Positive Results from Mesoblast’s Phase 2 Trial of Cell Therapy in Diabetic Kidney Disease


Mesoblast Limited has announced results from its Phase 2 clinical Trial that evaluated their Mesenchymal Precursor Cell (MPC) product, known as MPC-300-IV, in patients who suffer from diabetic kidney disease. In short, their cell product was shown to be both safe and effective. The results of their trial were published in the peer-reviewed journal EBioMedicine.  Researchers from the University of Melbourne, Epworth Medical Centre and Monash Medical Centre in Australia participated in this study.

The paper describes a randomized, placebo-controlled, and dose-escalation study that administered to patients with type 2 diabetic nephropathy either a single intravenous infusion of MPC-300-IV or a placebo.

All patients suffered from moderate to severe renal impairment (stage 3b-4 chronic kidney disease for those who are interested).  All patients were taking standard pharmacological agents that are typically prescribed to patients with diabetic nephropathy.  Such drugs include angiotensin-converting enzyme inhibitors (e.g., lisinopril, captopril, ramipril, enalapril, fosinopril, ect.) or angiotensin II receptor blockers (e.g., irbesartan, telmisartan, losartan, valsartan, candesartan, etc.).  A total of 30 patients were randomized to receive either a single infusion of 150 million MPCs, or 300 million MPCs, or saline control in addition to maximal therapy.

Since this was a phase 2 clinical trial, the objectives of the study were to evaluate the safety of this treatment and to examine the efficacy of MPC-300-IV treatment on renal function.  For kidney function, a physiological parameter called the “glomerular filtration rate” or GFR is a crucial indicator of kidney health.  The GFR essentially indicates how well the individual functional units within the kidney, known as “nephrons,” are working.  The GFR indicates how well the blood is filtered by the kidneys, which is one way to measure remaining kidney function.  The decline or change in glomerular filtration rate (GFR) is thought to be an adequate indicator of kidney function, according to the 2012 joint workshop held by the United States Food and Drug Administration and the National Kidney Foundation.

nephronanatomy

Diabetic nephropathy is an important disease for global health, since it is the single leading cause of end-stage kidney disease.  Diabetic nephropathy accounts for almost half of all end-stage kidney disease cases in the United States and over 40% of new patients entering dialysis treatment.  For example, there are almost 2 million cases of moderate to severe diabetic nephropathy in 2013.

Diabetic nephropathy can even occur in patients whose diabetes is well controlled – those patients who manage to keep their blood glucose levels at a reasonable level.  In the case of diabetic nephropathy, chronic infiltration of the kidneys by inflammatory monocytes that secrete pro-inflammatory cytokines causes endothelial dysfunction and fibrosis in the kidney.

Staging of chronic kidney disease (CKD) is based on GFR levels.  GFR decline typically defines the progression to kidney failure (for example, stage 5, GFR<15ml/min/1.73m2).  The current standard of care (renin-angiotensin system inhibition with angiotensin converting enzyme inhibitors or angiotensin II receptor blockers) only delays the progression to kidney failure by 16-25%, which leaves a large residual risk for end-stage kidney disease.  For patients with end-stage kidney disease, the only treatment option is renal replacement (dialysis or kidney transplantation), which incurs high medical costs and substantial disruptions to a normal lifestyle.  Due to a severe shortage of kidneys, in 2012 approximately 92,000 persons in the United States died while on the transplant list.  For those on dialysis, the mortality rate is high with an approximately 40% fatality rate within two years.

The main results of this clinical trial were that the safety profile for MPC-300-IV treatment was similar to placebo.  There were no treatment-related adverse events.  Secondly, patients who received a single MPC infusion at either dose had improved renal function compared to placebo, as defined by preservation or improvement in GFR 12 weeks after treatment.  Third, the rate of decline in estimated GFR at 12 weeks was significantly reduced in those patients who received a single dose of 150 million MPCs relative to the placebo group (p=0.05).  Finally, there was a trend toward more pronounced treatment effects relative to placebo in a pre-specified subgroup of patients whose GFRs were lower than 30 ml/min/1.73m2 at baseline (p=0.07).  In other words, the worse the patients were at the start of the trial, the better they responded to the treatment.

The lead author of this publication, Dr David Packham, Associate Professor in the Department of Medicine at the University of Melbourne and Director of the Melbourne Renal Research Group, said: “The efficacy signal observed with respect to preservation or improvement in GFR is exciting, especially given that this trial was not powered to show statistical significance. Patients receiving a single infusion of MPC-300-IV showed no evidence of developing an immune response to the administered cells, suggesting that repeat administration is feasible and may in the longer term be able to halt or even reverse progressive chronic kidney disease. I hope that this very promising investigational therapy will be advanced to rigorous Phase 3 clinical trials to test this hypothesis as soon as possible.”

Patients who received s single IV infusion of MPC-300-IV cells showed no evidence of developing an immune response to the administered cells.  This suggests that repeated administration of MPCs is feasible and might even have the ability to halt, or even reverse progressive chronic kidney disease.

Packham and his colleagues hope that this cell-based therapy can be advanced to a rigorous Phase 3 clinical trial to further test this treatment.

Rebooting Pancreatic Cells Can Normalize Blood Sugar Levels in Diabetic Mice


Type 1 diabetes results from the inability of the endocrine portion of the pancreas to secrete sufficient quantities of the hormone insulin. The cells that make insulin, beta cells, have been destroyed. Consequently, type 1 diabetics must inject themselves with insulin routinely in order to stay alive. Is there a better way?

A new strategy suggests that maybe pancreatic cells can be “rebooted” to produce insulin and that sure reprogramming could potentially help people with type 1 diabetes manage their blood sugar levels without the need for daily injections. This therapeutic approach is simpler and potentially safer than giving people stem cells that have been differentiated into pancreatic beta cells.

Philippe Lysy at the Cliniques Universitaires Saint Luc, which is part of the Catholic University of Louvain in Belgium, and his colleagues have reprogrammed pancreatic duct cells extracted from dead donors who were not diabetic at the time of death. The duct cells do not produce insulin, but they have a natural tendency to grow and differentiate into specific types of cells.

Lysy and his team grew the cells in the laboratory and encouraged them to become insulin-producing cells by exposing them to fatty particles. These fatty particles are absorbed into the cells after which they induce the synthesis of the MAFA transcription factor. MAFA acts as a genetic “switch” that binds to DNA and activates insulin production.

Implantation of these altered cells into diabetic mice showed that the cells were able to secrete insulin in a way that controls blood sugar levels. “The results are encouraging,” says Lysy.

Lysy’s colleague, Elisa Corritore, reported these results at this week’s annual meeting of the European Society for Pediatric Endocrinology in Barcelona, Spain. Lysy and others are preparing to submit their results for publication.

This work, if it continues to pan out, might lead to the harvesting of pancreatic ducts from deceased donors and converted in bulk into insulin-making cells. Such “off-the-shelf” cells could then be transplanted into people with type 1 diabetes to compensate for their inability to make their own insulin.

“We would hope to put the cells in a device under the skin that isolates them from the body’s immune system, so they’re not rejected as foreign,” says Lysy. He says devices like this are already being tested for their ability to house insulin-producing cells derived from stem cells.

Previous attempts to get round this problem have included embedding insulin-producing cells in a seaweed derivative prior to transplantation in order to keep them from being destroyed by the recipient’s immune system.

Lysy thinks that since insulin-producing cells originate from pancreatic tissue, they have an inherently lower risk of becoming cancerous after the transplant. This has always been a worry associated with tissues produced from embryonic stem cells, since these have the capability to form tumors if any are left in their original state in the transplanted tissue.

