Islets of youth

Turning the clock back on diabetes

Bill Snyder
Published: August, 2008

In the not-too-distant future, a child with type 1 diabetes will prick her finger, not to find out if she needs insulin, but to help scientists cure her disease.

Cells from her blood, a scrape of her skin, or another tissue will be “re-programmed” in the laboratory to create insulin-producing beta cells. They’ll be injected back into her body in an attempt to repair her damaged pancreas.

“I think we’ll be putting pancreatic beta cells that have been made in a dish into people within 10 years,” says Mark Magnuson, M.D., director of the Vanderbilt University Center for Stem Cell Biology.

Sounds like science fiction?

Magnuson and others might have agreed – until last year, when several provocative reports were published.

Cells in the pancreas of a 1-week-old mouse that express the Ptf1a transcription factor gene are revealed in this photograph. The Ptf1a gene has been genetically engineered to express a bacterial enzyme that produces a dark blue color. In both mice and humans, Ptf1a is essential for formation of the entire pancreas, including insulin-secreting beta cells. By tracing the “cell lineage,” or family history, of Ptf1a-expressing cells, scientists hope to learn more about how to maintain—or restore—the function of beta cells. At top right is the sausage-shaped spleen (light orange), and at bottom is the duodenum.
Photo by Fong Cheng Pan, Ph.D., research fellow, Department of Cell & Developmental Biology, Vanderbilt University. Courtesy of Christopher V. E. Wright, D.Phil.

By inserting various combinations of genes, scientists at Kyoto University in Japan and the University of Wisconsin, Madison, reported that they had “induced” human skin cells to revert to an embryonic-like state of “pluripotency” – capable of turning into any other kind of cell.

Injections of these so-called induced pluripotent stem (iPS) cells have been shown to improve symptoms of sickle cell anemia and Parkinson’s disease in experimental mice and rats.

Last year also provided evidence that the pancreas can be “coaxed” into repairing itself.

A team of Belgian and French researchers reported that, with the help of a factor called neurogenin3, injured adult mouse pancreas can generate new beta cells from immature “progenitor” cells.

Maureen Gannon, Ph.D., in her Vanderbilt lab. Behind her, from left: graduate student Kathryn Henley, research assistant Christine Pope, and Magda Bokiej, a student in the Medical Scientist (M.D./Ph.D.) Training Program.
Photo by Anne Rayner
“Everybody had been thinking for the past several years that … you wouldn’t make any new ‘baby’ insulin-producing cells from a progenitor,” says Vanderbilt developmental biologist Maureen Gannon, Ph.D. “And now there’s evidence that you can reactivate that program. That, to me, is really exciting.”

These findings are “hugely radical, unpredicted,” Magnuson adds. “They change the paradigm about the plasticity of every cell in the body … (implying that) you can follow the developmental path, go way back to the beginning and then come forward to whatever cell you like.”

Underlying problem

Reprogramming a patient’s cells to produce insulin would provide a welcome alternative to transplanting pancreatic tissue from other human or animal donors, a procedure limited both by the lack of donor tissue and by the need to suppress the patient’s immune system to prevent transplant rejection.

It also could avoid the need to harvest another, more controversial source of stem cells, those derived from human embryos.

However, the virus used by the Japanese scientists to insert the “reprogramming” genes also triggered formation of tumors in mice. “This is not a trivial issue,” cautions Alvin Powers, M.D., a leader in the study of pancreatic biology and islet transplantation who directs the Vanderbilt Diabetes Center.

And even if the pancreas can be induced to generate new beta cells, or if skin cells could be “re-programmed” to produce insulin, that does not solve the underlying problem of type 1 diabetes – a misguided attack by the body’s immune system that destroys the beta cells.

Christopher V.E. Wright, D.Phil., who directs the Vanderbilt Program in Developmental Biology, agrees.

