Induced Pluripotent Stem Cell-Based Model System of Hypertrophic Cardiomyopathy Provides Unique Insights into Disease Pathology


A research team at the Icahn School of Medicine at Mount Sinai led by Bruce Gelb created a model of hypertrophic cardiomyopathy (HCM) by using human induced pluripotent stem cells.

Patients who suffer from an extreme thickening of the walls of the heart exhibit HCM. This excessive heart thickening is associated with a several rare and common illnesses. There is a strong genetic component to the risk for developing HCM. Can stem cell-based model system be used to study the genetics of HCM?

The answer to this question seems to be yes, since laboratory-generated induced pluripotent stem cells lines that have been differentiated into heart cells that, in many cases, closely resemble human heart tissue. Studies with such stem cell-based model systems have reaped useful insights into disease mechanisms (see F Kamdar, et al., J Card Fail. 2015 Sep;21(9):761-70; Lee YK, Ng KM, Tse HF. J Biomed Nanotechnol. 2014 Oct;10(10):2562-85).

In this paper, Bruce Gelb and his colleagues examined a genetic disorder called cardiofaciocutaneous syndrome (CFC). CFC is caused by mutations in a gene called BRAF. It is a rare condition that affects fewer than 300 people worldwide, and causes head, face, skin, and muscular abnormalities, including abnormalities of the heart.

Gelb and his coworkers isolated skin cells from three CFC patients and reprogrammed them into induced pluripotent stem cells, which were then differentiated into heart cells. In this disease model system, the heart muscle cells enlarged, but this seemed to be due to the interaction of the heart muscle cells with heart-specific fibroblasts. Fibroblasts constitute a significant portion of total heart tissue, even though the heart muscle cells are responsible for the actual pumping activity of the heart. In their model system, Gelb and others observed that these fibroblast-like cells produce an excess of a protein growth factor called TGF-beta, which causes the cardiomyocytes to undergo hypertrophy or abnormal enlargement.

This model system has relevance for research on several related and more common genetic disorders, including Noonan syndrome, which is characterized by unusual facial features, short stature, heart defects, and skeletal malformations.

There is no cure for HCM in patients with these related genetic conditions, but if these findings are correct, then scientists might be able to treat HCM by blocking specific cell signals. This is something that scientists already know how to do. Approximately 40 percent of patients with CFC suffer from HCM (two of the three participants in this study had HCM). This suggests a pathogenic connection, though the link has never been adequately researched.

“We believe this is the first time the phenomenon has been observed using a human induced pluripotent stem cell model of the disease,” said Bruce Gelb.

Please see Rebecca Josowitz et al., “Autonomous and Non-Autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC -Derived Cardiomyocytes,” Stem Cell Reports, 2016; DOI: 10.1016/j.stemcr.2016.07.018.

Using Cord Blood Stem Cells to “Re-educate” White Blood Cells and Treat Hair Loss


Alopecia areata (AA) is an autoimmune disease that targets the hair follicles. It affects the quality of life and self-esteem of patients because they lose their hair. Is there a way to treat this disease without suppressing the immune system?

Yong Zhao and from Tianhe Stem Cell Biotechnologies in Shandong, China and his collaborators used a so-called “Stem Cell Educator therapy” in which they took the patient’s blood and circulated it through a closed-loop system that separated mononuclear cells from the whole blood, and then allowed those cells to briefly interact with adherent human cord blood-derived multipotent stem cells (CB-SC). After this interaction, the mononuclear cells were returned to the patient’s circulation. This procedure uses the cord blood cells to “educate” the white blood cells of the patient to not attack the patient’s hair follicles.

In an open-label, phase 1/phase 2 study, nine patients with severe AA received one treatment with the Stem Cell Educator therapy. These patients were about 20 years old and had lost their hair, on the average, about 5 years ago.

