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).

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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.”

Stem Cells For Better Drug Assays


Moving a drug from the laboratory to the clinic is terrifically expensive and slow. Even after extensive tests in cultured cells and laboratory animals, the drug may still fail in its clinical tests. Such failures cost drug companies massive amounts of money, and this drives up the cost of those drugs that succeed in clinical trials and secure FDA approval. If scientists could design drug assays that better predict whether a compound will succeed in human trials could help pharmaceutical companies identify the most promising drugs in which to invest their resources.

Fortunately, a study published this week in Cell Stem Cell might represent such a breakthrough. In this paper, researchers reported that a new stem cell-based assay was actually able to pinpoint a potential small molecule treatment for amyotrophic lateral sclerosis (ALS, also known as known as Lou Gehrig’s disease). In follow-up experiments, the drug promoted better cell survival better than two other drug candidates that had recently failed in phase III clinical trials.

One of the study’s co-authors, Clifford Woolf, the director of the F. M. Kirby Neurobiology Center at Boston Children’s Hospital, said that this new strategy “could either be used in the late preclinical stage to confirm the cellular actions of particular leads, or even better as a driver of early exploratory preclinical testing, revealing new targets and pathways.”

The laboratory of Lee Rubin at Harvard Medical School developed this new cell-bases assay by using embryonic stem cells derived from both healthy mice and those with a mutation in the gene SOD1. Mutations in SOD1 are known to cause ALS in people. After differentiating the stem cells into motor neurons, which are the cells that die off in ALS patients — the group exposed these embryonic stem cell-derived motor neurons to 5000 different small molecular-weight compounds. The cultured cells were also deprived of essential chemicals from the culture medium in order to accelerate their death.

In these experiments, a molecule called kenpaullone, which is an inhibitor of the enzyme GSK-3, stood out in their initial screen. GSK-3 controls cell growth and death, and kenpaullone strongly promoted the survival of both normal and mutated motor neurons and kept them morphologically healthy. In a different experiment, the group showed that the drug decreased levels of SOD1, which is thought to aggregate in the motor neurons of people with the disorder.

In further tests, Rubin and his colleagues treated motor neurons made from induced pluripotent stem cells derived from adult cells that had been taken from two ALS patients with kenpaullone. One of these ALS patients had mutations in the SOD1 gene, while the other harbored mutations in the TDP-43 gene. TDP-43 is yet another gene associated with ALS, since mutations in it also cause ALS. Rubin’s team found that the small molecule substantially boosted motor neuron survival by some 2- to 4-fold, and this effect was dose-dependent and was observed in both healthy and diseased cells.

In contrast, two drug candidates that recently failed phase III clinical trials were less effective when tested in the same assay. The drug dexpramipexole had no effect on patient-derived motor neurons, and olesoxime had a variable but only moderately positive effect.

It’s not clear how the SOD1 mutation causes the degeneration of motor neurons. According to Alysson Muotri, assistant professor of pediatrics and cellular and molecular medicine at the University of California, San Diego, who was not involved in the new study; knowing the pathological mechanism of inactive SOD1 on motor neurons could inform additional endpoints for the stem cell assays
.
To date, kenpaullone has only been tested in on cultured neurons and not in living mice to date. And, as with all cell culture assays, it is an open question as to “the extent to which changes in neurons in a dish phenocopy complex diseases that may take many years to manifest, and if rescue of the phenotype by a hit in a screen will translate into therapeutic benefit in patients,” Woolf noted.

However Muotri sees great potential for stem cell-based assays and their use for drug discovery. “Stem cell based screens will definitely speed up drug discovery, bringing more powerful candidates to clinical trial,” said Muotri. “I can see this going into personalized medicine—we will be testing drugs and doses in motor neurons derived from each patient to personalize treatment.”

Reference
Yang, Y. M., S. K. Gupta, K. J. Kim, B. E. Powers, A. Cerqueira, B. J. Wainger, H. D. Ngo, K. A. Rosowski, P. A. Schein, C. A. Ackeifi, et al. 2013. A small molecule screen in Stem-Cell-derived motor neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. Cell Stem Cell (April).

A New Automated Protocol to Prepare and Purify Induced Pluripotent Stem Cell Lines


Induced pluripotent stem cells (iPSCs) come from adult cells and not embryos. By genetically engineering adult cells to express a cadre of genes that are normally found in early embryonic cells, scientists can de-differentiate the adult cells into cells that resemble embryonic stem cells in many (although not all) ways.

