Stem Cells Faulty in Duchenne Muscular Dystrophy

Stem Cells Faulty in Duchenne Muscular Dystrophy

Thursday, 18 December 2014

Like human patients, mice with a form of Duchenne muscular dystrophy undergo progressive muscle degeneration and accumulate connective tissue as they age. Now, researchers at the Stanford University School of Medicine have found that the fault may lie at least partly in the stem cells that surround the muscle fibres.

They've found that during the course of the disease, the stem cells become less able to make new muscle and instead begin to express genes involved in the formation of connective tissue. Excess connective tissue – a condition called fibrosis – can accumulate in many organs, including the lungs, liver and heart, in many different disorders. In the skeletal muscles of people with muscular dystrophy, the fibrotic tissue impairs the function of the muscle fibres and leads to increasing weakness and stiffness, which are hallmarks of the disease.

The researchers discovered that this abnormal change in stem cells could be inhibited in laboratory mice by giving the animals a drug that is already approved for use in humans. The drug works by blocking a signalling pathway involved in the development of fibrosis. Although much more research is needed, the scientists are hopeful that a similar approach may one day work in 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 Thomas Rando, MD, PhD, professor of neurology and neurological sciences.

"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."

A paper describing the researchers' findings will be published Dec. 17 in Science Translational Medicine. Rando, the paper's senior author, is director of the Glenn Laboratories for the Biology of Aging and founding director of the Muscular Dystrophy Association Clinic at Stanford. Former postdoctoral scholar Stefano Biressi, PhD, is the lead author. Biressi is now at the Centre for Integrative Biology at the University of Trento in Italy.

A devastating disease

Duchenne muscular dystrophy is a devastating disease that affects about 1 in every 3,600 boys born in the United States. Patients usually experience severe, progressive muscle weakness that confines them to a wheelchair in early adolescence and eventually leads to paralysis. It's caused by mutations in the dystrophin gene, which encodes the dystrophin protein. The dystrophin protein serves to connect muscle fibres to the surrounding external matrix. This connection stabilizes the fibres, enhancing their strength and preventing injury. Sufferers are nearly always boys because the dystrophin gene is located on the X chromosome. (Girls would need to inherit two faulty copies, which is unlikely because male carriers often die in early adulthood.)

Under normal conditions, muscle stem cells respond to muscle damage by dividing into cells, one of which becomes new muscle, while the other remains a stem cell. However, in the mice missing the dystrophin gene, the muscle stem cells slowly assume a different fate. They begin to resemble fibroblasts instead of muscle-making machines.

To conduct the research, Biressi and Rando used a strain of laboratory mice in which the muscle stem cells were engineered to glow with a fluorescent light when treated with a drug called tamoxifen. They then bred the mice with another strain in which the dystrophin gene is mutated, and followed the fate of the cells over time.

The researchers found that the expression of myogenic genes, which are 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, while the expression of fibrotic genes had increased compared with that of control animals. The cells from the dystrophic animals were also oddly located: Rather than being nestled next to the muscle fibres, they had begun to move away into the spaces between tissues.

The role of a signalling pathway

Rando and Biressi knew that a similar, but much less pronounced, accumulation of connective tissue in muscle fibres occurs during normal aging. That process is governed by signalling 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. They wondered whether blocking the Wnt/TGF-beta pathway in the dystrophic mice would inhibit fibrosis in the animals' muscles.

The researchers turned to a drug called losartan, which is used to treat high blood pressure. Losartan inhibits the expression of the genes for TGF-beta types 1 and 2. The researchers thought it would probably interrupt the signalling pathway that leads the muscle stem cells astray.

Treating the mice with losartan, they found, did in fact prevent 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."

Next steps

Because TGF-beta type 1 plays many roles throughout the body, the researchers are now working to find ways to specifically inhibit TGF-beta type 2, which is involved in the transition of the muscle stem cells. 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."

Source: Stanford University Medical Center

Contact: Krista Conger

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A New Way to Make Muscle Cells from Human Stem Cells

A New Way to Make Muscle Cells from Human Stem Cells

Friday, 21 March 2014

Muscle cells are stained green in this micrograph

of cells grown from embryonic stem cells in the

lab of Masatoshi Suzuki at the University of

Wisconsin–Madison. Cell nuclei are stained blue;

the muscle fibres contain multiple nuclei. Nuclei

outside the green fibres are from non-muscle cells.

Suzuki has developed a new method of growing

stem cells into muscle cells that could be more

suitable for treating disease. Suzuki hopes to

experiment next with animals that model muscular

dystrophy and amyotrophic lateral sclerosis.

Credit: Masatoshi Suzuki. 

As stem cells continue their gradual transition from the lab to the clinic, a research group at the University of Wisconsin-Madison has discovered a new way to make large concentrations of skeletal muscle cells and muscle progenitors from human stem cells.

