Insulin-Producing Beta Cell Subtypes May Impact Diabetes Treatment


Researchers from the Oregon Stem Cell Center in Portland, Oregon have demonstrated the existence of at least four separate subtypes of human insulin-producing beta cells that may be important in the understanding and treatment of diabetes.

“This study marks the first description of several different kinds of human insulin producing beta cells,” said Markus Grompe of the Oregon Stem Cell Center at Oregon Health Science University. “Some of the cells are better at releasing insulin than others, whereas others may regenerate quicker. Therefore, it is possible that people with different percentages of the subtypes are more prone to diabetes. Further understanding of cell characteristics could be the key to uncovering new treatment options, as well as the reason why some people are diabetic and others are not.”

Diabetes mellitus affects more than 29 million people in the United States. There are two main types of diabetes mellitus, type I and type II. Type I diabetes mellitus is caused by insufficient production of insulin. Type II diabetes mellitus is caused by insulin resistance or the inability of the body to properly respond to the insulin produced by the body. Type I diabetes mellitus results from dysfunction or loss of insulin producing beta cells in the endocrine portion of the pancreas. Insulin is a hormone that helps the body keep normal blood sugar levels, and incorporate sugar in the bloodstream into cells to grow and repair tissues. Previously, only a single variety of beta cell was known to exist. However, using human pancreatic islets, or clusters of up to 4,000 cells, Grompe and colleagues identified a method to identify and isolate four distinct types of beta cells. They also found that hundreds of genes were differently expressed between cell subtypes and that these distinct beta cell subtypes produced different amounts of insulin.

All type 2 diabetics had abnormal percentages of the subtypes, suggesting a possible role in the disease process. Additional research is needed to determine how different forms of diabetes – and other diseases – affect the new cell subtypes, as well as how researchers may take advantage of these differences for medical treatment.

This work was published in: Craig Dorrell et al., “Human islets contain four distinct subtypes of β cells,” Nature Communications, 2016; 7: 11756 DOI: 10.1038/ ncomms11756.

Induced Pluripotent Stem Cells from Diabetic Foot Ulcer Fibroblasts


Dr. Jonathan Garlick is professor of Oral Pathology at Tufts University and has achieved some notoriety among stem cell scientists by publishing a stem-cell rap on You Tube to teach people about the importance of stem cells.

Garlick and his colleagues have published a landmark paper in the journal Cellular Reprogramming in which cells from diabetic patients were reprogrammed into induced pluripotent stem cells (iPSCs).

Garlick and his colleagues have established, for the first time, that skin cells from diabetic foot ulcers can be reprogrammed iPSCs. These cells can provide an excellent model system for diabetic wounds and may also used, in the future, to treat chronic wounds.

ESC and iPSCs differentiation to fibroblast fate. ESC and iPSC were differentiated and monitored at various stages of differentiation. Representative images show the morphology of ESC and 2 iPSC lines after days 1, 4, 7, 10, 14, 21 and 28 of differentiation. Early morphologic changes showed differentiation beginning at the periphery of colonies (day 1). At later stages cells acquired fibroblast features of elongated, stellate cells (day 10 at days 21 and 28 of differentiation.
ESC and iPSCs differentiation to fibroblast fate. ESC and iPSC were differentiated and monitored at various stages of differentiation. Representative images show the morphology of ESC and 2 iPSC lines after days 1, 4, 7, 10, 14, 21 and 28 of differentiation. Early morphologic changes showed differentiation beginning at the periphery of colonies (day 1). At later stages cells acquired fibroblast features of elongated, stellate cells (day 10 at days 21 and 28 of differentiation.

Garlick’s team at Tufts University School of Dental Medicine and the Sackler School of Graduate Biomedical Sciences at Tufts, have also used their diabetic-derived iPSCs to show that a protein called fibronectin is linked to a breakdown in the wound-healing process in cells from diabetic foot ulcers.

One of the goals of Garlick’s research is to develop efficient protocols to make functional cell types from iPSCs and to use them to generate 3D tissues that demonstrate a broad range of biological functions. His goal is to use the 3D model system to develop human therapies to replace or regenerate damaged human cells and tissues and restore their normal function.

In this paper, Garlick and his colleagues showed that not only can fibroblasts from diabetic wounds form iPSCs, but they can also participate in 3D skin-like tissues. This model system is more than a disease-in-a-dish system but disease-in-a-tissue system.

Fabrication of three-dimensional tissue construction. (A) A collagen gel embedded with human dermal fibroblasts is layered onto a polycarbonate membrane. (B) After dermal fibroblasts contract and remodel the collagen matrix, keratinocytes are then seeded onto it to create a monolayer that will form the basal layer of the tissue. (C) Tissues are raised to an air-liquid interface to initiate tissue development that mimics in vivo skin.
Fabrication of three-dimensional tissue construction. (A) A collagen gel embedded with human dermal fibroblasts is layered onto a polycarbonate membrane. (B) After dermal fibroblasts contract and remodel the collagen matrix, keratinocytes are then seeded onto it to create a monolayer that will form the basal layer of the tissue. (C) Tissues are raised to an air-liquid interface to initiate tissue development that mimics in vivo skin. From this site.

“The results are encouraging. Unlike cells taken from healthy human skin, cells taken from wounds that don’t heal – like diabetic foot ulcers – are difficult to grow and do not restore normal tissue function,” said Garlick. “By pushing these diabetic wound cells back to this earliest, embryonic stage of development, we have ‘rebooted’ them to a new starting point to hopefully make them into specific cell types that can heal wounds in patients suffering from such wounds.”

