Capricor Therapeutics Enrolls Patients in HOPE Clinical Trial


The Beverly Hills-based biotechnology company Capricor Therapeutics, Inc. (CAPR) has announced the enrollment of 25 patients for their randomized Phase 1/2 HOPE-Duchenne clinical trial.

“HOPE” stands for “Halt cardiomyOPathy progrEssion in Duchenne” Muscular Dystrophy. The HOPE trial will evaluate the company’s CAP-1002 investigational cardiac cell therapy in patients suffering from Duchenne muscular dystrophy (DMD)-associated cardiomyopathy. If all goes as planned, CAPR expects to the first data points from this trial in six months (first quarter of 2017).

DMD most seriously affects skeletal muscle, but the disease can also devastate heart muscle. In fact, the most common cause of death from DMD results from the consequences of the disease on heart muscle.

The HOPE trial will assess the safety and efficacy of CAP-1002 in these 25 patients.

In DMD patients, scar tissue gradually accumulates in the heart, which leads to a deterioration of cardiac function.

CAP-1002 consists of cells donated from the hearts of healthy volunteers. These “cardiosphere-derived cells” or CDCs, have been shown by work in the laboratory of Dr. Eduardo Marbán, Director of the Heart Institute at Cedars-Sinai Medical Center, to reduce scar tissue in damaged hearts and improve heart function in studies with laboratory animals. Furthermore, a clinical study with CDCs, the CADUCEUS study, showed that the reduction of heart scar tissue in patients given infusions of CDCs. Therefore CAD-1002 might be the only therapeutic agent that can potentially reduce scar tissue in the damaged heart.

The HOPE trial enrolled 25 boys with DMD who were at least 12 years of age at the time of screening and who show signs of DMD-associated cardiomyopathy. These boys all have significant scar tissue in at least four left ventricular segments, according to magnetic resonance imaging (MRI) scans.

Of these 25 subjects, 13 subjects were randomly assigned to receive CAP-1002 by means of intracoronary infusion into each of the three main coronary arteries in a single procedure.

The 12 subjects randomized to the control arm received usual care and received no such infusion.

Efficacy of CAD-1002 will be assessed by means of specified secondary outcome measures that include absolute and relative changes in cardiac scar tissue and cardiac function as measured by MRI, performance on the Six-Minute Walk Test (6MWT) and the Performance of the Upper Limb (PUL), and scoring on the Pediatric Quality of Life Inventory (PedsQL).

The HOPE trial is a multicenter study; it is being conducted at Cincinnati Children’s Hospital Medical Center in Cincinnati, Ohio, Cedars-Sinai Heart Institute in Los Angeles, Calif., and the University of Florida in Gainesville, Fla.

DMD is a genetically inherited condition. The dystrophin gene that is abnormal in DMD patients is on the X chromosome, and therefore, the vast majority of DMD patients are male. DMD afflicts approximately 20,000 boys and young men in the U.S. The dystrophin complex is a structural component of muscles, integral to the integrity of muscle fibers. Abnormalities in dystrophin leads to chronic skeletal and cardiac muscle damage.

AUF1 Gene Important Inducer of Muscle Repair


A new study in the laboratory of Robert J. Schneider at NYU Langone and his collaborators has uncovered a gene that plays integral roles in the repair of injured muscle throughout life. This investigation shows that this previously “overlooked” gene might play a pivotal role in “sarcopenia,” which refers to the loss of muscle tissues with age.

This collaboration between scientists at NYU Langone Medical Center and the University of Colorado at Boulder showed that the levels of a protein called AUF1 determine if stem cell populations retain the ability to regenerate muscle after injury and as mice age.

Changes in the activity of AUF1 have also been linked by past studies to human muscle diseases. More than 30 genetic diseases, known collectively as myopathies, show defective muscle regeneration and these anomalies cause muscles to weaken or waste away.

For example, muscular dystrophy is a disease in which abnormal muscles fail to function properly and undergo normal repair. Although the signs and symptoms of Duchenne Muscular Dystrophy vary, in some cases wildly, this disease develops in infants and affects and weakens the torso and limb muscles beginning in young adulthood. Sarcopenia, in healthy individuals occurs in older patients.

Skeletal muscles have a stem cell population set aside for muscle repair known as satellite cells. These cells divide and differentiate into skeletal muscle when skeletal muscle is damaged, and as we age, the capacity of muscle satellite cells to repair muscle decreases.

AUF1 is a protein that regulates muscle stem cell function by inducing the degradation of specific, targeted messenger RNAs (mRNAs). According to Robert Schneider, “This work places the origin of certain muscle diseases squarely within muscle stem cells, and shows that AUF1 is a vital controller of adult muscle stem cell fate.” He continued: “The stem cell supply is remarkably depleted when the AUF1 signal is defective, leaving muscles to deteriorate a little more each time repair fails after injury.”