The new method, described in the journal Stem Cells Translational Medicine, could be used to generate large numbers of muscle cells and muscle progenitors directly from human pluripotent stem cells. These stem cells, such as embryonic (ES) or induced pluripotent stem (iPS) cells, can be made into virtually any adult cell in the body.

Adapting a method previously used to make brain cells, Masatoshi Suzuki, an assistant professor of comparative biosciences in the School of Veterinary Medicine, has directed those universal stem cells to become both adult muscle cells and muscle progenitors.

Importantly, the new technique grows the pluripotent stem cells as floating spheres in high concentrations of two growth factors, fibroblast growth factor-2 and epidermal growth factor. These growth factors "urge" the stem cells to become muscle cells.

"Researchers have been looking for an easy way to efficiently differentiate stem cells into muscle cells that would be allowable in the clinic," says Suzuki. The novelty of this technique is that it generates a larger number of muscle stem cells without using genetic modification, which is required by existing methods for making muscle cells.

"Many other protocols have been used to enhance the number of cells that go to a muscle fate," says co-author Jonathan Van Dyke, a post-doctoral fellow in Suzuki's laboratory.

"But what's exciting about the new protocol is that we avoid some techniques that would prohibit clinical applications. We think this new method has great promise for alleviating human suffering."

Last year, Suzuki demonstrated that transplants of another type of human stem cells somewhat improved survival and muscle function in rats that model amyotrophic lateral sclerosis (ALS). Also known as Lou Gehrig's disease, ALS destroys nerves and causes a loss of muscle control. The muscle progenitors generated with Suzuki's new method could potentially play a similar role but with enhanced effect.

The new technique can also be used to grow muscle cells from iPS cells from patients with neuromuscular diseases like ALS, spinal muscular atrophy and muscular dystrophy. Thus, the technique could produce adult muscle cells in a dish that carry genetic diseases. These cells could then be used as a tool for studying these diseases and screening potential drug compounds, says Suzuki.

"Our protocol can work in multiple ways and so we hope to provide a resource for people who are exploring specific neuromuscular diseases in the laboratory."

The new protocol incorporates a number of advantages. First, the cells are grown in defined supplements without animal products such as bovine serum, enhancing the clinical safety for the muscle stem cells. Second, when grown as spheres, the cells grow faster than with previous techniques. Third, 40 to 60 percent of the cells grown using the process are either muscle cells or muscle progenitors, a high proportion compared to traditional non-genetic techniques of generating muscle cells from human ES and iPS cells.

Suzuki and his group hope that by further manipulating the chemical environment of the spheres of stem cells, they may increase that number, further easing the path toward human treatment.

Source: University of Wisconsin-Madison 

Contact: Masatoshi Suzuki

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Successful Regeneration of Human Skeletal Muscle in Mice Enables Accelerated Research in Muscular Dystrophy

Kennedy Krieger researchers develop valid and accurate model for FSHD

Tuesday, 28 January 2014

Researchers at the Kennedy Krieger Institute recently announced study findings showing the successful development of a humanized preclinical model for facioscapulohumeral muscular dystrophy (FSHD), providing scientists with a much needed tool to accelerate novel therapeutic research and development.

Published in Human Molecular Genetics, the study outlines the validity of a unique model that, for the first time, mirrors the gene expression and biomarker profile of human FSHD tissue. Previously, there has been no accepted preclinical model for FSHD, a complex and rare neuromuscular disorder that affects approximately 4 - 7 per 100,000 individuals. As a result, therapeutic development for the disorder has been stymied.

“The inability to mimic the FSHD’s genetic mechanism in preclinical models has been an ongoing challenge for the research community. Without an accurate model, making the leap to clinical research commonly fails,” said Kathryn Wagner MD, PhD, director of the Center for Genetic Muscle Disorders at the Kennedy Krieger Institute in Baltimore, MD.

“We believe this unique model will open the door to studying muscle regeneration over time and help better predict clinical response to therapeutic drugs.”

Inspired by cancer preclinical models developed with human tumour tissue, Dr. Wagner and her research team leveraged both basic science and clinical research resources available at Kennedy Krieger to successfully regenerate grafted muscle within the models. Human bicep muscle biopsies transplanted into models survived for over 41 weeks and retained features of normal and diseased tissue.

“This model is not only applicable to genetic muscle diseases for which we lack appropriate research models, but for other acquired muscle conditions,” said Wagner.

“Now there will be more research possibilities related to the overall impact of age and disease on the regenerative and growth capacity of human skeletal muscle.”

Source: Kennedy Krieger Institute 

Contact: Jennifer Burke

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Human Muscle Stem Cell Therapy Gets Help from Zebrafish

HSCI researchers improve therapy prospects

Thursday, 07 November 2013

Zebrafish in Research Lab for Animal Testing.

Harvard Stem Cell scientists have discovered that the same chemicals that stimulate muscle development in zebrafish can also be used to differentiate human stem cells into muscle cells in the laboratory, a historically challenging task that, now overcome, makes muscle cell therapy a more realistic clinical possibility.