Scientists in Garlick’s laboratory used these 3D tissues to test the properties of cells from diabetic foot ulcers and found that cells from the ulcers get are not able to advance beyond synthesizing an immature scaffold made up predominantly of a protein called fibronectin.  Fibronectin, unfortunately, seems to prevent proper closure of wounds.

Fibronectin Sigma

Fibronectin has been shown to be abnormal in other diabetic complications, such as kidney disease, but this is the first study that directly connects it to cells taken from diabetic foot ulcers.

Deriving more effective therapies for foot ulcers has been slow going because of a lack of realistic wound-healing models that mimic the extracellular matrices of human tissues. This scaffolding is critical for wound repair in skin, and other tissue as well.

The work in this paper builds on earlier experiments that showed that cells from diabetic ulcers have fundamental defects that can be simulated using laboratory-grown 3D tissue models. These 3D models will almost certainly be a good model system to test new therapeutics that could improve wound healing and prevent those limb amputations that result when treatments fail.

Garlick’s 3D model will allow him and other researchers to push these studies forward. Can they differentiate their cells into more mature cell types that can be studied in 3D models to see if they will improve healing of chronic wounds?

More than 29 million Americans have diabetes. Diabetic foot ulcers, often resistant to treatment, are a major complication. The National Diabetes Statistics Report of 2014 stated that about 73,000 non-traumatic lower-limb amputations in 2010 were performed in adults aged 20 years or older with diagnosed diabetes, and approximately 60 percent of all non-traumatic lower-limb amputations occur in people with diabetes.

This paper appeared in: Behzad Gerami-Naini, et al., Cellular Reprogramming. June 2016, doi:10.1089/cell.2015.0087.

Insulin-Secreting Beta Cells from Human Fat


In a study led by Martin Fussenegger of ETH Zurich, stem cells extracted from the fat of a 50-year-old test subject were transformed into mature, insulin-secreting pancreatic beta cells.

Fussenegger and his colleagues isolated stem cells from the fat of a 50-year-old man and used these cells to make induced pluripotent stem cells (iPSCs). These iPSCs were then differentiated into pancreatic progenitor cells and then into insulin-secreting beta cells but means of a “genetic software” approach.

Genetic software refers to the complex synthetic network of genes required to differentiate pancreatic progenitor cells into insulin-secreting beta cells. In particular, three genes, all of which expression transcription factors, Ngn3, Pdx1, and MafA, are particularly crucial for beta cell differentiation.

Fussenegger and his team designed a a protocol that would express within these fat-based stem cells the precise concentration and combination of these transcription factors. This feature is quite important because the concentration of these factors changes during the differentiation process. For example, MafA is not present at the start of beta cells maturation, but appears on day four on the final data of maturation when its concentration rises precipitously. The concentration of Ngn3 rises and then falls and the levels of Pdx1 rise at the beginning and towards the end of maturation.

The Zurich team used ingenious genetic tools to reproduce these vicissitudes of gene expression as precisely as possible. By doing so, they were able to differentiate the iPSC-derived pancreatic progenitor cells into insulin-secreting beta cells.

This work was published in Nature Communications 7, doi:10.1038/ncomms11247.

The fact that Fussenegger’s team was able to use a synthetic gene network to form mature beta cells from adult stem cells is a genuine breakthrough. The genetic network approach also seems to work better than the traditional technique of adding various chemicals and growth factors to cultures cells. “It’s not only really hard to add just the right quantities of these components (growth factors) at just the right time, it’s also inefficient and impossible to scale up,” said Fussenegger.

This new process can successfully transform three out of four fat stem cells into beta cells. Also the beta cells made with this method have the same microscopic appearance of natural beta cells in that they contain internal granules full of insulin. They also secrete insulin in response to increased blood glucose concentrations. Unfortunately the amount of insulin made by these cells is lower than that made by natural beta cells.

Pancreatic islet transplants have been performed in diabetic patients, but such transplantations also require treatment with potent antirejection drugs that have potent side effects.

“With our beta cells, there would likely be no need for this action (administering antitransplantation drugs), since we can make them using endogenous cell material taken from the patient’s own body. This is why our work is of such interest in the treatment of diabetes,” said Fussenegger.

Fussenegger and his group have made these beta cells in the laboratory, but they have yet to transplant them into a diabetic patient. However, the success of this synthetic genetic software technology might also be useful in the reprogramming of adult cells into other types of cells that are useful for therapeutic purposes.

Encapsulated Human Islet Cells Halt Diabetes for 6 Months in Mice


Researchers from the Massachusetts Institute of Technology (MIT), Boston’s Children Hospital, and several other institutions have successfully used human pancreatic islet cells encased in a porous capsule to halt Type 1 diabetes in mice without causing an adverse immune response for six months. These experiments been reported in two separate scientific journals.

The first study utilized a modified alginate material to encapsulate the pancreatic islet cells. Alginate is a material that originally was derived from brown algae, and has been used to encapsulate cells without harming them or preventing them from sensing and responding to biochemical signals.

Despite all these advantages to alginate, nonspecific kept theior immune responses against it eventually result in the build up of scar tissue around alginate capsules that renders any implanted cells placed inside them ineffective.

