – the same pathway by which mammalian skeletons form.
Intriguing links
Price has also worked out some of the molecular mechanisms and, intriguingly, they are reminiscent both of development in mammals and limb regeneration in amphibians. The link is retinoic acid, also known as vitamin A, long known as an important signalling molecule in both processes. Last year Price showed that retinoic acid and several of its receptor molecules are present in cells of growing antlers (Developmental Biology, vol 251, p 409). She is now performing experiments designed to find out whether blocking retinoic acid interferes with antler growth.
It is too early to say what impact the antler work will have on regenerative medicine, but Price’s co-author, Malcolm Maden of Kings College London, an expert on retinoic acid in development, says it at least confirms that mammals do retain some ancestral regenerative capacity. It also suggests they have no fundamental block to regeneration. “Maybe there’s more there that just needs awakening than we ever thought,” he says.
And one of those pathways may be about to be reactivated to treat emphysema, a growing cause of death and disability in developed countries. This degenerative lung disease is usually caused by smoking but can also be inherited. It involves the death of cells making up the walls of the alveoli, the tiny air sacs that give the lung its enormous surface area. There is no cure and the condition progresses inexorably, leaving patients short of breath on the slightest exertion.
In 1997 husband-and-wife team Donald and Gloria Massaro of Georgetown University in Washington DC showed that retinoic acid injections seemed to cause rats with emphysema to regenerate their alveoli walls. Some scientists have been unable to duplicate this effect, although Malcolm Maden is one who has. The regenerative process that retinoic acid kicks off is still unclear, though Maden says he believes it stimulates the lung walls to retrace the developmental pathway that gives rise to alveoli in the embryo.
Whatever the mechanism, clinical trials of the compound have already begun. Michael Roth, a professor of pulmonary medicine at the University of California, Los Angeles, has carried out a pilot study giving emphysema patients retinoic acid orally for three months. The results were inconclusive, although lab tests suggesting some improvement are due to published in the journal Chest. Roth is now carrying out a larger 150-patient trial involving a slightly different form of retinoic acid that may have a stronger effect.
That is not to say there are no potential downsides to regeneration. The main worry is that cell dedifferentiation and proliferation are also a hallmark of cancer. Would regenerative therapy trigger cells to multiply out of control? Maden doesn’t think so. “We don’t know anything about the stop signals for regeneration but they’re clearly there,” he says. “When a newt regrows its limb it knows when to stop. The mice lungs stop.”
These concerns, though, have not stopped clinical trials from going ahead. And while there is no guarantee that the trials will succeed, much remains to be learned about the basics of mammalian regeneration, and the new knowledge can only help. Meanwhile, researchers are starting to probe other natural regenerative pathways, in frog retinas for example, with a view to trying to reactivate them in mammals. It is no surprise that many regeneration researchers are confident their work represents the future of medicine. “At some point, whether it’s 15 or 40 years from now, in hospitals there will be departments of regenerative biology,” says Keating, “and you will be able to take medicines for regenerating just about everything.”
Good as new
Boosting the body’s natural regenerative capacity isn’t the only approach to replacing damaged or worn-out body parts. Three other technologies also look promising. Stem cell therapy: based on harvesting highly versatile stem cells, either from embryos or the patients themselves, manipulating them in some way and then transplanting them into the damaged organ. The most advanced work is on Parkinson’s disease.
One human patient has received a stem cell implant, and animal studies are well under way. Tissue engineering: growing replacement organs outside the body using a mixture of synthetic materials and cells. The commonest approach is to build a biodegradable “scaffold” and seed it with the correct cell types. It is already possible to grow arteries and there has been good progress with bladders and penises, but complex organs are more tricky.
Xenotransplantation: the idea is to harvest replacement organs from pigs genetically modified to be compatible with human tissue. Researchers are on their way to overcoming the biggest immunological barriers, although recipients would still have to take anti-rejection drugs for the rest of their lives. But the technology remains mired in controversy over the possibility that pig viruses might pass into humans.