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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ZenMaster

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Lab Creates Cells Used by Brain to Control Muscle Cells

Lab Creates Cells Used by Brain to Control Muscle Cells
Tuesday, 22 November 2011

 University of Central Florida researchers, for the first time, have used stem cells to grow neuromuscular junctions between human muscle cells and human spinal cord cells, the key connectors used by the brain to communicate and control muscles in the body.

Dr. Hickman has been working on this

project for more than a decade.

Credit: University of Central Florida.

The success at UCF is a critical step in developing "human-on-a-chip" systems. The systems are models that recreate how organs or a series of organs function in the body. Their use could accelerate medical research and drug testing, potentially delivering life-saving breakthroughs much more quickly than the typical 10-year trajectory most drugs take now to get through animal and patient trials.

"These types of systems have to be developed if you ever want to get to a human-on-a-chip that recreates human function," said James Hickman, a UCF bioengineer who led the breakthrough research.

"It's taken many trials over a number of years to get this to occur using human derived stem cells."

Hickman's work, funded through the National Institute of Neurological Disorders and Stroke (NINDS) at the National Institutes of Health, is described in the December issue of Biomaterials.

Hickman is excited about the future of his research because several federal agencies recently launched an ambitious plan to jump-start research in "human-on-a-chip" models by making available at least $140 million in grant funding.

The National Institutes of Health (NIH), the Defense Advanced Research Projects Agency (DARPA), and the Federal Drug Administration (FDA) are leading the research push.

The goal of the call for action is to produce systems that include various miniature organs connected in realistic ways to simulate human body function. This would make it possible, for instance, to test drugs on human cells well before they could safely and ethically be tested on living humans. The technique could potentially be more effective than testing in mice and other animals currently used to screen promising drug candidates and to develop other medical treatments.

Such conventional animal testing is not only slow and expensive, but often leads to failures that might be overcome with better testing options. The limitations of conventional testing options have dramatically slowed the emergence of new drugs, Hickman said.

The successful UCF technique began with a collaborator, Brown University Professor Emeritus Herman Vandenburgh, who collected muscle stem cells via biopsy from adult volunteers. Stem cells are cells that can, under the right conditions, grow into specific forms. They can be found among normal cells in adults, as well as in developing fetuses.

Nadine Guo, a UCF research professor, conducted a series of experiments and found that numerous conditions had to come together just right to make the muscle and spinal cord cells "happy" enough to join and form working junctions. This meant exploring different concentrations of cells and various timescales, among other parameters, before hitting on the right conditions.

"Right now we rely a lot on animal systems for medical research but this is a pure human system," Guo said.

"This work proved that, biologically, this is workable."

Besides being a key requirement for any complete human-on-a-chip model, such nerve-muscle junctions might themselves prove important research tools. These junctions play key roles in Amyotrophic lateral sclerosis, commonly known as Lou Gehrig's disease, in spinal cord injury, and in other debilitating or life threatening conditions. With further development, the team's techniques could be used to test new drugs or other treatments for these conditions even before more expansive chip-based models are developed.

Source: University of Central Florida

Contact: Barbara Abney
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
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Stem Cell Transplants in Mice Produce Lifelong Enhancement of Muscle Mass

Findings may have potential for future treatment of patients with chronic, degenerative muscle diseases
Thursday, 11 November 2010

A University of Colorado at Boulder-led study shows that specific types of stem cells transplanted into the leg muscles of mice prevented the loss of muscle function and mass that normally occurs with aging, a finding with potential uses in treating humans with chronic, degenerative muscle diseases.

The experiments showed that when young host mice with limb muscle injuries were injected with muscle stem cells from young donor mice, the cells not only repaired the injury within days, they caused the treated muscle to double in mass and sustain itself through the lifetime of the transplanted mice.

"This was a very exciting and unexpected result," said Professor Bradley Olwin of CU-Boulder's molecular, cellular and developmental biology department, the study's corresponding author.

Muscle stem cells are found within populations of "satellite" cells located between muscle fibres and surrounding connective tissue and are responsible for the repair and maintenance of skeletal muscles, said Olwin. The researchers transplanted between 10 and 50 stem cells along with attached myofibers – which are individual skeletal muscle cells – from the donor mice into the host mice.

"We found that the transplanted stem cells are permanently altered and reduce the aging of the transplanted muscle, maintaining strength and mass," said Olwin.

A paper on the subject was published in the Nov. 10 issue of Science Translational Medicine. Co-authors on the study included former CU-Boulder postdoctoral fellow John K. Hall, now at the University of Washington Medical School in Seattle, as well as Glen Banks and Jeffrey Chamberlain of the University of Washington Medical School.

Olwin said the new findings, while intriguing, are only the first in discovering how such research might someday be applicable to human health.

"With further research we may one day be able to greatly resist the loss of muscle mass, size and strength in humans that accompanies aging, as well as chronic degenerative diseases like muscular dystrophy."

Stem cells are distinguished by their ability to renew themselves through cell division and differentiate into specialized cell types. In healthy skeletal muscle tissue, the population of satellite stem cells is constantly maintained, said Olwin.

"In this study, the hallmarks we see with the aging of muscles just weren't occurring," said Olwin.

"The transplanted material seemed to kick the stem cells to a high gear for self-renewal, essentially taking over the production of muscle cells. But the team found that when transplanted stem cells and associated myofibers were injected to healthy mouse limb muscles, there was no discernable evidence for muscle mass growth.”

"The environment that the stem cells are injected into is very important, because when it tells the cells there is an injury, they respond in a unique way," he said.

"We don't yet know why the cells we transplanted are not responding to the environment around them in the way that the cells that are already there respond. It's fascinating, and something we need to understand."

At the onset of the experiments, the research team thought the increase in muscle mass of the transplanted mice with injured legs would dissipate within a few months. Instead, the cells underwent a 50 percent increase in mass and a 170 percent increase in size and remained elevated through the lifetime of the mice – roughly two years, said Olwin.

In the experiments, stem cells and myofibers were removed from three-month-old mice, briefly cultured and then transplanted into three-month-old mice that had temporarily induced leg muscle injuries produced by barium chloride injections.

"When the muscles were examined two years later, we found the procedure permanently changed the transplanted cells, making them resistant to the aging process in the muscle," he said.

"This suggests a tremendous expansion of those stem cells after transplantation," Olwin said. Fortunately, the research team saw no increase in tumours in the transplanted mice despite the rapid, increased growth and production of muscle stem cells.

As part of the research effort, the team used green fluorescent protein – which glows under ultraviolet light – to flag donor cells in the injected mice. The experiment indicated many of the transplanted cells were repeatedly fused to myofibers, and that there was a large increase in the number of satellite cells in the host mice.