The experiments in this study demonstrated that mice that lack AUF1 display accelerated skeletal muscle wasting as they age. These AUF1-depleted mice also showed impaired skeletal muscle repair following injury. When the molecular characteristics of these AUF1-depleted muscle satellite cells were examined, Schneider and his collaborators showed that auf1−/− satellite cells had increased stability and overexpression of so-called “ARE-mRNAs.” ARE mRNAs contain AU-rich elements at their tail-ends. AUF1 proteins bind to these ARE mRNAs and induce their degradation. In the absence of AUF1, muscle satellite cells accumulate ARE mRNAs. One of these ARE mRNAs includes that which encodes matrix metalloprotease, MMP9. Overexpression of MMP9 by aging muscle satellite cells causes degradation of the skeletal muscle matrix, which prevents satellite-cell-mediated regeneration of muscles. Consequently, the muscle satellite cells return to their quiescent state and fail to divide and repair skeletal muscle.

When Schneider and his coworkers and collaborators blocked MMP9 activity in auf1−/− mice, they found that they had restored skeletal muscle repair and maintenance of the satellite cell population.

These experiments suggest that repurposing drugs originally developed for cancer treatment that blocks MMP9 activity might be a way to dial down age-related sarcopenia.

“This provides a potential path to clinical treatments that accelerate muscle regeneration following traumatic injury, or in patients with certain types of adult onset muscular dystrophy,” said Schneider.

This work was published here: Devon M. Chenette et al., “Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity,” Cell Reports, 2016; DOI: 10.1016/j.celrep.2016.06.095.

Beta-Integrin Implicated In Slow Healing Of Aged Muscles


With age, the function and regenerative abilities of skeletal muscles decrease. Therefore, the elderly can find it difficult to recover from injury or surgery.

A new study from the laboratory of Chen-Ming Fan from Johns Hopkins University has shown that a protein called β1-integrin is crucial for muscle regeneration. β1-integrin seems to provide a promising target for therapeutic intervention to combat muscle aging or disease.

Muscle stem cells are the primary source of muscle regeneration after muscle injury from exercise, accidents, or surgery. These specialized adult stem cells lie dormant in the muscle tissue, and muscles even have them stored off to the side of the individual muscle fibers. Because of their location, these muscle stem cells are known as muscle “satellite cells.” After damage, these satellite cells awaken and proliferate, and go on to make new muscle fibers and restore muscle function. Some satellite cells return to dormancy, which allows the muscle to keep a reservoir of healing cells that can repair the muscle over and over again. Fan and her colleagues determined that proteins called integrins, and in particular, β1-integrin, are integral for maintaining the cycle of hibernation, activation, proliferation, and then return to hibernation, in muscle stem cells.

Integrins are cell surface proteins that provide tight connections between cells and the immediate external environment.

Integrin Dimer Structure: Globular domain structures of α and β subunits in a stable dimer. Ligand binding happens at the interface of the αI (left panel) or β-propeller (right panel) and the βI domain.
Integrin Dimer Structure: Globular domain structures of α and β subunits in a stable dimer. Ligand binding happens at the interface of the αI (left panel) or β-propeller (right panel) and the βI domain.

Without integrins, almost every stage of the regeneration is disrupted. Fan and her group predicted that defects in β1-integrin likely contribute to aging, which is associated with reduced muscle stem cell function and decreased quantities of muscle stem cells. This means that healing after injury or surgery is very slow, which can cause a long period of immobility and an accompanying loss of muscle mass. Inefficient muscular healing in the elderly is a significant clinical problem. Therapeutic approaches would be quite welcome by the aging population and their physicians. One way to improve muscle regeneration would be to stimulate muscle satellite cells in older individuals.

Fan and others determined that β1-integrin function is diminished in aged muscle stem cells. When they artificially activated integrins in aged mice, their regenerative abilities were restored to youthful levels. Improvement in regeneration, strength, and function were also seen when this treatment was applied to animals with muscular dystrophy, which underscores the potential importance of such an approach for the treatment of muscle disorders.

Muscle stem cells use β1-integrin to interact with many other proteins in the external environment of the muscle. Among this forest of proteins in the external environment of the muscle, Fan and her coworkers found one called fibronectin that might be the most relevant. They discovered that aged muscles contain substantially less fibronectin compared to young muscles. Like β1-integrin, eliminating fibronectin from young muscles makes them function as though they were old. However, restoring fibronectin to aged muscle tissue restores muscle regeneration to youthful levels. Fan’s group demonstrated a strong link between β1-integrin, fibronectin and muscle stem cell regeneration.

Taken together, the results show that aged muscle stem cells with compromised β1-integrin activity and aged muscles with insufficient amount of fibronectin both root causes of muscle aging. This makes β1-integrin and fibronectin very promising therapeutic targets.

This work appeared in the following journal: Michelle Rozo et al., “Targeting β1-integrin signaling enhances regeneration in aged and dystrophic muscle in mice,” Nature Medicine, 2016; DOI: 10.1038/nm.4116.

LIF Increases Muscle Satellite Expansion in Culture and Transplantation Efficiency


Transplantation of satellite stem cells, which are found in skeletal muscles, might potentially treat degenerative muscle diseases such as Duchenne muscular dystrophy. However, muscle satellite cells have an unfortunate tendency to lose their ability to be transplanted then they are grown in culture.

