Directly Reprogramming Gut Cells into Beta Cells to Treat Diabetes


Type 1 diabetes mellitus results from destruction of insulin-producing beta cells in the pancreas. Diabetics have to give themselves routine shots of insulin. The hope that stem cells offer is the production of cells that can replace the lost beta cells. “We are looking for ways to make new beta cells for these patients to one day replace daily insulin injections,” says Ben Stanger, MD, PhD, assistant professor of Medicine in the Division of Gastroenterology, Perelman School of Medicine at the University of Pennsylvania.

Some diabetics have had beta cells from cadavers transplanted into their bodies to replace the missing beta cells. Such a procedure shows that replacement therapy is, in principle possible. Therefore, transplanting islet cells to restore normal blood sugar levels in type 1 diabetics could treat and even cure disease. Unfortunately, transplantable islet cells are in short supply, and stem cell-based approaches have a long way to go before they reach the clinic. However, Stanger and his colleagues have tried a different strategy for treating type 1 diabetes. “It’s a powerful idea that if you have the right combination of transcription factors you can make any cell into any other cell. It’s cellular alchemy,” comments Stanger.

New research from Stanger and a postdoctoral fellow in his laboratory, Yi-Ju Chen that was published in Cell Reports, describes the production of new insulin-making cells in the gut of laboratory animals by introducing three new transcription factors. This experiment raises the prospect of using directly reprogrammed adult cells as a source for new beta cells.

In 2008, Stanger and others in Doug Melton’s laboratory used three beta-cell reprogramming factors (Pdx1, MafA, and Ngn3, collectively called PMN) to convert pancreatic acinar cells (the cells in the pancreas that secrete enzymes rather than hormones) into cells that had many of the features of pancreatic beta cells.

Following this report, the Stanger and his team set out to determine if other cells types could be directly reprogrammed into beta cells. “We expressed PMN in a wide spectrum of tissues in one-to-two-month-old mice,” says Stanger. “Three days later the mice died of hypoglycemia.” It was clear that Stanger and his crew were on to something. Further work showed that some of the mouse cells were making way too much extra insulin and that killed the mice.

When the dead mice were autopsied, “we saw transient expression of the three factors in crypt cells of the intestine near the pancreas,” explained Stanger.

They dubbed these beta-like, transformed cells “neoislet” cells. These neoislet cells express insulin and show outward structural features akin to beta cells. These neoislets also respond to glucose and release insulin when exposed to glucose. The cells were also able to improve hyperglycemia in diabetic mice.

Stanger and his co-workers also figured out how to turn on the expression of PMN in only the intestinal crypt cells to prevent the deadly whole-body hypoglycemia side effect that first killed the mice.

In culture, the expression of PMN in human intestinal ‘‘organoids,’ which are miniature intestinal units grown in culture, also converted intestinal epithelial cells into beta-like cells.

“Our results demonstrate that the intestine could be an accessible and abundant source of functional insulin-producing cells,” says Stanger. “Our ultimate goal is to obtain epithelial cells from diabetes patients who have had endoscopies, expand these cells, add PMN to them to make beta-like cells, and then give them back to the patient as an alternate therapy. There is a long way to go for this to be possible, including improving the functional properties of the cells, so that they more closely resemble beta cells, and figuring out alternate ways of converting intestinal cells to beta-like cells without gene therapy.”

This is hopefully a grand start to what might be a cure for type 1 diabetes.

Isolation of Pancreatic Stem Cells


There has been a robust debate as to whether or not the pancreas has a stem cell population. Several studies suggested that the pancreatic duct cells could differentiate into hormone-secreting pancreatic cells. Unfortunately, when the cells of the pancreatic duct are marked, they clearly never contribute to regeneration of the pancreas. According to an article that appeared in the journal Developmental Cell by Oren Ziv, Benjamin Glaser, and Yuval Dor entitled “The Plastic Pancreas,” tying off the pancreatic duct kills off the acinar cells, but it leads to a large increase in the number of hormone-secreting beta cells. Something seems to be contributing cells to the adult pancreas. However when lineage studies tried to confirm that the pancreatic duct cells formed the new cells, it failed to find any connection between the new cells in the pancreas and the duct.