The basic premise of the work looks solid, says Juan Dominguez-Bendala, director of stem cell development for Translational Research at the University of Miami Miller School of Medicine’s Diabetes Research Institute in Florida. “However, until a peer-reviewed manuscript is published and all the details of the work become available to the scientific community, it is difficult to judge if this advance represents a meaningful leap in the state of the art.”

Lysy expects it will take between three and five years before the technique is ready to be tested in human clinical trials.

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.

Encapsulated Stem Cells to Treat Diabetes


A research group from the Sanford-Burnham Medical Research Institute in La Jolla, San Diego, California has used pluripotent stem cells to make insulin-secreting pancreatic beta cells that are encapsulated in a porous capsule from which they secrete insulin in response to rising blood glucose levels.

“Our study critically evaluates some of the potential pitfalls of using stem cells to treat insulin-dependent diabetes,” said Pamela Itkin-Ansari, an adjunct assistant professor with a joint appointment at UC San Diego. “We have shown that encapsulated hESC-derived pancreatic cells are able to produce insulin in response to elevated glucose without an increase in the mass or their escape from the capsule. This means that the encapsulated cells are both fully functional and retrievable.”

For this particular study, Itkin-Ansari and her colleagues used glowing cells to ensure that their encapsulated cells stayed in the capsule. To encapsulate the cells, this group utilized a pouch-like encapsulation device made by TheraCyte, Inc. that features a bilaminar polytetrafluoroethylene (PTFE) membrane system. This pouch surrounds the cells and protects from the immune system of the host while giving cells access to nutrients and oxygen.

With respect to the cells, making insulin-secreting beta cells from embryonic stem cell lines have met with formidable challenges. Not only are beta cells differentiated from embryonic stem cells poorly functional, but upon transplantation, they tend to be fragile and poorly viable.

To circumvent this problem, encapsulation technology was tapped to protect donor cells from the ravages of the host immune system. However, an additional advance made by Itkin-Ansari and her colleagues is that when they encapsulated islet-precursor cells, derived from embryonic stem cells, these cells survived and differentiated into pancreatic beta cells. In fact, islet progenitor cells turn out to be the ideal cell type for encapsulation, since they are heartier, and differentiate into beta cells quite efficiently when encapsulated.

In their animal model tests, these cells remained encapsulated for up to 150 days. Also, as an added bonus, because the progenitor cells develop glucose responsiveness without significant changes in mass, they do not outgrow their capsules.

In order to properly get this protocol to work in humans, Itkin-Ansari and her group has to scale up the size of their capsules and the number of cells packaged into them. Another nagging question is, “How long will an implanted capsule last in a human patient?

“Given the goals and continued successful results, I expect to see the technology become a treatment option for patients with insulin-dependent diabetes,” said Itkin-Ansari.

To date, Itkin-Ansari and others have been able to successfully treat diabetic mice. The problem with these experiments is that they mice were made diabetic by treatment with a drug called beta-alloxan, which destroys the pancreatic beta cells. Human type 1 diabetic patients have an immune system that is sensitized to beta cells. Even though the encapsulation shields the beta cells from contact with the immune system, will this last in human patients with an aggressive immune response against their own beta cells? It seems to me that induced pluripotent cells made from the patient’s own cells would be a better choice in this case than an embryonic stem cell line.

Nevertheless, this is a fine piece of research for diabetic patients.

Umbilical Cord Stem Cells Normalize Blood Glucose Levels in Diabetic Mice


Diabetes mellitus results from an insufficiency of insulin (Type 1 diabetes) or an inability to properly respond to insulin (Type 2 diabetes). Type 1 diabetes is caused by an attack by the patient’s own immune system on their pancreatic beta cells, which synthesize and secrete insulin. It is a disease characterized by inflammation in the pancreas. This suggests that abatement of inflammation in the pancreas might provide relief and delay the onset of diabetes.

Mesenchymal stem cells isolated from umbilical cord connective tissue, which is also known as Wharton’s jelly (WJ-MSCs), have the ability to reverse inflammatory destruction and might provide a way to delay or even reverse the onset of Type 1 diabetes.

To test this possibility, Jianxia Hu, Yangang Wang, and their colleagues took 60 non-obese diabetic mice and divided them into four groups: a normal control group, a normal diabetic group, a WJ-MSCs prevention group that was treated with WJ-MSCs before the onset of diabetes, and a WJ-MSCs treatment group that was treated with WJ-MSCs after the onset of diabetes.

After their respective treatments, the onset time of diabetes, levels of fasting plasma glucose (FPG), fed blood glucose levels and C-peptide (an indication of the amount of insulin synthesized), regulation of cytokines, and islet cells were examined and evaluated.

After WJ-MSCs infusion, fasting and fed blood glucose levels in WJ-MSCs treatment group decreased to normal levels in 6-8 days and were maintained for 6 weeks. The levels of fasting C-peptide of the WJ-MSC-treated mice was higher compared to diabetic control mice. In the WJ-MSCs prevention group, WJ-MSCs protected mice from the onset of diabetes for 8-weeks, and the fasting C-peptide in this group was higher compared to the other two diabetic groups.

Other comparisons between the WJ-MSC-treated group and the diabetic control group, showed that levels of regulatory T-cells (that down-regulate autoinflammation), were high and levels of pro-inflammatory molecules such as IL-2, IFN-γ, and TNF-α. The degree of inflammation in the pancreas was also examined, and pancreatic inflammation was depressed, especially in the WJ-MSCs prevention group.

These experiments show that infusions of WJ-MSCs can down-regulate autoimmunity and facilitate the recovery of islet β-cells whether given before or after onset of Type 1 Diabetes Mellitus. THis suggests that WJ-MSCs might be an effective treatment for Type 1 Diabetes Mellitus.

See March 2014 edition of the journal Endocrine.

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.

A New Way to Treat Kidney Disease and Heart Failure


St. Michael’s Hospital in Toronto, Ontario is the site of new research that uses bone marrow stem cells to treat chronic kidney disease and heart failure in rats.

Darren Yuen and Richard Gilbert of St. Michael’s Hospital were the first to show in 2010 that enriched stem cells improved heart and kidney functions in rats afflicted with both diseases. Their work generated concerns about the side effects of returning such stem cells to the body.

Since 2010, Yuen and Gilbert have found that enriched bone marrow stem cells secrete stromal cell–derived factor-1α (SDF-1α), a chemokine that is made by ischemic tissue but is rapidly degraded by dipeptidyl peptidase-4 (DPP-4), in culture dishes.  Injection of SDF-1α into rats has many of the same positive effects as when the stem cells themselves are injected into rats.  Even more remarkably, if a drug that inhibits the enzyme DPP-4 is given (sitagliptin) produced many improvements as well.

“We’ve shown that we can use these ‘hormones’ to replicate the beneficial effects of the stem cells in treating animals with chronic kidney disease and heart failure,” said Yuen, who practices as a nephrologist. “In our view, this is a significant advance for stem cell therapies because it gets around having to inject stem cells.”

Yuen said that they do not yet know what kind of hormone the cells are secreting, but identifying the hormone would be the first step toward the goal of developing a synthetic drug.

Chronic kidney disease (CKD) is much more prevalent than was once believed, with recent estimates suggesting that up to five percent of the Canadian population may be affected with this condition.

The number of people with CKD and end-stage renal failure is expected to rise as the population ages and more people develop Type 2 diabetes. People with kidney disease often develop heart disease, and many of them die from heart failure rather than kidney failure.