The work, published this week in the journal Cell, began with a discovery by Boston Children's Hospital researchers, led by Leonard Zon, MD, and graduate student Cong (Tony) Xu, who tested 2,400 different chemicals in cultures of zebrafish embryo cells to determine if any could increase the numbers of muscle cells formed. Using fluorescent reporter fish in which muscle cells were visible during their creation, the researchers found six chemicals that were very effective at promoting muscle formation.

Zon shared his results with Harvard Department of Stem Cell and Regenerative Biology professor Amy Wagers, PhD, and Mohammadsharif Tabebordbar, a graduate student in her laboratory, who tested the six chemicals in mice. One of the six, called forskolin, was found to increase the numbers of muscle stem cells from mice that could be obtained when these cells were grown in laboratory dishes. Moreover, the cultured cells successfully integrated into muscle when transplanted back into mice.

Inspired by the successful application of these chemicals in mice, Salvatore Iovino, PhD, a joint postdoctoral fellow in the Wagers lab and the lab of C. Ronald Kahn, MD, at the Joslin Diabetes Center, investigated whether the chemicals would also affect human cells and found that a combination of three chemicals, including forskolin, could induce differentiation of human induced pluripotent stem (iPS) cells, made by reprogramming skin cells. Exposure of iPS cells to these chemicals converted them into skeletal muscle, an outcome the Wagers and Kahn labs had been striving to achieve for years using conventional methods. When transplanted into a mouse, the human iPS-derived muscle cells also contributed to muscle repair, offering early promise that this protocol could provide a route to muscle stem cell therapy in humans.

The interdisciplinary, cross-laboratory collaboration between Zon, Wagers, and Kahn highlights the advantage of open exchange between researchers.

"If we had done this screen directly on human iPS cells, it would have taken at least 10 times as long and cost 100 times as much," said Wagers.

"The zebrafish gave us a big advantage here because it has a fast generation time, rapid development, and can be easily and relatively cheaply screened in a culture dish."

"This research demonstrates that over 300 million years of evolution, the pathways used in the fish are conserved through vertebrates all the way up to the human," said Wagers' fellow HSCRB professor Leonard Zon, chair of the Harvard Stem Cell Institute Executive Committee and director of the stem cell program at Boston Children's Hospital.

"We can now make enough human muscle progenitors in a dish to allow us to model diseases of the muscle lineage, like Duchenne muscular dystrophy, conduct drug screens to find chemicals that correct that disease, and in the long term, efficiently transplant muscle stem cells into a patient."

In a similar biomedical application, Kahn, who is chief academic officer at the Joslin, plans to apply the new ability to quickly produce muscle stem cells for diabetes research. His lab will generate iPS-derived muscle cells from people who are at risk for diabetes and people who have diabetes to identify alterations that lead to insulin resistance in the muscle.

Going forward, Zon plans to apply this platform of cross-species discovery to other stem cell lines, including those involved in blood and eye development.

"We have a new system to use to study tissue development, and it's not just muscle that can be studied, every single organ can be studied in the zebrafish system," he said.

Source: Harvard University 

Contact: B. D. Colen

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Scientists Distinguish Signatures of Old versus Young Stem Cells

Scientists Distinguish Signatures of Old versus Young Stem Cells

Friday, 28 June 2013

A chemical code scrawled on histones — the protein husks that coat DNA in every animal or plant cell — determines which genes in that cell are turned on and which are turned off. Now, Stanford University School of Medicine researchers have taken a new step in the deciphering of that histone code.

Professor of neurology Thomas Rando, 

MD, PhD.. Credit: Stanford University 

Medical Center. 

In a study to be published June 27 in Cell Reports, a team led by Thomas Rando, MD, PhD, professor of neurology and neurological sciences and chief of the Veterans Affairs Palo Alto Health Care System's neurology service, has identified characteristic differences in "histone signatures" between stem cells from the muscles of young mice and old mice. The team also distinguished histone-signature differences between quiescent and active stem cells in the muscles of young mice.

"We've been trying to understand both how the different states a cell finds itself in can be defined by the markings on the histones surrounding its DNA, and to find an objective way to define the 'age' of a cell," said Rando, who is also director of Stanford's Glenn Laboratories for the Biology of Aging and deputy director of the Stanford Center on Longevity.

While all cells in a person's body share virtually the same genes, these cells can be as different from one another as a nerve cell is from a fat cell. This is because only a fraction of a cell's genes are actually "turned on" — actively involved in the production of one or another protein. A muscle cell produces the proteins it uses to be a muscle cell, a liver cell produces those it needs in order to be a liver cell and so forth. Rando's team thinks the same kinds of on/off differences may distinguish old stem cells from young stem cells.

In human cells, the DNA in which genes are found doesn't float loose inside the cell nucleus but is, rather, packaged inside protein "husks" called histones. Chemical marks on the histones, which sheathe our chromosomal DNA in each cell's nucleus, act as "stop" and "go" traffic signals. These signals tell the complex molecular machinery that translates genes' instructions into newly produced proteins which genes to read and which ones to skip.

