Beta Stem Cells: Searching for the Diabetic's Holy Grail

Diabetics have few practical therapeutic options. Daily insulin injections, while life-saving, are not without problems. Millions could benefit from islet cell transplantation, but only a few thousand healthy pancreases (where islet cells are located) become available each year. The solution to this dilemma has been clear for some time: the creation of new, healthy beta cells. Researchers want to cultivate beta cells, the insulin-producing cell found in the Islet of Langerhans, that would grow like fury and produce insulin in response to glucose. While some researchers have created long-lived cell lines from beta cells1 and others have engineered non-beta cells to make insulin,2 no one has completed the whole package-no infinitely expandable, transplantable glucose-responsive cell exists. Researchers are looking for the right stem cell and the right conditions to grow it to the right differentiation stage. Here, adult stem cells share the limelight with embryonic stem cells (ESCs), and not simply because they enjoy a more favorable political climate. One recent review commented that using cells already heading toward the beta cell pathway might be more productive than starting with totally undifferentiated embryonic cells.3 At the Very Beginning Despite this view favoring adult stem cells, ESC cultures are an obvious place to look for pancreatic stem cells, and of course they are there-by definition, all kinds of stem cells exist in ESCs. But teasing out just the right one can be tricky, and some cell types, like muscle cells, are easier to pull out of the mix than others. Several groups have reported somewhat encouraging results with ESCs. Earlier this year, National Institutes of Health researchers reported inducing insulin production in murine ESCs, though insulin production was poor (as little as 1/50th of the amount produced by a beta cell) and they failed to cure diabetic mice.4 Taking a different approach, Spanish scientists working with murine ESCs used gene-trapping technology to isolate insulin-producing cells. In this scheme, researchers transfected an antibiotic resistance gene adjoined to an insulin promoter into ESCs, providing a selection for insulin-producing cells.5 When allowed to form three-dimensional structures, the cells subsequently isolated increased insulin production to therapeutic levels.6 Last August, Israeli scientists found insulin-producing cells among human embryoid bodies-the three dimensional structures that ESCs spontaneously form when they stop growing.7 Like the murine stem cells, these cells didn't make much insulin. Furthermore, the investigators couldn't control the process. Insulin-producing cells arose randomly and sporadically in a fraction of the embryoid bodies. Seeking Real Pancreatic Stem Cells Another obvious pancreatic stem cell source is in the pancreas itself. In the past few years, several groups have claimed to have demonstrated their existence here.8,9 Last year, pathology professor Ammon Peck, University of Florida College of Medicine, Gainesville, announced that his group had generated pancreatic islets in vitro from dissociated adult murine ductal cells.8 In a multi-step process, Peck went from what he calls islet-producing stem cells (IPSCs) to islet progenitor cells (IPCs), which grew into islet-like structures with partially differentiated endocrine cells. While Peck could increase the pancreatic islet mass some 10,000-fold by growing IPCs in culture, the cells never fully matured. To effect final differentiation, Peck transplanted the immature cells into mice to marshal those signals present in vivo that were missing from his culture system. Peck had success with this approach. He says that insulin production from the transplanted cells was much greater than the amount that the same cells produced in his culture conditions. In addition, enough insulin was made to cure the recipient diabetic mice that were entirely dependent on insulin; they were taken off daily injections a week after transplantation. Peck further explains that he has reproduced these results with human beta cells in the laboratory. Ixion Biotechnology, in Alachua, Fla., which has licensed the technology from the University of Florida, plans to use this approach on large animal models, such as dogs and pigs. Treasure Everywhere ©2000 J. Berndt Susan Bonner-Weir Susan Bonner-Weir, associate professor of Medicine at Harvard Medical School and Joslin Diabetes Center, who has been researching beta cell growth in rodent model animals for years, recently found herself awash with human pancreatic duct cells. Her colleagues across the hall, who were preparing islet cultures for transplantation, were