Generating iPSCs from human adult cells is tedious and not terribly efficient, but there are ways to increase the efficiency of iPSC generation (see here). Additionally, iPSCs can show a substantial tendency to form tumors, but this tendency is cell line-specific (see here and here). Furthermore, there are ways to screen iPSC lines for tumorgenicity.

Because iPSCs are directly from the patient’s cells, the chances of rejection by the immune system are less likely (see here). Therefore, many stem cells scientists believe that iPSCs may represent one of the best future possibilities for regenerative medicine. However, a hurdle in iPSC development is the ability to generate and evaluation iPSC lines in a rapid, but reliable manner. Once adult cells are induced to become iPSCs, the iPSC cultures are a mixed bag of iPSCs, undifferentiated adult cells that failed to make the transition to iPSCs, and partially reprogrammed cells. Selecting the iPSCs by merely eye-balling the cells through the microscope is tricky and fraught with errors. If the scientist wants to select iPSCs for toxicity studies and not partially differentiated cells, selecting the wrong cells for the experiment can be fatal to the experiment itself.

Scientists from the New York Stem Cell Foundation (NYSCF) Research Institute have developed a protocol for iPSC generation and evaluation is automated and efficient, and may bring us closer to the goal of using iPSCs in the clinic some day. This protocol is the culmination of three and a half years of work. This protocol uses a technology called “fluorescence activated cell sorting” or FACS to identify fully reprogrammed cells. FACS sorts the cells according to their expression of two specific cell surface molecules and the absence of another cell surface molecule. This negative selection for a cell surface molecule found in partially reprogrammed cells but not iPSCs is a very powerful technique for purifying iPSCs.

David Kahler, the NYSCF director of laboratory automation, said, “To date, this protocol has enabled our group to derive (and characterize over) 228 individual iPS cell lines, representing one of the largest collections derived in a single lab.” Kahler continued: “This standardized method means that these iPS cells can be compared to one another, an essential step for the use in drug screens and the development of cell therapies.”

This particular cell selection technique provides the basis for a new technology developed by NYSCF, the Global Stem Cell Array, which is a fully automated, robotic platform to generate cell lines in parallel.

Underway at the NYSCF Laboratory, the Array reprograms thousands of adult cells from kin and blood samples taken from healthy donors and diseased patients into iPSC lines. Sorting and characterizing cells at an early stage of reprogramming allows efficient development of iPSC clones and derivation of adult cell types.

“We are excited about the promise this protocol holds to the field. As stem cells move towards the clinic, Kahler’s work is a critical step to ensure safe, effective treatments for everyone.” said Susan L. Solomon, who is the Chief Executive Officer of NYSCF.

Using Hydrogels to Direct Stem Cell Differentiation and Proliferation


Stem cells are incredible healers that can undergo self-renewal. However, getting them to do this in culture is often tedious and difficult. Stem cells requires their own specific set of surroundings to grow and self renew and recapitulating those surroundings takes a deep understanding of stem cell niches.

Fortunately there is a way to grow stem cells in a three-dimensional culture system that more closely resembles their native milieu, and this system utilizes biomaterials. One such biomaterial is a hydrogel; a water-loving material that consists of a polymer that is highly dissolvable in water. By varying the degree of cross-links between polymer chains, the properties of the hydrogel can vary and these diverse characteristics can adapt the hydrogel to particular stem cell cultures systems.

A research team and Case Western Reserve University in Cleveland, Ohio has created several different hydrogels.  Some consist of molecules that compose a highly crosslinked three-dimensional checkerboard, while others are less highly crosslinked.  The different degree of crosslinking changes the spaces within the hydrogel, and when stem cells are grown in these hydrogels, the spacing differences affect stem cell behaviors such as proliferation and differentiation.

Eben Alsberg, associate professor of biomedical engineering, said this about his research project; “We think that control over local biomaterial properties may allow us to guide the formation of complex tissues.  With this system, we can regulate cell proliferation and cell-specific differentiation into, for example bone-like or cartilage-like cells.”

The degree of crosslinking between the soluble molecules of a hydrogel influences the rigidity of the hydrogel.  Also the porosity of the hydrogel is decreased when the degree of crosslinking increases.  Alsberg used a hydrogel of oxidized methacrylated alginate with an 8-arm (polyethylene glycol) amine .  When the methacrylated alginate was reacted with the polyethylene glycol, it generated crosslinks that conveyed an organized internal structure to the hydrogel.