In 2005, Rando and his colleagues published a study in Nature showing that stem cells in several tissues of older mice, including muscle, seemed to act younger after continued exposure to younger mice's blood. Their capacity to divide, differentiate and repopulate tissues, which typically declines with an organism's advancing age, resembled those of their stem-cell counterparts in younger animals.

This naturally led to curiosity about exactly what is happening inside a cell to rejuvenate it, said Rando. One likely place to look for an answer was histones, to see if changes in the patterns of the chemical marks on them might reveal any secrets, at the cellular level, of the aging process we all experience — and, perhaps, whether there might be anything we can do about it. Rando and his colleagues also wanted to learn more about what kinds of difference in these patterns accompany a cell's transition from one level of activity to another.

To do that, Rando and his team looked at satellite cells, an important class of stem cells that serve as a reserve army of potential new muscle tissue. Under normal circumstances, these rather rare stem cells sit quietly adjacent to muscle fibres. But some signal provided by muscular injury or degeneration prompts satellite cells to start dividing and then to integrate themselves into damaged fibres, repairing the muscle tissue. The investigators profiled the histone markings of mice that are as old, in mouse years, as young human adults, as well as mice whose human counterparts would be 70 to 80 years old.

The researchers harvested satellite cells from both healthy and injured muscle tissue of young mice and from healthy tissue of old mice; extracted these cells' DNA with the histone coatings intact; and used tagged antibodies targeting the different kinds of marks to find which spots on those histones were flagged with either "stop" or "go" signals.

"Satellite cells can sit around for practically a lifetime in a quiescent state, not doing much of anything. But they're ready to transform to an activated state as soon as they get word that the tissue needs repair," Rando said.

"So, you might think that satellite cells would be already programmed in a way that commits them solely to the 'mature muscle cell' state." The researchers expected that in these quiescent stem cells, the genes specific for other tissues like skin and brain would be marked by "stop" signals.

Instead, they found, in quiescent satellite cells taken from the younger mice, copious instances in which histones in the vicinity of genes ordinarily reserved for other tissues were marked with both "stop" and "go" signals, just as genes associated with development to mature-muscle status were.

"We weren't looking for that, and we certainly weren't expecting it," Rando said.

"We figured all the muscle genes would be either poised for activity — marked with both 'on' and 'off' signals — or 'on,' and that all the other genes would be turned off. But when you look at these satellite cells the way we did, they seem ready to become all kinds of cells. It's a mystery," he said, suggesting that it could mean stem cells thought to be committed to a particular lineage may be capable of becoming other types of tissue entirely.

"Maybe their fates are not permanently sealed," he said.

"The door is not locked. Who knows what could happen if they're given the right signals?" The Rando lab is now beginning to test this proposition.

Oddly, activated satellite cells from injured muscle tissue featured far more gene-associated "stop" signals than did quiescent ones.

"As a cell goes from quiescent to activated state, you might expect to see more genes marked by 'on' signals," Rando said.

"We found the opposite. The dominant pattern when cells become activated is a big increase in repressive marks across the genome. Apparently it's not until then that a satellite cell makes the effort to turn off all of its non-muscle options."

The differences between quiescent and activated cells, Rando's team found, are mirrored by those between young and old quiescent satellite cells.

"With age, there's an uptick in repressive markers. A lot more genes are locked in the 'off' position," he said.

The meaning of this is not yet clear, he added.

"In a division-capable cell, as opposed to the non-dividing, differentiated muscle cells that activated satellite cells may someday become, it may be important to maintain a high level of repression with age. Maybe this increase in repression is a kind of tumour-suppression mechanism, keeping aging satellite cells — which could have accumulated some dangerous mutations over the passing months and years — in check."

The description of the histone-code differences between young and old cells constitutes a yardstick allowing investigators to ask which of these differences are important in aging and in rejuvenation, Rando said.

"We don't have the answers yet. But now that we know what kinds of changes occur as these cells age, we can ask which of these changes reverse themselves when an old cell goes back to becoming a young cell" — as appeared to be the case when tissues of older mice were exposed to blood from younger mice.

Rando's group is now looking to test whether the signatures they've identified in satellite cells generalize to other kinds of adult stem cells as well.

Source: Stanford University Medical Center 

Contact: Bruce Goldman

Reference:

Chromatin Modifications as Determinants of Muscle Stem Cell Quiescence and Chronological Aging

Ling Liu, Tom H. Cheung, Gregory W. Charville, Bernadette Marie Ceniza Hurgo, Tripp Leavitt, Johnathan Shih, Anne Brunet, and Thomas A. Rando
Cell Reports, 27 June 2013, 10.1016/j.celrep.2013.05.043

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Stem-cell Approach Shows Promise for Duchenne Muscular Dystrophy

Stem-cell Approach Shows Promise for Duchenne Muscular Dystrophy

Monday, 14 January 2013

Researchers have shown that transplanting stem cells derived from normal mouse blood vessels into the hearts of mice that model the pathology associated with Duchenne muscular dystrophy (DMD) prevents the decrease in heart function associated with DMD.

