Digestive Cells Converted into Insulin-Secreting Cells

By switching off a single gene, Columbia Medical Center scientists have converted cells from the digestive tract into insulin-secreting cells. This suggests that drug treatments might be able to convert gut cells into insulin-secreting cells.

Senior author Domenico Accili said this of this work: “People have been talking about turning one cell into another for a long time, but until now we hadn’t gotten to the point of creating a fully functional insulin-producing cell by the manipulation of a single target.”

Accili’s work suggests that lost pancreatic beta cells might be replaced by retraining existing cells rather than transplanting new insulin-secreting cells. For nearly two decades, scientists have been trying to differentiate a wide variety of stem cells into pancreatic beta cells to treat type 1 diabetes. In type 1 diabetes, the patient’s insulin-producing beta cells are destroyed, usually by the patient’s own immune system. The patient becomes dependent on insulin shots in order to survive.

Without insulin, cells have no signal to take up sugar and metabolize it. Also muscles and the liver do not take up amino acids and make protein, and the body tends to waste away, ravaged by high blood sugar levels that progressively and relentlessly damage it without the means to repair this damage.

Insulin-producing beta cells can be made in the lab from several different types of stem cells, but the resulting beta cells often do not possess all the properties of naturally occurring beta cells.

This led Accili and others to attempt to transform existing cells into insulin-secreting beta cells. In previous work, Accili and others demonstrated that mouse intestinal cells could be converted into insulin-secreting cells (see Talchai C, et al., Nat Genet. 2012 44(4):406-12), This recent paper demonstrates that a similar technique also works in human intestinal cells.

The gene of interest, FOXO1, is indeed present in human gut endocrine progenitor and serotonin-producing cells. In order to determine in FOXO1 inhibition could induce the formation of insulin-secreting cells, Accili and others used human induced pluripotent stem cells (iPSCs) and small “gut organoids,” which are small balls of gut tissue that grow in culture.

Inhibition of FOXO1 by either introducing a mutant version of the gene that encoded a protein that soaked up all the wild-type protein or by using viruses that forced the expression of a small RNA that prevented the expression of the FOXO1 gene caused loss of FOXO1 activity. FOXO1 inhibition promoted the generation of insulin-positive cells within the gut organoids that express all the genes and proteins normally found in mature pancreatic β-cells. These transdifferentiated cells also released “C-peptide,” which is a byproduct of insulin production, in response to drugs that drive insulin secretion (insulin secretagogues). Furthermore, these cultured insulin-secreting cells and survive when transplanted into mice where they continue to secrete insulin in response to increased blood sugar concentrations.

The findings of Accili and his colleagues provide some evidence that gut-targeted FOXO1 inhibition or transplantation of cultured gut organoids made from iPSCs could serve as a source of insulin-producing cells to treat human diabetes.

This is a remarkable piece of research, but there is one thing that troubles me about it. If the patient’s immune system has been sensitized to beta cells, making new beta cells will simply give the immune system something else to attack. It seems to me that retraining to immune system needs to be done first before replacement of the beta cells can ever hope to succeed.

Human Stem Cells Elucidate the Mechanisms of Beta-Cell Failure in Diabetes

Wolfram syndrome is a rare form of diabetes characterized by high blood sugar levels that result from insufficient levels of the hormone insulin.  The chronically high blood sugar levels cause degeneration of the optic nerve, leading to progressive vision loss (optic atrophy).  Wolfram syndrome patients often also have abnormal pituitary glands that release abnormally low levels of the hormone vasopressin (also known as antidiuretic hormone or ADH), which causes hearing loss caused by changes in the inner ear (sensorineural deafness), urinary tract problems, reduced amounts of the sex hormone testosterone in males (hypogonadism), or neurological or psychiatric disorders.

Diabetes mellitus is typically the first symptom of Wolfram syndrome, usually diagnosed around age 6. Nearly everyone with Wolfram syndrome who develops diabetes mellitus requires insulin replacement therapy. Optic atrophy is often the next symptom to appear, usually around age 11. The first signs of optic atrophy are loss of color vision and peripheral (side) vision. Over time, the vision problems get worse, and people with optic atrophy are usually blind within approximately 8 years after signs of optic atrophy first begin.