"We expected the cells to go in, repopulate and repair damaged muscle and to dissipate," Olwin said. "It was quite surprising when they did not.”

"It is our hope that we can someday identify small molecules or combinations of small molecules that could be applied to endogenous muscle stem cells of humans to mimic the behaviour of transplanted cells," Olwin said.

"This would remove the need for cell transplants altogether, reducing the risk and complexity of treatments."

But Olwin said it is important to remember that the team did not transplant young cells into old muscles, but rather transplanted young cells into young muscles.

The research has implications for a number of human diseases, Olwin said. In muscular dystrophy, for example, there is a loss of a protein called dystrophin that causes the muscle to literally tear itself apart and cannot be repaired without cell-based intervention. Although injected cells will repair the muscle fibres, maintaining the muscle fibres requires additional cell injections, he said.

"Progressive muscle loss occurs in a number of neuromuscular diseases and in muscular dystrophies," he said.

"Augmenting a patient's muscle regenerative process could have a significant impact on aging and diseases, improving the quality of life and possibly improving mobility."

Olwin said the research team is beginning experiments to see if transplanting muscle stem cells from humans or large animals into mice will have the same effects as those observed in the recent mouse experiments.

"If those experiments produce positive results, it would suggest that transplanting human muscle stem cells is feasible," he said.

Source: University of Colorado at Boulder
Contact: Bradley Olwin
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ZenMaster

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Salamander Regeneration Trick Replicated in Mouse Muscle Cells

Salamander Regeneration Trick Replicated in Mouse Muscle Cells
Friday, 06 August 2010

Researchers in Helen Blau's lab have

found a way to help mouse cells

regenerate tissues in the same way

that newts do.

Tissue regeneration a la salamanders and newts seems like it should be the stuff of science fiction. But it happens routinely. Why can't we mammals just re-grow a limb or churn out a few new heart muscle cells as needed? New research suggests there might be a very good reason: Restricting our cells' ability to pop in and out of the cell cycle at will — a prerequisite for the cell division necessary to make new tissue — reduces the chances that they'll run amok and form potentially deadly cancers.

Now scientists at the Stanford University School of Medicine have taken a big step toward being able to confer this regenerative capacity on mammalian muscle cells; they accomplished this feat in experiments with laboratory mice in which they blocked the expression of just two tumour-suppressing proteins. The finding may move us closer to future regenerative therapies in humans — surprisingly, by sending us shimmying back down the evolutionary tree.

"Newts regenerate tissues very effectively," said Helen Blau, PhD, the Donald E. and Delia B. Baxter Professor and a member of Stanford's Institute for Stem Cell Biology and Regenerative Medicine.

"In contrast, mammals are pathetic. We can regenerate our livers, and that's about it. Until now it's been a mystery as to how they do it."

Blau is the senior author of the research, which will be published in Cell Stem Cell on Aug. 6. Kostandin Pajcini, PhD, a former graduate student, and Jason Pomerantz, MD, a former postdoctoral scholar in Blau's laboratory, are primarily responsible for the work and are first author and co-senior author, respectively.

Although there has been a lot of discussion about using adult or embryonic stem cells to repair or revitalize tissues throughout the body, in this case the researchers were not studying stem cells. Instead, they were investigating whether myocytes, run-of-the mill muscle cells that normally do not divide, can be induced to re-enter the cell cycle and begin proliferating. This is important because most specialized, or differentiated, cells in mammals are locked into a steady state that does not allow cell division. And without cell division, it is not possible to get regeneration.

In contrast, the cells of some types of amphibians are able to replace lost or damaged tissue by entering the cell cycle to give rise to more muscle cells. While doing so, the cells maintain their muscle identity, which prevents them from straying from the beaten path and becoming other, less useful cell types.

Pomerantz and Blau wondered if it could be possible to coax mammalian cells to follow a similar path. To do so, though, they needed to pinpoint what was different between mammalian and salamander cells when it comes to cell cycle control. One aspect involves a class of proteins called tumour suppressors that block inappropriate cell division.

Previous research had shown that a tumour suppressor called retinoblastoma, or Rb, plays an important role in preventing many types of specialized mammalian cells, including those found in muscle, from dividing willy-nilly. However, the effect of blocking the expression of Rb in mammalian cells has been inconsistent: In some cases it has allowed the cells to hop back into the cell cycle; in others, it hasn't.

The researchers employed some evolutionary detective work to figure out that another tumour suppressor called ARF might be involved. Like Rb, ARF works to throw the brakes on the cell cycle in response to internal signals. An examination of the evolutionary tree provided a key clue. They saw that ARF first arose in chickens. It is found in other birds and mammals, but not in animals like salamanders nestled on the lower branches. Tellingly, it's also missing in cell lines that begin cycling when Rb is lost, and it is expressed at lower-than-normal levels in mammalian livers — the only organ that we humans can regenerate.

Based on previous investigators' work with newts, Blau said it "seemed to us that they don't have the same limitations on growth. We hypothesized that maybe, during evolution, humans gained a tumour suppressor not present in lower animals at the expense of regeneration."

Sure enough, Pajcini and Pomerantz found that blocking the expression of both Rb and ARF allowed individual myocytes isolated from mouse muscle to dedifferentiate and begin dividing. When they put the cells back into the mice, they were able to merge with existing muscle fibres — as long as Rb expression was restored. Without Rb the transplanted cells proliferated excessively and disrupted the structure of the original muscle.

"These myocytes have reached the point of no return," said Blau.

"They can't just start dividing again. But here we show that temporarily blocking the expression of just two proteins can restore an ancient ability to contribute to mammalian muscle."

The key word here is "temporarily." As is clear from the mouse experiments, blocking the expression of tumour suppressors in mammalian cells can be a tricky gambit. Permanently removing these proteins can lead to uncontrolled cell division. But, a temporary and well-controlled loss — as the researchers devised here — could be a useful therapeutic tool.

The research required some sophisticated technology to separate individual myocytes from one another for study. To do so, Pajcini travelled to Munich to learn how to optimize a technique normally used on cryopreserved and fixed tissue sections — "laser micro-dissection catapulting" — for use with living cells. But the effort paid off when he was able to prove conclusively that once the expression of the two proteins was blocked, individual live cells were, in fact, dividing in culture.

Next, the researchers would like to see if the technique works in other cell types, like those of the pancreas or the heart, and whether they can induce it to happen in tissue at sites of injury. If so, it may be possible to trigger temporary cell proliferation as a means of therapy for a variety of ailments.

