The Long Road to Beta Cells

In 2000, researchers in Canada reported a possible breakthrough in the treatment of type 1 diabetes. By transfusing insulin-producing cells from donated pancreases into patients, the researchers provided what looked like cures. Within a week after the procedure, all of the first six patients were liberated from daily insulin injections.

For a time, the team that developed the procedure, at the University of Alberta in Edmonton, appeared to have succeeded where many others had failed.

But after a year or two, the transplants began to falter. Last year, the latest report on the Edmonton protocol found that of 36 patients, 21 initially were able to ditch their insulin needles. Two years after transplantation, though, 16 of those patients were back on insulin. Another report on 65 patients found that only 6 were insulin-independent at 5 years post-transplant.

While ultimately disappointing, the Edmonton protocol is “clearly orders of magnitude better than previous attempts at [pancreatic-cell] transplantation,” two diabetes researchers wrote in 2006 in the New England Journal of Medicine.

Since the 1970s, researchers had been implanting pancreatic cells from cadavers into type 1 diabetes patients with little luck. By the mid-1990s, after some 450 transplants worldwide, the success rate hovered at a dismal 2 percent.

The field was ready to give up. But the Edmonton team, led by transplant surgeon James Shapiro, studied every previous transplantation and theorized why the earlier procedures had failed: Patients weren’t receiving enough cells and the older immune-suppressing drugs given after the infusions were actually hurting the implanted cells. Shapiro and his team engineered a more efficient way of extracting islets—the working zones of the pancreas—from cadavers and replaced the older immune-suppressing drugs with newer ones.

Islets contain beta cells, which secrete insulin in response to glucose, maintaining healthy concentrations of blood sugar. In type 1 diabetes, the immune system attacks and destroys islets. After 60 to 80 percent of the small, round structures disappear, symptoms arise, starting with severe thirst and hyperglycemia. Over the long term, toxic acids can build up in the blood and cause blindness, kidney failure, nerve damage, and accelerated blood vessel hardening. Early death from heart attack or stroke often results.

Between 1 million and 2 million people in the United States have type 1 diabetes, with about 30,000 new cases each year. While the disease is often called juvenile diabetes, it can strike at any age. Type 2 diabetes is a more common yet distinct disease in which beta cells generally continue making insulin, but the body’s other cells lose the ability to use it.

When Shapiro began treating patients with the Edmonton protocol in 1999, he infused huge numbers of islets into the large vein that feeds the liver. There, the cells nest and deposit insulin directly into the blood, each clump acting like a little pancreas.

The early success of the protocol “galvanized the medical community,” says Alan Colman, an embryonic stem cell researcher and executive director of Singapore Stem Cell Consortium. Stem cell researchers noted the results because they provided “proof of principle,” says Colman, that cell replacement could treat type 1 diabetes.

After scientists first grew human embryonic stem cells in the laboratory in 1998, many researchers waxed about an imminent era of cellular-replacement therapy, where the blank slate embryonic cells would be transformed into an array of tissues useful for treating Parkinson’s disease, heart failure, diabetes, and other conditions.

To date, though, there is little evidence that cell replacement works in people. The Edmonton protocol is the sole exception. For cell replacement to ultimately succeed in type 1 diabetes, though, researchers first must fix the long-term failure of the cell transfusions. (One prominent diabetes researcher recently published data suggesting how to do so.) Second, clinicians will require a vast supply of insulin-making cells. With a chronic donor shortage, very few pancreases become available for islet or whole-organ transplants. (Whole pancreas transplants often cure type 1 diabetes, but in 2006, just 1,367 people in the United States received pancreas or pancreas-kidney transplants, many for conditions other than diabetes.)

Enter embryonic stem cells. When cared for properly, the cells double in population every 18 hours or so. If a magic formula to coax embryonic cells to form beta cells could be found, scientists could conjure bottomless vats of potential diabetes cures.

But the road from embryonic cell to beta cell is proving to be long and treacherous. Dead ends and wrong turns plague the travelers. Since 2001, several research teams have announced the creation of insulin-producing cells only to watch their results evaporate under scrutiny.