In order to generate enough cells for transplantation, the cells are isolated from the body and then they must be grown in culture. However, in order to properly grow in culture, the cells must be prevented from differentiating because fully differentiated cells stop growing and die soon after transplantation. Several growth factors, cytokines, and chemicals have been used in muscle satellite cell culture systems. Unfortunately, the optimal culture conditions required to maintain the undifferentiated state, inhibit differentiation, and enhance eventual transplantation efficiency have not yet been established satisfactorily.

Because it is impossible to extract enough satellite cells for therapeutic purposed from biopsies, these cells must be expanded in culture. However this very act of culturing satellite cells renders them inefficient for clinical purposes. How can we break away from this clinical catch-22?

Shin’ichi Takeda from the National Center of Neurology and Psychiatry and his colleagues have used growth factors to maintain muscle satellite cell efficiency during cell culture. In particular, Takeda and others used a growth factor called leukemia inhibitory factor (LIF). LIF effectively maintains the undifferentiated state of the satellite cells and enhances their expansion and transplantation efficiency. LIF is also thought to be involved in muscle regeneration.

This is the first study on the effect of LIF on the transplantation efficiency of primary satellite cells,” said Shin’ichi Takeda of the National Center of Neurology and Psychiatry. “This research enables us to get one step closer to the optimal culture conditions for muscle stem cells.”

The precise mechanisms by which LIF enhances transplantation efficiency remain unknown. Present work is trying to determine the downstream targets of LIF. Identifying the precise mechanisms by which LIF enhances satellite cell transplantation efficiency would help to clarify the functional importance of LIF in muscle regeneration, and, even more importantly, further its potential application in cell transplantation therapy.

The reference for this paper is: N. Ito et al., “Enhancement of Satellite Cell Transplantation Efficiency by Leukemia Inhibitory Factor,” Journal of Neuromuscular Diseases, 2016; 3 (2): 201. DOI: 10.3233/JND-160156.

Dying Muscles Leave “Ghost Fibers” that Direct Muscle Regeneration


When muscles are injured, they die off in order to make room for the growth of replacement muscles. However, it turns out that these moribund muscle leave behind small evanescent fibers that have been called “ghost fibers.” Ghost fibers seem to be remnants of the gooey stuff that provides the substratum upon which muscle cells sit. This gooey foundation is called “extracellular matrix” or ECM. The ECM consists of acid sugars called “glycosaminoglycans,” which are given the unfortunate abbreviation of GAGs, proteins to which GAGs are attached called “proteoglycans,” and proteins that glue cells to the ECM, such as fibronectin, laminin, and collagen IV. Cells adhere to the ECM by means of receptors embedded in their cell membranes called integrins.

Extracellular matrix

Dying muscle cells leave collagen fibers in their wake and these collagen fibers constitute these so-called ghost fibers. However, these ghost fibers provide the structure into which new muscle cells are inserted. A new study by research teams at the Carnegie Institution for Science and the National Institute of Child Health and Human Development that was published in the journal Cell Stem Cell has established that ghost fibers guide new muscle cells to grow in place and ultimately heal muscle injury in laboratory mice.

Ghost Fibers
Ghost Fibers

Chen-Ming Fan at the Carnegie Institute of Washington in Baltimore, Maryland and his colleagues, in collaboration with and Jennifer Lippincott-Schwartz and her colleagues from the NIH disabled the hind limb muscles of laboratory mice by means of physical injury (laceration), or the administration of toxins. These insults to the skeletal muscles caused the injured muscle fibers to die and disintegrate. They also confirmed that as the skeletal muscle disappeared, they left networks of collagen ghost fibers in their wake.

Then, this team utilized three-dimensional, time-lapse intravital imaging to directly visualize the process of muscle regeneration in live mice. What they saw was stunning. The extracellular matrix remnants or ghost fibers left by the injured skeletal directed muscle stem/progenitor cell behavior during muscle regeneration. The two-photon imaging and second-harmonic generation microscopy employed by this team enabled them to precisely observe the muscle stem and precursor cells in individual mice orient themselves along the ghost fibers and grow new muscle tissue.

The muscle stem cells were quiescent and did not move in uninjured muscle tissue. Only when muscle cells were injured did the muscle stem cells come to life, move to the site of injury and begin the healing process. Both the cell division of these muscle stem cells and their migration were oriented along the longitudinal axes of the ghost fibers.

ImageJ=1.49m unit=inch
ImageJ=1.49m
unit=inch

If the ghost fibers were artificially reoriented, then the muscle progenitors migrated and divided in different planes and gave rise to disorganized regenerated muscle fibers.

From these results, Fan and his team concluded that “the ghost fiber (1) is a key determinant for patterning muscle stem cell behavior and (2) provides the foundation for proportional regeneration. They concluded that “ghost fibers are autonomous, architectural units necessary for proportional regeneration after tissue injury.” They continued, “This finding reinforces the need to fabricate bioengineered matrices that mimic living tissue matrices for tissue regeneration therapy.”

