One step into Chris Wright’s Vanderbilt office, and it’s clear that this guy is fond of frogs. Perched on a long low bookcase are all manner of them — wooden, ceramic, stuffed. The figures join a striking series of models that show, in hand-painted detail, stages of the developing frog embryo.
The décor pays homage to the animal that gave Wright his scientific start. It was in studies of the frog embryo — a system long-favored for developmental biology research — that Wright discovered a gene critical to the development of the pancreas. The findings launched a path of inquiry that has landed Wright in the thick of the push to cure diabetes.
It’s not a place he set out to be. The pressure of finding something that will benefit the millions of patients suffering from diabetes can be daunting, says Wright, professor of Cell & Developmental Biology, but “it’s also invigorating.
“Our research really has a chance to help people; it’s not just an academic question whose answer might be in the textbooks, if you’re lucky.”
Wright’s team and a handful of other laboratories are seeking the set of genes that control the development of the specialized cells of the pancreas. “If we can identify the factors that make a pancreas,” Wright says, “we might be able to coerce embryonic stem cells or other cells to turn into pancreas.”
Success could mean unlimited supplies of insulin-producing cells for transplantation therapy. And transplantation appears to be as close to a cure for diabetes as we’ve come.
Only one kind of cell in the body — the pancreatic beta cell — can sense blood sugar and respond by secreting insulin. Destruction of these precious cells by a person’s own immune system gives rise to type 1 diabetes.
Although glucose testing and insulin injections can stand in for the lost beta cells, they cannot begin to match the minute-to-minute control of blood sugar normally exerted by these cells. While intensive insulin therapy reduces the long-term complications of diabetes, it does not eliminate these complications and often results in dangerous episodes of hypoglycemia.
So why not simply replace the lost beta cells with new ones? That is the premise of transplantation therapy as a cure for diabetes.
One option is to transplant an entire pancreas, and indeed, pancreas transplantation has proven successful in restoring normal blood sugar levels. Unfortunately, the procedure carries high morbidity and mortality rates, restricting it as a therapy to those patients with diabetes and significant end-stage organ disease like renal failure.
The majority of patients with type 1 diabetes need a safer alternative. Perhaps, investigators reasoned, the surgical complications of full pancreas transplants could be avoided by transplanting only the “islets of Langerhans,” named for the German pathologist who first described the characteristic cell islands in the pancreas. Islets are home to the insulin-producing beta cells, along with several other hormone-releasing cell types.
The idea showed early promise. In 1972, Paul Lacy at Washington University in St. Louis reported that islet transplantation could cure diabetes in rats. Investigators raced to apply the procedure to human beings. But of the hundreds of attempts made over the next 20-plus years, less than 10 percent resulted in insulin independence.
More encouraging results began to surface in the late 1990s, with groups in Miami, Minneapolis, and Milan achieving longer periods of islet cell survival, higher percentages of insulin independence, and, for those transplant patients who still required insulin, avoidance of the dangerous blood sugar extremes.
The breakthrough report came in July 2000 from a group at the University of Alberta in Edmonton, Canada. Dr. James Shapiro and colleagues announced that each of seven islet transplant recipients — patients with type 1 diabetes and a history of severe hypoglycemia — were insulin independent, the longest for 15 months and counting. Their strategy used a novel combination of non-steroid immunosuppressant drugs — ones that were kinder to the fragile islets and that were less likely to induce insulin resistance in the transplant recipients. And it used islets from two, and in one case three, donor pancreases.
The Edmonton group “made a leap forward in terms of immunosuppression and standardization of islet isolation techniques, culminating in a very high success rate,” says Dr. Christopher Marsh, chief of the organ transplantation service at the Scripps Clinic in La Jolla, Calif., and co-director of the Organ and Cell Transplantation Center at Scripps Green Hospital.
The approach, now known as the “Edmonton protocol,” triggered a surge in interest in islet cell transplantation and prompted the National Institutes of Health and the Juvenile Diabetes Research Foundation (JDRF) to fund larger clinical trials of the protocol and variations of it.
Since the Edmonton group’s original publication, more than 150 patients worldwide have received islet transplants as part of these trials, according to the JDRF.