The MIT group tested modified alginate derivatives that would not elicit this nonspecific immune response. From a library of over 800 alginate derivatives, the group came upon one particular alginate derivative called triazolethiomorpholine dioxide (TMTD).

To test TMTD capsules in diabetic mice, The MIT group teamed up with Harvard researcher Doug Melton, who supplied human pancreatic beta cells derived from human embryonic stem cells. Once the TMTD-encased beta cells were implanted into mice suffering from type 1 diabetes, the cells immediately began producing insulin in response to increases in blood glucose levels. Diabetic, laboratory mice with these islet cell TMTD-encased implants kept their blood glucose levels within a healthy range for 174 days, which was the whole length of the study.

“Encapsulation therapies have the potential to be groundbreaking for people with Type 1 Diabetes. These treatments aim to effectively establish long-term insulin independence and eliminate the daily burden of managing the disease for months, possibly years, at a time without the need for immune suppression,” said Julia Greenstein, an executive with the Juvenile Diabetes Research Foundation, who funded these two studies.

See here and here.

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.

Some Types of Obesity Might be Caused by a Faulty Immune System


When we think of our Immune systems, we normally entertain visions of white blood cells that fight off invading viruses and bacteria. However, recent work suggests that our immune systems may also being fighting a war against fat.

When laboratory mice are engineered to lack a specific type of immune cell, they become obese and show signs of high blood pressure, high cholesterol, and diabetes. Even though these findings have yet to be replicated in humans, they are already helping scientists understand the triggers of metabolic syndrome, a cluster of conditions associated with obesity.

A new study “definitely moves the field forward,” says immunologist Vishwa Deep Dixit of the Yale School of Medicine, who was not involved in the work. “The data seem really solid.”

Scientists have known for some time now that there is a correlation between inflammation—a heightened immune response—and obesity. Fat cells have the ability to release inflammatory molecules, which complicates these findings, since it is difficult to distinguish if the inflammation causes weight gain or is a side effect of weight gain.

Immunologist Yair Reisner of the Weizmann Institute of Science in Rehovot, Israel, came upon this new cellular link between obesity and the immune system while he was studying autoimmune diseases. Reinser was interested in an immune molecule called perforin, which kills diseased cells by boring a hole in their outer membrane. Reisner’s group suspected that perforin-containing dendritic cells might also be destroying the body’s own cells in some autoimmune diseases. To test their hypothesis, Reisner and his colleagues engineered mice that lacked perforin-wielding dendritic cells. Then they waited to see whether they developed any autoimmune conditions.

“We were looking for conventional autoimmune diseases,” Reisner says. “Quite surprisingly, we found that the mice gained weight and developed metabolic syndrome.”

Mice lacking the dendritic cells with perforin had high levels of cholesterol, early signs of insulin resistance, and molecular markers in their bloodstreams associated with heart disease and high blood pressure. Furthermore, the immune systems of these laboratory animals revealed that they also had a peculiar balance of T cells—a type of white blood cell that directs immune responses.

Reisner and his colleagues report online in the journal Immunity that when they removed these T cells from the mice, the absence of dendritic cells no longer caused the animals to become obese or develop metabolic syndrome.

The results, according to Reisner, suggest that the normal role of the perforin-positive dendritic cells is to keep certain populations of T cells under control. In the same way that perforin acts to kill cells infected with viruses, it can be directed to kill subsets of unnecessary T cells. When the brakes are taken off those T cells, they cause inflammation in fat cells, which leads to altered metabolism and weight gain.

“We are now working in human cells to see if there is something similar going on there,” Reisner says. “I think this is the beginning of a new focus on a new regulatory cell.” If these results turn out to be true in humans, they could point toward a way to use the immune system to treat obesity and metabolic disease.

Daniel Winer, an endocrine pathologist at the University of Toronto in Canada and the lead author of a January Diabetes paper that links perforin to insulin resistance, says the new results overlap with his study. Winer and his group found that mice whose entire immune systems lack perforin developed the early stages of diabetes when fed a high-fat diet. This new paper builds on that by homing in on perforin-positive dendritic cells and showing the link even in the absence of a high-fat diet. “It provides further evidence that the immune system has an important role in the regulation of both obesity and insulin resistance.”

Even if the results hold true in humans, however, a treatment for Type 2 diabetes, obesity or metabolic disease are far off. Dixit said. “Talking about therapeutics at this point would be a bit of a stretch.” Injecting perforin into the body could kill cells beyond those T cells that promoting obesity. We can’t live without any T cells at all, since they are vital to fight diseases and infections.

However, research on what these T cells are recognizing when they seek out fat cells and cause inflammation in fat tissue could eventually reveal drug targets.

University of Iowa Team Creates Insulin-Producing Cells from Skin Cells


A research team from the University of Iowa has designed a protocol that can create insulin-producing cells that help normalize blood-sugar levels in diabetic mice from skin cells. This discovery represents one of the first steps toward developing patient-specific cell replacement therapy for Type 1 diabetes. This research, which was led by Nicholas Zavazava from the department of internal medicine, was published in the journal PLoS ONE.

Zavazava and his coworker used human skin cells taken from punch biopsies and reprogrammed them to into induced pluripotent stem cells. These induced pluripotent stem cells were then differentiated in culture into pancreatic insulin-producing beta cells.

In culture, Zavazava’s cell made insulin in response to increased concentrations, but when they were implanted into diabetic mice, these cells responded to glucose, secreted insulin and worked to lower the blood-sugar levels in the mice to normal or near-normal levels.