All these patients experienced improved hair regrowth and quality of life after receiving Stem Cell Educator therapy.  Furthermore, analyses of immune cells from the blood of treated patients showed that the types of immune cells that attack tissues decreased and the number of cells that regulate the immune response increased. Also, investigations of hair follicles in the treated patients revealed that the restored hair follicles expressed a ring of transforming growth factor beta 1 (TGF-β1) around the hair follicles. TGF-β1 is a secreted molecule that down-regulates the immune response and prevents immune cells from attacking your own tissue. The fact that the hair follicles secreted all this TGF-β1 shows that the restored hair follicles had steeled them against the immune system.

How did the cord blood cells do this? By culturing white blood cells with cord blood cells in cell culture, Zhao and others showed that the human cord blood-derived multipotent stem cells induced white blood cells to increase their expression of molecules that are known to tame self-destructive white blood cells. Thus the cord blood stem cells secrete regulatory molecules that change the character of the immune cells so that they no longer attack the hair follicles.

These clinical data demonstrate the safety and efficacy of the Stem Cell Educator therapy for the treatment of AA. This is a very innovative approach that can produce lasting improvement in hair regrowth in subjects with moderate or severe AA.

Muscle Wasting in Muscular Dystrophy Due to Defective Muscle Stem Cells, But Can Be Treated with Blood Pressure Drug


By utilizing a mouse model of Duchenne muscular dystrophy (DMD), researchers at Stanford University School of Medicine have compared gene expression differences between muscle stem cells from DMD mice and muscle stem cells from non-DMD mice. Muscle stem cells from DMD mice express connective-tissue genes associated with fibrosis and muscle weakness as opposed to those from non-DMD mice.

DMD mice, just like their human counterparts, experience progressive muscle degeneration and accumulate connective tissue within the muscle as they age. This new study strongly suggests that the stem cells that surround the muscle fibers might be responsible for this defect. During the course of the disease, muscle stem cells in DMD mice become less able to make new muscle and instead begin to express genes involved in the formation of connective tissue. Excess connective tissue causes scarring (a condition called fibrosis), and these excess scars can accumulate in other organs besides muscle, including the lungs, liver and heart. In the skeletal muscles of people with muscular dystrophy, scarring impairs muscle function and leads to increasing weakness and stiffness, which are hallmarks of the disease.

In addition to this discovery, Thomas Rando, professor of neurology at Stanford University Medical School, and his colleagues showed that these abnormal changes in muscle stem cells could be prevented in laboratory mice by giving the animals a drug that is already approved for use in humans. This drug blocks a signaling pathway involved in the development of fibrosis. Of course more work is required, but scientists are hopeful that a similar approach may one day help treat children with muscular dystrophy.

“These cells are losing their ability to produce muscle, and are beginning to look more like fibroblasts, which secrete connective tissue,” said Dr. Rando. “It’s possible that if we could prevent this transition in the muscle stem cells, we could slow or ameliorate the fibrosis seen in muscular dystrophy in humans.”

Rando and his coworkers published their findings in Science Translational Medicine. Rando, who is the senior author of this paper, is also the director of the Glenn Laboratories for the Biology of Aging and is also the founding director of the Muscular Dystrophy Association Clinic at Stanford. Rando’s former postdoctoral scholar Stefano Biressi, who is presently at the Centre for Integrative Biology at the University of Trento in Italy, is the lead author of this paper.

DMD is a truly devastating disease that affects about 1 in every 3,600 boys born in the United States. The hallmark of this disease is the severe, progressive muscle weakness that confines patients to a wheelchair by early adolescence and eventually leads to paralysis. Mutations in the dystrophin gene cause DMD. The dystrophin gene encodes the Dystrophin protein, which connects muscle fibers to the surrounding external matrix, which stabilizes the fibers, enhances their strength and prevents their injury. Mutations in the dystrophin gene cause production of defective copies of the dystrophin protein. Without functional copies of Dystrophin, the unanchored muscle is unstable, weak, and subject to constant injury. DMD patients are almost always boys because the dystrophin gene is located on the X chromosome. Girls must inherit two faulty copies of the dystrophin gene to contract DMD, which is unlikely because male carriers often die in early adulthood.