Pancreas

Recent experiments from Chris Wright’s lab suggest that the acinar cells are a population of progenitor cells that divide and differentiate into different kinds of pancreatic cell types after injury to the pancreas. A similar result was observed in work by Desai and others. If that’s not odd enough for you, another set of experiments from Pedro Herrera research group has shown once all the insulin-secreting beta cells are killed off, the adjacent glucagon-secreting cells transdifferentiate into insulin-secreting beta cells. Therefore, something interesting is afoot in the pancreas.

All these experiments were done with rodents. Whether or not they are transferable to human remains uncertain. Nevertheless, a fascinating paper in EMBO Journal from Hans Clevers lab at the Hubrecht Institute, Utrecht, Netherlands haws succeeded in culturing pancreatic precursor cells.

Here’s how they did it. Clevers and his crew took the pancreatic duct of mice and partially tied it off. In order to stem cells from the digestive tract to grow, they must upregulate a signaling pathway called the “Wnt” pathway. The Wnt pathway is quiet in the pancreas, but one the pancreas is injured, the Wnt pathway swings into gear and the cells begin to divide.

When Clevers and company dropped pancreatic duct tissue into culture, Wnt signaling activity soared and the cells grew into a mini-organ (organoid) that resembled and tiny pancreas in a culture dish. In fact, a single cell taken from the pancreatic duct could be cultured into an organoid.

Establishment of the pancreas organoids from adult pancreatic ducts. (A) Scheme representing the isolation method of the pancreatic ducts and the establishment of the pancreatic organoid culture. The pancreatic ducts were isolated from adult mouse pancreas after digestion, handpicked manually and embedded in matrigel. Twenty-four hours after, the pancreatic ducts closed and generated cystic structures. After several days in culture, the cystic structures started folding and budding. (B) Representative serial DIC images of a pancreatic organoid culture growing at the indicated time points. Magnifications: × 10 (days 0, 2, 4, 6, and 8) and × 4 (day 10 onwards). (C) Growth curves of pancreas cultures originated from isolated pancreatic ducts cultured as described in Materials and methods. Note that the cultures followed an exponential growth curve within each time window analysed. Graphs illustrate the number of cells counted per well at each passage from passages P1–P3 (left), P5–P7 (middle) and P10–P12 (right). The doubling time (hours) is indicated in each graph. Data represent mean±s.e.m., n=2. (D) Representative DIC images of XGAL staining in WT (left), Axin2-LacZ (middle) and Lgr5-LacZ (right) derived pancreas organoids.
Establishment of the pancreas organoids from adult pancreatic ducts. (A) Scheme representing the isolation method of the pancreatic ducts and the establishment of the pancreatic organoid culture. The pancreatic ducts were isolated from adult mouse pancreas after digestion, handpicked manually and embedded in matrigel. Twenty-four hours after, the pancreatic ducts closed and generated cystic structures. After several days in culture, the cystic structures started folding and budding.  (B) Representative serial DIC images of a pancreatic organoid culture growing at the indicated time points. Magnifications: × 10 (days 0, 2, 4, 6, and 8) and × 4 (day 10 onwards). (C) Growth curves of pancreas cultures originated from isolated pancreatic ducts cultured as described in Materials and methods. Note that the cultures followed an exponential growth curve within each time window analysed. Graphs illustrate the number of cells counted per well at each passage from passages P1–P3 (left), P5–P7 (middle) and P10–P12 (right). The doubling time (hours) is indicated in each graph. Data represent mean±s.e.m., n=2. (D) Representative DIC images of XGAL staining in WT (left), Axin2-LacZ (middle) and Lgr5-LacZ (right) derived pancreas organoids.

This experiment shows that there are techniques for growing unlimited quantities of pancreatic cells.  The therapeutic possibilities of this technology is tremendous.  In Clever’s own words, “We have found a way to activate the Wnt pathway to produce an unlimited expansion of pancreatic stem cells isolated from mice.  By changing the growth conditions we can select two different fates for the stem cells and generate large numbers of either hormone-producing beta cells or pancreatic duct cells.”

Can this work with human pancreatic duct cells?  That is the $64,000 question.   Clevers and his groups will almost certainly try to answer this questions next.  If Clevers and his crew can get this to work, then the possibilities are vast indeed.