Radio Interview About my New Book


I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

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.

Stem Cells Analyze the Cause and Treatment of Diabetes


A research group that is part of the New York Stem Cell Foundation or NYSCF has generated patient-specific beta cells (the cells that secrete insulin in the pancreas), and these cultured beta cells accurately recapitulate the features of maturity-onset diabetes of the young or MODY.

In this research, NYSCF scientists and researchers from the Naomi Berrie Diabetes Center of Columbia University used skin cells from MODY patients to produce induced pluripotent stem cells (iPSCs) that were differentiated in the culture dish to form insulin-secreting pancreatic beta cells (if that sounds like a lot of work that’s because it is).

Other laboratories have succeeded in generating beta cells from embryonic stem cells and iPSCs, but questions remain as to whether or not these cells accurately recapitulate genetically-acquired forms of diabetes mellitus.

Senior co-author of this study, Dieter Egli, a senior research fellow at NYSCF, said: “We focused on MODY, a form of diabetes that affects approximately one in 10,000 people. While patients and other models have yielded important clinical insights into this disease, we were particularly interested in its molecular aspects – how specific genes can affect responses to glucose by the beta cell.”

MODY is a genetically inherited form of diabetes mellitus, and the most common form of MODY, type 2, results from mutations in the glucokinase or GCK gene. Glucokinase is a liver-specific enzyme and it adds a phosphate group to sugar so that the sugar can be broken down to energy by means of a series of reactions known as “glycolysis.” Glucokinase catalyzes the first step of glycolysis in the liver and in pancreatic beta cells. Mutations in GCK increase the sugar concentration in order for GCK to properly metabolize sugar, and this increases blood sugar levels and increases the risk for vascular complications.

The steps of the enzymatic pathway glycolysis, which is used by cells to degrade sugar to energy.
The steps of the enzymatic pathway glycolysis, which is used by cells to degrade sugar to energy.

MODY patients are usually misdiagnosed as type 1 or type 2 diabetics, but proper diagnosis can greatly alter the treatment of this disease. Correctly diagnosing MODY can also alert family members that they too might be carriers or even susceptible to this disease.

NYSCF researchers worked with skin cells from two patients from the Berrie Center who had type 2 MODY. After reprogramming these skin cells to become iPSCs, they differentiated the cells into beta cells, These cells had the impaired GCK activity, but in order to compare them to something, the NYSCF group also made iPSCs with a genetically engineered version of GCK that was impaired in the same way as the GCK gene in these two patients, and another cell line with normal versions of the GCK gene. They used these iPSCs to make cultured beta cells.

“Our ability to create insulin-producing cells from skin cells, and then to manipulate the GCK gene in these cells using recently developed molecular methods, made it possible to definitely test several critical aspects of the utility of stem cells for the study of human disease,” said Haiqing Hua, lead author of this paper and a postdoctoral fellow in the Division of Molecular Genetics.

The beta cells made from these iPSCs were transplanted into mice and these mice were given an oral glucose tolerance test. An oral glucose tolerance test is used to diagnose diabetes mellitus. The patient fasts for 12 hours and then is given a concentrated glucose concentration (4 grams per kilogram body weight), which the patient drinks and then the blood glucose level is examined at 30-minute intervals. The blood glucose levels of diabetic patients will rise and only go down very sluggishly whereas the blood glucose levels of a nondiabetic patient will rise and then decrease as their pancreatic beta cells start to make insulin. Insulin signals cells to take up glucose and utilize it, which lowers the blood glucose levels. A reading of over 200 milligrams per deciliters

When mice with the transplanted beta cells made from iPSCs were given oral glucose tolerance tests, the beta cells from MODY patients   showed decreased sensitivity to glucose.  In other words, even in the presence of high blood sugar levels, the beta cells made from iPSCs that came from MODY patients secreted little insulin.  However, high levels of amino acids, which are the precursors of proteins, also induces insulin secretion, and in this case, beta cells from MODY patients secreted sufficient quantities of insulin.

When the iPSCs made from cells taken from MODY patients were subjected to genetic engineering techniques that repaired the defect in the GCK gene, these iPSCs differentiated into beta cells that responded normally to high blood glucose levels and secreted insulin when the blood glucose levels rose.

By making beta cells from MODY patients and then correcting the genetic defect in them and returning them to normal glucose sensitivity, NYSCF scientists showed that this type of diagnosis could lead to cures for MODY patients.

“These studies provide a critical proof-of-principle that genetic characteristics of patient-specific insulin-producing cells can be recapitulated through use of stem cell techniques and advanced molecular biological manipulation.  This opens up strategies for the development of new approaches to the understanding, treatment, and, ultimately, prevention of more common types of diabetes,” said Rudolph Leibel of the Columbia University Medical Center.

Betatrophin, a New Liver Protein that Increases the Number of Insulin-Making Cells


Douglas Melton’s laboratory at the Harvard University Stem Cell Institute in Cambridge, Massachusetts has discovered a liver hormone that stimulates the growth of insulin-secreting beta cells in the pancreas. This discovery could very well lead to new treatments for diabetes.

This hormone, betatrophin, was induced in mice by treating them with a peptide that binds to insulin receptors. The insulin-occupied insulin receptors were unable to bind insulin, and that caused the animals to be resistant to insulin. Under these conditions, the livers of these mice produced betatrophin, which caused the animals’ insulin-secreting pancreatic β cells to proliferate. Melton and others searched for genes that showed increased activity under these insulin-resistant conditions, which allowed Melton and colleagues to isolate and identify betatrophin.

According to Melton and his co-workers, “Transient expression of betatrophin in mouse liver significantly and specifically promotes pancreatic β cell proliferation, expands β cell mass, and improves glucose tolerance” (from the abstract of the paper).

Further experiments showed that when eight-week-old mice injected with betatrophin there was an average 17-fold rise in the proliferation of their insulin-secreting pancreatic β cells. Melton and others published these results in the journal Cell. Fortunately, betatrophin is also found in the human liver, according to Melton and others.

“It’s rare that one discovers a new hormone, and this one is interesting because it’s so specific,” says Melton. “It works only on β cells and it’s so robust and so potent.”

Pancreatic β cells replicate rapidly during embryonic and neonatal stages in both mice and humans, but beta cell growth decreases dramatically in adults. A decrease in the function of beta cells late in life is the main cause of type 2 diabetes. Type 2 diabetes is a metabolic disorder that affects more than 300 million people worldwide. In the United States alone, the two forms of diabetes — type 2 and type 1— account for US$176 billion in direct medical costs each year.

Melton hypothesized that injections of betatrophin once a month, or even once a year, could potentially induce enough activity in pancreatic β cells to provide the same level of blood-sugar control for people with type 2 diabetes as do daily insulin injections. According to Melton, betatrophin would cause fewer complications, since the body would make its own insulin. He also hopes that betatrophin will be able to help people with type 1 diabetes.

Matthias Hebrok, director of the University of California, San Francisco, Diabetes Center, says that the work “is a great advance”. “The findings are very interesting,” he says, although he would like to see the experiments repeated in older mice. “Do mice that are on their way to becoming diabetic at an advanced age truly have an increase in proliferative capacity upon treatment with betatrophin?” he asks. This is a fair question.

Henrik Semb, managing director of the Danish Stem Cell Center in Copenhagen, says that “the identification of a factor, betatrophin, that stimulates mouse β-cell replication with remarkable efficiency is a very important discovery, because it provides the starting point for further studies to elucidate the underlying mechanism of β-cell replication.”