Mind you, these induced pluripotent stem cell-derived beta cells were not as effective as pancreatic cells in controlling blood sugar levels, according to Zavazava in a UI news release. However, Zavazava and his team views the cells’ response in mice as an “encouraging first step” toward the goal of generating effective insulin-producing cells that potentially could be used to not just treat but cure Type 1 diabetes in humans.

“This raises the possibility that we could treat patients with diabetes with their own cells,” Zavazava said. “That would be a major advance, which will accelerate treatment of diabetes.”

Zavazava is also a member of UI’s Fraternal Order of Eagles Diabetes Research Center. This center is one of several groups whose aim is to create an alternative source of insulin-producing cells that can replace the pancreatic beta cells that die off in people with Type 1 diabetes.

According to the UI news release, this study is the first to use human induced pluripotent stem cells instead of embryonic stem cells to generate insulin-producing pancreatic beta cells. This protocol has the advantage of creating beta cells from a patient’s own cells include. This would eliminate the need to wait for a donor pancreas, since pancreas transplants are an option for treating Type 1 diabetes, but the demand for transplants is much greater than the availability of organs from deceased donors. The use of induced pluripotent stem cells would also eliminate the need for transplant patients to take immunosuppressive drugs. Finally, the use of induced pluripotent stem cells would also avoid the ethical concerns with treatments based on embryonic stem cells.

Preventing the Onset of Type 1 Diabetes


Diabetes researchers at Saint Louis University have discovered a way to prevent the onset of Type I diabetes mellitus in diabetic mice. This strategy involves inhibiting the autoimmune processes that result in the destruction of the insulin-secreting pancreatic beta cells.

Type I diabetes is a life-long disease that results from insufficient production of the vital anabolic hormone insulin. In most cases of Type I diabetes mellitus, the body’s immune system destroys insulin-producing beta cells, and this insulin deficiency causes high blood sugar levels, also known as hyperglycemia. Treatments for the disease require daily injections of insulin.

Dr. Thomas Burris, chair of the university’s pharmacological and physiological science department, and his colleagues, have published their results in the journal Endocrinology. IN this paper, they report a procedure that could potentially prevent the onset of the disease rather than just treating the symptoms

“None of the animals on the treatment developed diabetes even when we started treatment after significant beta cell damage had already occurred,” Burris explained in a prepared statement. “We believe this type of treatment would slow the progression of type I diabetes in people or potentially even eliminate the need for insulin therapy.”

A group of immune cells known as lymphocytes come in two main forms: B lymphocytes, which secrete the antibodies that bind to foreign cells and neutralize them, and T cells, which recognize foreign substances and regulate the immune response. There are several different types of T lymphocytes, but for the purposes of this discussion, two specific subtypes of T lymphocytes seem to be responsible for the onset of Type I diabetes. T “helper cells” that have the CD4 protein on their surfaces, and T “cytotoxic “ cells have the CD8 protein on their cell surfaces seem to play a role in the onset of Type I diabetes, but a third subtype of T lymphocyte has remained a bit of an enigma for some time. This subtype of T lymphocytes is a subcategory of CD4 T cells and secretes a protein called “interleukin 17,” and is, therefore, known as TH17.

Dr. Burris and his collaborators from the Department of Molecular Therapeutics at the Scripps Research Institute have been examining TH17 cells for some time and they came upon a pair of nuclear receptors that play a crucial role in the development of TH17 cells. Could hamstringing the maturation of TH17 cells delay the onset of Type 1 diabetes mellitus?

Burris and others targeted these receptors by using drugs that bound to them and prevented them from working. This prevented the maturation of the TH17 T lymphocytes. When two nuclear receptors, Retinoid-related orphan receptors alpha (ROR-alpha) and Gamma-t (ROR-gamma-t) were inhibited, they prevented the autoimmune response that destroyed the beta cells.

To block these ROR alpha and gamma t receptors, Burris and others used a selective ROR alpha inhibitor and a gamma t inverse agonist called SR1001 that was developed by Dr. Burris. These drugs significantly reduced diabetes in the mice that were treated with it.

These findings show that TH17 cells play a significant role in the onset of Type I diabetes, and suggest that the use of drugs like these that target this cell type may offer a new treatment for the illness.

According to the American Diabetes Association, only 5% of people with diabetes have the Type I form of the disease, which was previously known as juvenile diabetes because it is usually diagnosed in children and young adults. The organization said that over one-third of all research they conduct is dedicated to projects relevant to type 1 diabetes.

Gestational Diabetes Affects the Quality of Umbilical Cord Mesenchymal Stromal Cells


The laboratories of Drs. Jene Choi and Chong Jai Kim from the University of Ulsan College of Medicine in Seoul, South Korea have collaboratively shown that the therapeutic quality of umbilical cord mesenchymal stem cells is profoundly affected by gestational diabetes. Their work was published in a recent issue of the journal Stem Cells and Development and has profound implications for regenerative medicine.

Choi and Kim and their coworkers collected umbilical cords from mothers who had been given birth by Cesarian section and had also been diagnosed with gestational diabetes and mothers who had also just given birth by Cesarian section and showed normal blood sugar control. These umbilical cord tissues were processed and the mesenchymal stem cells from the cord tissue were isolated and cultured. These cells were grown and then subjected to a rather extensive battery of tests. These tests were a reflection of the ability of these to perform in regenerative treatments.