Muscular Dystrophy is a Stem Cell-Based Disease


Michael Rudnicki, who has done pioneering work in muscle stem cell biology and muscle regeneration, and whose work has been featured several times on this blog, has struck again. Rudnicki, who serves as director of the Regenerative Medicine Program at The Ottawa Hospital and a professor at the University of Ottawa and holds the prestigious Canada Research Chair in Molecular Genetics, teamed up with workers from the Sprott Centre for Stem Cell Research and the Sinclair Centre for Regenerative Medicine to investigate the role of muscle-specific stem cells in patients who suffer from Duchenne muscular dystrophy. This new earth-shaking study, which was published in the journal Nature Medicine (November 16, 2015), has changed the way we think about muscular dystrophy and will almost certainly force people to rethink the treatments and cures for this dreadful disease.

According to this new study, Duchenne muscular dystrophy directly affects muscle stem cells, and is, largely a disease of muscle stem cells.

Rudicki said: “For nearly 20 years, we’ve thought that the muscle weakness observed in patients with Duchenne muscular dystrophy is primarily due to problems in their muscle fibers, but our research shows that it is also due to intrinsic defects in the function of their muscle stem cells. This completely changes our understanding of Duchenne muscular dystrophy and could eventually lead to far more effective treatments.”

Muscular dystrophy comes in several different forms, but the predominant sign of muscular dystrophy is progressive muscle weakness. Altogether, muscular dystrophy refers to a group of more than 30 genetic diseases, all of which cause progressive weakness and degeneration of skeletal muscles used during voluntary movement. Approximately half of all who suffer from muscular dystrophy have Duchenne muscular dystrophy (DMD). Because muscular dystrophy results from mutations in the dystrophin gene, which is on the X chromosome, the vast majority of muscular dystrophy patients are male. Girls can be carriers of muscular dystrophy and can be mildly affected.

Interestingly, somewhere around one-third of boys who suffer from DMD have no family history of the disease. Because the dystrophin gene is so large, spontaneous mutations in it are probably relatively common.

The signs and symptoms typically appear between the ages of 2 and 3, and may include frequent falls, difficulty getting up from a lying or sitting position, trouble running and jumping, a strange, shuffling way of walking or having a tendency to walk on their toes, calf muscles that are abnormally large, muscle pain and stiffness, and some learning disabilities.

Becker muscular dystrophy (BMD) has signs and symptoms that are largely similar to those of DMD, but BMD tends to be a milder form of the disease that progresses more slowly. Symptoms typically begin in the teens but, some patients may not experience symptoms until their mid-20s and some may not experience symptoms until later.

There are also several different types of muscular dystrophy-type diseases. Steinert’s disease or myotonic muscular dystrophy, which is characterized by an inability to relax muscles at after contractions, is the most common form of adult-onset muscular dystrophy. The first muscles to be affected are the muscles of the face and neck. Facioscapulohumeral muscular dystrophy affects the muscles of the face and shoulders, where symptoms first begin. When patients with facioscapulohumeral raise their arms, their shoulder blades noticeably protrude. This disease may first manifest itself in children, teenagers as late as age 40. This disease tends to affect one side more than the other.

Limb-girdle muscular dystrophy affects the muscles of the shoulders and hips. There are over 20 inherited forms of this disease, and because this condition is not due to mutations in dystrophin, but to mutations in genes that encode proteins that interact with dystrophin, the inheritance of limb-girdle muscular dystrophy is not sex-linked. Some forms of this disease are recessive and some are dominant. Patients with this type of muscular dystrophy usually trip more often because they have trouble raising the front part of their feet. Some autosomal recessive forms of the disorder are now known to be due to a deficits in proteins called sarcoglycans or dystroglycan.

Congenital muscular dystrophy is extremely varible and is probably a cluster of several different diseases caused by mutations in different genes. Some of types of congenital muscular dystrophy show sex-linked inheritance while others do not. Most cases of congenital muscular dystrophy result from the absence of a muscle protein called merosin, which is found in the connective tissue that surrounds muscle fibers. Other types of congenital muscular dystrophy have normal merosin and still others result from abnormal motor neuron migration. Clinically, this disease is also extremely variable and can manifest itself at birth or before age 2, progress slowly or rapidly, and cause mild disability or severe impairment.

Muscular dystrophy affects all ethnic groups and occurs globally. It affects around 1 in every 3,500 to 6,000 male births each year in the United States.  DMD affects approximately one in 3,600 boys.

Because DMD results from mutations in the dystrophin gene, the vast majority of muscular dystrophy research was based on a simple model in which the Dystrophin protein played a structural role in the structural integrity of muscle fibers. Abnormal versions of the Dystrophin protein caused the muscle fibers to become damaged and die as a result of contraction.  Dystrophin anchors the cytoskeleton of the muscle fibers, which are essential for muscle contraction, to the muscle cell membrane, and then to the extracellular matrix outside the cell that serves as a foundation upon which the muscle cells are built.

gb-2001-2-4-reviews3006-3

However in this current study, Rudnicki and his team discovered that muscle stem cells also express the dystrophin protein. This is a revelation because Dystrophin was thought to be protein that ONLY appeared in mature muscle. However, in this study, it became exceedingly clear that in the absence of Dystrophin, muscle stem cells generated ten-fold fewer muscle precursor cells, and, consequently, far fewer functional muscle fibers. Dystrophin is also a component of a signal transduction pathway that allows muscle stem cells to properly ascertain if they need to replace dead or dying muscle.  Muscle stem cells repair the muscle in response to injury or exercise by dividing to generate precursor cells that differentiate into muscle fibers.