University of Illinois comparative biosciences 

professor Suzanne Berry-Miller, veterinary 

clinical medicine professor Robert O’Brien 

and their colleagues developed a method 

that enhanced cardiac function in a mouse

model of Duchenne muscular dystrophy. 

Credit: L. Brian Stauffer. 

Their findings appear in the journal Stem Cells Translational Medicine.

Duchenne muscular dystrophy is a genetic disorder caused by a mutation in the gene for dystrophin, a protein that anchors muscle cells in place when they contract. Without dystrophin, muscle contractions tear cell membranes, leading to cell death. The lost muscle cells must be regenerated, but in time, scar tissue replaces the muscle cells, causing the muscle weakness and heart problems typical of DMD.

The U.S. Centers for Disease Control and Prevention estimates that DMD affects one in every 3,500 males. The disease is more prevalent in males because the dystrophin mutation occurs on the X chromosome; males have one X and one Y chromosome, so a male with this mutation will have DMD, while females have two X chromosomes and must have the mutation on both of them to have the disease. Females with the mutation in one X chromosome sometimes develop muscle weakness and heart problems as well, and may pass the mutation on to their children.

Although medical advances have extended the lifespans of DMD patients from their teens or 20s into their early 30s, disease-related damage to the heart and diaphragm still limits their lifespan.

"Almost 100 percent of patients develop dilated cardiomyopathy," in which a weakened heart with enlarged chambers prevents blood from being properly pumped throughout the body, said University of Illinois comparative biosciences professor Suzanne Berry-Miller, who led the study.

"Right now, doctors are treating the symptoms of this heart problem by giving patients drugs to try to prolong heart function, but that can't replace the lost or damaged cells," she said.

Treatment with stem cells derived from 

blood vessels spurred nestin-positive 

stem cells already present in the heart 

to form new cardiac muscle cells (see 

arrows). Credit: Suzanne Berry-Miller. 

In the new study, the researchers injected stem cells known as aorta-derived mesoangioblasts (ADM) into the hearts of dystrophin-deficient mice that serve as a model for human DMD. The ADM stem cells have a working copy of the dystrophin gene.

This stem cell therapy prevented or delayed heart problems in mice that did not already show signs of the functional or structural defects typical of Duchenne muscular dystrophy, the researchers report.

Berry-Miller and her colleagues do not yet know why the functional benefits occur, but proposed three potential mechanisms. They observed that some of the injected stem cells became new heart muscle cells that expressed the lacking dystrophin protein. The treatment also caused existing stem cells in the heart to divide and become new heart muscle cells, and the stem cells stimulated new blood vessel formation in the heart. It is not yet clear which of these effects is responsible for delaying the onset of cardiomyopathy, Berry-Miller said.

"These vessel-derived cells might be good candidates for therapy, but the more important thing is the results give us new potential therapeutic targets to study, which may be activated directly without the use of cells that are injected into the patient, such as the ADM in the current study," Berry-Miller said.

"Activating stem cells that are already present in the body to repair tissue would avoid the potential requirement to find a match between donors and recipients and potential rejection of the stem cells by the patients."

After injecting dystrophin-deficient mouse hearts

with normal, blood-vessel-derived stem cells,

researchers saw an increase in cell division among

nestin-positive stem cells (shown in green) in the heart.

These stem cells are found in the hearts of adult

humans, rats, and mice, but their function is not yet

known. An increase in formation of new heart muscle

cells from the nestin-positive stem cells was also

detected. Credit: Suzanne Berry-Miller. 

Despite the encouraging results that show that stem cells yield a functional benefit when administered before pathology arises in DMD mouse hearts, a decline in function was seen in mice that already showed the characteristics of dilated cardiomyopathy. One of these characteristics is the replacement of muscle tissue with connective tissue, known as fibrosis.

This difference may occur, Berry-Miller said, as a result of stem cells landing in a pocket of fibrosis rather than in muscle tissue. The stem cells may then become fibroblasts that generate more connective tissue, increasing the amount of scarring and making heart function worse. This shows that the timing of stem cell insertion plays a crucial role in an increase in heart function in mice lacking the dystrophin protein.

She remains optimistic that these results provide a stepping-stone toward new clinical targets for human DMD patients.

"This is the only study so far where a functional benefit has been observed from stem cells in the dystrophin-deficient heart, or where endogenous stem cells in the heart have been observed to produce new muscle cells that replace those lost in DMD, so I think it opens up a new area to focus on in pre-clinical studies for DMD," Berry-Miller said.