dismayed to find that these cultures were full of proliferating ductal epithelial cells. But, one man's trash is another man's treasure. The ductal cells, normally thrown away, represented a gold mine to Bonner-Weir as a potential source for human pancreatic stem cells. By judiciously applying growth-promoting substances and factors known to direct beta cell development, Bonner-Weir stimulated the ductal epithelial cells to grow and differentiate, a stem cell's two defining properties.9 This outcome seems to have taken even Bonner-Weir by surprise. "We were able to do it. Now we're trying to find out how it works." A unique aspect of this work was growing the adult ductal cells until they assumed the properties of an early duct cell. Once Bonner-Weir got the cells expanding and expressing the IPF1/ PDX-1 protein, a transcription factor necessary for endocrine cell development, she switched to differentiation-inducing factors. The cells then formed into what she calls cultivated human islet buds, or CHIBS, and once that step was accomplished, the cells produced insulin in response to glucose-the acid test for a useful beta cell. Bonner-Weir doesn't believe her cells are stem cells in the true sense of the phrase; she doesn't think that a pancreatic stem cell has yet been isolated. She favors the idea that she is reprogramming duct epithelial cells into a more primitive, multipotent state, which is receptive to signals that put the cells along the pathway to beta cells. Whatever their origins, the CHIBS haven't been produced in sufficient numbers to be useful in transplantation. From a single pancreas, Bonner-Weir gets 30,000 to 35,000 islets, far short of the current islet transplant protocol of 400,000-500,000 islets per patient. Bonner-Weir is trying to boost islet production, by testing the limit on how far she can grow the cells before they start regressing to a totally undifferentiated cell by trying to improve the differentiation phase's efficiency. Genetic Engineering Courtesy of Susan Bonner-Weir Left, Buds of insulin-producing beta cells stained red with dithizone; Right, A similar structure is sectioned to show islet tissue budding from a ductal cyst. Fred Levine, associate professor of pediatrics, University of California, San Diego, Cancer Center and Whittier Institute, is applying the armamentarium of genetic engineering to beta cell production. By commandeering the cells to produce growth and differentiation factors, rather than exposing the cells to gene products, Levine essentially creates beta stem cells. He refers to his cultures as "genetically engineered human pancreatic stem cells" to differentiate them from naturally occurring stem cells. Starting with adult human beta cells, Levine immortalizes them by transfecting the cells with the SV40 T antigen and the oncogene K-ras, which stimulates growth. While most people tread carefully not to overgrow, afraid of losing the differentiation function, Levine is unconcerned because he induces the cells to differentiate by inserting the transcription factor PDX-1. Finally, he creates the three-dimensional environment conducive to insulin production.10 This approach has worked in Levine's hands with another pancreatic endocrine cell, the delta cell. Here, he created permanent and stable lines,11 but so far, the beta cell has proven more recalcitrant. Beta cell cultures grow and differentiate to glucose-responsive insulin producers, but they're not indefinitely stable like the delta cell. Levine wonders why one lineage is stable and the other isn't. "We don't know the answer. If we can figure it out, maybe we can fix it," he says. Nonetheless, Levine is making the case for these cultures as cell transplantation sources-minus the oncogenes. Levine has used another genetic engineering trick, the cre-lox recombinase system, to remove the oncogenes after they've done their job and before the cells are transplanted. Levine transplanted his oncogene-less cultures into nude mice and found that they were glucose-responsive in vivo, yet not tumorigenic. As researchers press on to find a way to produce sufficient islet cells for transplantation, many questions remain unanswered. How best to strike the balance between growth and differentiation is not known, nor is the location of pancreatic stem cells. Some question their existence at all. If they don't exist, then the intriguing possibility arises that some cell types could be transformed into others-a process called transdifferentiation. If transdifferentiation exists, that could open up a whole new world of tissue engineering. Laura DeFrancesco (defrancesco1@earthlink.net) is a freelance writer in Pasadena, Calif. References 1. G.M. Beattie et al., "Sustained proliferation of PDX-1+ cells derived from human islets," Diabetes, 48:1013-9, 1999. 