Preliminary results from at least one JDRF-sponsored transplant center support the idea that islets from a single donor pancreas might even cure diabetes. Using one cadaveric pancreas per diabetes patient would stretch the islet supply, but not enough to provide islet transplantation to all the patients who could benefit from it.
Last year in the United States, fewer than 2000 pancreases were recovered from donors. More than a million patients have type 1 diabetes.
“What you need is more tissue,” says Vanderbilt’s Wright. “Where do you get more tissue?”
Islet tissue could potentially come from another species, like pig, an area of research called xenotransplantation. Or insulin-producing cells could be grown in the laboratory, as genetically engineered cells or as the products of stem cells — embryonic or adult.
None of these options are clear-cut. Xenotransplantation must overcome concerns that pig-specific pathogens will infect the human recipients and potentially endanger public health. Genetic engineering of cells requires an appropriate cell starting point and knowledge of all the genes that are necessary for glucose-regulated insulin secretion. Likewise, turning stem cells into pancreatic cells requires identification of the complete set of factors that will elicit that conversion.
Securing a plentiful supply of islets or an alternative source of insulin-producing cells is just one of the hurdles looming for islet transplantation. Equally vexing is the long-term immunosuppression required to prevent attack of the transplanted cells.
Immunosuppressive drugs can blunt the immune system’s attack on transplanted tissues, as well as its attack on beta cells, the hallmark of type 1 diabetes. But even the Edmonton protocol’s newer, less toxic drug cocktail has “side effects that are not justified lifelong in juveniles,” says Dr. Allen Spiegel, former director of the National Institute of Diabetes & Digestive & Kidney Diseases. Immunosuppression puts patients at increased risk for infections of all types, for lymphomas and related malignancies, and for renal toxicity.
One appealing way to leap over this obstacle, Spiegel says, would be to induce “tolerance” of the transplanted cells, without requiring chronic drugs and without suppressing immunity overall. The Immune Tolerance Network, a $144 million project co-funded by the National Institute of Allergy and Infectious Diseases, the NIDDK and the JDRF, is working toward this goal. “There are some encouraging results, but a lot of work remains to be done,” Spiegel says.
It might also be possible to sheathe islets, or cell clusters, in materials that protect them from immune system attack but that allow passage of nutrients, glucose and insulin. Encapsulation strategies include ultrathin polymer membranes — microencapsulation — and porous matrixes with cells or islets dispersed inside — macroencapsulation.
“What we like about encapsulation is that it will eliminate the need for long-term immunosuppressive drug therapy,” says Taylor Wang, Centennial Professor of Mechanical Engineering and Applied Physics at Vanderbilt, and a former astronaut whose 1985 space shuttle experiments involving water and oil droplets turned out to have implications for encapsulating islets.
Wang and colleagues developed an encapsulation method — materials and bioreactor system — and successfully reversed diabetes in a mouse model by transplanting encapsulated rat or pig islets. But their capsules failed in dogs. The investigators have since worked through nearly 20 different engineering parameters, adjusting each in order to optimize performance.
Now, bolstered by a new grant from NASA, they are preparing to test the improved capsules in larger animals, and assuming success, in human beings. “We are starting to see the light at the end of the tunnel,” says Wang. “You can get a little bit edgy though. Is it really the light, or a train coming at me?”
As investigators make progress in shielding transplanted cells from host immune attack, islet transplantation will become all the more desirable as a treatment and cure for diabetes. Translating it into a widespread option, however, will depend on securing a suitable and plentiful source of insulin-producing tissue.
Even if xenotransplantation proves workable, the number of donor animals required to meet patient demand for islets would likely be excessive. Insulin-releasing cells that could be produced in unlimited quantities in the laboratory — or pharmaceutical factory — are a more desirable option.
Toward that end, an international group of scientists, assembled together as the NIH-supported Beta Cell Biology Consortium, are working toward the ultimate goal of converting human stem cells into functional beta-like cells or complete pancreatic islets.
Stem cells, the basic building blocks of the body’s many different tissues, offer promise for a variety of ills. They come in two types: embryonic, which normally populate the early embryo and give rise to all the tissues of the body, and adult, which are found in and serve to replenish “mature” tissues — the best known are the bone marrow-residing cells that renew the blood supply. Embryonic stem cells are considered more versatile than their adult stem cell relatives, in terms of the cell types they can become, but they are more controversial.