Mutations in the WFS1 gene cause more than 90 percent of the cases of Wolfram syndrome type 1.  The WFS1 gene encodes a protein called wolframin that regulates the amount of calcium in cells.  A proper calcium balance is important for a whole host of cellular processes, including cell-to-cell communication, the tensing (contraction) of muscles, and protein processing.  Wolframin protein is found in many different tissues, such as the pancreas, brain, heart, bones, muscles, lung, liver, and kidneys.  Inside cells, wolframin is in the membrane of a cell structure called the endoplasmic reticulum that is involved in protein production, processing, and transport. Wolframin is particularly important in the pancreas, where it helps process proinsulin into mature hormone insulin, the hormone that helps control blood sugar levels.

WFS1 gene mutations lead to the production of a sub-functional versions of wolframin.  As a result, calcium levels within cells are not properly regulated and the endoplasmic reticulum does not work correctly.  When the endoplasmic reticulum does not have enough functional wolframin, the cell triggers its own cell death (apoptosis).  In the pancreas, the cells that make insulin (beta cells) die off, which causes diabetes mellitus.  The gradual loss of cells along the optic nerve eventually leads to blindness, and the death of cells in other body systems likely causes the various signs and symptoms of Wolfram syndrome type 1.

A certain mutation in the CISD2 gene also causes Wolfram syndrome type 2. The CISD2 gene provides instructions for making a protein that is in the outer membrane of cell structures called mitochondria,the energy-producing centers of cells.  Even though the function of the CISD2 protein is unknown, CISD2 mutations produce nonfunctional CISD2 protein that causes mitochondria to eventually break down. This accelerates the onset of cell death.  Cells with high energy demands such as nerve cells in the brain, eye, or gastrointestinal tract are most susceptible to cell death due to reduced energy, and people with mutations in the CISD2 gene have ulcers and bleeding problems in addition to the usual Wolfram syndrome features.

Some people with Wolfram syndrome do not have an identified mutation in either the WFS1 or CISD2 gene. The cause of the condition in these individuals is unknown.

Now that you have a proper introduction to Wolfram syndrome, scientists from the New York Stem Cell Foundation and Columbia University Medical Center have produce induced pluripotent stem cells (iPSCs) from skin samples provided by Wolfram syndrome patients.  All of the patients who volunteered for this study were recruited from the Naomi Berrie Diabetes Center and had childhood onset diabetes and required treatment with injected insulin, and all had vision loss.  Control cell lines that did not have mutations in WFS1 were obtained from Coriell Research for Medical Research.

These skin samples contained cells known as fibroblasts and these were reprogrammed into induced pluripotent stem cells.  In order to show that these cells were truly iPSCs, this group implanted them underneath the kidney capsule of immuno-compromised mice, and they formed the teratoma tumors so characteristic of these cells.

When these iPSCs were differentiated into insulin-secreting pancreatic beta cells, Linshan Shang and her colleagues discovered that the beta cells made from cells that did not come from Wolfram syndrome patients secreted normal levels of insulin.  However, those beta cells made from iPSCs derived from Wolfram patients failed to secrete normal quantities of insulin either in culture or when transplanted into the bodies of laboratory animals.  Further investigations of these cells showed these beta cells showed elevated levels of stress in the endoplasmic reticulum as a result of an accumulation of unfolded proteins.

What on earth is endoplasmic reticulum protein-folding stress?  First some cell biology.  When the cell needs to make a protein that will be secreted, embedded in a membrane or vesicle. that protein begins its life on ribosomes (protein synthesis factories of the cell) in the cytoplasm, but later those ribosomes are dragged to a cellular structure called the endoplasmic reticulum.  While on the surface of the endoplasmic reticulum, the ribosome completes the synthesis of the protein and extrudes the protein into the interior of the endoplasmic reticulum or embeds the protein into the endoplasmic reticulum membrane.  From there, the protein is trafficked in a vesicle to another subcellular structure called the Golgi apparatus, were it undergoes further modification, and from the Golgi apparatus, the protein goes to the membrane, secretory vesicle or other places.