“What is the nature of the autoimmune problem in diabetes?” he asks. Is the immune system of these patients dysfunctional, such that it mistakes normal tissue for a germ and attacks it? Or could the beta cell be displaying the wrong “badge” on its surface, one that attracts “friendly fire?”

One way to answer these questions is to figure out the steps that lead to the development of the beta cell, and then to try to determine whether that differentiation program is “messed up” in the patient with diabetes.

That’s where the embryo may help.

During a part of embryonic development called gastrulation, groups of cells migrate into three distinct layers: the outer layer or ectoderm, which will develop into the nervous system and skin; a middle layer or mesoderm, which will become the musculature and other internal organs; and an inner layer, or endoderm, which will form the stomach, intestines, liver – and the pancreas.

The human pancreas secretes digestive enzymes and, from cells clustered in the islets of Langerhans, several important hormones, including insulin.

One of insulin’s main jobs is to ensure that fuel – primarily glucose – gets from the bloodstream into the tissues. Diabetes, characterized by a sustained and dangerous rise in blood levels of glucose, occurs when insulin production is unable to keep up with demand.

Whereas in type 1 diabetes there is a loss of beta cells, in type 2 diabetes, the most common form of the disease, the tissues of the body have become “resistant” to insulin. The beta cells also have lost their ability to produce sufficient levels of the hormone.

Wright believes developmental biology may hold the keys to unlocking the mystery of this ancient disorder.

“One of my strongest beliefs is that developmental biology and cancer biology and aging and all forms of inherited disease are basically the same process,” he says.

“Because the study of developmental biology involves trying to understand the generation of life, it uses and develops completely novel principles and tools and ways of looking at things to understand how multiple signaling pathways are used by cells to talk to each other in complicated ways.

“And because of that, it ends up being one of the most pioneering of disciplines.”

Pioneering discipline

Until the early 1980s, the mechanisms of development had been shrouded in mystery. Then, as the new tools of molecular biology became widely available, came several pivotal discoveries.

Among them: the discovery of the “homeobox,” from the Greek word for similar, a specific short sequence of DNA shared by a set of regulatory-switch genes in the fruit fly genome that determine which embryonic segments will become the future head, thorax and abdomen. Nearly identical sequences were found in the genes of vertebrates, including mice and humans.

Colors hint at the “cell lineage” in the developing pancreatic tissue of a mid-gestational mouse embryo. Antibodies linked to fluorescent molecules that absorb and re-emit light of different wavelengths detect hormone-producing endocrine tissue (green), epithelial duct and associated progenitor cells (blue), or cells (red) that specifically express pancreas specific transcription factor-1a.
Image by Fong Cheng Pan, Ph.D., research fellow, Cell & Developmental Biology, Vanderbilt. 
Courtesy of Christopher V.E. Wright, D.Phil.
 
At the time, Wright was a freshly minted biochemist from Oxford University who had just joined the laboratory of pioneering developmental biologist Edward De Robertis, M.D., Ph.D., at UCLA.

“I had somehow a gut feeling that the homeobox genes were a huge breakthrough,” he recalls.

De Robertis set Wright to work on the frog Xenopus laevis. By 1988, they had discovered the first homeobox gene expressed exclusively in the endoderm.

The gene, eventually named pdx1, for pancreatic and duodenal homeobox factor 1, is essential for development of the pancreas – as well as for maintenance of the adult beta cell. The pdx1 gene encodes a protein, called a transcription factor, which turns on other genes.

Wright’s career was launched at a time when scientists were just learning the “language” of the cell. He came to Vanderbilt in 1990 to work with Brigid Hogan, Ph.D., now chair of Cell Biology at Duke University, who helped pioneer methods for introducing extra genetic material into mice embryos.

Another technique, gene targeting, enabled the Vanderbilt team – which by then included Magnuson, Roland Stein, Ph.D., and Patricia Labosky, Ph.D. -- to study what happens to the pancreas when pdx1 is “knocked out” of embryonic stem cells in the mouse.