First umbilical cord mesenchymal stem cells (UCMSCs) from mothers with gestational diabetes (GD) did not grow as well as UCMSCs from mother who did not have GD.  As you can see in the graphs below, these are not small growth differences.  The UCMSCs from non-GD mothers (on the left) grow substantially better than those from GD mothers.  This result is also consistent for different cell lines.  This also means that transplanted cells would not grow very well if they were used for therapeutic purposes.

Umbilical cord mesenchymal stromal cells (UC-MSCs) derived from gestational diabetes mellitus (GDM) patients exhibit retarded growth proliferation. The growth of 7.5×103 UC-MSCs isolated from patients with normal pregnancies (A) and GDM (B) was monitored over a period of 12 days. GDM-UC-MSCs consistently showed decreased proliferation compared with normal pregnant women (N-UC-MSCs). Points represent the mean values from three independent experiments; bars denote standard deviation (SD).
Umbilical cord mesenchymal stromal cells (UC-MSCs) derived from gestational diabetes mellitus (GDM) patients exhibit retarded growth proliferation. The growth of 7.5×103 UC-MSCs isolated from patients with normal pregnancies (A) and GDM (B) was monitored over a period of 12 days. GDM-UC-MSCs consistently showed decreased proliferation compared with normal pregnant women (N-UC-MSCs). Points represent the mean values from three independent experiments; bars denote standard deviation (SD).

Secondly, UCMSCs from GD mothers showed a greater tendency to undergo premature senescence.  When MSCs are grown in culture, they usually grow rather well for several days and then the cells go to sleep and they stop growing.  This is called culture senescence and it is due to intrinsic properties of the cells.  When the cells go into senescence tends to be a cell line-specific property, but one thing is certain; the sooner cells become senescent, the few cells they will generate in culture.  The UCMSCs from GD mothers go into senescence early and easily and this is one of the reasons they grow so poorly relative to normal cells – because they are running to their beds to take a nap (so to speak).  Such cells are usually not good candidates for regenerative medicine.

Third, UCMSCs from GD mothers show poor lineage-specific differentiation.  MSCs have the ability to differentiate into fat cells, bone cells, and cartilage cells if particular well-established protocols are used.  However, UCMSCs from GD mothers showed inefficient differentiation and that is one of the things that MSCs must do if they are to repair bone or cartilage problems or if they are to help make smooth muscle for new blood vessels formation. 

Stem cell differentiation potentials are largely different between normal and GDM-affected UC-MSCs. Three different cell lines of normal and GDM-affected pregnancies were cultured in a control medium or induction medium for 5 days. Upregulation of the expression of the adipogenic-specific gene PPARγ (A) and the osteogenic genes alkaline phosphatase (ALP) (B), osteocalcin (OC) (C), and collagen type 1 alpha 1 (Col1α1) (D) was evaluated by real-time RT-PCR and normalized to GAPDH. All assays were performed in triplicate; bars denote SD (*P<0.05).
Stem cell differentiation potentials are largely different between normal and GDM-affected UC-MSCs. Three different cell lines of normal and GDM-affected pregnancies were cultured in a control medium or induction medium for 5 days. Upregulation of the expression of the adipogenic-specific gene PPARγ (A) and the osteogenic genes alkaline phosphatase (ALP) (B), osteocalcin (OC) (C), and collagen type 1 alpha 1 (Col1α1) (D) was evaluated by real-time RT-PCR and normalized to GAPDH. All assays were performed in triplicate; bars denote SD (*P<0.05).

The figure above shows the disparity between these established UCMSC cell lines.  The dark, solid bars indicate non-induced cells that were grown in normal culture media, and the striped bars are cells grown in media that designed to induce the differentiation of these cells into either bone, fat, or cartilage cells.  The cell lines with “N” in their name are from non-GD mothers and those with “D” in their designations are from GD mothers.  These assays are for genes known to be strongly induced when cells begin to differentiate into fat (PPARgamma), bone (ALP or osteocalcin or collagen 1 alpha 1).  As you can clearly see, the Ns outdo the Ds every time.

Finally, when the mitochondria, the compartments in cells that generate energy, from these two cell populations were examined it was exceedingly clear that UCMSCs from GD mothers had mitochondria that were abnormal and did not make every very well.  Mitochondria from UCMSCs taken from GD mothers showed decreased expression of the energy-making components.  Thus the energy-making pathways in these cell compartments were sub-par from a structural perspective.  Functional assays for mitochondria showed that mitochondria from UCMSCs from GD mothers consistently underperformed those from UCMSCs taken from non-GD mothers.  Also, when markers of mitochondrial dysfunction were measured (reactive oxygen species and indicators of mitochondrial damage from reactive oxygen species), such markers were consistently higher in mitochondria from UCMSCs from GD mothers relative to those from non-GD mothers.  This shows that the energy-making or powerhouses of the cells are dysfunctional in UCMSCs from GD mothers.  Without the ability to properly make energy from food molecules, the cells have a diminished capacity to heal damaged tissues and organs.

Several studies have established a positive link between mitochondrial dysfunction and accelerated aging.  Therefore, these cells, because they have more extensive indications of mitochondrial damage, may show profound accumulation of mitochondrial damage and accelerated aging.

In summary, this study shows that integral biological properties of human UC-MSCs differ according to obstetrical conditions.  These data also stress the importance of maternal–fetal conditions in biological studies of hUC-MSCs and the development of future therapeutic strategies using hUC-MSCs.