β-cell replication has proved difficult to control in humans, but producing enough betatrophin for testing in human clinical trials will take about two years, according to Melton, who is also working to identify the hormone’s receptor and its mechanism of action.

References: Yi, P., Park, J.-S. & Melton, D. A. Cell http://dx.doi.org/10.1016/j.cell.2013.04.008 (2013).

No Evidence of Regeneration of Insulin-Making Cells in the Pancreas


Type 1 diabetics and severe type 2 diabetics show reduction of insulin secretion as a result of destruction of the specific cells in the pancreas that produce insulin. These cells, the so-called beta cells, suffer destruction from the patient’s immune system (type 1 diabetes) or from overwork (type 2 diabetes). The holy grail of diabetes treatment is the regeneration of lost beta cells.

pancreas beta cells

Several reports have marshaled evidence that the pancreatic beta cells do regenerate, but the constant assault by the immune system eventually destroys all the beta cells. Other reports have argued that a stem cell population in the ductal system of the pancreas can replenish the beta cells. Thus, augmenting beta cell regeneration seemed to be simply a matter of employing the already-present regenerative properties of the pancreas.

Unfortunately, a recent study seems to put the kibosh on any hope that the pancreatic beta cells regenerate. This new study was published in the Journal of Clinical Investigation. In this paper, researchers at Children’s Hospital of Pittsburgh report were unable to find signs of new beta cell production in several common models of pancreatic injury (see Xiao, X., et al. 2013. No evidence for beta-cell neogenesis in murine adult pancreas. J Clin Invest., 123(5):2207-17).

“Overall, the paper puts one more nail in what was already becoming an increasingly tight coffin for what had been the prevailing hypothesis about β-cell neogenesis in adult mice,” said Fred Levine, who studies β-cell regeneration at Sanford Burnham Medical Research Institute in La Jolla, California and was not involved in the study. Still, Levine cautions that this negative result does not completely rule out adult regeneration of β-cells in other injury models.

To detect the formation of new beta cells, George Gittes and his colleagues used an old cell tracking method, but applied it in a different manner. They used two fluorescent tags in transgenic mice: a red tag that targets a protein in the cell membrane of most cells in the body, except for insulin producing cells, and a green tag that only tagged pancreatic beta cells. Gittes team looked for cells that turned on their insulin genes for the first time during a 40–48 hour window. The cells, therefore, would express both tags and, as a result, appeared yellow.

The yellow transition was detected in embryonic mice, where neogenesis (new beta cell production) is expected to occur. But when the researchers examined adult cells, they saw no yellow cells—meaning no evidence of neogenesis. They repeated this experiment in several common models of pancreatic damage. For example, the pancreatic duct ligation model (PDL damages other pancreatic cell types but not β-cells. The absence of detectable neogenesis in these models “puts pressure on us to find models in which there is neogenesis,” said Gittes. But he remains “very confident” that there are other models in which neogenesis occurs.

In fact, several models not tested in this paper have shown evidence of neogenesis, including one of Gittes’ own. In 2011, in which his team found evidence of neogenesis in a mice who beta cells were engineered to express diphtheria toxin receptors, that led to their death (see Criscimanna, A., et al. 2011. Duct cells contribute to regeneration of endocrine and acinar cells following pancreatic damage in adult mice. Gastroenterology, 141(4):1451-62). In 2010, two other research groups, including one headed by Levine, also demonstrated neogenesis in adult mice through trandifferentiation of preexisting α-cells in pancreatic islets into β-cells (Thorel, F., et al. 2010. Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-cell loss. Nature,464(7292):1149-54 and Chung, C.H., et al. 2010. Pancreatic β-cell neogenesis by direct conversion from mature α-cells. Stem Cells, 28(9):1630-8).

“Overall, I believe that the pathway by which β-cell regeneration occurs…is likely to vary depending on the stimulus for regeneration,” said Levine. Therefore, the current work does not rule out neogenesis, even from duct cells, in other models. “I would argue that the old cliché, ‘Absence of evidence is not evidence of absence’ should be kept in mind when evaluating studies like this.”

Human Amniotic Epithelial Cells – Remarkable Possibilities for a Small Price


My apologies to my readers for my inactivity. Many deadlines make for less blogging. Nevertheless, I hope to get back to a more regular blogging schedule once things quiet down a bit.

Today’s entry is about a fascinating group of cells found in the extraembryonic membranes of the fetus known as the amnion. The amniotic sac is a thin, transparent pair of membranes that is actually rather tough. This sac holds the fetus until shortly before birth. In inner membrane of the amnion sac contains the amniotic fluid and fetus and the outer membrane, the chorion, surrounds the amnion and is part of the placenta.

The amniotic membrane contains a remarkable cell type known as amniotic epithelial cells or hAECs (the “h” is for human). Upon isolation after birth, the amnion membrane and manually separated from the chorion membrane and washed in a saline (salt) solution in order to remove all the blood. Then the epithelial cells are liberated from the basement membrane upon which they sit by a product called TrypZean. TrypZean is a recombinant trypsin, which is very clean and devoid of animal products. Trypsin is one of the enzymes in your digestive system that degrades proteins. By expressing the human trypsin gene in bacteria and purifying the protein, Sigma-Aldrich corporation can sell it for a profit to scientists for various procedures.

A single amnion membrane can yield in the vicinity of 120 million viable hAECs, which can be maintained in serum-free culture conditions. After being grown for some time, hAECs will have normal chromosome compositions and will also maintain chromosomes that have nice, long ends (telomeres). This indicates that the cells are healthy and dying while they grow in culture (see Murphy et al., Current Protocols in Stem Cell Biology, 2010; Chapter 1: Unit 1E.6). .

In culture,. hAECs do not grow like weeds. Mesenchymal stem cells (MSCs) tend to grow better than their hAEC brethren, but hAECs possess a remarkable ability to differentiate into a wide variety of different cell types. Sivakami Ilancheran in the laboratory of Martin Pera at the University of Monash in Clayton, Australia showed that hAECs were able to differentiate into heart muscle, skeletal muscle, bone, fat cells, pancreatic cells, liver, and at least two kinds of nerve cells. Also, when injected into mice, hAECs never formed tumors (Ilancheran et al., Biology of Reproduction 77 (2007): 577-88). Murphy and others have also shown that hAECs can be isolated after collection and stored for clinical therapies.

Given that hAECs are accessible, what are they good for? When it comes to regenerative medicine, preclinical studies with hAECs have produced very solid results that may pave the way for other studies.

HAECs can differentiate into lung cells and this feature makes them an attractive candidate for lung diseases. Lung diseases cause inflammation of the lung and scarring that decreases overall lung capacity. Cystic fibrosis, acute respiratory distress syndrome, chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary edema, and pulmonary hypertension are all lung diseases that could potentially be treated with hAECs.

In animal models of lung disease, particular chemicals are given to the animal that damage the lung. The wounded lung tissue initiates inflammation that brings white blood cells into the lung that augment the lung damage, which results in lung scarring. If hAECs are given to mice whose lungs have been damaged by the anti-cancer drug bleomycin, the signs of inflammation and the genes normally expressed during inflammation fade away. There is also less scarring in the lungs and the functional recovery of these animals is significantly better than those animals that do not receive hAECs (Murphy et al., Cell Transplantation 2011 20(6): 909-23). In fact, hAECs can differentiate into lung cells and integrate into lung tissue. The significance of this is not lost on respiratory specialists who treat patients with cystic fibrosis. Cystic fibrosis patients lack a functional copy of a ion transport protein and poor ion transport cause the production of thick, sticky mucous that clogs up the lung pathways and causes patients to suffocate to death. However, hAECs can differentiate into lung cells that express this ion transporter. Therefore, hAECs could be a potential treatment for cystic fibrosis. Clearly hAECs have great potential for tissue engineering applications with lung disease.