Source: University of Illinois at Urbana-Champaign 

Contact: Diana Yates

Reference:

Injection of vessel derived stem cells-prevent dilated cardiomyopathy and promote angiogenesis and endogenous cardiac stem cell proliferation in mdx/utrn-/-but not aged mdx mouse models for Duchenne muscular dystrophy

Ju Lan Chun, Robert O'Brien, Min Ho Song, Blake F. Wondrasch and Suzanne E. Berry

Stem Cells Trans Med, December 27, 2012, doi:10.5966/sctm.2012-0107

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Complexity of Human Genome Unravelled

ENCODE data describes function of human genome’s ‘dark matter’

Wednesday, 05 September 2012

The Human Genome Project produced an almost complete order of the 3 billion pairs of chemical letters in the DNA that embodies the human genetic code — but little about the way this blueprint works. Now, after a multi-year concerted effort by more than 440 researchers in 32 labs around the world, a more dynamic picture gives the first holistic view of how the human genome actually does its job.

During the new study, researchers linked more than 80 percent of the human genome sequence to a specific biological function and mapped more than 4 million regulatory regions where proteins specifically interact with the DNA. These findings represent a significant advance in understanding the precise and complex controls over the expression of genetic information within a cell. The findings bring into much sharper focus the continually active genome in which proteins routinely turn genes on and off using sites that are sometimes at great distances from the genes themselves. They also identify where chemical modifications of DNA influence gene expression and where various functional forms of RNA, a form of nucleic acid related to DNA, help regulate the whole system.

"During the early debates about the Human Genome Project, researchers had predicted that only a few percent of the human genome sequence encoded proteins, the workhorses of the cell, and that the rest was junk. We now know that this conclusion was wrong," said Eric D. Green, M.D., Ph.D., director of the National Human Genome Research Institute (NHGRI), a part of the National Institutes of Health.

"ENCODE has revealed that most of the human genome is involved in the complex molecular choreography required for converting genetic information into living cells and organisms."

NHGRI organized the research project producing these results; it is called the Encyclopedia of DNA Elements or ENCODE. Launched in 2003, ENCODE's goal of identifying all of the genome's functional elements seemed just as daunting as sequencing that first human genome. ENCODE was launched as a pilot project to develop the methods and strategies needed to produce results and did so by focusing on only 1 percent of the human genome. By 2007, NHGRI concluded that the technology had sufficiently evolved for a full-scale project, in which the institute invested approximately $123 million over five years. In addition, NHGRI devoted about $40 million to the ENCODE pilot project, plus approximately $125 million to ENCODE-related technology development and model organism research since 2003.

William Noble, professor of genome sciences and computer
science, in the data centre at the William H. Foege Building, 

University of Washington, Seattle, Washington. Noble, an 

expert on machine learning, and his team designed artificial 

intelligence programs to analyse ENCODE data. These 

computer programs can learn from experience, recognize 

patterns, and organize information into categories 

understandable to scientists. The centre houses systems for a 

wide variety of genetic research. The computer centre has the 

capacity to store and analyse a tremendous amount of data, 

the equivalent of a 670-page autobiography of each person on 

earth, uncompressed. The computing resources analyse over 

4 pentabytes of genomic data a year. Credit: Clare McLean, 

courtesy of University of Washington. 

The scale of the effort has been remarkable. Hundreds of researchers across the United States, United Kingdom, Spain, Singapore and Japan performed more than 1,600 sets of experiments on 147 types of tissue with technologies standardized across the consortium. The experiments relied on innovative uses of next-generation DNA sequencing technologies, which had only become available around five years ago, due in large part to advances enabled by NHGRI's DNA sequencing technology development program. In total, ENCODE generated more than 15 trillion bytes of raw data and consumed the equivalent of more than 300 years of computer time to analyse.

"We've come a long way," said Ewan Birney, Ph.D., of the European Bioinformatics Institute, in the United Kingdom, and lead analysis coordinator for the ENCODE project.

"By carefully piecing together a simply staggering variety of data, we've shown that the human genome is simply alive with switches, turning our genes on and off and controlling when and where proteins are produced. ENCODE has taken our knowledge of the genome to the next level, and all of that knowledge is being shared openly."

The ENCODE Consortium placed the resulting data sets as soon as they were verified for accuracy, prior to publication, in several databases that can be freely accessed by anyone on the Internet. These data sets can be accessed through the ENCODE project portal as well as at the University of California, Santa Cruz genome browser, the National Center for Biotechnology Information, and the European Bioinformatics Institute.

"The ENCODE catalogue is like Google Maps for the human genome," said Elise Feingold, Ph.D., an NHGRI program director who helped start the ENCODE Project.

"Simply by selecting the magnification in Google Maps, you can see countries, states, cities, streets, even individual intersections, and by selecting different features, you can get directions, see street names and photos, and get information about traffic and even weather. The ENCODE maps allow researchers to inspect the chromosomes, genes, functional elements and individual nucleotides in the human genome in much the same way."

The coordinated publication set includes one main integrative paper and five related papers in the journal Nature; 18 papers in Genome Research; and six papers in Genome Biology. The ENCODE data are so complex that the three journals have developed a pioneering way to present the information in an integrated form that they call threads.