Brigham and Women’s Hospital Researchers Reverse Type 1 Diabetes in Diabetic Mice


Brigham and Women’s Hospital (BWH) is a Harvard University-affiliated institution with a robust research program. In particular, several BWH are interested in mesenchymal stem cells and their ability to suppress inflammation and mediate healing in injured organs.

To that end, a research team led by Robert Sackstein from BWH’s Departments of Dermatology and of Medicine and Reza Abdi from BWH’s Department of Medicine and Transplantation Research Center, has published a stupendous report in the journal Stem Cells. In this paper, Sackstein and his coworkers used mesenchymal stem cells (MSCs) to successfully treat laboratory animals that suffered from type 1 diabetes.

Type 1 diabetes is, to a large extent, a disease of the immune system, since a large majority of type 1 diabetes patients have immune cells that recognize the insulin-secreting beta cells as foreign and these immune cells attack and obliterate them. MSCs are a type of adult stem cell that has shown potent immune-suppressing and anti-inflammatory effects in animal and human clinical studies. Previous preclinical trials with diabetic-prone mice have demonstrated that intravenous administration of MSCs can tamp down pancreatic injury and reduce the blood sugar levels without insulin administration. However, these effects were modest and temporary.

Sackstein and his team suggested that if more MSCs could be inserted into the pancreatic islets, then more islets would be spared from immune destruction. This would yield a more complete reversal of diabetes.

MSCs tend to lack a key cell surface adhesion molecule called HCELL. HCELL mediates the homing of cells in the bloodstream to inflammatory sites. Unfortunately, direct injection of MSCs directly into pancreatic islets is not clinically feasible because the pancreas is fragile and the damage caused by injection would cause the release of hydrolytic enzymes that would degrade the rest of the pancreas and other tissues as well. In order to move intravenously administered MSCs to the sites of the immune attack, Sackstein and others engineered MSCs that expressed the HCELL homing molecule. The presence of HCELL on the surfaces of these MSCs directed them to the inflamed pancreatic islets.

The BWH team found that administering these HCELL-bearing MSCs into diabetic mice caused the MSCs to lodge in the islets. These cells decreased inflammation in the pancreas and durably normalized blood sugar levels in the mice, which eliminated the need for insulin administration; in other words they caused a sustained reversal of diabetes

Sackstein concluded that while further studies of the effects of MSCs are warranted, this preclinical study represents an important step in the potential use of mesenchymal stem cells in the treatment of type 1 diabetes and other immune-related diseases.

Young Blood Vessels Rejuvenate Aged Insulin-Producing Beta Cells


Professor Per-Olof Berggren Rolf Luft at the Research Center for Diabetes and Endocrinology at Karolinska Institutet has led an important study in collaboration with Alejandro Caicedo at University of Miami Miller School of Medicine and Hong Gil Nam at DGIST from the Republic of Korea that demonstrates that young capillary vessels can rejuvenate aged pancreatic islets. This study was published in the Proceedings of the National Academy of Sciences, USA and challenges prevailing views on the causes of age-dependent impaired glucose balance regulation, which often, in older patients, develops into type 2 diabetes mellitus.  These results suggest that treating inflammation and fibrosis in the small blood vessels of the pancreatic islets might provide a new way to treat age-dependent dysregulation of blood glucose levels.

“This is an unexpected but highly important finding, which we expect will have a significant impact on diabetes research in the future. The results indicate that beta cell function does not decline with age, and instead suggest that islet function is threatened by an age-dependent impairment of vessels that support them with oxygen and nutrients”, says Berggren.

Pancreatic beta cells are in the pancreatic islets and secrete the hormone insulin, which regulates blood glucose levels and also is one of the most important anabolic hormones of the human body.  Ageing may lead to a progressive decline in glucose regulation which may contribute to the onset of diabetes.  Generally, it has been assumed that this is due to reduced capacity of the beta cell to secrete insulin or multiply.

“In the study we challenged the view that the age-dependent impairment in glucose homeostasis is solely due to intrinsic, dysfunction of islet cells, and hypothesized that it is instead affected by systemic aging factors”, says first author Joana Almaça at the Diabetes Research Institute, University of Miami.

Even though the common wisdom in modern medicine is that insulin-producing pancreatic beta cell lose function through the constant demands on them,  Berggren and his collaborators showed that mouse and human beta cells are fully functional at advanced age.  When they replaced the islet vasculature in aged islet grafts with young capillaries, the investigators found that the islets were rejuvenated and glucose homeostasis fully restored.

“While expanding beta cell mass may still be desirable for future diabetes therapy, improving the local environment of the otherwise healthy aged beta cell could prevent age-associated deterioration in glucose homeostasis and thereby promote healthy ageing, which is conceptually novel and highly exciting”, says Per-Olof Berggren.

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.

Mesoblast Clinical Trial Shows Stem Cell Treatments Improve Glycemic Control in Type 2 Diabetics


Mesoblast Ltd has announced the results of their clinical trial in type 2 diabetics at the annual meeting of the American Diabetes Association.

Mesoblast has developed a proprietary adult stem cell they call a mesenchymal precursor cell or MPC, which they are attempting show can be used as an “off the shelf” medical product. MPCs seem to act like immature mesenchymal stem cells that can modulate the immune response and have greater flexibility.