That controversy might be avoided by identifying human adult pancreatic stem cells — cells that spin off renewed pancreatic cells, including beta cells. Evidence from several laboratories suggests that these adult stem cells exist, but no one has yet isolated the actual cells.
“In the next couple of years, one may unequivocally identify the adult pancreatic stem cell,” says Douglas Melton, a Harvard investigator studying the development of the pancreas. “The problem is whether it can be replicated, to any appreciable extent, to be useful in a clinical context. Can you coax it to grow outside the body, to make a pile of cells?”
More promising, Melton says, is the embryonic stem cell. “Here, two of the problems have already been solved. This cell is available, and it grows like a weed. You can make virtually an infinite amount.
“That leaves one big problem: how do you direct differentiation into pancreatic cells?”
This is where studies of pancreas development come in — finding the gene switches that turn an undefined bit of embryonic tissue into the specialized cells of the pancreas. Wright’s observations in the frog embryo identified one of the first.
In the mid-1980s, Wright was plugging away as a postdoctoral fellow at UCLA, looking for new members of the “homeobox” gene family — genes that had just been identified as being critical for proper pattern formation in the fruit fly. Of the several they identified, one had an interesting pattern of expression in the developing frog embryo; it was turned on in the area that would give rise to the pancreas and part of the intestines.
Wright launched his Vanderbilt laboratory with the intention of studying the function of the gene, now called pdx1, in mammals, specifically mice. “I was sure it would be exciting,” he recalls. He was right. Mice without the pdx1 gene failed to develop a pancreas.
The pdx1 gene is a transcription factor — it turns on other genes, a cascade of which eventually turn undifferentiated embryonic tissue into the pancreas.
In recent studies, Wright and colleagues have characterized the action of another transcription factor, a gene called PTF1p48 (p48 for short).
The group reported last summer in Nature Genetics a novel and powerful cell marking method that they used to track cells in the mouse that express the p48 gene, starting very early in embryonic pancreas formation. The method relied on genetic manipulations to introduce an inherited marker — a blue color that could be followed in cells that turned on the p48 gene, and in all the cells that came from those cells.
A simple way to think about the technique, Wright says, is to picture the crowd at a football stadium and to imagine that somewhere in the stadium, for a limited time, a man gave away unique blue hats and asked people to wear them. “Now we can follow the people who got hats, no matter where they go,” Wright says. “Whether they go to get a hot dog or leave the stadium entirely, we can find them.”
Wright and colleagues found that cells expressing the p48 gene, blue hat-wearing cells, formed the pancreas. When the investigators knocked out the p48 gene, they found that cells, which would have normally formed the pancreas, became intestinal cells instead.
“The really important point is that these cells without p48 don’t just die; they go on to behave as a different tissue,” Wright says. “That is very powerful information when you are thinking about manipulating stem cells in the laboratory. Because you know now — at least for some genes — that you can put them in or take them away and you don’t kill the cells; you manipulate what they’re going to become. And that’s exactly what we want to do therapeutically.”
The next step, he says, is to see whether introducing the p48 gene into cells that would normally become intestinal cells changes their fate and causes them to become pancreas cells instead.
“If we can do that,” Wright says, “we’re a big step further towards knowing that p48 is one of the gene triggers that you might want to put into an embryonic or other stem cell to make pancreas.”
But one of how many? Wright wonders. “We’re just sort of clutching and still trying to get some basic understanding. It’s like climbing an ice wall with ice picks: you’d better have a good hold before you start going up too high.”
Even knowing the complete set of factors that will convert stem cells into pancreas doesn’t address the logistical problems of applying that information on a large scale to generate products for human transplantation. Simple manipulation of laboratory growth conditions, for example, may not be sufficiently rigorous to pass FDA muster.
“We have to understand very deeply the physiological behavior of those cells,” Wright adds. “How long do we spend characterizing those cell types before we say it’s appropriate to put them into people? I don’t know the answer to that.”
At the end of the day, it is the fundamental academic question that drives Wright’s quest for answers. Relaxing in his office of frog companions, he ponders the intricacies of pancreas development. “I’m still fascinated by how a piece of tissue buds out, goes through a branching process, makes the right numbers of cells of different types, and generates an organ that works so beautifully,” he says. “It’s an amazing thing.”