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.

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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.

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.”

Making Pancreatic Beta Cells from Embryonic Stem Cells


Type 1 diabetes results from an inability to produce sufficient quantities of the hormone insulin. Without insulin, the body does not receive the signal to take up sugar from the blood, and the result is high blood sugar levels, which are damaging to tissues, and a general wasting of tissues because they cannot take up enough sugar to feed them.

Pancreatic beta cells

The cells in the pancreas that produce insulin are the beta cells, and animal studies have shown that transplantation of new beta cells into diabetic animals can reverse and even in some cases cure the diabetic animals. Therefore researchers have tried to make beta cells from pluripotent stem cells in order to make a source of beta cells for transplantation.

Unfortunately, beta cell production in the laboratory has been fraught with problems. The cells produced by differentiation of embryonic stem cells do not have the characteristics of mature beta cells and they produce little insulin and are not glucose responsive (D’Amour, et al., (2006) Nat Biotechnol 24, 1392-1401).

A different strategy, however, works much better. Instead of differentiating stem cells into beta cells, differentiate them into those cells that will form beta cells and other types of pancreatic cells in the embryo – immature endocrine cell precursors – and then transplant those into the pancreas of diabetic mice. In this case, the endocrine cell precursors differentiate in the bodies of the mice into pancreatic beta cells that greatly resemble normal beta cells.

Why don’t embryonic stem cells for beta cells in culture? This question was pursued by a collaboration between research team led by Maike Sander at UC San Diego and a company called ViaCyte, Inc.

When it comes to endocrine precursors transplanted into mice, Dr. Sander noted that, “We found that the endocrine cells retrieved from transplanted mice are remarkably similar to primary human endocrine cells.” He continued, “This shows that hESCs (human embryonic stem cells) can differentiate into endocrine cells that are almost indistinguishable from their primary human counterparts.”

Well, ESCs can make perfectly fine beta cells in the mouse body, but not in culture. What’s up with that?

Sander and her colleagues examined the gene expression patterns of embryonic stem cells as they were differentiated and compared them with the gene expression patterns in those cells that were transplanted into mice and allowed to differentiate inside the body of the mouse.

What Sander and her team found was astounding. As cells progress through their developmental program, particular genes are brought on-line and expressed, and then turned off as the cells passed through each stage of endocrine cell differentiation. The cellular machinery that shuts off genes after they have been activated consists of a family of proteins that remodel chromatin known as the Polycomb group (PcG). PcG-mediated repression of genes silenced those genes that were only turned on temporarily once they were no longer required.

Two major Polycomb repressive complexes (PRCs) have been described. The PRC2 complex contains the histone methyltransferase enhancer of zeste homologue 2 (EZH2), which together with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) catalyses the trimethylation of histone H3 at lysine K27 (H3K27me3). The EZH2 SET domain confers this activity. Multiple forms of the PRC1 complex exist and these contain combinations of at least four PC proteins (CBX2, CBX4, CBX7 and CBX8), six PSC proteins (BMI1, MEL18, MBLR, NSPC1, RNF159 and RNF3), two RING proteins (RNF1 and RNF2), three PH proteins (HPH1, HPH2 and HPH3) and two SCML proteins (SCML1 1 and SCML2). Some results have suggested that PRC1 complexes are recruited by the affinity of chromodomains in chromobox (Cbx) proteins to the H3K27me3 mark. PRC1 recruitment results in the RNF1 and RNF2-mediated ubiquitylation of histone H2A on lysine 119, which is thought to be important for transcriptional repression. PC, Polycomb; PSC, Posterior sex combs ; SCML, Sex combs on midleg .
Two major Polycomb repressive complexes (PRCs) have been described. The PRC2 complex contains the histone methyltransferase enhancer of zeste homologue 2 (EZH2), which together with embryonic ectoderm development (EED) and suppressor of zeste 12 homolog (SUZ12) catalyses the trimethylation of histone H3 at lysine K27 (H3K27me3). The EZH2 SET domain confers this activity. Multiple forms of the PRC1 complex exist and these contain combinations of at least four PC proteins (CBX2, CBX4, CBX7 and CBX8), six PSC proteins (BMI1, MEL18, MBLR, NSPC1, RNF159 and RNF3), two RING proteins (RNF1 and RNF2), three PH proteins (HPH1, HPH2 and HPH3) and two SCML proteins (SCML1 1 and SCML2). Some results have suggested that PRC1 complexes are recruited by the affinity of chromodomains in chromobox (Cbx) proteins to the H3K27me3 mark. PRC1 recruitment results in the RNF1 and RNF2-mediated ubiquitylation of histone H2A on lysine 119, which is thought to be important for transcriptional repression. PC, Polycomb; PSC, Posterior sex combs ; SCML, Sex combs on midleg .

In the transplanted endocrine precursors, Sanders and his team noted an orderly progression of genes that were turned on and then turned off once as needed. However, in the embryonic stem cells that were differentiated into beta cells in culture, they discovered that these cells failed to express the majority of genes critical for endocrine cell function. The main reason for this appeared to be that the PcG-mediated repression of genes was not fully eliminated when particular genes had to be expressed at specific developmental stages. Thus these cultured cells failed to fully eliminate PcG-mediated repression on endocrine-specific genes, which contributes to the abnormality of the culture-derived beta cells.

Sander commented: “This information will help devise protocols to generate functional insulin-producing beta cells in vitro. This will be important not only for cell therapies, but also for identifying disease mechanisms that underlie the pathogenesis of diabetes.”