"Because ENCODE has generated so much data, we, together with the ENCODE Consortium, have introduced a new way to enable researchers to navigate through the data," said Magdalena Skipper, Ph.D., senior editor at Nature, which produced the freely available publishing platform on the Internet.

Since the same topics were addressed in different ways in different papers, the new website will allow anyone to follow a topic through all of the papers in the ENCODE publication set by clicking on the relevant thread at the Nature ENCODE explorer page. For example, thread number one compiles figures, tables, and text relevant to genetic variation and disease from several papers and displays them all on one page. ENCODE scientists believe this will illuminate many biological themes emerging from the analyses.

In addition to the threaded papers, six review articles are being published in the Journal of Biological Chemistry and two related papers in Science and one in Cell.

The ENCODE data are rapidly becoming a fundamental resource for researchers to help understand human biology and disease. More than 100 papers using ENCODE data have been published by investigators who were not part of the ENCODE Project, but who have used the data in disease research. For example, many regions of the human genome that do not contain protein-coding genes have been associated with disease. Instead, the disease-linked genetic changes appear to occur in vast tracts of sequence between genes where ENCODE has identified many regulatory sites. Further study will be needed to understand how specific variants in these genomic areas contribute to disease.

"We were surprised that disease-linked genetic variants are not in protein-coding regions," said Mike Pazin, Ph.D., an NHGRI program director working on ENCODE.

"We expect to find that many genetic changes causing a disorder are within regulatory regions, or switches, that affect how much protein is produced or when the protein is produced, rather than affecting the structure of the protein itself. The medical condition will occur because the gene is aberrantly turned on or turned off or abnormal amounts of the protein are made. Far from being junk DNA, this regulatory DNA clearly makes important contributions to human health and disease."

Identifying regulatory regions will also help researchers explain why different types of cells have different properties. For example why do muscle cells generate force while liver cells break down food? Scientists know that muscle cells turn on some genes that only work in muscle, but it has not been previously possible to examine the regulatory elements that control that process. ENCODE has laid a foundation for these kinds of studies by examining more than 140 of the hundreds of cell types found in the human body and identifying many of the cell type-specific control elements.

Despite the enormity of the dataset described in this historic collection of publications, it does not comprehensively describe all of the functional genomic elements in all of the different types of cells in the human body. NHGRI plans to invest in additional ENCODE-related research for at least another four years. During the next phase, ENCODE will increase the depth of the catalog with respect to the types of functional elements and cell types studied. It will also develop new tools for more sophisticated analyses of the data.

Source: National Human Genome Research Institute

Contact: Omar McCrimmon 

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ZenMaster

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Advance Understanding of Spinal Muscular Atrophy with Stem Cells

Cedars-Sinai's Regenerative Medicine Institute research sheds new light on cell death in a common, lethal genetic disease in children, suggesting paths for potential treatment
Thursday, 21 June 2012 

Cedars-Sinai's Regenerative Medicine Institute has pioneered research on how motor-neuron cell-death occurs in patients with spinal muscular atrophy, offering an important clue in identifying potential medicines to treat this leading genetic cause of death in infants and toddlers.

The study, published in the June 19 online issue of PLoS ONE, extends the institute's work to employ pluripotent stem cells to find a pharmaceutical treatment for spinal muscular atrophy or SMA, a genetic neuromuscular disease characterized by muscle atrophy and weakness.

"With this new understanding of how motor neurons die in spinal muscular atrophy patients, we are an important step closer to identifying drugs that may reverse or prevent that process," said Clive Svendsen, PhD, director of the Cedars-Sinai Regenerative Medicine Institute.

Svendsen and his team have investigated this disease for some time now. In 2009, Nature published a study by Svendsen and his colleagues detailing how skin cells taken from a patient with the disorder were used to generate neurons of the same genetic makeup and characteristics of those affected in the disorder; this created a "disease-in-a-dish" that could serve as a model for discovering new drugs.

As the disease is unique to humans, previous methods to employ this approach had been unreliable in predicting how it occurs in humans. In the research published in PLoS ONE, to the team reproduced this model with skin cells from multiple patients, taking them back in time to a pluripotent stem cell state (iPS cells), and then driving them forward to study the diseased patient-specific motor neurons.

Children born with this disorder have a genetic mutation that doesn't allow their motor neurons to manufacture a critical protein necessary for them to survive. The study found these cells die through apoptosis – the same form of cell death that occurs when the body eliminates old, unnecessary as well as unhealthy cells. As motor neuron cell death progresses, children with the disease experience increasing paralysis and eventually death. There is no effective treatment now for this disease. An estimated one in 35 to one in 60 people are carriers and about in 100,000 new-borns have the condition.

"Now we are taking these motor neurons (from multiple children with the disease and in their pluripotent state) and screening compounds that can rescue these cells and create the protein necessary for them to survive," said Dhruv Sareen, director of Cedars-Sinai's Induced Pluripotent Stem Cell Core Facility and a primary author on the study.

"This study is an important stepping stone to guide us toward the right kinds of compounds that we hope will be effective in the model – and then be reproduced in clinical trials."