“Muscle stem cells that lack dystrophin cannot tell which way is up and which way is down,” said Dr. Rudnicki. “This is crucial because muscle stem cells need to sense their environment to decide whether to produce more stem cells or to form new muscle fibres. Without this information, muscle stem cells cannot divide properly and cannot properly repair damaged muscle.”

Even though Rudnicki used mice as a model system in these experiments, the Dystrophin protein is highly conserved in most vertebrate animals. Therefore, it is highly likely that these results will also apply to human muscle stem cells.

Treatment for DMD patients is limited to steroids to decrease muscle inflammation and muscle cell death, and physical therapy to increase muscle use and prevent muscle atrophy. These approaches only delay the progression of the disease and alleviate symptoms. Gene therapy experiments and trials are in progress and even show some promise, but Rudnicki’s work tell us that gene therapy approaches must target muscle stem cells as well as muscle fibers if they are to work properly.

“We’re already looking at approaches to correct this problem in muscle stem cells,” said Dr. Rudnicki. “I’m not sure if we will ever cure Duchenne muscular dystrophy, but I’m very hopeful that someday in the future, we will have new therapies that correct the ability of muscle stem cells to repair the muscles of afflicted patients and turn this devastating, lethal disease into a chronic but manageable condition.”

This paper has received high praise from the likes of Ronald Worton, who was one of the co-discovers of the dystrophin gene with Louis Kunkel in 1987.  Worton later served as Vice-President of Research at The Ottawa Hospital from 1996 to 2007.

“When we discovered the gene for Duchenne muscular dystrophy, there was great hope that we would be able to develop a new treatment fairly quickly,” said Dr. Worton, who is now retired. “This has been much more difficult than we initially thought, but Dr. Rudnicki’s research is a major breakthrough that should renew hope for researchers, patients and families.”

Human Muscle Satellite Cells Isolated and Characterized


A research group from the University of California, San Francisco have isolated and characterized human muscle stem cells. In addition, they have established that these stem cells can robustly replicate and repair damaged muscles when they are grafted onto an injured site. These remarkable findings might open the door to potential treatments for patients with severe muscle injuries, paralysis or genetic diseases that adversely affect skeletal muscles (e.g., muscular dystrophy).

Jason Pomerantz, MD is an assistant professor of plastic and reconstructive surgery at UCSF, and served as the managing author of this work. “We’ve shown definitively that these are bona-fide stem cells that can self-renew, proliferate and respond to injury,” said Pomerantz.

Badly damaged muscles can suffer terrible depletion of their native populations of stem cells or even obliteration of the stem cell niches and populations. Since such muscles have lost the very things that can heal them, these muscles will not be able to heal the damage they have sustained. This very fact represents a terrible hurdle for physicians who specialize in patients who have been crippled by muscle injury and paralysis. One of the worse cases is those conditions that cause damage or paralysis in the critical small muscles of the face, hand and eye, according to Pomerantz.

When muscles are badly damaged, they can lose the native populations of stem cells that are needed to heal. This has posed a major roadblock for treating patients crippled by muscle injury and paralysis, particularly in the critical small muscles of the face, hand and eye, Pomerantz said.

Fortunately, there have been remarkable surgical advances in restoring nerves in damaged muscles. Unfortunately, if the healing process takes too long, the stem cell pool is exhausted and the regenerative capacity is attenuated and eventually. Such injured muscles fail to connect to the nerve tissue and without accompanying motor and sensory nerves, skeletal muscles then to degenerate.

“This is partly why we haven’t had major progress in treating these patients in 30 years,” Pomerantz said. “We know we can get the axons there, but we need the stem cells for there to be recovery.”

A group of stem cells called “satellite cells” line the borders of muscle fibers and, in mice, can function as stem cells and contribute to muscle growth and repair. Until now, however, it wasn’t clear whether human satellite cells worked the same way. It was also terribly unclear how to isolate muscle satellite cells from human tissue samples or even adapt them to help treat patients with muscle damage.

Muscle satellite cells in section

Pomerantz and colleagues tackled this problem used muscle tissue from surgical biopsies of muscles of the head, trunk and leg. Then they used antibody staining to show that human satellite cells can be identified by the expression of the transcription factor PAX7 in combination with the cell-surface proteins CD56 and CD29. Pomerantz and his colleagues use this molecular signature to isolate populations of human satellite cells from these patient biopsies. Then they grafted these satellite cells into mice with damaged muscles whose own muscle stem-cell populations had been depleted. Five weeks after the transplantation, these human cells had successfully integrated into the mouse muscles and divided to produce families of daughter stem cells; effectively replenishing the stem cell niche and repairing the damaged muscle tissue.

This characterization of human muscle stem cells and the ability to transplant them into injured muscles has varied and wide-ranging implications for patients who are presently suffering from muscle paralysis, whose damaged muscles have lost the ability to regenerate. Additionally, protocols that allow us to isolate and manipulate human stem cells also may have applications for understanding why our muscles lose their regenerative capacity during normal aging or in the case of genetic diseases such as muscular dystrophy.