Methacrylated alginate
Methacrylated alginate
Poly (ethylene glycol) amine
Poly (ethylene glycol) amine

By playing with the mixture and the proportion of methacrylated alginate to polyethylene glycol, Alsberg and his coworkers made a second set of crosslinks that were light-activated.  when they made these molecules in checkerboard masks, patterns of alternating single and double crosslinked spaces emerged that ranged, in size, from 25 to 200 micrometers across.  These little molecule cubbyholes resulted from molecules that were evenly singly and doubly crosslinked.

When human stem cells isolated from fat were grown in the singly and doubly-crosslinked regions of the hydrogel, the cells grew better and differentiated better in the hydrogels with larger spaces.  The larger the spaces, the better the growth and the clusters they formed, and the more efficiently the cells differentiated.

Alsberg commented on his results: “Potentially, what’s happening is the single-crosslinked regions allow better nutrient transport and provide more space for cells to interact and, because it’s less restrictive, there’s space for new cells and matrix production.  CLuster formation, in turn, may influence proliferation and differentiation.  Differences in mechanical properties between regions likely also regulate the cell behaviors.

Alsberg and his team would ultimately like to understand how micropatterning influences stem cell fate decisions.  By using biomaterials to  produce particular micropatterns, Alsberg and his colleagues hope to engineer multiple tissues composed of multiple cell types by using a single stem cell source whose differentiation is directed and control by the structures into which the cells grow and differentiation.

Treating Heart Patients with “Smart” Stem Cells


By aggressively treating heart attack patients soon after their episodes, clinicians have been able to reduce early mortality from heart attacks. However, the survival of these patients tends to create a whole new set of issues for them and their hearts. Chronic heart failure is a common aftermath of a heart attack for heart attack survivors. (see Kovacic JC and Fuster V., Clin Pharmacol Ther 2011;90:509-18).

Since the heart muscle (myocardium) has only a limited capacity to regenerate after a heart attack, multifaceted treatments have emerged that are designed to relieve symptoms and improve the patient’s clinical status. In particular, therapies target impaired contractility of the heart and the ability of the heart to handle the workload without enlarging. However, these treatments do not address the loss of heart muscle that underlies all heart attacks (see McMurray JJ. Systolic heart failure. N Engl J Med 2010;362:228-38). To address the loss of contracting heart tissue, stem cells, traditionally isolated from bone marrow, have been used in several clinical trials. However, the results of these studies have been highly variable, since most bone marrow stem cells placed in a heart after a heart attack, die soon after implantation.

To improve the ability of bone marrow stem cells to repair the heart, Andre Terzic from the Mayo Clinic Center for Regenerative Medicine has designed a special cocktail to induce mesenchymal stem cells from bone marrow to become more heart-friendly. This cocktail consisted of the following growth factors: TGFβ1, BMP-4, Activin-A, retinoic acid, IGF-1, FGF-2, α-thrombin and IL-6. Mesenchymal stem cells were cultured for 10 days in this cocktail and then tested for heart-specific genes.

Terzic calls this procedure “cardiopoiesis,” and when he subjected bone marrow mesenchymal stem cells (BM-MSCs) to this procedure, they expressed a cadre of genes that is normally found in developing heart cells (Nkx2-5, MEF2C, GATA4, TBX5, etc.). In an earlier publication, Terzic and his colleagues transplanted BM-MSCs from heart patients into the hearts of mice that had suffered a heart attack and compared the effects of these cells on the heart, with BM-MSCs that had undergone this guided cardiopoiesis protocol. The results were astounding. Not only did the function of the hearts that had received the guided cardiopoiesis M-MSCs much more normal than those had had received the untreated BM-MSCs, but post-mortem examination of the hearts showed that the hearts that had received guided cardiopoiesis BM-MSCs contained human heart muscle cells integrated into the heart muscle tissue (Atta Behfar, et al., J Am Coll Cardiol. 2010 August 24; 56(9): 721–734). Therefore, this procedure, cried out for a clinical trial, and data from such a trial has already been reported.