Source: Stanford University Medical Center
Contact: Krista Conger
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/

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Scientists Develop New Way to Grow Adult Stem Cells in Culture

Scientists Develop New Way to Grow Adult Stem Cells in Culture Sunday, 18 July 2010

Researchers at the Stanford University Medical Center have developed a technique they believe will help scientists overcome a major hurdle to the use of adult stem cells for treating muscular dystrophy and other muscle-wasting disorders that accompany aging or disease. They've found that growing muscle stem cells on a specially developed synthetic matrix that mimics the elasticity of real muscle allows them to maintain their self-renewing properties.

"Cells don't normally exist in contact with a rigid cell culture dish," said Helen Blau, PhD, the Donald E. and Delia B. Baxter Professor and member of Stanford's Institute for Stem Cell Biology and Regenerative Medicine.

"They sit on soft tissue. By mimicking this environment we can really influence their function and allow them to self-renew in ways we've never been able to achieve before."

Adult stem cells already exist in the body, and are important in regenerating tissues like blood, muscles and neurons in the brain. However, scientists have struggled to produce them in quantities needed for therapies because the cells differentiate and lose their "stemness" as soon as they are placed in a tissue culture dish. This new method of growing the cells creates a way to study the behaviour of many types of adult stem cells in culture and may revolutionize the ability to produce these cells for future therapies, say the researchers.

Blau is the senior author of the research, which will be published online July 15 in Science Express. Postdoctoral scholar Penney Gilbert, PhD, and graduate student Karen Havenstrite share first authorship of the work.

Self-renewal, or the ability to become both another stem cell and a differentiating daughter cell, is a defining trait of stem cells. This ability is necessary for a small number of cells to, for example, fully reconstitute the pantheon of blood cell types necessary to regenerate a patient's immune system after chemotherapy or to successfully contribute to the long-term generation of new, healthy muscle tissue. Until now, however, all attempts to grow these and some other adult stem cells, like blood stem cells, in culture have resulted in the cells differentiating into more specialized — but less therapeutically useful — progenitor cells. This differentiation constitutes a major obstacle to treating muscle-wasting diseases, for using cord blood or for treating blood cancers.

The researchers wondered if the way the cells are normally grown in culture could be the problem. After all, as Blau pointed out, cells are used to rubbing shoulders comfortably with their neighbours on all sides rather than being splayed out and anchored on a rigid plastic culture dish that is 100,000-fold less elastic than true muscle.

To find out if the cells would be happier on a softer, more giving surface, they had to develop an entirely new culture system. They turned to a material called hydrogel, which is made up of a latticework of polyethylene glycol polymers filled with water. Decreasing the percentage of polymer molecules in the mix makes the resulting matrix more elastic and wobbly; increasing it makes it more dense and rigid.

Hydrogel is often used as scaffolding to grow cells in two- and three-dimensional arrays useful in tissue engineering. But because it can swell over time, it was not possible to accurately calibrate the amount of proteins and other components needed to maintain the cells in this type of experiment. Gilbert and Havenstrite tinkered with the system until they came up with a version that maintains a constant volume, making it possible to test the effects of gels of different elasticity that all contained the same amount of protein. They then patterned the gel into hundreds of tiny wells and added one freshly isolated muscle stem cell per well.

After letting the cells grow for one week, the researchers found that the softer, or more pliant, gels mimicking the elasticity of muscle tissue had many more cells than the less elastic gels. Closer investigation using an algorithm they developed for automated cell tracking showed that it was not because the cells were dividing more quickly, but because not as many were dying during the culture period. The computer program, which they have called the Baxter Algorithm to honour the Baxter Foundation that funded this portion of the work, reduced the time needed to analyze the cell division data by more than 90 percent.

"This in itself is a huge advance," Blau said of the software.

"Until now it's been pretty impossible to do these studies without spending half a year or more manually scoring pictures or movies of cells in culture. Now we can figure out exactly how the cells divide and move, who begets who. As a result we can begin to study all types of variables."

After studying the dynamics of the muscle stem cells' division and dying, the researchers began to study specific aspects of their biology. They found that the cells grown on the softer surface were less likely than those grown on the harder surfaces to express a gene associated with differentiation called myogenin. They were also as able as freshly isolated muscle stem cells to contribute to the development of leg muscles when transplanted into recipient mice.

"Testing their function in animals like this is extremely important," said Blau.

"It's really the only way to confirm their 'stemness.'"

To prove definitively that the stem cells were self-renewing, Gilbert and Blau turned to a "doublet" experiment. In this test, Gilbert allowed just one cell to divide just one time, resulting in two daughter cells. There are three potential combinations for these resulting doublets: two stem cells, one stem cell and one progenitor cell, or two progenitor cells. The first two represent self-renewal; the last does not. (A progenitor cell is one that can go on to differentiate into more specialized cells.)

Gilbert found that one-third of cells grown on the muscle-mimicking substrate expressed a muscle-stem-cell-specific gene (indicating that at least one of the two cells was a stem cell) but that only 6 percent of those grown on the plastic surface did so. Furthermore, when Gilbert transplanted five doublets (for a total of 10 cells) into mice from the muscle-mimicking substrate, the cells made themselves at home and began to contribute to muscle fibre development in three of 12 recipient animals. When she repeated the experiment with doublets grown on hard surfaces, none of the animals demonstrated similar engraftment.

"Clearly the cells grown on the more-elastic surfaces have better survival and self-renewing properties than those grown on standard tissue culture dishes," said Blau.

"We conducted our experiments with muscle stem cells, but I expect this will be true for other types of adult stem cells as well."

In addition to exploring this possibility in the future, the researchers will also investigate how their findings may help advance therapies for conditions like muscular dystrophy.

"Researchers really had no way to grow these cells in the laboratory before," said Blau.

"These findings may allow us one day to replenish the muscles of patients with muscular dystrophy and other muscle-wasting diseases with healthy stem cells."

Source: Stanford University Medical Center
Contact: Krista Conger
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/

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Improving Clinical Use of Stem Cells to Repair Heart Damage

Improving Clinical Use of Stem Cells to Repair Heart Damage
Wednesday, 14 July 2010

Presenting at the UK National Stem Cell Network annual science conference today (13 July), Professor Michael Schneider describes a new approach to treating heart attack and cardiomyopathy using stem cells.

Professor Schneider, British Heart Foundation Professor at Imperial College London, said:

"Recent clinical trials using stem cells to treat heart damage have been successful in terms of safety but unfortunately the bone marrow stem cells used tend to give only a small improvement in how well the heart is pumping.”

"We really want to use stem cells from the patients themselves that we know can give rise to beating heart cells and these are not found in bone marrow. The good news is that we're now finding ways to identify and purify such cells."