Source: Cedars-Sinai Medical Center 

Contact: Nicole White

Reference:

Inhibition of Apoptosis Blocks Human Motor Neuron Cell Death in a Stem Cell Model of Spinal Muscular Atrophy

Dhruv Sareen, Allison D. Ebert, Brittany M. Heins, Jered V. McGivern, Loren Ornelas, Clive N. Svendsen

PLoS ONE, 19 Jun, 2012, 201210.1371/journal.pone

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Researchers Develop New Muscular Dystrophy Treatment Approach Using Human Stem Cells

Researchers Develop New Muscular Dystrophy Treatment Approach Using Human Stem Cells

Saturday, 05 May 2012

Researchers from the University of Minnesota's Lillehei Heart Institute have effectively treated muscular dystrophy in mice using human stem cells derived from a new process that – for the first time – makes the production of human muscle cells from stem cells efficient and effective.

The research, published today in Cell Stem Cell, outlines the strategy for the development of a rapidly dividing population of skeletal myogenic progenitor cells (muscle-forming cells) derived from induced pluripotent (iPS) cells. iPS cells have all of the potential of embryonic stem (ES) cells, but are derived by reprogramming skin cells. They can be patient-specific, which renders them unlikely to be rejected, and do not involve the destruction of embryos.

This is the first time that human stem cells have been shown to be effective in the treatment of muscular dystrophy.

According to U of M researchers – who were also the first to use ES cells from mice to treat muscular dystrophy – there has been a significant lag in translating studies using mouse stem cells into therapeutically relevant studies involving human stem cells. This lag has dramatically limited the development of cell therapies or clinical trials for human patients.

The latest research from the U of M provides the proof-of-principle for treating muscular dystrophy with human iPS cells, setting the stage for future human clinical trials.

"One of the biggest barriers to the development of cell-based therapies for neuromuscular disorders like muscular dystrophy has been obtaining sufficient muscle progenitor cells to produce a therapeutically effective response," said principal investigator Rita Perlingeiro, Ph.D., associate professor of medicine in the Medical School's Division of Cardiology.

"Up until now, deriving engraftable skeletal muscle stem cells from human pluripotent stem cells hasn't been possible. Our results demonstrate that it is indeed possible and sets the stage for the development of a clinically meaningful treatment approach."

Upon transplantation into mice suffering from muscular dystrophy, human skeletal myogenic progenitor cells provided both extensive and long-term muscle regeneration which resulted in improved muscle function.

To achieve their results, U of M researchers genetically modified two well-characterized human iPS cell lines and an existing human ES cell line with the PAX7 gene. This allowed them to regulate levels of the Pax7 protein, which is essential for the regeneration of skeletal muscle tissue after damage. The researchers found this regulation could prompt naïve ES and iPS cells to differentiate into muscle-forming cells.

Up until this point, researchers had struggled to make muscle efficiently from ES and iPS cells. PAX7 – induced at exactly the right time – helped determine the fate of human ES and iPS cells, pushing them into becoming human muscle progenitor cells.

Once Dr. Perlingeiro's team was able to pinpoint the optimal timing of differentiation, the cells were well suited to the regrowth needed to treat conditions such as muscular dystrophy. In fact, Pax7-induced muscle progenitors were far more effective than human myoblasts at improving muscle function. Myoblasts, which are cell cultures derived from adult muscle biopsies, had previously been tested in clinical trials for muscular dystrophy, however the myoblasts did not persist after transplantation.

"Seeing long-term maintenance of these cells without major adverse side effects is exciting," said Perlingeiro.

"Our research proves that these differentiated stem cells have real staying power in the fight against muscular dystrophy."

According to John Wagner, M.D., scientific director of clinical research at the University's Stem Cell Institute and renowned blood and marrow transplant expert,

"This research is a phenomenal breakthrough. Dr. Perlingeiro and her collaborators have overcome one of the most significant obstacles to moving stem cell therapies into the treatment of children with devastating and life threatening muscular dystrophies."

The U of M researchers say alternative methods of Pax7 induction will need to be investigated before this study can be turned into a human clinical trial. Their method of delivering the Pax7 protein involved genetic modification of cells with viruses and because viruses sometimes cause mutations, they add risk to a clinical trial. But the U of M researchers are committed to developing a safe and effective clinical protocol, and are actively testing alternate methods of delivering Pax7.

Source: University of Minnesota Academic Health Center 

Contact: Caroline Marin

Reference:

Human ES- and iPS-Derived Myogenic Progenitors Restore DYSTROPHIN and Improve Contractility upon Transplantation in Dystrophic Mice

Radbod Darabi, Robert W. Arpke, Stefan Irion, John T. Dimos, Marica Grskovic, Michael Kyba, Rita C.R. Perlingeiro

Cell Stem Cell, Volume 10, Issue 5, 610-619, 4 May 2012, 10.1016/j.stem.2012.02.015

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ZenMaster

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