In this trial, Mesoblast was banking of the ability of administered MPCs to suppress inflammation. Type 2 diabetes results from an insensitivity of tissues to secreted insulin. Consequently, cells do not receive enough of the insulin signal to take up sugar and make protein, glycogen, and fat. Another prominent feature of type 2 diabetes is chronic, low-level inflammation, which is largely due to the chronically high blood glucose concentrations that damages cells, blood vessels, nerves, and connective tissue. By treating type 2 diabetics with MPCs, Mesoblast was hoping to ascertain the ability of MPCs to quell chronic inflammation.

The trial was conducted across 18 sites in the US. 61 patients with type 2 diabetes received either one intravenous infusion of 0.3, 1.0 or 2.0 millions MPCs per kilogram body weight over 12 weeks. One group of patients were given a placebo. Patients had suffered from diabetes an average of 10 years and had poor control with the drug metformin (Glucophage), which is one of the most widely-used drugs for type 2 diabetes.

The results were largely positive:
When it comes to safety, there were no safety issues observed during the 12-week study period. The MPC cell infusions were well tolerated (with a maximal dose of 246 million cells). With regard to efficacy, there were dose-dependent improvement in glycemic control as evidenced by a decrease at all time points after week 1 in hemoglobin A1c (HbA1c) in MPC- treated patients compared with an increase in HbA1c in placebo treated subjects. HbA1c is a blood test that determines how much damage the high sugar levels are doing to the body. The test uses the blood protein hemoglobin to assess the damage that high glucose levels are doing to the rest of the body. In this clinical trial, significant reductions in HbA1c were observed after 8 weeks in the 2 M/kg MPC group compared to placebo (p<0.05) which was sustained through 12 weeks. The reduction in HbA1c was most pronounced in subjects with baseline HbA1c ≥ 8% (i.e. those patients with relatively poorer glucose control).

Fasting insulin levels were reduced in the 1 million and 2 million/kg groups compared to placebo (P<0.05), and reduced levels of inflammatory cytokines TNF-alpha and IL-6 (which are made at high levels during inflammation) were observed at 12 weeks in MPC groups compared to placebo.

The scientists and physicians involved in this clinical trial concluded there was sufficient evidence to support further evaluation into the use of MPCs in the treatment of type 2 diabetes and its complications. They also thought that there were grounds for exploring other therapeutic venues in which MPCs might prove useful.

Mesoblast Chief Executive Silviu Itescu said: “We are very pleased with these results which are consistent with an immunomodulatory mechanism by which our MPCs may have glucose-lowering effects in patients with type 2 diabetes. We are evaluating whether similar effects may be seen with the use of MPCs in the treatment of kidney disease and other complications of type 2 diabetes.”

While it is improbable in the extreme that this one-time treatment will improve the long-term clinical outcomes of diabetics, it is possible that repeated treatments will provide better Glycemic control for poorly controlled diabetics, and that these repeated treatments will produce long-term improvements in the health of these patients.

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.

Directly Reprogramming Gut Cells into Beta Cells to Treat Diabetes


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

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

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

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

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

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

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

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

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

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

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

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.

Brown Fat Stem Cells for Treating Diabetes and Obesity


Mammals have two main types of fat: brown fat and white fat.  Brown fat is especially abundant in newborns and in mammals undergoing hibernation.  The primary function of brown fat is to produce body heat so that the animal does not shiver.  In contrast to white fat cells, which contain a single lipid droplet, brown fat cells contain numerous smaller droplets and a higher number of mitochondria, and it is these mitochondria and their high iron content that makes this fat tissue brown.  Brown fat also contains more small blood vessels than white fat, since it has a greater need for oxygen than most tissues.

Recently, researchers at the University of Utah School of Medicine have identified stem cells from brown fat.  The principal researcher of this project, Amit Patel, associate professor of medicine, refuted an old dogma – that adult humans do not possess brown fat.  Children have large amounts of brown fat that is highly metabolically active.  Children can eat a great deal and not gain any weight, relative to adults.  Adults, on the other hand, have an abundance of white fat, and accumulation of white fat leads to weight gain and cardiovascular disease (something not seen in brown fat).  As people age, the amount of white fat increases and the amount of brown fat decreases, which contributes to the onset of diabetes and high cholesterol.

As Patel put it, “If you have more brown fat, you weigh less, you’re metabolically efficient, and you have fewer instances of diabetes and high cholesterol.  The unique identification of human brown fat stem cells in the chest of patients aged 28-34 years is profound.  We were able to isolate the human stem cells, culture and grow them, and implant them into a pre-human model which has demonstrated positive effects on glucose levels.”