Lungs are not the only organ that hAECs can help heal. These cells can also differentiate into pancreatic insulin-making cells. In the laboratory, Wei and coworkers succeeded in stimulating hAECs to secrete insulin and express the main sugar transport protein found in pancreatic insulin-secreting cells (Wei et al., Cell Transplantation 2003 12(5): 545-552). When transplanted into diabetic mice, hAECs normalize their blood sugar levels and their weights returned to normal. This shows that hAECs might represent a major breakthrough in the management of diabetes.

Clearly these cells, which come from a tissue that is normally thrown out after birth, are brimming with possibilities for regenerative medicine. Hopefully more research will produce even more possibilities.

Using Tissue-Specific Mesenchymal Stem Cells to Make Insulin-Producing Beta Cells from Embryonic Stem Cells


Embryonic stem cell lines are made from four-five-day-old human embryos. At this stage of development, the embryo is a sphere of cells with two distinct cell populations; an outside layer of flat trophoblast cells and an inner clump of round inner cell mass (ICM) cells. The embryo consists of ~100 cells four days after fertilization, and ~150 cells five days after fertilization.

Trophoblast

Embryonic stem cell (ESC) derivation involves the removal of the trophoblast cells (which are collectively called the trophectoderm) and the isolation of the ICM cells. There are several ways to remove the trophectoderm, but the most commonly-used technique is “immunosurgery,” which uses antibodies that bind to proteins on the surfaces of the trophectoderm, and serum to initiate destruction of the trophoblast cells. The isolated ICM cells are then cultured, and if they grow, they may produce an embryonic stem cell line.

Immunosurgery was first perfected by Davor Solter and Barbara B. Knowles on mouse embryos. They used an antiserum that was raised in rabbits when the rabbits were immunized against mouse spleen tissue. When mouse embryos were incubated with this antiserum plus serum from mice, all the cells of the mouse embryo died. However, if they used the rabbit antiserum and serum from guinea pig, then only the trophoblast cells were destroyed. For human embryonic stem cell derivation, the rabbits are immunized again human red blood cells, and this rabbit antiserum is used with guinea pig serum. The serum contains proteins called “complement,” which bind to cells that have antibodies attached to them and bore holes in those cells, thus destroying them.

When ICM cells are cultured, they are placed on a layer of mouse cells that have been treated with a chemical called mitomycin C to prevent them from dividing. These non-dividing cells act as “feeder cells” that keep the ICM cells from differentiating. Because ICM cells are grown on animal cells, they cannot be used for clinical purposes, since they will possess animal proteins can carbohydrates on their surfaces, which would be attacked by the patient’s immune system. However, several ESC lines have been derived without animal products, and it is possible to make ESC lines that would be safe or human use.

ESC derivation

ESC derivation results in the destruction of human embryos. There is not two ways about it. Even though there are potential ways around this problem, the majority of ESC lines were made literally over the dead bodies of very young human beings. All the rationalization in the world (the embryo is too young, too small, too inchoate, too unwanted, going to die anyway, in the wrong place at the wrong time) do not undo the fact that the embryo is a very young human being, and making an ESC line from it ends his/her life.

Getting the ESC line to differentiate in what you want it to be is another problem. If any undifferentiated cells remain after differentiation, they can cause tumors. Therefore, there is a need to ensure that differentiation is efficient and complete. To this end, Doug Melton’s lab at Harvard University has published a remarkable paper in the journal Nature that uses mesenchymal stem cells from particular organs to direct the differentiation of ESC lines.

Melton’s lab, in particular Julie M. Sneddon and Malgorzata Borowiak (say that fast five times), established 16 lines of tissue-specific mesenchymal stem cells (MSCs) from embryonic, neonatal and adult mouse intestine, liver, spleen, and pancreas and human pancreas too. Then they cultured mouse ESCs on these MSC lines to determine if they could drive the ESCs to differentiate into pancreas cells. In the embryo, pancreatic precursors express several genes in a nested, hierarchical fashion. First, they express Sox17, which is a common endodermal marker, and then pancreatic progenitors all express Pdx1. Of these pancreatic progenitors, some express Ngn3 and these will become endocrine rather than exocrine cells, and othe the Ngn3-expressing cells, a few will become beta cells that make insulin.

Melton and his co-workers tried to determine if any of these genes was up-regulated in their ESC lines if that were co-cultured with their established MSC lines. They discovered that four lines – MSC1, 2, 3, & 4, all affected gene expression when co-cultured with ESCs. MSC 1 and 2 induced and increase in Sox17 expression and MSC 3 and 4 increased the expression of Ngn3 in ESCs.

These changes in gene expression were due to increased cell proliferation of cells actually expressing these genes and not due to differential survival. Also, no combination of growth factors could achieve the same results as the accompanying MSC lines. Thus there is more going on here than the MSCs just secreting the right growth factors. The MSCs must be making contact with the ESCs and inducing them to differentiate into a particular cell type.

Next Melton and his colleagues determined if this interaction with MSCs caused the ESCs to lose their ability to self-renew. The answer was a clear “no.” Even though these ESC lines were expressing genes characteristic of endodermal or pancreatic tissue, they did not lose their ability to differentiate into pancreatic tissue when appropriately induced to do so, and they also id not lose their ability to self-renew and grow competently in culture.

In a more stringent test, these ESCs that had been grown on tissue-specific MSCs were implanted into mice. As Melton points out in the paper, the “most efficient published protocols for in vitro differentiation of pluripotent cells to beta-cells yield only a small percentage (typically 0-15%) of insulin-positive cells, and these do not secrete insulin in a glucose-responsive manner.” Could the MSC-conditioned ESCs do any better?

Before implantation, the ESCs were differentiated into endodermal progenitors (Sox17-expressing cells), and co-cultured with MSCs for at least 3-7 passages. Then they were differentiated into beta cells and transplanted into mice. There were a few important controls that were used; Just saline, implantations of MSCs alone, and ESCs that had been differentiated into beta cells, but had never been passaged on MSCs. Finally, human pancreatic islets were used as a positive control.

The results were interesting to say the least. The saline and MSC alone implantations showed no insulin production with or without glucose. Likewise the human pancreatic islets made insulin in a glucose-dependent manner (no surprise there). The ESC-derived beta cells that had never been passaged on MSCs made insulin, and even showed some ability to respond to glucose and make more insulin after glucose ingestion. However, the beta cells derived from ESCs that had been passaged on MSCs made insulin in a glucose-dependent manner. The experiment produced a wide range of variability since the number of transplanted cells differed between each trial, but the implanted beta cells derived from ESCs passaged on tissue-specific MSCs definitely performed the best, and even did as well or better than the implanted human beta cells in some cases.

a, Schematic depicting implantation of human ESC-derived progenitors. b, Immunofluorescence staining of human ESC-derived endoderm, passaged seven times on mesenchyme and engrafted for 3 months (top panel) or further differentiated to Pdx1+ stage and then engrafted for 2 months (bottom panel). c, Glucose-tolerance test of animals implanted with PBS or mesenchyme only, human islets or Pdx1+ pancreatic progenitors derived from unpassaged (P0), or passaged (P4 or P7) human endoderm. d, Fasting- and glucose-induced (45 min glucose) plasma human C-peptide levels. Pairs of bars represent two time points per animal; data represent mean of two technical replicates ± s.d.
a, Schematic depicting implantation of human ESC-derived progenitors. b, Immunofluorescence staining of human ESC-derived endoderm, passaged seven times on mesenchyme and engrafted for 3 months (top panel) or further differentiated to Pdx1+ stage and then engrafted for 2 months (bottom panel). c, Glucose-tolerance test of animals implanted with PBS or mesenchyme only, human islets or Pdx1+ pancreatic progenitors derived from unpassaged (P0), or passaged (P4 or P7) human endoderm. d, Fasting- and glucose-induced (45 min glucose) plasma human C-peptide levels. Pairs of bars represent two time points per animal; data represent mean of two technical replicates ± s.d.