If the proteins in the endoplasmic reticulum cannot fold properly, they clump and build up inside the endoplasmic reticulum, and this induces the ERAD or Endoplasmic Reticulum-Associated Protein Degradation response.  The players in the ERAD response are shown below.  As you can see, this response is rather complicated, but if it fails to properly clear the morass of unfolded proteins in the endoplasmic reticulum, then the cell will undergo programmed cell death.

ERAD Response

However, this research team did not stop there.  When they treated the cultured beta cells made from cells taken from Wolfram syndrome patients with a chemical called 4-phenyl butyric acid, the stress on the cells was relieved and the cells survived.  This experiment shows that relieving this unfolded protein stress is a potential target for clinical intervention.

“These cells represent an important mechanism that causes beta-cell failure in diabetes.  This human iPS cell model represents a significant step forward in enabling the study of this debilitating disease and the development of new treatments,” said Dieter Egli, the principal investigator of the study, and senior research fellow at the New York Stem Cell Foundation.

Because all forms of diabetes mellitus ultimately result from an inability of the pancreatic beta cells to provide sufficient quantities of insulin in response to a rise in blood sugar concentrations, this Wolfram patient stem cell model enables an analysis of a more specific pathway that leads to beta-cell failure in more prevalent forms of diabetes.  Furthermore, this strategy enables the testing of strategies to restore beta-cell function that may be applicable to all types of diabetes.

Susan L. Solomon of the New York Stem Cell Foundation, said, “Using stem cell technology, we were able to study a devastating condition to better understand what causes the diabetes syndromes as well as discover possible new drug targets.”

Rudolph L. Leibel, a professor of diabetes research and co-author of this study, said, “This report highlights again the utility of close examination of rare disorders as a path to elucidating more common ones.  Our ability to create functional insulin-producing cells using stem cell techniques on skin cells from patients with Wolfram’s syndrome has helped to uncover the role of ER stress in the pathogenesis of diabetes.  The use of drugs that reduce such stress may prove useful in the prevention and treatment of diabetes.”

The ERAD response seems to play a role in the survival of insulin-producing beta cells in both type 1 and type 2 diabetes.  The ERAD response opposes the stress of the immune assault in type 1 diabetes and the metabolic stress of high blood glucose levels in both types of diabetes.  When the ERAD response fails, cell death ensues and this reduces the number of insulin-producing cells.

New 3D Method Used to Grow Miniature Pancreas

Researchers from the University of Copenhagen, in collaboration with an international team of investigators, have successfully developed an innovative three-dimensional method to grow miniature pancreas from progenitor cells. The future goal of this research is to utilize this model system to fight against diabetes. This research was recently published in the journal Development.

The new method allows the cell material from mice to grow vividly in picturesque tree-like structures.
The new method allows the cell material from mice to grow vividly in picturesque tree-like structures.

The new method takes cell material from mice and grows them in vividly picturesque tree-like structures.  The cells used were mouse embryonic pancreatic progenitors, and they were grown in a compound called Matrigel with accompanying cocktails of growth factors.  In vitro maintenance and expansion of these pancreatic progenitors requires active Notch and FGF signaling, and therefore, this culture system recapitulated the in vivo conditions that give rise to the pancreas in the embryo.

Professor Anne Grapin-Botton and her team at the Danish Stem Cell Centre, in collaboration with colleagues from the Ecole Polytechnique Fédérale de Lausanne in Switzerland, have developed a three-dimensional culture method that takes pancreatic cells and vigorously expands them. This new method allows the cell material from mice to grow vividly into several distinct picturesque, tree-like structures. The method offers tremendous long-term potential in producing miniature human pancreas from human stem cells. Human miniature pancreas organoids would be valuable as models to test new drugs fast and effectively, without the use of animal models.

“The new method allows the cell material to take a three-dimensional shape enabling them to multiply more freely. It’s like a plant where you use effective fertilizer, think of the laboratory like a garden and the scientist being the gardener,” says Anne Grapin-Botton.

In culture, pancreatic cells neither thrive nor develop if they are alone. A minimum of four pancreatic cells, growing close together is required for these cells to undergo organoid development.

“We found that the cells of the pancreas develop better in a gel in three-dimensions than when they are attached and flattened at the bottom of a culture plate. Under optimal conditions, the initial clusters of a few cells have proliferated into 40,000 cells within a week. After growing a lot, they transform into cells that make either digestive enzymes or hormones like insulin and they self-organize into branched pancreatic organoids that are amazingly similar to the pancreas,” adds Anne Grapin-Botton.