“This gives us hope that we will be able to extract healthy stem cells from other muscles in the patient’s body and transplant them at the site of injury,” Pomerantz said. “If replenishing a healthy muscle stem cell pool facilitates reinnervation and recovery, it would be a significant leap forward.”

These findings appeared the Sept. 8 edition in the open access Cell Press journal, Stem Cell Reports.

Fat-Derived Stem Cells Form Muscle in Muscular Dystrophy Mice


Stem cell therapy for Duchenne muscular dystrophy (DMD) has been plagued by poor cell engraftment into diseased muscles. Additionally, there are no reports to date describing the efficient generation of muscle progenitors from fat-derived stem cells (ADSCs) that can contribute to muscle regeneration.

A study by Cheng Zhang and others from Sun Yat-sen University in Guangzhou, China, Guangdong Province has examined the ability of progenitor cells differentiated from ADSCs using forskolin, basic fibroblast growth factor, the glycogen synthase kinase 3β inhibitor 6-bromoindirubin-3′-oxime as well as the supernatant of ADSC cultures to form workable muscle cells.

When these fat-derived stem cells were treated as described above, they formed a proliferative population of muscle progenitors from ADSCs that had characteristics similar to muscle satellite cells. Furthermore, in culture, these cells were capable of terminal differentiation into multinucleated myotubes.

When these fat-derived stem cells were transplanted into mice that had an inherited type of DMD, the progenitor cells successfully engrafted in skeletal muscle for up to 12 weeks, and generated new muscle fibers, restored dystrophin expression, and contributed to the satellite cell compartment.

These findings highlight the potential application of ADSCs for the treatment of muscular dystrophy. They also illustrate the ability of ADSCs to differentiate into functional skeletal muscle cells when treated properly in culture. These same cells might serve as a treatment for DMD patients.

This article was published in Hum. Mol. Genet. (2015) doi: 10.1093/hmg/ddv316.

Lab-Grown Muscle FIbers Aid in Studying Muscular Dystrophy


Skeletal muscle is the most abundant tissue in the human body, but, strangely, growing large quantities of it in the laboratory have proven rather challenging. While it is possible to reprogram other mature cells into heart muscle cells, or neurons, differentiating cells into skeletal muscle cells has simply not worked. So where do we go from here?

A new study from Brigham and Women’s Hospital (BWH) published in Nature Biotechnology has identified and even mimicked integral cues in the development of skeletal muscle. They used these cues to grow millimeter-long muscle fibers that are capable of contracting in the laboratory. This new method for growing functional muscle fibers in the laboratory potentially offer a better model for studying muscle diseases such as muscular dystrophy and for testing new treatments for these diseases.

Previous studies have used genetic modification techniques to grow small numbers of skeletal muscle cells in the laboratory. However, this new technique, which is the result of a collaboration between BWH and Harvard Stem Cell Institute, has produced a way to grow large numbers of skeletal muscle cells for use in clinical applications.

Olivier Pourquié of Harvard Medical School said, “We took the hard route: we wanted to recapitulate all of the early stages of muscle cell development that happen in the body and recreate that in a dish in the lab. We analyzed each stage of early development, and generated cell lines that glowed green when they reached a each stage. Going step by step, we managed to mimic each stage of development and coax cells toward muscle cell fate.”

The team found that a combination of secreted factors are important at the very early stages of embryonic development to stimulate muscle differentiation. By recapitulation this cocktail in the laboratory, Pourquié and his colleagues were able to mature muscle fibers in the laboratory from mouse or human pluripotent stem cells. Additionally, they produced muscle fibers in mice afflicted with muscular dystrophy by using muscle satellite cells. It is unknown if this method could help humans who suffer from muscular dystrophy, as more research is needed.

“This has been the missing piece: the ability to produce muscle cells in the lab could give us the ability to test out new treatments and tackle a spectrum of muscle diseases,” Pourquié said.

This new method also has the potential to help researchers study other muscle diseases, such as sarcopenia, or degenerative muscle loss and cachexia, the wasting away of muscle that typically occurs during severe illness.

Gene Controls Proliferation of Muscle Stem Cells


Fortunately, skeletal muscles have a high potential for regeneration, unlike other organs. When injured, muscle stem cells, known as satellite cells and located between the individual muscle fibers, rapidly begin to proliferate and subsequently replace the damaged muscles cells. New research from researchers from the Max Planck Institute for Heart and Lung Research in Bad Nauheim, Germany, have shown that a protein called Prmt5 plays a key role in regulating the activity of muscle satellite cells. These data gave rise to new studies that would like to examine the impact of Prmt5 in muscle disorders.

Satellite cells in skeletal muscles are small, spherical stem cells in between the individual muscles fibers. Normally, these cells remain almost completely inactive, but when a muscle is immediately begin to proliferate and heal the injury by replacing damaged muscles fibers.

satellite_cells

When satellite cells react to an injury, they undergo a transition from their inactive state to one of increased activity. This transition must be finely balanced because uncontrolled proliferation of satellite cells in healthy muscle tissue increases the risk of tumor formation. Conversely, muscle regeneration is impeded if the satellite cells are not activated fast enough when muscles are injured.

satellite cells
satellite cells

Now a research team headed by Thomas Braun from the Max Planck Institute for Heart and Lung Research in Bad Nauheim has now identified a gene that plays a decisive role in regulating the activity of satellite cells. Braun and his colleagues isolated muscle satellite cells from laboratory mice and identified 120 genes that are instrumental for the function of these cells.