2. A.T. Cheung et al., "Glucose-dependent insulin release from genetically engineered K cells," Science, 290:1959-62, 2000. 3. P. Serup et al., "Islet and stem cell transplantation for treating diabetes," British Medical Journal, 322,29-32; 2001. 4. N. Lumelsky et al., "Differentiation of embryonic stem cells to Insulin-secreting structures similar to pancreas islets," Science, 292:1389-94, 2001. 5.M.G. Klug et al., "Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts," Journal of Clinical Investigation, 98:216-24, 1999. 6. B. Soria, "Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice," Diabetes, 49:157-62, 2000. 7. S. Assady et al., "Insulin production by human embryonic stem cells," Diabetes, 50:1691-7, August 2001. 8. V.K. Ramiya et al., "Reversal of insulin-dependent diabetes using islet cells generated in vitro from pancreatic stem cells," Nature Medicine, 6:278-82, 2000. 9. S. Bonner-Weir, "In vitro cultivation of human islets from expanded ductal tissue," Proceedings of the National Academy of Sciences, USA, 97:7999-04, 2000. 10. D.D. de la Tour et al., "b-cell Differentiation from a Human Pancreatic Cell Line In Vivo and In Vitro," Molecular Endocrinology, 15:476-83, 2001. 11. P. Itkin-Ansari et al., "PDX-1 and cell-cell contact act in synergy to promote cell development in a human pancreatic endocrine precursor cell line," Molecular Endocrinology, 14:814-22, 2000. The Role of Blood Vessels in Insulin Production While efforts to culture beta cells have been successful, getting them to produce therapeutic levels of insulin has not been easy. Recent work from Harvard University suggests that blood could hold a key. Douglas Melton, Thomas Dudley Cabot professor in the natural sciences, has shown that vascular endothelium induces insulin production by cells in the beta cell lineage.1 This work expands blood's role from its typically considered source of sustenance to an initiator of differentiation. There is symmetry at work here. It is known that the pancreas and other organs send out signals in the form of growth factors to draw blood vessels to them. Melton's work suggests this activity is a two-way street. As the blood vessels approach, they in turn send out a signal that induces the precursor cells to complete differentiation. Behind the discovery are two key observations. First, Melton observed that in murine embryos, pancreas development occurs only where blood vessels are in contact with the endoderm. In addition, only those islet buds in close contact with blood vessels fuse to form the pancreas. "Whenever we found evidence for pancreas differentiation, ... there was a blood vessel there," Melton says. Melton conducted three experiments demonstrating the role of blood vessels in pancreatic development. First, he showed that culturing murine embryonic endoderm in the presence of blood vessel endothelium induced the endoderm to express Pdx-1, a marker for pancreatic differentiation, and to produce insulin. Without the endothelium close by, the endodermal cells did neither. In a separate experiment with frog embryos, Melton showed that removing the tissue that gives rise to the blood vessels around the pancreas prevented the pancreas from developing. Finally, Melton created transgenic mice that were engineered to overexpress vascular endothelial growth factor, which triggers blood vessel development, under the control of the Pdx-1 promoter. This gave cells already programmed to go down the beta cell path the capacity to signal for more blood vessels. He found that these mice had hypervascularized pancreases and three times the normal amount of islet tissue. What exactly is being produced in vascular endothelium that causes beta cells to differentiate is not known, but Melton is confident that they will track it down. He states that his intuition tells him that similar signaling systems will be found in other organs, such as the liver or lung, which rely on the blood to carry out their function in a similar fashion to endocrine organs. In fact, a report posted at the same time as Melton's shows that vascular endothelium promotes liver organogenesis in murine embryos.2 -Laura DeFrancesco References 1. E. Lammert et al., "Induction of pancreatic differentiation by signals from blood vessels," Science Express, Sept. 27, 2001 (www.scienceexpress.org, Article 10.1126/science.1064344). 2. K. Matsumoto et al., "Liver organogenesis promoted by endothelial cells prior to vascular function," Science Express, Sept. 27, 2001 (www.scienceexpress.org, Article 10.1126/science.1063889).

Beta Stem Cells: Searching for the Diabetic's Holy Grail

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