Since then, Wright and his colleagues have continued to elaborate the role that pdx1 plays in pancreas development. Among their findings:

-- When one of the two copies of the pdx1 gene normally inherited from one’s parents is inactivated in mice, the animals exhibit a pre-diabetic state in which blood glucose levels are higher than normal. Similarly, in humans, certain mutations in the gene are associated with increased risk for developing a form of type 2 diabetes.

-- Both pdx1 and another regulatory gene for pancreas specific transcription factor-1a (Ptf1a) signal progenitor cells to become pancreas. When Ptf1a is inactivated, however, these cells instead form the lining of the duodenum.

Two other transcription factors -- HNF6 and FoxM1 – studied by Gannon and her colleagues play important roles in development and maintenance of pancreatic function.

HNF6’s role is time sensitive: unless its gene is turned off at a critical stage, the pancreatic islets fail to develop normally in mice. FoxM1, on the other hand, is essential for expanding the population of insulin-producing beta cells after birth. Mice lacking the FoxM1 gene are born with normal pancreases but slowly lose beta cells and end up with diabetes.

These factors “are all connected, but we haven’t filled in all the lines,” says Gannon, associate professor of Medicine, Molecular Physiology & Biophysics, and Cell & Developmental Biology.

Brand new view

Meanwhile, Magnuson had become interested in a variation of the knock-out technique that uses a DNA-cutting enzyme called Cre recombinase.

Because the enzyme cuts at precise locations in the DNA, this method enabled Magnuson and his colleagues to inactivate various genes involved in insulin action and glucose regulation in specific tissues, notably the pancreas and liver – to figure out exactly what they do. A major goal now is to learn all the steps needed to direct a stem cell to become a beta cell.

Wright visualizes a day when scientists will be able to create “personalized” pluripotent stem cells from the tissues of a patient with diabetes, and then kick them forward to see if they develop into beta cells completely normally, or display abnormalities at a specific stage of formation.

“That is what stem cell biology has done for us so far,” adds Magnuson, the Earl W. Sutherland Jr. Professor of Molecular Physiology & Biophysics. “It has given us a brand new view of what is possible.”

It also has spurred collaboration across diverse research disciplines.

For example, Vanderbilt scientists as diverse as David Piston, Ph.D., who helped pioneer the use of fluorescence imaging to study living beta cells, Richard O’Brien, Ph.D., who studies diabetes-related genes, and Guoqiang Gu, Ph.D., an expert on Cre recombinase, compare notes with Powers, Magnuson, Wright, Gannon, Stein and their colleagues in a weekly Beta Cell Biology Interest Group.

On a regional level, Stein recently organized the first annual meeting of the Upper Midwest Islet Club at Vanderbilt to foster communication between senior and junior investigators, with “a decided focus” on graduate students, post-doctoral fellows and new faculty members. The goal: to inspire the next generation of researchers.

Vanderbilt also is the coordinating center for the international Beta Cell Biology Consortium, established in 2001 in response to a congressional mandate to capitalize on the advances of the previous two decades.

Currently the consortium facilitates collaboration among 30 principal investigators from nine countries, three of whom reported earlier this year that, in the mouse at least, “you can reactivate the embryonic program and make new insulin-producing cells in adult pancreases from a progenitor cell,” Gannon says.

While collaboration is no guarantee of faster progress, “I would not be at all surprised if five years yields a completely novel way of looking at cells,” says Wright, whose vision of the future draws from the science fiction comic books of his youth.

“I think there will be a way of either looking at a normal cell or labeling a cell in some very clever way, and then probing the cell, with something like a ray gun,” he continues, his eyes twinkling.

That futuristic firing will provide “extremely high-precision data telling us what is going on inside all the cells -- all the protein-protein interactions, metabolic pathways and which genes are being switched on and off, and in real time.”

“You ought to be able to pull a trigger,” Wright envisions, “and get really exciting information directly from each nucleus.”

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