Next, they switched off one of these genes, Prmt5, in the satellite cells of adult mice. “In healthy mice, switching off Prmt5 in the satellite cells had no effect on the muscles. But when the mice had a muscle injury, the results were completely different”, says Ting Zhang, the study’s lead author. No signs of regeneration were observed in Prmt5-deficient mice, but the muscles of control mice that had an active Prmt5 gene healed normally. “Instead of growing new muscle tissue, the mice without Prmt5 eventually developed clear signs of fibrosis”.

Braun and others further examined how Prmt5 regulates muscle regeneration. In mice without Prmt5, the number of satellite cells was noticeably reduced. Prmt5 seems to regulate proliferation activity of satellite cells. Furthermore, these results indicated that Prmt5 also prevents satellite cells from dying prematurely and plays a key role in transforming them into functional muscle fibers.

Braun and his colleagues hope their study will help them gain a better understanding of muscle disorders in humans. “The loss of muscle tissue in the absence of Prmt5 shows clear parallels to degenerative muscle disorders such as Duchenne muscular dystrophy”, says Johnny Kim, a member of Braun’s working group. In fact, the Bad Nauheim team now hopes that in the future, mice lacking the Prmt5 gene can serve as models for this particular disorder. “But we also want to study the etiological effects of Prmt5 regarding the genesis of muscular hypertrophies and certain tumor types,” Kim adds.

A “SMARTer” Way to Isolate Mesenchymal Stem Cells


The Singapore-MIT Alliance for Research and Technology or SMART employs a team of engineers and life scientists to design technologies that address problems in science and medicine. In particular, a SMART team has devised a new technique to identify mesenchymal stem cells from bone marrow cells on the basis of cell size, cell stiffness, and the deformation of the nucleus.

Mesenchymal stem cells (MSCs) constitute less than one percent of the total cells in bone marrow. Therefore, isolating these cells from the morass of cells that are in the bone marrow is somewhat of a challenge. Most of the procedures for isolating MSCs from bone marrow utilize cell surface proteins found on the surfaces of MSCs, but there are no few cell surface proteins that are unique to only MSCs. Therefore, such isolation procedures tend to be tedious and not completely efficient. Because MSCs can differentiate into cells that produce bone, cartilage, fat, or muscle, they have proven invaluable for tissue repair therapies.

This new study by the SMART team has identified three physical characteristics of MSCs that can distinguish them from other immature cells found in the bone marrow. These physical characteristics should help them invent devices that could rapidly isolate MSCs, and facilitate the isolation of sufficient numbers of stem cells to treat patients.

Presently there are no sure-fire ways to quickly and efficiently separate MSCs from bone marrow cells that have already begun to differentiate into other cell types, but share the same molecules on the cell surface. This caveat may explain why experimental results vary among labs, and why stem-cell treatments now in clinical trials are not as effective as they could be, said Krystyn Van Vliet, an MIT associate professor of materials science and engineering and biological engineering and a senior author of the paper, that appeared in the Proceedings of the National Academy of Sciences.

“Some of the cells that you’re putting in and calling stem cells are producing a beneficial therapeutic outcome, but many of the cells that you’re putting in are not,” Van Vliet said. “Our approach provides a way to purify or highly enrich for the stem cells in that population. You can now find the needles in the haystack and use them for human therapy.”

In bone marrow, MSCs exist alongside other immature cells, such as osteogenic cells, which have already begun the developmental path toward becoming cartilage- or bone-producing cells. Currently, researchers try to isolate MSCs based on protein markers found on the cell surfaces, but these markers are not specific to MSCs. Therefore isolation techniques that rely on cell surface proteins can also co-isolate other types of immature cells that are more differentiated.

“Conventional cell-surface markers are frequently used to isolate different types of stem cells from the human bone marrow, but they lack sufficient ‘resolution’ to distinguish between subpopulations of mesenchymal stromal cells with distinct functions,” Lee said.

The researchers set out to find biophysical markers for multipotency (the ability to differentiate into several different cell types). They hypothesized that cell size might be a factor, since fetal bone marrow stem cells, which tend to have a higher percentage of MSCs, are usually small in diameter.

Jongyoon Han, an MIT professor of electrical engineering and biological engineering, had previously invented a device that captures circulating tumor cells based on their size. The SMART team used Han’s machine to isolate bone marrow cells based on size and discovered that none of the larger cells were multipotent, but not all of the smaller cells were multipotent. Therefore, size alone in insufficient to distinguish MSCs.

After measuring several other physical traits, the SMART team observed that two other physical characteristics could be combined with cell size to completely distinguish MSCs from other stem cells: stiffness of the cell, and the degree of fluctuation in the cell’s nuclear membrane.

“You don’t need more than these three, but you also can’t use fewer than these three,” Van Vliet said. “We now have a triplet of characteristics that identifies populations of cells that are going to be multipotent versus populations of cells that are only going to be able to become bone or cartilage cells.”