“It’s turning out to be an extraordinarily difficult cell to make,” says Colman, who knows how to persevere—he worked on the team that in 1997 cloned Dolly the sheep from a skin cell after 277 failed attempts.

At a recent meeting organized by the private New York Stem Cell Foundation, however, three teams reported progress down the beta cell road. “I think it’s going to be possible … to turn human embryonic stem cells into fully functional beta cells,” says Douglas Melton, a diabetes researcher at the Harvard Stem Cell Institute and the Howard Hughes Medical Institute in Cambridge.

Dead ends

In 2001, a team headed by a National Institutes of Health researcher, Ron McKay, announced success in making insulin-producing cells from mouse embryonic stem cells.

But Melton and his Harvard team found something peculiar when they tried to replicate the results. While insulin appeared on the surface of the cells, the team found no evidence that the cells churned out the messenger RNA needed to make insulin. Puzzled, the researchers grew more batches of the putative insulin-making cells—but this time, unlike McKay, they used a growth medium devoid of insulin. Now there was no insulin on the surface of the cells either. In 2003, the Harvard group concluded that the cells were not making insulin; they were simply sucking in small amounts from their surroundings. The work was a dead end.

Beginning in 2000 and continuing through this year, other research groups periodically announced potential short-cuts: Cells in the human spleen, liver, bone marrow, or near the pancreas that also could be grown into insulin-making cells. The announcements unfailingly generated excitement—if such cells existed, they could provide a quicker route to a cure, obviating the scarce and finicky embryonic cells.

But Melton and other top stem cell researchers reject such claims. “Routinely, I open a newspaper and find a report on a new cell that can replace a pancreatic beta cell,” says Melton. “There isn’t any … reason to believe those reports are correct.”

Most organs—including the brain, heart, skin, eyes, liver, and bone marrow—harbor small reservoirs of organ-specific, or adult, stem cells. From these, a trickle of new tissue repairs normal wear and tear. But the pancreas behaves differently, says Melton. There is no adult stem cell for the pancreas, he argues. Instead, he says, new beta cells arise from existing beta cells.

To support his claim, Melton points to work he published in the September Journal of Clinical Investigation with investigators at the Hebrew University in Jerusalem. The team engineered a strain of mice whose beta cells contain a “Trojan horse” gene for the diphtheria toxin. When activated by an antibiotic such as doxycycline, the toxin destroys its host beta cell. Feeding the mice doxycycline-spiked water makes them diabetic.

“The surprise was, when we removed the doxycycline, the animals recovered,” says Melton. Necropsies of the once-diabetic animals revealed that their pancreases held almost as many beta cells as normal mice. To see where these new cells came from, the team shoehorned another gene, a marker, into newborn animals’ beta cells. This marker appears in any new beta cells spun off from the originals. After repeating the experiments, the team found that all of the new beta cells displayed the marker—they all came from existing beta cells, not from some pancreatic adult stem cell.

In the May Developmental Cell, a team at Children’s Hospital of Philadelphia reported similar results (SN: 6/2/07, p. 350).

“There is no evidence for beta cells coming from adult stem cells,” says Melton. Instead, “during the life of a type 1 diabetic, beta cells are constantly replicating and then they’re being smacked down by the immune system.”

Jake Kushner, who led the work in Philadelphia, says that “if you could understand the biology of the beta cells that do grow, maybe you could make them grow” faster to treat diabetes. Melton says NIH and other funders should stop paying for work focused on finding pancreatic adult stem cells—he thinks it’s a false short-cut. He says researchers should instead look for treatments that increase the replication rate of existing beta cells.

Melton’s mouse experiments may also explain the Edmonton protocol’s long-term failures. When the diabetic animals received the two immune-suppressing drugs used in transplants, sirolimus and tacrolimus, the mice failed to spontaneously recover. Melton surmises that the very drugs that suppress the immune system also prevent beta cells from replicating—and such replication apparently contributes to the early success of the cell transfusions. Melton says that Edmonton protocol practitioners “would be well advised to look for immunosuppresants that don’t block beta cell regeneration.”