By decelerating the fibrotic activity of muscle stem cells in DMD patients, it is possible to delay or even fix the scarring observed in human DMD patients. Normally, muscle stem cells are stimulated when muscles are damaged, and they divide into new cells, some of which form new muscle. In DMD mice, however, muscle stem cells the lack a functional copy of the dystrophin gene slowly begin to resemble fibroblasts instead of muscle-making stem cells.

In this study, Biressi and Rando used a strain of laboratory mice in which the muscle stem cells express a glowing protein when they are treated with a drug called tamoxifen. These glowing mice were then mated with another mouse strain that had a defective copy of the dystrophin gene. These DMD mice now had muscle stem cells that glowed when treated with tamoxifen, which allowed Biressi, Rando and others to trace the movements and activities of muscle stem cells. They discovered that the expression of myogenic genes associated with the regeneration of muscle in response to injury was nearly completely lacking in many of the muscle stem cells in the mice after just 11 months. However, the expression of fibrotic genes had increased compared with that of control animals. The muscle stem cells from the DMD animals were also oddly located, since instead of being nestled next to the muscle fibers where they normally are found, they had begun to move away into the spaces between tissues.

Such increased fibrosis is also observed during normal aging and this process is governed by signaling proteins, which include the Wnt and TGF-beta protein families. Wnt plays a critical role in embryonic development and cancer; TGF-beta controls cell division and specialization. Rando and Biressi hypothesized that inhibiting the Wnt/TGF-beta pathway in DMD would inhibit fibrosis in the animals’ muscles.

To do this, they turned to a blood pressure medicine called losartan. Losartan inhibits the expression of the genes for TGF-beta types 1 and 2, and therefore, might interrupt the signaling pathway that leads the muscle stem cells astray. When DMD mice were treated with losartan, the drug prevented the muscle stem cells from expressing fibrosis-associated genes and partially maintained their ability to form new muscle.

“This scar tissue, or fibrosis, leaves the muscle less elastic and impairs muscle function,” Rando said. “So we’d like to understand why it happens, and how to prevent it. It’s also important to limit fibrosis to increase the likelihood of success with other possible therapies, such as cell therapy or gene therapy.”

TGF-beta-1 is an important signaling molecule throughout the body. Therefore, researchers are now working to find ways to specifically inhibit TGF-beta-2, which is involved in the transition of the muscle stem cells from muscle makers to scar producers. They’re also interested in learning how to translate the research to other diseases.

“Fibrosis seems to occur in a vicious cycle,” Rando said. “As the muscle stem cells become less able to regenerate new muscle, the tissue is less able to repair itself after damage. This leads to fibrosis, which then further impairs muscle formation. Understanding the biological basis of fibrosis could have a profound effect on many other diseases.”

Cartilage Production From Fat-Based Stem Cells Without Exogenous Growth Factors


Making cartilage from fat-based stem cells would be so much more attractive if we didn’t have to use exogenous sources of growth factors. Nevertheless, fat-based stem cells remain quite attractive as a source of cartilage since these cells can be grown in culture to large numbers and can also be readily differentiated into chondrocytes if they are stimulated with the growth factor transforming growth factor-β1 (TGF-β1). Using exogenous TGF-β1, however, has side undesirable effects. Is there another way?

Maybe. A new study by Loran Solorio and Eben Alsberg at Case Western Reserve University has used a culture medium containing TGF-β1-loaded microspheres to make cartilage from fat-based stem cells in culture. This technique can make cartilage without any exogenous growth factors, since all growth factors required for cartilage production are found within the culture system.

In this study, Solorio and Alsberg used exogenous TGF-β1 to induce cartilage formation in fat-based stem cells that were grown in sheets. These sheets of cells made cartilage after 3 weeks. Once it was clear that their experimental system worked well, they used TGF-β1-loaded gelatin microspheres to deliver the growth factor. By tweaking the quantity of microspheres and the concentration of TGF-β1 required for this to work, Solorio and Alsberg showed that the use of TGF-β1-loaded microspheres could induce cartilage formation as well as exogenous TGF-β1. Staining for cartilage-specific molecules and detailed microscopic observation of the cartilage showed that it was indeed, good, solid cartilage.