A, Human-specific troponin-I (green) in the anterior wall of naive- versus CP-treated hearts, respectively, co-localized with ventricular myosin light chain (MLC2v, red). Bar, 100 μm. B, Human troponin-I staining of naïve versus CP hMSC treated hearts, counterstained with α-Actinin (red), demonstrated engraftment of human cells. Cell cycle activation, documented by Ki-67 expression (yellow, arrows), noted in human troponin positive and endogenous cardiomyocytes. C, Confocal evaluation of collateral vessels from CP hMSC treated hearts demonstrated human-specific CD-31 (PECAM-1) staining. D, Human lamin staining (arrows) co-localized with nuclei of smooth muscle in vessels from CP hMSC treated but not saline or naïve treated hearts. Bar, 20 μm for B-D.
A, Human-specific troponin-I (green) in the anterior wall of naive- versus CP-treated hearts,
respectively, co-localized with ventricular myosin light chain (MLC2v, red). Bar, 100 μm.
B, Human troponin-I staining of naïve versus CP hMSC treated hearts, counterstained with
α-Actinin (red), demonstrated engraftment of human cells. Cell cycle activation,
documented by Ki-67 expression (yellow, arrows), noted in human troponin positive and
endogenous cardiomyocytes. C, Confocal evaluation of collateral vessels from CP hMSC
treated hearts demonstrated human-specific CD-31 (PECAM-1) staining. D, Human lamin
staining (arrows) co-localized with nuclei of smooth muscle in vessels from CP hMSC
treated but not saline or naïve treated hearts. Bar, 20 μm for B-D.

In a paper from February 2013 (Bartunek J, et al., Journal of the American College of Cardiology (2013), doi: 10.1016/j.jacc.2013.02.071), Terzic and his team has reported on the administration of BM-MSCs into the hearts of 34 heart patients. Of these patients, 21 were implanted with their own BM-MSCs that had undergone guided cardiopoiesis and the other 12 received standard therapy for heart patients with no transplanted cells.

The results from this study were striking to say the least. According to Terzic, “The benefit to patients who received cardiopoietic stem cell delivery was significant.” Cardiologist Charles Murry wrote in an editorial, “Six months after treatment, the cell therapy group had a seven percent absolute improvement in EF (ejection fraction) over baseline, versus a non-significant change in the control group. The improvement in EF is dramatic, particularly given the duration between the ischemic injury and cell therapy. It compared favorably with our most potent therapies in heart failure.”

This clinical trial, known as the C-CURE trial, which stands for Cardiopoietic Stem Cell Therapy in Heart Failure. was an international, multi-center trial that treated enrolled patients from hospitals in Belgium, Serbia, and Switzerland. This trial represents the culmination of almost a decade of work by Terzic and others. “Discovery of rare stem cells that could inherently promote heart regeneration provided a critical clue. In following this natural blueprint, we further developed the know-how needed to convert patient-derived stem cells into cells that can reliably repair a failing heart.”

For this trial, Mayo Clinic partnered with Cadio3 Biosciences, which is a bio-science company in Mort-Saint-Guilbert, Belgium. This company provided advance product development, manufacturing scale-up, and clinical trial execution.  Adaptation of this exciting new technology to the clinic could mean a new exciting fix for heart patients.

Allergy-Associated White Blood Cell Triggers Stem Cell-Mediated Muscle Repair


White blood cells help our bodies ward off invasions from microorganisms, but they serve other purposes too. Once white blood cell in particular, the “eosinophil” helps us when we are infected by multicellular parasites (worms and the like). Eosinophils, however, also play a more unpleasant role, and that is in allergies. When we suffer from allergies, eosinophils multiply and move to our lungs and other places, where they mediate inflammation and tissue damage. Thus eosinophils are the white blood cells we all love to hate.

However, researchers at the University of California, San Francisco (UCSF) have generated new data that, in their view, suggests that eosinophils also play an integral role in muscle regeneration.

Ajay Chawla, Associate Professor at the Cardiovascular Research Institute at UCSF and

Eosinophil
Eosinophil

lead researcher for this study, said, “Eosinophils are needed for the rapid clearance of necrotic debris, a process that is necessary for timely and complete regeneration of tissues.”

Chawla’s laboratory showed that eosinophils serve double duty when it comes to muscle repair. First, they remove the cellular debris that results from damaged tissues. Secondly, eosinophils secrete a protein called “Interleukin 4” or IL-4. IL-4 triggers a specific type of stem cell to replicate and repair muscle tissue.

Interleukin-4
Interleukin-4

According to Chawla, “Without eosinophils you cannot regenerate muscle.”

These eosinophil-activated stem cells are known as “fibro/adipogenic progenitors” (FAP). Until recently, the general thinking surrounding FAPs was that they could only form fat tissue (see Natarajan A., Lemos DR, and Rossi FM, Cell Cycle 2010 9(11): 2045-6).  However, when FAPs are exposed to IL-4, they begin to differentiate into muscle fibers.

“They wake up the cells in muscle that divide and form muscle fibers,” said Chawla

“Bites from venomous animals, many toxicants, and parasitic worms all trigger somewhat similar immune responses that cause injury. We want to know if eosinophils and FAPs are universally employed in these situations as a way to get rid of debris without triggering severe reactions such as anaphylactic shock,” said Chawla.