In vitro differentiation of brown adipose derived stem cells (BADSCs). (A) Gene expression profile comparing undifferentiated BADSCs to undifferentiated white adipose derived stem cells derived from subcutaneous adipose tissue. Genes in red are associated with brown fat phenotype. (B) Gene expression profile comparing undifferentiated brown adipose derived stem cells to differentiated brown adipocytes. Biological replicates performed in triplicate from a single clone were used for gene expression profile. (C) Transmission electron microscopy of 21 day brown adipocyte differentiation induced with fibronectin type III domain containing 5 (FNDC5) demonstrate multiocular intracytoplasmic lipid vacuoles and mitochondria (arrows). (D) Alizarian red staining of brown adipose derived stem cells induced to undergo osteogenesis. (E) Alcian blue staining of brown adipose derived stem cells directionally differentiated into chondrocytes. (F) Fatty acid binding protein 4 (FABP4) immunocytochemistry of brown adipose derived stem cells induced to undergo white adipogenesis. (G) Undifferentiated BADSCs. (H) Western blot 21 days post FNDC5 induction. Lane 1 brown adipose derived stem cells directionally differentiated into brown adipocytes. Lane 2 non- FNDC5 cells.
In vitro differentiation of brown adipose derived stem cells (BADSCs). (A) Gene expression
profile comparing undifferentiated BADSCs to undifferentiated white adipose derived stem cells
derived from subcutaneous adipose tissue. Genes in red are associated with brown fat phenotype. (B)
Gene expression profile comparing undifferentiated brown adipose derived stem cells to differentiated
brown adipocytes. Biological replicates performed in triplicate from a single clone were used for gene
expression profile. (C) Transmission electron microscopy of 21 day brown adipocyte differentiation
induced with fibronectin type III domain containing 5 (FNDC5) demonstrate multiocular
intracytoplasmic lipid vacuoles and mitochondria (arrows). (D) Alizarian red staining of brown
adipose derived stem cells induced to undergo osteogenesis. (E) Alcian blue staining of brown adipose
derived stem cells directionally differentiated into chondrocytes. (F) Fatty acid binding protein 4
(FABP4) immunocytochemistry of brown adipose derived stem cells induced to undergo white
adipogenesis. (G) Undifferentiated BADSCs. (H) Western blot 21 days post FNDC5 induction. Lane 1
brown adipose derived stem cells directionally differentiated into brown adipocytes. Lane 2 non-
FNDC5 cells.

This new discovery of finding brown fat stem cells may help in identifying potential drugs that may increase the body’s own ability to make brown fat or find novel ways to directly implant brown fat stem cells into patients.

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.

Long-Lasting Blood Vessels Regenerated from Reprogrammed Human Cells


Researchers from Massachusetts General Hospital (MGH) in Boston, MA have used human induced pluripotent stem cells to make vascular precursor cells to produce functional blood vessels that lasted as long as nine months.

Rakesh Jain, director of the Steele Laboratory for Tumor Biology at MGH and his team derived human induced pluripotent stem cells (iPSCs) from adult cells of two different groups of patients. One group of individuals were healthy and the second group had type 1 diabetes. Remember that iPSCs are derived from adult cells through the process of genetic engineering. By introducing specific genes into these adult cells, the adult cells are de-differentiated into an embryonic-like state. The embryonic-like cells can be cultured and grown into a cell line that can be differentiated into various cell types in the laboratory. These differentiated cells types can then be transplanted into laboratory animals for regenerative purposes.

“The discovery of ways to bring mature cells back to a ‘stem-like’ state that ca differentiate into many different types of tissue has brought enormous potential to the field of cell-based regenerative medicine, but the challenge of deriving functional cells from these iPSCs still remains,” said Rakesh. “our team has developed an efficient method to generate vascular precursor cells from human iPSCs and used them to create networks of engineered blood vessels in living mice.”

The ability to regenerate or repair blood vessels could be a coup for regenerative medicine. Cardiovascular disease, for example, continues to be the number one cause of death in the United States and other conditions caused by blood vessel damage (e.g., the vascular complications of diabetes) continue to cause a great deal of morbidity and mortality each year. Also, providing a blood supply to newly generated tissue remains one of the greatest barriers to building solid organs through tissue engineering.

Some studies have used iPSCs to build endothelial cells (the cells that line blood vessels), and connective tissue cells that provide structural support. These cells, unfortunately, tend to not produce long-lasting vessels once they are introduced into laboratory animals. A collaborator with Jain, Dai Fukumura, stated, “The biggest challenge we faced during the early phase of this project was establishing a reliable protocol to generate endothelial cell lines that produced great quantities or precursor cells that could generate durable blood vessels.”

Jain’s group adapted a protocol that was originally designed to derived endothelial cells from human embryonic stem cells. They isolated cells based on the presence of more than one cell surface protein that marked out vascular potential. Then they expanded this population of cells using a culture system developed with embryonic stem cells that had been differentiated into endothelial cells. Further experiments confirmed that only those iPSCs that expressed all three cell surface proteins on their surfaces had the potential to form blood vessels.

To test the capacity of those cells to generate blood vessels, they implanted them onto the surface of the brain of mice in combination with mesenchymal precursors that generate smooth muscles that surround blood vessels.

Within two weeks after transplantation, the implanted cells had formed entire networks of blood vessels with blood flowing through them that has also fused with the already existing blood vessels. These engineered blood vessels continued to function for as long as 280 days in the living animal. Implantation under the skin, however, was a different story. It took 5 times the number of cells to get them to form blood vessels and they were short-lived. This is similar to the results observed in other studies.

Because type 1 diabetes can ravage blood vessels, Jain’s team made iPSCs from patients with type 1 diabetes to determine if iPSCs from such patients would generate functional blood vessels. Similarly to the cells generated from healthy individuals, vascular precursors generated from type 1 diabetics were able to form long-lasting blood vessels. However, these same cell lines showed variability in their ability to form vascular precursors. The reason for this is uncertain.

Rekha Samuel, one of the lead authors of this paper, said “The potential applications of iPSC-generated blood vessels are broad – from repairing damaged vessels supplying the heart or brain to preventing the need to amputate limbs because of the vascular complications of diabetes. But first we need to deal with such challenges as the variability of iPSC lines and the long-term safety issues involved in the use of these cells, which are being addressed by researchers around the world. We need better ways of engineering the specific type of endothelial cell needed for specific organs and functions.”

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.