Around 1000 patients have been treated in approximately 20 trials worldwide, mostly using bone marrow stem cells or derivatives of bone marrow cells to repair damage caused by heart attack. There has also been a significant body of work looking at ways of producing beating heart cells from stem cells. The best proven approaches to creating new beating heart cells are using embryonic stem cells, induced pluripotent cells and heart-derived stem cells.

Professor Schneider continued:

"Using heart-derived stem cells to treat heart attack and cardiomyopathy has some advantages over embryonic and induced pluripotent cells as they are potentially safer. It's also notable that of these three cell types, it's only heart-derived cells that are in current human clinical trials for this sort of treatment.”

"The biggest challenge is to make an ideal product for transplant, which would be either a mixture of heart muscle- and blood vessel-forming cells or a pure population of some sort of precursor that could give rise to both muscle and blood vessel cells."

Professor Schneider's team have discovered a way to identify heart stem cells to purify them for transplant. They first developed the method in mice and although the identifying markers are quite different in human cells, they have been able to successfully map their knowledge from mice onto humans.

Professor Schneider said:

"We've developed a method to identify cells that have three important characteristics: They are definitely stem cells; they have the right molecular machinery turned on in order to become heart muscle or blood vessel; and they don't yet have any of the full characteristics of heart muscle or blood vessel cells such as producing cardiac myosin - an important protein in heart muscle cells."

The next stage of the research is to develop this technique into a method for extracting, purifying and multiplying heart stem cells in the clinic to be used to repair heart damage arising from heart attack or cardiomyopathy. Professor Schneider's laboratory uses advanced robotics, automated microscopy and other high-throughput methods to screen many thousands of experimental conditions in order to devise the best ways to grow the cells and instruct them to go down the route of becoming heart muscle.

The British Heart Foundation, the European Research Council, the European Union (through the EU FP7 CardioCell consortium), the Leducq Foundation and the Medical Research Council funded the research.

Source: Biotechnology and Biological Sciences Research Council
Contact: Nancy Mendoza
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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at
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MyoD Helps Stem Cells Proliferate in Response to Muscle Injury

MyoD Helps Stem Cells Proliferate in Response to Muscle Injury Monday, 04 January 2010 The master regulator of muscle differentiation, MyoD, functions early in myogenesis to help stem cells proliferate in response to muscle injury, according to researchers at Case Western Reserve University. The study appears online Jan. 4 in the Journal of Cell Biology. MyoD is a transcription factor that activates muscle-specific genes as myoblast precursors differentiate and fuse to form mature muscle fibres. But MyoD is also expressed at an earlier stage of myogenesis when quiescent stem cells rapidly expand in number to generate the myoblasts needed to repair tissue damage. The transcription factor's function in this proliferative phase is unknown. The team found that MyoD bound to the promoter of CDC6, a gene that initiates DNA replication, suggesting that MyoD might activate Cdc6 expression in muscle stem cells to promote their re-entry into the cell cycle and rapid proliferation. Indeed, Cdc6 was expressed shortly after MyoD in stimulated muscle progenitors, and knocking down MyoD reduced Cdc6 production and slowed cells' entry into S phase. MyoD works in conjunction with transcription factors from the E2F family. E2F3a activated the CDC6 promoter with MyoD, but was replaced by the repressive family member E2F4 as myoblasts began to differentiate. Senior author Nikki Harter now wants to investigate how the transcription factors cooperate to control Cdc6 expression — initial results suggest that MyoD recruits E2F3a to the promoter region. The researchers also propose that a related protein, Myf5, might control Cdc6 transcription in MyoD's absence, acting as a backup mechanism to ensure that muscle stem cells expand to repair tissue damage. ......... ZenMaster

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New Strategy for Mending Broken Hearts?

Patch created to repair damaged heart tissue Wednesday, 14 October 2009 By mimicking the way embryonic stem cells develop into heart muscle in a lab, Duke University bioengineers believe they have taken an important first step toward growing a living "heart patch" to repair heart tissue damaged by disease. In a series of experiments using mouse embryonic stem cells, the bioengineers used a novel mould of their own design to fashion a three-dimensional "patch" made up of heart muscle cells, known as cardiomyocytes. The new tissue exhibited the two most important attributes of heart muscle cells – the ability to contract and to conduct electrical impulses. The mould looks much like a piece of Chex cereal in which researchers varied the shape and length of the pores to control the direction and orientation of the growing cells.

The researchers grew the cells in an environment much like that found in natural tissues. They encapsulated the cells within a gel composed of the blood-clotting protein fibrin, which provided mechanical support to the cells, allowing them to form a three-dimensional structure. They also found that the cardiomyocytes flourished only in the presence of a class of "helper" cells known as cardiac fibroblasts, which comprise as much as 60 percent of all cells present in a human heart. "If you tried to grow cardiomyocytes alone, they develop into an unorganized ball of cells," said Brian Liau, graduate student in biomedical engineering at Duke's Pratt School of Engineering. Liau, who works in the laboratory of assistant professor Nenad Bursac, presented the results of his latest experiments during the annual scientific sessions of the Biomedical Engineering Society in Pittsburgh. "We found that adding cardiac fibroblasts to the growing cardiomyocytes created a nourishing environment that stimulated the cells to grow as if they were in a developing heart," Liau said. "When we tested the patch, we found that because the cells aligned themselves in the same direction, they were able to contract like native cells. They were also able to carry the electrical signals that make cardiomyocytes function in a coordinated fashion."

"The addition of fibroblasts in our experiments provided signals that we believe are present in a developing embryo," Liau said. The need for helper cells is not uncommon in mammalian development. For example, he explained, nerve cells need "sheathe" cells known as glia in order to develop and function properly. Bursac believes that the latest experiments represent a proof-of-principle advance, but said there are still many hurdles to overcome before such patches could be implanted into humans with heart disease. "While we were able to grow heart muscle cells that were able to contract with strength and carry electric impulses quickly, there are many other factors that need to be considered," Bursac said. "The use of fibrin as a structural material allowed us to grow thicker, three-dimensional patches, which would be essential for the delivery of therapeutic doses of cells. One of the major challenges then would be establishing a blood vessel supply to sustain the patch." The researchers plan to test their model using non-embryonic stem cells. For use in humans, this is important for many reasons, both scientifically and ethically, Bursac said. Recent studies have demonstrated that some cells from human adults have the ability to be reprogrammed to become similar to embryonic stem cells. "Human cardiomyocytes tend to grow a lot slower than those of mice," Bursac said. "Since it takes nine months for the human heart to complete development, we need to find a way to get the cells to grow faster while maintaining the same essential properties of native cells." If they could use a patient's own cells, the patch would also evade an immune system reaction, Bursac added. .........