This publication is the first demonstration of the self-assembly of fat-derived stem cells into high-density cell sheets capable of forming cartilage in the presence of TGF-β1-releasing microspheres. The incorporation of these microspheres might bypass the need for extended culture of the stem cells, potentially allowing stem cells sheets to be implanted more rapidly into defects to regenerate cartilage in a living organism.

Laser-Activation of Dental Stem Cells Spurs Dentine Regeneration


A variety of experiments, clinical trials, and strategies have attempted to exploit stem cells as therapeutic agents in regenerative medicine. However, once stem cells are removed from their niches within the body and grown in artificial culture systems their properties can change. Such culture-acquired changes can often compromise the therapeutic potential of some stem cells. For this reason, the development of relatively simple but effective stem cell isolation and manipulation techniques represents someone of the prominent technical hurdles to the clinical use of stem cells.

Several laboratories have used exogenous factors to direct the differentiation of tissue-resident stem cells, but these exogenous factors can often cause unwanted side effects. For this reason, simpler manipulation techniques are always a welcome addition to the armamentarium of stem cell scientists.

To that end, Ashok B. Kulkarni from the National Institute of Dental and Craniofacial Research in Bethesda, MD and David J. Mooney from the Harvard School of Engineering and their colleagues and co-workers have used non-ionizing, low-power laser (LPL) treatments to activate host stem cells and promote tissue regeneration. This is a minimally invasive treatment that directs stem cells already present in tissues to heal damaged tissues.

LPL treatment was used to activate human dental stem cells in a laboratory culture system. Upon LPL treatment, the dental stem cells began to synthesize a powerful growth factor called transforming growth factor–β1 (TGF-β1). The endogenous synthesis of TGF-β1 and its receptor drove the dental stem cells to form dentin tubes.

When Kulkami and Mooney used an assay in animals called a “pulp capping model,” they discovered that LPL-activated dental stem cells were able to regenerate dentin after laser activation. To further demonstrate that these regenerative effects were the result of TGF-β1, Kalkami and Mooney and others made cells that did not have a functional TGF-β receptor II. This mutation completely abrogated the effects of LPL treatments. Also, if the dental stem cells were incubated with a TGF-βRI inhibitor, the effects of LPL on the dental stem cells was attenuated.

Thus, there is a simple and non-invasive way to activate a resident stem cell population in our bodies. Furthermore, the mechanisms by which LPL activates these stem cells has been defined as TGF-β mediated. These experiments also outlines the mechanism by which resident stem cells might be harnessed by means of light-activated endogenous cues for clinical regenerative applications. Exciting, huh?

A Molecular Switch that Determines Stem Cell Or Neuron


A University of California, San Diego School of Medicine research team has provided new information about a well-known protein that provides the switch for cells to become neurons. This protein is part of a regulatory circuit that can push an immature neural cell to become a functional neuron.

Postdoctoral fellow Chih-Hong Lou and his colleagues worked with principal investigator Miles F. Wilkinson, who is a professor in the Department of Reproductive Medicine, and is also a member of the UC San Diego Institute for Genomic Medicine. These data were published in the February 13 online issue of the journal Cell Reports. These data may also elucidate a still poorly understood process – neuron specification – and might significantly accelerate the development of new therapies for specific neurological disorders, such as autism and schizophrenia.

Wilkinson, Lou and others discovered that the conversion of immature cells to neurons is controlled by a protein called UPF1. UPF1 works in a pathway called the “nonsense-mediated RNA decay” or NMD pathway. The NMD pathway provides a quality control mechanism that eliminates faulty messenger RNA (mRNA) molecules.

mRNA molecules are synthesized from DNA in the nucleus of cells and are exported to the cytoplasm where they are translated by ribosomes into protein. All proteins are encoded by stretches of DNA known as genes and the synthesis of an RNA copy of this stretch of DNA is called transcription. After the transcription of a messenger RNA molecule, is goes to the cytoplasm and is used as the template for the synthesis of a specific protein. Occasionally, mistakes are made in the transcription of mRNAs, and such aberrant mRNAs will either be translated into junk protein, or are so damaged that they cannot be recognized by ribosomes. Such junk mRNAs will gum up the protein synthesis machinery, but cells have the NMD pathway that degrades junk mRNAs to prevent the collapse of the protein synthesis machinery.