Shapiro, who developed the Edmonton protocol, agrees that “the drugs we use are not ideal. We can certainly improve on them.” He adds that his team is now testing a new drug regimen that employs lower doses of immune-suppressing drugs. “We’re continuing to improve it,” he says.

Backing up

While a few researchers continue searching for pancreatic stem cells, the leaders in the field have backed up. “I don’t believe [adult stem cells] are really going to work. Let’s move to the embryonic stem cell and start from the beginning,” says Emmanuel Baetge of San Diego–based Novocell.

The beta-cell creation strategy now in vogue seeks to retrace the development of the pancreas from embryo to organ.

To visualize this journey, imagine that the drive from Washington, D.C., to New York City represents the trip from embryonic stem cell to beta cell. Along the route there are obvious stops—Baltimore, Philadelphia, Newark—corresponding to well-defined fetal tissue types.

The first stop, call it Baltimore, is endoderm, a thin layer that appears a few days after fertilization. It eventually forms most of the digestive tract, including the pancreas.

In 2005, Baetge’s team published a simple formula for turning human embryonic stem cells into endoderm. The team also identified a marker that distinguishes endoderm from what Baetge calls its “somewhat evil twin,” extra-embryonic endoderm. This tissue looks and acts almost exactly like endoderm, but it doesn’t grow into a pancreas. Instead, it forms the yolk sac that feeds the embryo. “It was a major problem,” says Baetge, often leaving him and other researchers thinking they’d reached Baltimore when in fact they’d veered left to Pittsburgh.

With Baetge’s formula, researchers can reliably concoct endless dishes of real endoderm in just 2 days.

The next stop, Philadelphia, corresponds to a cell type that, in fetuses, buds into the beginnings of a pancreas. Baetge and Colman say they can turn about half of a dish of embryonic stem cells into these cells. Melton’s group can also make them. The cells are committed to becoming a pancreas, but they don’t produce insulin. They “represent a major intermediate population,” says Colman. “And we can reliably produce them.”

Then, in October 2006 in Nature Biotechnology, Baetge and his Novocell colleagues published a five-step recipe to drive embryonic cells almost all the way to Manhattan. Drawing from the developmental biology literature, the researchers determined which growth factors and other molecules to sprinkle on the cells to move them along. Baetge says the cells at the end of the journey produce insulin and other pancreatic hormones. However, they do not respond to glucose—a key shortcoming. “What we have is a not fully formed, but betalike” cell, Baetge says. It’s like being stuck in Newark.

Colman says his lab has not been able to reproduce Novocell’s recipe, despite trying with nine embryonic stem cell lines. A member of Colman’s team will soon travel to Novocell to observe the technique. “This is sincere, and it’s been sealed by a tribal oath and a pint of bitters,” the Scot says of the scientist exchange.

When Baetge tried implanting the betalike cells, the Newark cells, into diabetic mice, the animals remained diabetic. So he backed up and tried again with less-developed cells, or Philadelphia cells. The idea: Let the animal’s body tell the immature cells how to grow into fully functional beta cells. At the New York Stem Cell Foundation meeting, Baetge reported new, unpublished work that suggests this is exactly what happens. In the experiment, Baetge’s team implanted a million or more Philadelphia cells under the skin, near the kidney, or in the fat of 24 diabetic mice. In two of the mice, the graft didn’t take. But in the others, the cells switched on a critical beta-cell gene, churned out insulin in response to glucose, and cured the animals. “By transplanting [cells of] this earlier state and allowing the in vivo environment to finish the job, we can make structures that look very much like islets,” says Baetge. “We think that’s a remarkable efficiency.”

Whether a similar strategy will work in people is, of course, unknown. Also unknown: whether researchers will ever be able to conjure beta cells in the lab. “Perhaps we’re all asking too much too quickly,” says Colman. Fetuses take 8 weeks to begin producing insulin, a figure Baetge’s recipe cuts to 12 days.

Melton is taking a slower road. His team spent 3 years building a system to screen thousands of drugs and growth factors that might push embryonic cells along. “Our approach is perhaps too slow, but it’s certainly systematic,” he says. He’s convinced such painstaking methods are the best route to beta cell, a journey that he cautions will take “some years. We’re just taking our first baby steps.”

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