ZenMaster

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Age Affects Function of Embryonic Muscle Stem Cell Genes

Adult satellite cells have distinct genetic requirements Thursday, 25 June 2009 Scientists working at the Carnegie Institution's Department of Embryology, with colleagues, have overturned previous research that identified critical genes for making muscle stem cells. It turns out that the genes that make muscle stem cells in the embryo are surprisingly not needed in adult muscle stem cells to regenerate muscles after injury. The finding challenges the current course of research into muscular dystrophy, muscle injury, and regenerative medicine, which uses stem cells for healing tissues, and it favours using age-matched stem cells for therapy. The study is published in the June 25 advance on-line edition of Nature. Previous studies have shown that two genes Pax3 and Pax7, are essential for making the embryonic and neonatal muscle stem cells in the mouse. Lead researcher Christoph Lepper, a predoctoral fellow in Carnegie's Chen-Ming Fan's lab and a Johns Hopkins student, for the first time looked at these two genes in promoting stem cells at varying stages of muscle growth in live mice after birth. As Christoph explained: "The paired-box genes, Pax3 and Pax7 are involved in the development of the skeletal muscles. It is well established that both genes are needed to produce muscle stem cells in the embryo. A previous student, Alice Chen, studied how these genes are turned on in embryonic muscle stem cells (also published in Nature). I thought that if they are so important in the embryo, they must be important for adult muscle stem cells. Using genetic tricks, I was able to suppress both genes in the adult muscle stem cells. I was totally surprised to find that the muscle stem cells are normal without them."

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This cross section of hind limb muscle tissue is from a mouse five days after injury. The uninjured cells are at top and stained red. The blue cells below are regenerating muscles cells. They were labelled with a blue stain and formed from muscle stem cells. Credit: Christoph Lepper.

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The researchers then looked at whether the same was true upon injury, after which the repair process requires muscle stem cells to make new muscles. For this, they injured the leg muscles between the knee and ankle. They were again surprised that these muscle stem cells, without the two key embryonic muscle stem cell genes, could generate muscles as well as normal muscle stem cells. They even performed a second round of injury and found that the stem cells were still active. The scientists then wondered when these genes become unnecessary for muscle stem cells to regenerate muscles. It turned out that these embryonic genes are important to muscle stem cell creation up to the first three weeks after birth. What makes the muscle stem cells different after three weeks? The scientist believe that these two embryonic muscle stem cell genes also tell the stem cells to become quiet as the organism matures. After that time is reached, they "hand over" their jobs to a different set of genes. The researchers suggest that since the adult muscle stem cells are only activated when injury occurs (by trauma or exercise), they use a new set of genes from those used during embryonic development, which proceeds without injury. The scientists are eager to find these adult muscle stem cell genes. "We are just beginning to learn the basics of stem cell biology, and there are many surprises," remarked Allan Spradling, director of Carnegie's Department of Embryology. "This work illustrates the importance of carrying out basic research using animal models before rushing into the clinic with half-baked therapies." Reference: Adult satellite cells and embryonic muscle progenitors have distinct genetic requirements Christoph Lepper, Simon J. Conway & Chen-Ming Fan Nature advance online publication 25 June 2009, doi:10.1038/nature08209 ......... ZenMaster

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Stem Cells That Repair Injured Muscles

Research effort may have important implications for muscular dystrophy Saturday, 07 March 2009 A University of Colorado at Boulder research team has identified a type of skeletal muscle stem cell that contributes to the repair of damaged muscles in mice, which could have important implications in the treatment of injured, diseased or aging muscle tissue in humans, including the ravages of muscular dystrophy. The newly identified stem cells are found within populations of satellite cells located between muscle fibres and the surrounding connective tissue that are responsible for the repair and maintenance of skeletal muscles, said Professor Bradley Olwin of CU-Boulder's molecular, cellular and developmental biology department. When muscle fibres are stressed or traumatized, satellite cells divide to make more specialized muscle cells and repair the muscle, said Olwin. The stem cell population identified by the CU team within the satellite cells – dubbed "satellite-SP" cells – were shown to renew the satellite cell population after injection into injured muscle cells, contributing to recovery of muscle tissue in the laboratory mice. "This research shows how satellite cells can maintain their populations within injured tissues," said Olwin. "The hope is this new method will allow us to repair damaged or diseased skeletal muscle tissue." A paper on the subject was published in the March 5 issue of the journal Cell Stem Cell. Co-authors on the study included the MCD biology department's Kathleen Tanaka, John Hall and Andrew Troy, as well as Dawn Cornelison from the University of Missouri and Susan Majka from the University of Colorado Denver. Stem cells are distinguished by their ability to renew themselves through cell division and differentiate into specialized cell types. In healthy skeletal muscle tissue, the population of satellite cells is constantly maintained, leading the CU-Boulder team to believe that at least some of the satellite cell population in the mouse study included stem cells. For the study, the researchers injected 2,500 satellite-SP cells into a population of satellite cells within injured mouse muscle tissue. They found that 75 percent of the satellite cells that reproduced were derived from the previous satellite-SP cells injected into the tissue. The results demonstrated the injected satellite-SP cells were renewing the satellite cell pool, Olwin said. "The key point here is we are not just repairing the tissue," said Olwin. "We injected a permanent, self-renewing population of stem cells. One advantage of using this technology is that we can use a relatively small number of stem cells and do the job with a small number of injections – in this case, only one." The research has implications for a number of human diseases, he said. In muscular dystrophy, the loss of a protein called dystrophin causes the muscle to literally tear itself apart, a process that cannot be repaired without cell-based intervention. Although injected cells will repair the muscle fibres, maintaining the muscle fibres requires additional cell injections. Reference: Syndecan-4-Expressing Muscle Progenitor Cells in the SP Engraft as Satellite Cells during Muscle Regeneration Kathleen Kelly Tanaka, John K. Hall, Andrew A. Troy, D.D.W. Cornelison, Susan M. MajkaandBradley B. Olwin Cell Stem Cell, Volume 4, Issue 3, 217-225, 6 March 2009, doi:10.1016/j.stem.2009.01.016 ......... ZenMaster

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Single Adult Muscle Stem Cell Can Self Renew