UPF1 mechanism

A second function for the NMD pathway is to degrade a specific group of normal mRNAs to prevent the production of particular proteins. This NMD function is physiologically important, but until now it had not been clear why it is important.

Wilkinson and others have discovered that UPF1, in combination with a particular class of microRNAs, acts as a molecular switch to determine when immature (non-functional) neural cells take the plunge and differentiate into non-dividing (functional) neurons. In particular, UPF1 directs the degradation of a specific mRNA that encodes for a protein in the TGF-beta signaling pathway, which promotes neural differentiation. The destruction of this mRNA prevents the proper functioning of the TGF-beta signaling pathway and neural differentiation fails to occur. Therefore, Wilkinson, Lou and co-workers identified, for the first time, a molecular pathway in which NMD drives a normal biological response.

NMD also promotes the decay of mRNAs that encode proliferation inhibitors, which Wilkinson said might explain why NMD stimulates the proliferative state characteristic of stem cells. There are many potential clinical ramifications for these findings,” Wilkinson said. “One is that by promoting the stem-like state, NMD may be useful for reprogramming differentiated cells into stem cells more efficiently.

Wilkinson continued: “Another implication follows from the finding that NMD is vital to the normal development of the brain in diverse species, including humans. Humans with deficiencies in NMD have intellectual disability and often also have schizophrenia and autism. Therapies to enhance NMD in affected individuals could be useful in restoring the correct balance of stem cells and differentiated neurons and thereby help restore normal brain function.”

Co-authors on this paper include Ada Shao, Eleen Y. Shum, Josh L. Espinoza and Rachid Karam, from the UCSD Department of Reproductive Medicine; and Lulu Huang, from Isis Pharmaceuticals.

Funding for this research came, in part, from National Institutes of Health (grant GM-58595) and the California Institute for Regenerative Medicine.

Taiwanese Group Identifies Stem Cell-Based Drug to Rejuvenate Aged Hearts


A southern Taiwan-based National Cheng Kung University research team led by Patrick Ching-Ho Hsieh has discovered that a molecule called prostaglandin E2 can regenerate aged hearts in rodents.

This discovery provides a useful new perspective on heart regeneration and presents an effective option for heart disease patients other than heart transplant.

According to Hsieh, congestive heart disease and other cardiovascular diseases are a leading cause of morbidity and mortality throughout the world. There are some six million patients with congestive heart failure in the US alone and some 400,000 in Taiwan. Despite intensive drug, surgical and other medical interventions, 80 percent of all heart patients die within 8 years of diagnosis.

Even though several experiments and clinical trials have established that heart regeneration can take place, the means by which the heart regenerates is still not completely clear, and there are also no drugs to stimulate heart regeneration by the resident stem cell population in the heart.

Now, after seven years of hard work, Hsieh’s team has identified the critical time period and the essential player that directs heart repair.

Hsieh and his colleagues used genetically engineered mice that Hsieh had developed as a postdoctoral research fellow at Harvard Medical School. By using this transgenic mouse strain, Hsieh and others showed that the self-repair process of the heart begins 7 days after injury and peaks at 10 days after injury.

The “director” of this self-repair process is the molecule PGE2. PGE2 regulates heart-specific stem cell activities.

PGE2

“More importantly, both young and old mice have significant improvements for cardiac remodeling if you treat both of them [with] PGE2,” said Hsieh.

Hsieh’s team also established that PGE2 decreases expression of a gene associated with aging, TGF-beta1. PGE2 also rejuvenates the micro-environment of the aged cells, according to Hsieh.