Melton notes at the end of his paper that this technique worked rather well for coaxing ESCs to form pancreatic derivatives, but it could very well be applicable to other systems as well. Also, it could probably work with induced pluripotent stem cells, which have many (though not all) of the characteristics of ESCs and can be made without killing human embryos. Thus another technique for increasing ESC differentiation seems to be on the table.

Stem Cells from Pancreas Discovered


While it is abundantly clear, that the pancreas has some ability to regenerate, it is unclear if the pancreas has a stem cell population or simply makes new cells through cell division. However, a research team from Bundoora, Victoria, Australia has identified what they think is a stem cell from pancreas that can differentiate into insulin-producing beta cells. Such a discovery might means that type diabetics could potentially regrow their beta cells.

This work comes from scientists from the Walter and Eliza Hall Institute. In particular, Ilia Banakh and Len Harison from the institute’s Molecular Medicine division have identified stem cells from the pancreas, and have also developed a technique to differentiate these cells into insulin-producing beta cells.

Patients with type 1 diabetes mellitus make insufficient quantities of the hormone insulin. Insulin is the most important anabolic hormone of the body. Insulin signals to cells to take up glucose and use it, but it also signals to muscles to make muscle proteins and to fat cells to store more fat. Damage to the beta cells in the pancreas causes a decrease in insulin production to point that the body does not have sufficient quantities of insulin to properly control blood sugar levels. Type 1 diabetics must give themselves shots of insulin daily.

This new work by Banakh and Harrison advances recent studies that found cells with stem cell-like characteristics. However, according to Harrison, “But what Dr. Banakh has done is pinpoint the cell of origin of the insulin-producing cells and show that the number of these cells and their ability to turn into insulin-producing cells increases in response to pancreas injury. This is exciting, because it means that the potential to regenerate insulin-producing cells is there in all of us, even as adults.”

Professor Harrison continued: “In the long run, we hope that people with type 1 diabetes might be able to regenerate their own insulin-producing cells. This would mean they could make their own insulin and regain control of their blood sugar levels, curing their diabetes. Of course, this strategy will only work if we can devise ways to overcome the immune attack on the insulin-producing cells, that causes diabetes in the first place.”

This work confirms the presence of a stem cell population in the pancreas.  In this study, the authors and their co-workers discovered a so-called  “side population” (SP) of cells that have the ability to pump a dye that binds DNA from the cell.  Cell with this capability have stem cell-like  properties.  However, when the pancreas was injured, the number of these side population cells increased substantially (enriched >10-fold).  Even though these SP cells expressed neither insulin protein nor RNA, when they were cultured in serum-free culture medium with defined factors, the SP cells grew and differentiated into islet hormone-expressing cells that secreted insulin in response to glucose.

To extend this work, Harrison and Banakh and others transplanted these SP cells into diabetic mice encased in vascularized chambers, and they maintained their insulin-producing capacities.

Thus these SP cells in the adult pancreas expand in response to β cell injury and are a source of β cell progenitors with potential for the treatment of diabetes.

Embryonic Stem Cell-Derived Beta Cells Cure Diabetic Mice


Stem cell scientists in Canada have collaborated with biotechnology industries to successfully reverse diabetes mellitus in mice by means of stem cell treatments. This is certainly a medical breakthrough that might lead to treatments in human patients.

The lead researcher, Timothy Kieffer, who is a professor at the University of British Columbia, and scientist from the New Jersey-based company BetaLogics showed that stem cell transplants can restore insulin production and reverse diabetes mellitus in mice.

Beta cells reside in an organ called the pancreas, which is behind the stomach. The pancreas has an “exocrine” function, which means that it secretes materials such as digestive enzymes and bicarbonate ions into a duct, and an endocrine function, which means that it secretes hormones directly into the bloodstream. The exocrine function of the pancreas is accomplished by clusters of cells known as “acinar cells.” Acinar cells cluster around a tiny branch of the pancreatic duct, and they secrete digestive enzymes and bicarbonate ions into the pancreatic duct, which are released into the upper portion of the small intestine (duodenum). These enzymes degrade fats, proteins, nucleic acids, and carbohydrates in the small intestine, which prepares the complex molecules in food for digestion. The endocrine functions are carried out by islands of cells dispersed throughout the pancreas that are away from the pancreatic duct, but clustered around blood vessels. These “pancreatic islets” as they are called secrete hormones that regulate the metabolism of food-derived molecules in our bodies.

There are five types of cells in pancreatic islets: alpha cells, beta cells, delta cells, epsilon cells and PP cells. Alpha cells secrete a hormone called glucagon, which mobilizes store sugar stores in the body and releases them into the bloodstream, this raising blood sugar levels. Beta cells secrete insulin, which stimulates the uptake and metabolism of bloodstream sugar, thus lowering blood sugar levels. Delta cells secrete somatostatin, which regulates growth hormone release by the anterior pituitary, but also affects the release of many hormones in the digestive system and inhibits the release of glucagon and insulin. The epsilon cells secrete a hormone called ghrelin, which is a potent appetite stimulant. The PP cells secrete PP or pancreatic peptide, which helps the pancreas to self-regulate its secretory activities, both exocrine and endocrine.

Once glucose levels rise in the blood, the beta cells release insulin, and when glucose levels in the blood decrease, insulin secretion decreases. This “feedback loop” is essential for proper regulation of blood glucose levels, and beta cells that are immature do not properly respond to rises in blood glucose levels. In this study, however, the research effort completely recreated the insulin/sugar feedback loop that enables insulin levels to automatically rise or fall according to the blood glucose levels.

Damage to the beta cells results in insufficient insulin production and poor regulation of the blood sugar levels. Damage to the beta cells results in type 1 diabetes mellitus, and without the secretion of sufficient quantities in insulin after a meal, the cells do not receive the signal to take up sugar, and are starved from energy. Meanwhile, extremely high sugar levels in the blood react with molecules in the organs of the body, which causes long-term damage to the nervous system, eyes, kidneys, and peripheral tissues. Consequently, type 1 diabetics are at increased risk for amputations, blindness, heart attack, stroke, nerve damage and kidney failure.

Regular injections of insulin are the most common treatment for type 1 diabetes mellitus, but experimental transplants of healthy pancreatic cells from human donors have shown to be effective. Unfortunately, such a treatment is severely limited by the availability of donors.

In this experiment, human embryonic stem cells were differentiated into beta cells and implanted into the diabetic mice. After the stem cell transplant, the diabetic mice were weaned off insulin. Three to four months later, the mice were able to maintain healthy blood sugar levels even after being fed large quantities of sugar. Transplanted cells removed from the mice after several months had all the markings of normal insulin-producing pancreatic cells.

These experiments, however, have one very large caveat. In the words of Kiefer: “We are very excited by these findings, but additional research is needed before this approach can be tested clinically in humans. The studies were performed in diabetic mice that lacked a properly functioning immune system that would otherwise have rejected the cells. We now need to identify a suitable way of protecting the cells from immune attack so that the transplant can ultimately be performed in the absence of any immunosuppression.”