Single Adult Muscle Stem Cell Can Self Renew Monday, 15 December 2008 The first demonstration that a single adult stem cell can self-renew in a mammal was reported at the American Society for Cell Biology (ASCB) 48th Annual Meeting, Dec. 13-17, 2008 in San Francisco. The transplanted adult stem cell and its differentiated descendants restored lost function to mice with hind limb muscle tissue damage. The adult stem cells used in the study, conducted at Stanford University, were isolated from a mixed population of satellite cells in the skeletal muscle of mice. The skeletal adult muscle stem cells (MusSC), which live just under the membrane that surrounds muscle fibres, normally respond to tissue damage by giving rise to progenitor cells that become myoblasts, fusing into myofibers to repair the tissue damage. The scientists transplanted the MusSC into special immune-suppressed "nude" mice whose muscle satellite cells had been wiped out in a hind limb by irradiation. The mice would only be able to repair injury if the transplanted MuSC "took." The scientists, Alessandra Sacco and Helen Blau, had genetically engineered the transplanted MusSC to express Pax7 and luciferase proteins. As a result, every transplanted cell glowed under ultraviolet light and was easy to trace. "To be able to detect the presence of the cells by bioluminescence was really a breakthrough," says Blau. "It taught us so much more. We could see how the cells were responding, and really monitor their dynamics." Through luminescent imaging as well as quantitative and kinetic analyses, Sacco and Blau tracked each transplanted stem cell as it rapidly proliferated and engrafted its progeny into the irradiated muscle tissue. The scientists then injured the regenerated tissue, setting off massive waves of muscle cell growth and repair, and subsequently showed that the MuSC and descendents rescued the second animal's lost muscle healing function. After isolating the luciferase-glowing muscle stem cells from the transplanted animal, the scientists duplicated, or cloned, the cells in the lab. Like the original MuSC, the cloned copies were intact and capable of self-renewal. "We are thrilled with the results," says Sacco. "It's been known that these satellite cells are crucial for the regeneration of muscle tissue, but this is the first demonstration of self-renewal of a single cell." The ability to isolate and then transplant skeletal adult muscle stems cells could have a wide impact in treating not only a variety of muscle wasting diseases such as muscular dystrophy but also severe muscle injuries or loss of function from aging and disuse. In other experiments, the researchers transplanted between 10 and 500 luciferase-tagged MuSC into the leg muscles of mice. These cells also proliferated and engrafted, forming new myofibers and fusing with injured fibres. Unlike tumour cells, the transplanted stem cells achieved homeostasis, growing to a stable, constant level and ceasing replication. After demonstrating that the transplanted stem cells proliferated and fully restored the animal's lost function, Sacco and Blau recovered new stem cells from the transplant with full stem cell potency, meeting the final "gold standard" test for adult multipotent stem cells. ......... ZenMaster

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Heart Regenerates after Infarction

First trials with mice Monday, 15 December 2008 Up until today scientists assumed that the adult heart is unable to regenerate. Now, researchers and cardiologists from the Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch and the Charité – Universitätsmedizin Berlin (Germany) have been able to show that this dogma no longer holds true. Dr. Laura Zelarayán and Assistant Professor Dr. Martin W. Bergmann were able to show that the body’s own heart muscle stem cells do generate new tissue and improve the pumping function of the heart considerably in an adult organism, when they suppress the activity of a gene regulator known as beta-catenin in the nucleus of the heart cells. The gene regulator beta-catenin plays an important role in the development of the heart in embryos. Dr. Zelarayán and Dr. Bergmann could now show that beta-catenin is also important for the regeneration of the adult heart. They suppressed this factor in the nucleus of the heart cells in mice. This way they activated heart precursor cells (stem cells) to turn on the regeneration of heart in adult mice. Four weeks after blocking beta-catenin, the pumping function of the heart of the animals had improved and the mice survived an infarction much better than those animals with a functioning beta-catenin gene. An important contribution to this project has been a transgenic mouse line generated by Professor Walter Birchmeier`s (MDC) laboratory. Markers identified for Heart Muscle Stem Cells In addition, the researchers have proven that heart muscle stem cells exist. So far, these cells had not been characterized clearly. They could demonstrate that two markers for heart cells – the structural protein alpha myosin heavy chain and the transcription factor Tbx5 - are also expressed on heart precursor cells. "The evidence of cells with these markers in the adult heart demonstrates that stem cells dating back from heart development survive in niches in the adult heart", Dr. Bergmann explains. The researchers in Germany collaborated with scientists in the Netherlands and Belgium. For this research, Dr. Bergmann was awarded the Wilhelm P. Wintersteinpreis this summer. The research group of Dr. Bergmann, a guest researcher at the MDC who recently became Deputy of the Department of Cardiology at the Asklepios Clinic St. Georg in Hamburg, belongs to the research group of Professor Rainer Dietz (MDC and Charité). Reference: Beta-catenin downregulation attenuates ischemic cardiac remodeling through enhanced resident precursor cell differentiation Laura Zelarayán, Claudia Noack, Belaid Sekkali, Jana Kmecova, Christina Gehrke, Anke Renger, Maria-Patapia Zafiriou, Roel van der Nagel, Rainer Dietz, Leon J. de Windt, Jean-Luc Balligand and Martin W. Bergmann PNAS, online December 10, 2008, doi: 10.1073/pnas.0808393105 ......... ZenMaster

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Neurons and Muscle Cells Need Stabilizing Force for Effective Communication

Neurons and Muscle Cells Need Stabilizing Force for Effective Communication Thursday, 09 October 2008 You cannot raise a finger without your brain directing muscle cells, and scientists have figured out another reason that usually works so well. A neuron sends a message, or neurotransmitter, to the muscle cell to tell it what to do. To get the message, the receiving cell must have a receptor. Oddly, the unstable protein rapsyn is responsible for anchoring the receptor so it is properly positioned to catch the message. Medical College of Georgia scientists have found what keeps rapsyn in proper conformation. It is a heat shock protein, one of a large family of molecular chaperones that make sure proteins get where they are needed and do what they should, says Dr. Lin Mei, chief of developmental neurobiology at MCG and Georgia Research Alliance Eminent Scholar in Neuroscience. Hsp90β helps stabilize rapsyn so receptors can get and stay where needed, according to research published in the Oct. 9 issue of Neuron. Dr. Mei suspects that other hsp siblings have a similar caretaker role in neuron-to-neuron communication in the brain. Scientists knew rapsyn's role in getting neuromuscular receptors to aggregate and stay where needed, but they didn't know what stabilized it. "It makes you wonder how to control this naughty boy which is very important," says Dr. Mei, the study's corresponding author. They found hsp90β wherever rapsyn clustered in muscle cells. When they disrupted its activity or expression, they realized hsp90β's stabilizing role in forming and maintaining receptor clusters, says Dr. Shiwen Luo, postdoctoral fellow in Dr. Mei's lab and the study's first author. Rapsyn and the receptor apparently interact, and then hsp90β comes along to help stabilize the relationship. Rapsyn mutations have been implicated in muscular dystrophies including congenital myasthenia gravis. MCG researchers are looking now to see if a mutated rapsyn still interacts with hsp90β. They used a type of acetylcholine nicotinic receptor at the neuromuscular junction as a model for their studies of brain development and communication. The junction is 1,000 times larger than connections, or synapses, between two neurons but structurally similar. Fundamentals include presynaptic terminals that release neurotransmitters picked up by receptors on the postsynaptic side. Terminals and receptors must be lined up well, whether it's a muscle cell or neuron getting the message. "In central nervous system synapses and at the neuromuscular junction, receptors have to be concentrated at the right spot to receive the neurotransmitter released," says Dr. Mei. If receptors are in the wrong place, the message can be weak or even lost. At the neuromuscular juncture, communication is usually straightforward, with primarily one neurotransmitter and one principal receptor. "Whenever you tell a muscle to move, it moves. If you want your muscles to think, you wouldn't be able to pick up a pin," says Dr. Mei. In the brain, where neurons have thousands of synapses, it is more of a negotiation. "Signals have to be integrated in the neuron for it to decide what to do." ......... ZenMaster