The scientists used this system to discover that the cells of the pancreas are sensitive to their physical environment, and are influenced by such seemingly insignificant factors as the stiffness of the gel and contact with other cells.

An effective cellular therapy for diabetes is dependent on the production of sufficient quantities of functional beta-cells. Recent studies have enabled the production of pancreatic precursors but efforts to expand these cells and differentiate them into insulin-producing beta-cells have proved a challenge.

“We think this is an important step towards the production of cells for diabetes therapy, both to produce mini-organs for drug testing and insulin-producing cells as spare parts. We show that the pancreatic cells care not only about how you feed them but need to be grown in the right physical environment. We are now trying to adapt this method to human stem cells,” adds Anne Grapin-Botton.

Adult Stem Cells to Cure Diabetes?

Type 1 diabetics must inject themselves with insulin on a daily basis in order to survive. Without these shots, they would die.

Insulin injection

In most cases, type 1 diabetics have diabetes because their immune systems have attacked their insulin-producing cells and have destroyed them. However, a recent study at the University of Missouri has revealed that the immune system-dependent damage to the pancreas in type 1 diabetics goes beyond direct damage to the insulin-producing cells in the pancreas, The immune response also destroys blood vessels that feed tissues within the pancreas. This finding could provide the impetus for a cure that includes a combination of drugs and stem cells.

Habib Zaghouani and his research team at the University of Missouri School of Medicine discovered that “type 1 diabetes destroys not only insulin-producing cells but also blood vessels that support them,” explained Zaghouani. “When we realized how important the blood vessels were to insulin production, we developed a cure that combines a drug we created with adult stem cells from bone marrow. The drug stop the immune system attack, and the stem cells generate new blood vessels that help insulin-producing cells to multiply and thrive.”

Type 1 diabetes or juvenile diabetes, can lead to numerous complications, including cardiovascular disease, kidney damage, nerve damage, osteoporosis and blindness. The immune response that leads to type 1 diabetes attacks the pancreas, and in particular, the cell clusters known as the islet of Langerhans or pancreatic islets. Pancreatic islets contain several hormone-secreting cells types, but the one cell type in particular attacked by the immune system in type 1 diabetics are the insulin-secreting beta cells.

Pancreatic islets
Pancreatic islets

Destruction of the beta cells greatly decreases the body’s capability to make insulin, and without sufficient quantities of insulin, the body’s capability to take up, utilize and store sugar decelerates drastically, leading to mobilization of fats stores, the production of acid, wasting of several organs, excessive water loss, constant hunger, thirst, urination, acidosis (acidification of the blood), and eventually coma and death if left untreated.

The immune system not only destroys the beta cells, it also causes collateral damage to small blood vessels (capillaries) that carry blood to and from the pancreatic islets. This blood vessel damage led Zaghouani to examine ways to head this off at the pass and heal the resultant damage.

In previous studies, Zaghouani and others developed a drug against type 1 diabetes called Ig-GAD2. Treatment with this drug stops the immune system from attacking beta cells, but, unfortunately too few beta cells survived the onslaught from the immune system to reverse the disease. In his newest study, Zaghouani and his colleagues treated non-obese diabetic (NOD) with Ig-GAD2 and then injected bone marrow-based stem cells into the pancreas in the hope that these stem cells would differentiate into insulin-secreting beta cells.

“The combination of Ig-GAD2 and bone marrow [stem] cells did result in production of new beta cells, but not in the way we expected,” explained Zaghouani. “We thought the bone marrow [stem] cells would evolve directly into beta cells. Instead, the bone marrow cells led to growth of new blood vessels, and it was the new blood vessels that facilitated reproduction of the new beta cells. In other words, we discovered that to cure type 1 diabetes, we need to repair the blood vessels that allow the subject’s beta cells to grow and distribute insulin throughout the body.”

Zaghouani would lie to acquire a patent for his promising treatment and hopes to translate his preclinical research discovery from mice to larger animals and then to humans. In the meantime, his research continues to be funded by the National Institutes of Health and the University of Missouri.