Type 1 diabetes usually results from the immune system of the diabetic patient attacking their own beta cells. Replacing the beta cells mere gives the immune system something that it already recognizes to attack. Therefore, replacing the beta cells with new beta cells from any other source is potentially problematic.

There is a possibility that the beta cells could be implanted inside a porous encasement that is not accessible to the immune system, but can still secrete insulin into the bloodstream in response to increase blood sugar levels. Such a strategy would circumvent the immune system problems.

Stem Cells Derived from Fat Show Promise for Regenerative Medicine


A detailed review article in the June issue of Plastic and Reconstructive Surgery, the official medical journal of the American Society of Plastic Surgeons, has examined the safety and clinical efficacy of fat-derived stem cells. Stem cells from fat, also known as ACSs, are a promising source of cells for use in plastic surgery and regenerative medicine, according to this review, but there are still many questions that remain about them. Much more research is needed in order to completely establish the safety and effectiveness of ASC-based therapies in human patients. The review article was written by ASPS Member Surgeon Rod Rohrich, MD of University of Texas Southwestern Medical Center, Dallas, and his colleagues (Dr. Rohrich is Editor-in-Chief of Plastic and Reconstructive Surgery).

ASCs are very easily procured from humans, since simple procedures such as liposuction can provide more than enough material for therapies. On the average, one gram of fat yields about 5,000 stem cells, whereas the yield from the same quantity of bone marrow is about 1,000 cells (B. M. Strem, K. C. Hicok, M. Zhu et al., “Multipotential differentiation of adipose tissue-derived stem cells,” Keio Journal of Medicine, vol. 54, no. 3, pp. 132–141, 2005.). Once isolated from the fat, ASCs have the capacity to form fat cells, but also bone, cartilage and muscle cells.

From a therapeutic standpoint, ASCs promote the development of new blood vessels (angiogenesis). ASCs are also not recognized by the immune system and they seem to staunch inflammation. According the Dr. Rohrich and is co-authors, “Clinicians and patients have high expectations that ASCs may well be the answer to curing many recalcitrant diseases or to reconstruct anatomical defects.”

Fortunately, there is great interest in ASCs, and this means that the number of studies that examine ASCs or utilize them for experimental treatments have soared. Unfortunately, there is continued concern about the “true clinical potential” of ASCs. In the words of this new article, “For example, there are questions related to isolation and purification of ASCs, their effect on tumor growth, and the enforcement of FDA regulations.”

Rohrich and others conducted a rather in-depth review of all known clinical trials of ASCs. Thus far, most studies have been performed in Europe and Korea, and only three in the United States, to date. This reflects the stringency of FDA regulations.

Most ASC clinical trials to date have been examined the use of ASCs in plastic surgery. In this case, plastic surgeon-researchers have used ASCs for several types of soft tissue augmentation (breast augmentation, especially after implant removal and regeneration of fat in patients with abnormal fat loss or lipodystrophy). Studies exploring the use of ASCs to promote healing of difficult wounds have been reported as well. ASCs have also been used as in so-called soft tissue engineering or tissue regeneration. In these cases, the results have been inconclusive.

Other medical specialties have also made use of ASCs as treatments for other types of medical conditions. For example, ASCs have been studied for used to treat certain blood and immunologic disorders, heart and vascular problems, and fistulas (abnormal connection between an organ, vessel, or intestine and another structure). There are some other studies that have examined the use of ASCs for generating new bone for use in reconstructive surgery. A few studies have reported promising preliminary results in the treatment of diabetes, multiple sclerosis, and spinal cord injury. Perhaps one of the most encouraging results was the complete absence of serious adverse events related to ASCs in any of these studies.

These results are encouraging, but all of these applications are in their infancy. Globally speaking, less than 300 patients have been treated with ASCs, and no standardized protocol exists for the preparation or clinical applications of ASCs. Additionally, there is no consensus as to the number of ASCs required per treatment, or how many treatments are required for the patient to show clinical improvement. Thus Rohrich and his colleagues have taken a “proceed with caution approach.” They conclude that “further basic science experimental studies with standardized protocols and larger randomized controlled trials need to be performed to ensure safety and efficacy of ASCs in accordance with FDA guidelines.”

Stem Cell Research Provides New Insights into Insulin Production


Insulin is a protein hormone made by the beta cells of the pancreatic islets. It signals to the liver, skeletal muscles, and fat tissue to take up glucose and store it as glycogen (a polymer of glucose), or to convert it into fat. Insulin also induces the uptake of amino acids by muscles and the liver to form protein. This makes insulin one of the most important anabolic (building) hormones in the body.

Without sufficient quantities of insulin, blood sugar levels soar, since cells do not have the signal to take up sugar. Large quantities of sugar are quite damaging to cells and tissues, and the accumulating damage causes blindness, kidney failure, heart failure, circulatory and peripheral nerve troubles and other ailments.

This pathological condition is known as diabetes mellitus, and treatment of it requires routine injections of insulin. In order to actually treat insulin, we must somehow replace the deleted or damaged beta cells. Stem care cell treatment can potentially do this, but the details are still being worked out.

Danish stem cell scientists have provided some insights into ways to convert stem cells into pancreatic beta cells. By examining pancreatic development in mice, Palle Serup and his research group discovered a new gene called “Mind Bomb-1” that plays a role in pancreatic beta cell formation.

Accord to Dr., Serup, “To get stem cells to develop into insulin-producing beta cells, it is necessary tp know what signaling mechanisms normally control the creation of beta cells during fetal development. This is what our new research results can contribute. When we know the signaling paths, we can copy then in test tubes and thus in time convert stem cells to beta cells.” Dr. Serup is a member of the Danish Stem Cell Center or DanStem at the University of Copenhagen.

In a collaboration with researchers at DanStem, the Danish Hagedorn Research Institute, and other international partners in Japan, Germany, South Korea and the United States, these new findings were published in the April edition of the Proceedings of the National Academy of Sciences.

Previous work has established that during the early hours of the development of the pancreas, a signaling pathway that utilizes the “Notch” protein prevents pancreatic cells from differentiating into endocrine (hormone-making) cells and promotes the continued growth and proliferation of a kind of generic, all-purpose pancreas precursor cell. These all-purpose pancreatic precursor cells are called multipotent progenitor cells or MPCs, and they express two genes: Nkx6-1, and Ptf1.

A bit later, Nkx6 and Ptf1a start to antagonize each other such that cells that express Nkx6 cannot express and Ptf1 and Ptf1-expressing cells cannot express Nkx6. This antagonism between these two genes segregates the developing pancreas into two domains. The bit that is furthest away from the ductal system expresses Ptf1a+ and form “acinar progenitors.” The acinar cells are the clusters that make all the digestive enzymes released by the pancreas the bicarbonate ions. The portion of the developing pancreas that is closet to the ductal system expresses Nkx6-1, and makes the pancreatic duct and β-cell progenitors (see Russ HA, Efrat S. Pediatr Endocrinol Rev. 2011 Dec;9(2):590-7).

This sounds simple, but there are still several gaps that have yet to be filled in. For example, the signals that regulate patterning of the incipient pancreas and cause the segregation of the cells from one end to the other. Also, what dictates the formation of β-cell progenitors as opposed to ductal cells is also presently unknown.