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Stem Cells Restore Muscle in Muscular Dystrophy

Muscle stem cell transplant boosts diseased muscle function and replenishes stem cell pool Friday, 11 July 2008 Researchers at the Joslin Diabetes Center have demonstrated for the first time that transplanted muscle stem cells can both improve muscle function in animals with a form of muscular dystrophy and replenish the stem cell population for use in the repair of future muscle injuries. By injecting purified stem cells isolated from adult skeletal muscle, researchers have shown they can restore healthy muscle and improve muscle function in mice with a form of muscular dystrophy. Those muscle-building stem cells were derived from a larger pool of so-called satellite cells that normally associate with mature muscle fibers and play a role in muscle growth and repair. In addition to their contributions to mature muscle, the injected cells also replenished the pool of regenerative cells normally found in muscle. Those stem cells allowed the treated muscle to undergo subsequent rounds of injury repair, they found. "I'm very excited about this," said lead author Amy J. Wagers, Ph.D., Principal Investigator in the Joslin Section on Developmental and Stem Cell Biology, principal faculty member at the Harvard Stem Cell Institute and Assistant Professor of Stem Cell and Regenerative Biology at Harvard University. "This study indicates the presence of renewing muscle stem cells in adult skeletal muscle and demonstrates the potential benefit of stem cell therapy for the treatment of muscle degenerative diseases such as muscular dystrophy." "Our work shows proof-of-concept that purified muscle stem cells can be used in therapy," said Wagers, noting that in some cases the stem cells replaced more than 90 percent of the muscle fibers. Such an advance would require isolation of stem cells equivalent to those in the mouse from human muscle, something Wagers said her team is now working on. The study was designed to test the concept that skeletal muscle precursor cells could function as adult stem cells and that transplantation of these cells could both repair muscle tissue and regenerate the stem cell pool in a model of Duchenne muscular dystrophy, she said. Duchenne muscular dystrophy is the most common form of the disease and is characterized by rapidly progressing muscle degeneration. The disease is caused by a genetic mutation and there is currently no cure. The data from this new study demonstrate that regenerative muscle stem cells can be distinguished from other cells in the muscle by unique protein markers present on their surfaces. The authors used these markers to select stem cells from normal adult muscle and transferred the cells to diseased muscle of mice carrying a mutation in the same gene affected in human Duchenne muscular dystrophy. Satellite cells were first described decades ago and have since generally been considered as a homogeneous group, Wagers said. While anatomically they look similar under a microscope, they nonetheless show considerable variation in their physiology and function. In a previous study, Wagers' identified a set of five markers that characterize the only subset of satellite cells responsible for forming muscle, which they also refer to as skeletal muscle precursors or SMPs. In the new study, the researchers analyzed the stem cell and regenerative properties of those SMPs. When engrafted into muscle of mice lacking dystrophin, purified SMPs contributed to up to 94 percent of muscle fibers, restoring dystrophin expression and significantly improving muscle structure and contractile function, they report. (The dystrophin gene encodes a protein important for muscle integrity. Mice lacking dystrophin, also known as mdx mice, are a model for Duchenne muscular dystrophy, the most prevalent form of muscular dystrophy.) "Importantly, high-level engraftment of transplanted SMPs in mdx animals shows therapeutic value — restoring defective dystrophin gene expression, improving muscle histology, and rescuing physiological muscle function," the researchers said. "Moreover, in addition to generating mature muscle fibers, transplanted SMPs also re-seed the satellite cell niche and are maintained there such that they can be recruited to participate in future rounds of muscle regeneration.” "Taken together, these data indicate that SMPs act as renewable, transplantable stem cells for adult skeletal muscle. The level of myofiber reconstitution achieved by these myogenic stem cells exceeds that reported for most other myogenic cell populations and leads to a striking improvement of muscle contraction function in SMP-treated muscles. These data thus provide direct evidence that prospectively isolatable, lineage-specific skeletal muscle stem cells provide a robust source of muscle replacement cells and a viable therapeutic option for the treatment of muscle degenerative disorders." Wagers noted however that there may be complications in the delivery of cell therapy in humans, particularly for those with conditions influencing skeletal muscle throughout the body. Even so, the new findings present an "opportunity to understand what happens [to these regenerative cells] in disease and identify factors and pathways that may boost their activity," she said. "We may get a handle on drugs that could target muscle impairment" not only in those with muscular dystrophies, but also in elderly people suffering from the muscle wasting that comes with age. "Once the healthy stem cells were transplanted into the muscles of the mice with muscular dystrophy, they generated cells that incorporated into the diseased muscle and substantially improved the ability of the treated muscles to contract," said Wagers. "At the same time, the transplantation of the healthy stem cells replenished the formerly diseased stem cell pool, providing a reservoir of healthy stem cells that could be re-activated to repair the muscle again during a second injury." According to the paper, these cells provide an effective source of immediately available muscle regenerative cells as well as a reserve pool that can maintain muscle regenerative activity in response to future challenges. "This work demonstrates, in concept, that stem cell therapy could be beneficial for degenerative muscle diseases," Wagers said. Wagers also said the study will lead to other studies in the near-term that will identify pathways that regulate these muscle stem cells in order to figure out ways to boost the normal regenerative potential of these cells. These could include drug therapies or genomic approaches, she said. In the long-term, the idea will be to replicate these findings in humans. "This is still very basic science, but I think we're going to be able to move forward in a lot of directions. It opens up many exciting avenues," she said. The Wagers Lab at Joslin studies both hematopoietic stem cells, which constantly maintain and can fully regenerate the entire blood system, as well as skeletal muscle stem cells, involved in skeletal muscle growth and repair. The work is aimed particularly at defining novel mechanisms that regulate the migration, expansion, and regenerative potential of these two distinct adult stem cells. About Joslin Diabetes Center Joslin Diabetes Center is the world's largest diabetes clinic, diabetes research center and provider of diabetes education. Joslin is dedicated to ensuring people with diabetes live long, healthy lives and offers real hope and progress toward diabetes prevention and a cure for the disease. Founded in 1898 by Elliott P. Joslin, M.D., Joslin is an independent non-profit institution affiliated with Harvard Medical School. Reference: Highly Efficient, Functional Engraftment of Skeletal Muscle Stem Cells in Dystrophic Muscles Massimiliano Cerletti, Sara Jurga, Carol A. Witczak, Michael F. Hirshman, Jennifer L. Shadrach, Laurie J. Goodyear, and Amy J. Wagers Cell, Vol 134, 37-47, 11 July 2008 ......... ZenMaster