In this present article, Serup and his colleagues discovered that deleting Mind Bomb-1 activity from the developing pancreas preventing the segregation of MPCs into Nkx6-expressing and Ptf1a-expressing cells. Instead the Nkx6-1-expressing cells were replaced by Ptf1-expressing cells. This prevented the formation of beta cells.

Interestingly, Serup and his team found that once the Notch protein acts early during pancreatic development, it actually acts again to help establish the segregated pancreas with Nkx6-1-expressing cells at one end and Ptf1a-expressing cells at the other. This shows that Notch is not only necessary early on, but also later for beta cell formation.

According the Serup, “Our research contributes knowledge about the next step in development and the signaling involved in the communication between cells – an area that has not been extensively described. This new knowledge about the ability of the so-called “Notch” signaling first to inhibit and then to stimulate the creation of hormone-producing cells is crucially important to being able to control stem cells better when working with them in test tubes.”

Osiris’ Prochymal Mesenchymal Stem Cell Formulation is Safe for Diabetes Treatments


The biotechnology company called Osiris Therapeutics, Inc. has developed an adult mesenchymal stem cell formulation it calls “Prochymal.” Osiris scientists have been busy subjecting Prochymal to a battery of clinical trials that include testing Prochymal as a treatment for chronic obstructive pulmonary disease, Crohn’s disease, myocardial infarction, and acute graft-versus-host disease. Now Osiris is in the process of testing Prochymal as a treatment for newly diagnosed diabetes mellitus.

This clinical trial transferred mesenchymal stem cells from unrelated adult donors into 63 pediatric and adult type diabetics to determine if such a transfer can slow the progression of this debilitating disease. Patients will randomly receive either the stem cells or a placebo. Thus far, no patients who have received the mesenchymal stem cell infusion have shown any adverse reactions, despite receiving the cells from unrelated donors and without any drugs to suppress the immune system. Additionally, no significant differences in insulin levels were observed between the placebo and the experimental group after one year of receiving the mesenchymal stem cell infusion. However, patients who had received Prochymal showed fewer severely low blood glucose concentrations hypoglycemic events) than those who had been given the placebo. The test is still ongoing, and all patients will be observed for another year.

The rationale behind this trial resides in the unique ability of mesenchymal stem cells to down-regulate the immune response. Because type 1 diabetes typically results from the patient’s immune system attacking and destroying the insulin-secreting beta cells found in the pancreatic islets, an influx of mesenchymal stem cells might be able to decelerate the destruction of the beta cells. This suppression of beta cell destruction might lead to the regeneration of the beta cells, since several stem cell populations in the pancreas and pancreatic ducts can differentiate into beta cells. Since, Prochymal is specifically designed to control inflammation, promote tissue regeneration and prevent the formation of scar tissue; it is a prime candidate agent to reduce the loss of beta cells at the onset of type 1 diabetes.

Jay Skyler, professor and medicine and deputy researcher of the Diabetes Research Institute at the University Of Miami Miller School Of Medicine commented, “This groundbreaking study in an important first step in the use of stem cells to potentially alter the course of type 1 diabetes. The ability to safely use stem cells from unrelated donors is an important finding of this study and provides new possibilities for further development and stem cell therapies for type 1 diabetes.”

Co-culturing Immune Cells with Umbilical Cord Stem Cells Reverses Type 1 Diabetes in a Small Study


Type 1 diabetes results from an inability to make sufficient quantities of insulin. Insulin is made by specific cells in the pancreatic islets (also known as the islet of Langerhans). Most type 1 diabetics have suffered destruction of their pancreatic beta cells. Beta cell destruction can result from physical trauma to the pancreas, which causes the digestive enzymes of the pancreas to destroy the beta cells. For example, pancreatitis, pancreatic surgery, or certain industrial chemicals can cause diabetes. Also, particular drugs can also cause temporary diabetes, such as corticosteroids, beta blockers, and phenytoin. Rare genetic disorders (Klinefelter syndrome, Huntington’s chorea, Wolfram syndrome, leprechaunism, Rabson-Mendenhall syndrome, lipoatrophic diabetes, and others) and hormonal disorders (acromegaly, Cushing syndrome, pheochromocytoma, hyperthyroidism, somatostatinoma, aldosteronoma) also increase the risk for diabetes.

Additionally, viral infections of pancreas can cause the immune response to destroy pancreatic cells, and this wipes out enough beta cells to cause the onset of type 1 diabetes. The coxsackievirus family of viruses is a family of enteric viruses that are cause infections that are sometimes associated with the onset of type 1 diabetes, as are mumps and congenital rubella. In most cases, genetic factors cause the immune system to view the pancreatic beta cells as foreign invaders, and the beta cells are attacked and destroyed. Researchers have found at least 18 genetic loci that are designated IDDM1 – IDDM18 that are related to type 1 diabetes. The IDDM1 region contains the “HLA genes” that encode proteins called “major histocompatibility complex”. HLA genes encode cell-surface proteins that act as “bar codes” for the immune system. When cells have the proper bar codes on their cell surfaces, the immune system recognizes those cells as being a part of the body in which they reside, and the immune system leaves them alone. Any cells that do not have the right bar codes are attacked and destroyed, which is known as the “graft versus host response.” Therefore, it is fair to say that HLA genes affect the immune response. New advances in genetic research are identifying other genetic components of type 1 diabetes. Other chromosomes and genes continue to be identified.

A recent paper attempts to cure type 1 diabetes by using umbilical cord stem cells. Umbilical cord stem cells (UCSCs) have the ability to greatly calm down the immune system. UCSCs secrete a wide variety of molecules that prevent immune cells from reacting to and destroying other cells, and also have many cell surface proteins that bind to the surfaces of immune cells and put them to sleep (see Abdi et al., Diabetes 2008;57:1759-67 & Aguayo-Mazzucato C. and Bonner-Weir S, Nature Reviews Endocrinology 2010;6:726-36).

Animal experiments have shown that co-culturing UCSCs with circulating immune cells alters the immune response against pancreatic beta cells and greatly increases the ability of the animal to regulate blood glucose levels (Zhao et al., PLoS ONE 2009;4:e4226). The UCSCs seem to “re-educate” the immune cells so that they do not recognize the pancreatic islets are foreign anymore. Therefore, Yong Zhao and his colleagues in Theodore Mazzone’s laboratory at the University of Chicago, IL, and collaborators at the General Hospital of Jinan Military Command, Shandong, China, used human UCSCs to re-educate immune cells in human type 1 diabetic patients.  See here for this paper.

To do this, they circulated the blood of each patient through a close-loop system that separated the immune cells (lymphocytes) from whole blood. Thee lymphocytes were then co-cultured with UCSCs for 2-3 hours and then returned to the patients.

The results were remarkable. Six patients in group A, who all had some residual beta cell function showed successively improved insulin production 12-24 weeks after treatment. They also showed a reduced need for insulin shots, and overall improvement of their fasting blood glucose levels. Six patients in group B, who had no residual beta cell function, showed increased production of insulin production 12 week after treatment. This is an incredible finding because those without beta cells essentially grew new ones that were not attacked by the immune system. The group B group also saw successively reduced requirements for injected insulin. The patients in the control, whose immune cells did not undergo re-education by UCSCs showed no improvement.

Furthermore, the patients whose immune cells were re-educated by the UCSCs, did not experience any adverse effects. This procedure seems to be quite safe and feasible.

There is a word of caution here. These patients must be followed over several years to establish that the re-education of the lymphocytes is maintained over time. Also, this study is quite small and despite the amazing results, a larger study is needed. All the same, this is an incredible result that reverses type 1 diabetes, and even though caution is needed, embryonic stem cells were not required to do this.