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New Source of Heart Stem Cells Discovered

Finding offers hope of recapitulating developmental events to regenerate tissue Sunday, 22 June 2008 Researchers at Children's Hospital Boston are continuing to document the heart's earliest origins. Now, they have pinpointed a new, previously unrecognized group of stem cells that give rise to cardiomyocytes, or heart muscle cells. These stem cells, located in the surface of the heart, or epicardium, advance the hope of being able to regenerate injured heart tissue. This finding, published online by the journal Nature on June 22, comes on the heels of parallel cardiac stem cell discoveries in 2006, at both Children's and Massachusetts General Hospital. Then, the Children's team found that a specific stem cell or progenitor, marked by expression of a gene called Nkx2-5, forms many components of the heart: heart muscle cells, vascular smooth muscle cells, and the endothelial cells lining blood vessels in the heart's left-sided chambers. The team at MGH found a related progenitor, marked by expression of the Isl1 gene, which produces these same cell-types in the right-sided heart chambers. Now, researchers at Children's have shown that heart muscle cells can also be derived from a third type of cardiac progenitor, located within the epicardium and identifiable through its expression of a gene called Wt1. "There's a lot of interest in finding places to obtain new cardiomyocytes, because in heart failure, you lose cardiomyocytes, so the only way to reverse heart failure is to make more of these cells," said William Pu, MD, a paediatric cardiologist at Children's who was the study's senior investigator. Although epicardial cells are known to give rise to smooth muscle and endothelial cells during coronary vessel formation, nobody previously thought that epicardial cells might turn into cardiomyocytes. "I couldn't believe it at first, myself," said Bin Zhou, MD, a research fellow in Pu's laboratory and the study's first author. The results were independently corroborated by researchers from the University of California, San Diego. Using a different genetic marker, Tbx18, the UCSD team also showed that cardiomyocytes can be derived from the epicardium, and their study will be published in the same issue of Nature. Pu and Zhou showed that a specific population of cells in the epicardium, marked by Wt1 expression, not only differentiated into cardiomyocytes, but also smooth muscle cells, endothelial cells and fibroblasts (found in connective tissue). "If you're going to regenerate a tissue, you need to regenerate the whole tissue, not just the cardiomyocytes," said Pu. "This progenitor population contains all the potential to regenerate multiple tissue types within the heart." In recent years, the scientific literature has described many progenitors for cardiomyocytes, Pu added, but the markers used frequently did not play a direct role in heart development. For example, Sca-1 and c-Kit are markers that most stem cells express throughout the body, with no cardiac or developmental specificity. "I think our best chance of getting a cell to do what we want is to modify what it was designed to do," Pu elaborated. "Some of these other progenitors were isolated in the adult heart, but we don't know what they do in the normal heart, and what they're related to in the embryo. However, we clearly know what progenitors expressing Wt1, Nkx2-5, and Isl1 do in the foetus: they can make fibroblasts, blood vessels, and cardiomyocytes. Therefore we think we have a good shot, in the adult heart, of recapitulating these events." Pu considers his and Zhou's discovery to be a fortunate accident. They were trying, instead, to study a different gene, GATA4, by deleting it in the epicardium. "The tool we created for that experiment irreversibly marks the cells involved, so you can see where their descendants are headed in normal development," Pu explained. "Unexpectedly, we saw that these epicardial cells were becoming cardiomyocytes — it was a lucky observation."

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Pu and Zhou tagged the Wt-1 expressing epicardial cells with a fluorescent red protein, and then allowed the cells to differentiate. The image shows a descendent cardiomyocyte (green) that carries the same red marker, and another cell that arose from different origins. (The blue stain indicates cell nuclei). Credit: Bin Zhou, MD (Children's Hospital Boston).

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Using an enzyme called Cre recombinase, Pu and Zhou labelled epicardial cells in live mouse embryos with red fluorescent protein (RFP). Each time the Wt1 gene in these cells was activated, RFP lit up. Since the marker is inherited by descendants of the Wt1-expressing cell, the researchers could identify these descendants by looking for RFP. "If the marker shows up in a cardiomyocyte, then I know that cardiomyocyte came from the Cre-expressing progenitor," said Pu. At the moment, scientists are still trying to figure out whether and how the Wt1-expressing progenitors relate to the progenitors reported in 2006. "What we think is that very early on, our particular progenitor expresses Nkx2-5 and Isl1, but quickly loses expression of both and starts expressing Wt1," said Pu. "Think of a lineage hierarchy with Nkx2-5 and Isl1 at the top, and Wt1 as a branch. These two lineages separate pretty early, before the heart is present in the embryo. However, the Wt1-expressing progenitor may retain some of the developmental capabilities of the progenitors expressing Nkx2-5 and Isl1." Pu and Zhou now want to know whether the epicardium in an adult mouse could be induced to make cardiomyocytes. "If so, obviously this would be much more translatable to human studies," Pu said. Other ongoing questions are whether this newly-discovered progenitor is truly multipotent (able to turn into all other cell types), how multipotency is controlled, and whether this can be used therapeutically to benefit adults with heart failure. The fact that the Wt1-expressing progenitors can also differentiate into fibroblasts in the developing heart suggests that they can contribute to scar formation in the adult heart after injury, Pu added. "But if we can turn the progenitors away from making scars, and instead turn them towards making cardiomyocytes, that would be pretty exciting." Put another way, Pu and Zhou would love to learn what controls a progenitor's choice — to become a fibroblast, a smooth muscle cell, or a cardiomyocyte — and develop ways of "biasing progenitors to make the choice or choices we want," says Pu. "We still don't know how we can manipulate these progenitors, and there's no way to predict which ones will be useful. But I think having more choices is good, because then hopefully one of them will work." Reference: Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart Bin Zhou, Qing Ma, Satish Rajagopal1, Sean M. Wu, Ibrahim Domian, José Rivera-Feliciano, Dawei Jiang, Alexander von Gise, Sadakatsu Ikeda1, Kenneth R. Chien & William T. Pu Nature advance online publication 22 June 2008, doi:10.1038/nature07060 ......... ZenMaster

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