These features seem to correspond to what is already known about stem cells, Van Vliet said. In contrast to cells that have already committed to their final fate, immature cells have genetic material that moves around inside the nucleus, producing more fluctuations of the nuclear cell membrane. Stem cells also have a less rigid internal cytoskeletal structure than those of highly differentiated cells, which makes them seem less stiff.

The researchers then tested the regenerative abilities of MSCs isolated on the basis of these three characteristics in mice. They found that immature MSCs could help repair both muscle and bone injuries, but cells identified as osteogenic stromal cells were able to repair bone but not muscle.

“We have provided the first demonstration that subpopulations of mesenchymal stromal cells can be identified and highly enriched for bone growth and muscle repair,” Lee said. “We envision that this approach would also be important in the selection and purification of bone marrow-derived stem cells for tissue repair in human patients suffering from a range of tissue-degenerative diseases.”

“This is potentially a big step forward in establishing a marker-free way of identifying mesenchymal stem cells with maximum differentiation capacity,” said Jochen Guck, a professor of cellular machines at the Dresden University of Technology. “Biophysical markers have long been discussed and sought as an alternative to antibody labeling. What sets this work apart from others is that it clearly said that no single marker (at least of those tried) alone is predictive enough, but that a combination of them is required.”

The SMART team is now working on high-speed methods for separating MSCs. Creating more pure populations of such cells should lead to more effective stem-cell treatments for tissue injuries, Van Vliet said.

“Instead of putting in 30 percent of the cells that you want, and 70 percent filler, you’re putting in 100 percent of the cells that you want,” she explains. “That should lead to more reliable patient outcomes, because you’re not going to have this variability from batch to batch, or patient to patient, in how many of each cell population are present.”

Van Vliet and Poon also hope to initiate a clinical trial that utilizes the osteogenic cells isolated in this study, which could potentially prove useful for treating bone injuries.

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


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

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

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

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

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

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

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

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

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

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

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

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

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

Gene Editing in iPS Cells Corrects Genetic Mutations That Cause Muscular Dystrophy


Induced pluripotent stem cells or iPSCs have many of the same characteristics as embryonic stem cells. One such feature is the ability to be grown in culture and manipulated like genuine tissue culture cells.

To that end a research group at the Center for iPS Cell Research and Application (CiRA) have used iPSCs made from the cells of patients with Duchenne muscular dystrophy (DMD) to show that such mutations can be efficiently fixed.

This research, which was published in Stem Cell Reports, demonstrates how a new group of engineered nucleases, such as TALEN and CRISPR, can edit the genome of iPS cells generated from skin cells isolated from a DMD patient. After being genetically fixed, these iPSCs were differentiated into skeletal muscles, and it was clear that the mutation responsible for DMD had disappeared.

DMD is a severe muscular degenerative disease caused by loss-of-function mutations in the dystrophin gene. DMD affects 1 in 3500 boys and normally leads to death by early adulthood. The treatments for this disease are largely palliative.

However, the capability to edit the genomes of mutant cells is a formerly unknown option that was once only for the realms of science fiction. Two nucleated called TALEN and CRISPR have quickly become invaluable tools in molecular biology. These enzymes allow scientists to cleave genes at specific locations and then modify the cut ends to generate a specifically chosen genomic sequence. However, these programmable nucleases are not perfect and often mistakenly edit similar sequences that vary a few base pairs from the target sequence. This makes them unreliable for clinical use because of the potential for creating new, undesired mutations.

For precisely this reason, iPSCs are ideal model systems because they provide researchers an abundance of patient cells on which to test the programmable nucleated, and determine the optimal conditions that minimize off-target modifications. CiRA scientists used this very feature to generating iPS cells from a DMD patient. Then they utilized several different TALENs and CRISPRs to modify the genome of the iPS cells, which were then differentiated into skeletal muscle cells. In all cases, dystrophin protein expression was restored, and in some cases, the dystrophin gene was fully corrected.

One of the reasons for the success in this project was the development of a computational protocol that minimized the risk of off-target editing. The CiRA team built a database that contained all possible combination of sequences up to 16 base pairs long. Among these, they isolated those sequences that only appear once in the human genome. DMD can be caused by several different mutations. For example, in the case of the patient used in this study, it was the result of the deletion of exon 44. After building a histogram of unique sequences that appeared in a genomic region that contained this exon, the CiRA group found a cluster of unique sequences in exon 45.

The head researcher for this project, Akitsu Hotta, who headed the project and holds joint positions at CiRA and the Institute for Integrated Cell-Materials Sciences at Kyoto University, said:  “Nearly half the human genome consists of repeated sequences. So even if we found one unique sequence, a change of one or two base pairs may result in these other repeated sequences, which risks the TALEN or CRISPR editing an incorrect region. To avoid this problem, we sought a region that hit high in the histogram.”

This paper provides a proof-of-principle for using iPS cell technology to treat DMD in combination with TALEN or CRISPR. The group now aims to expand this protocol to other diseases.  First author Lisa Li explains, “We show that TALEN and CRISPR can be used to correct the mutation of the DMD gene. I want to apply the nucleases to correct mutations for other genetic-based diseases like point mutations”.

“In Body” Muscle Regeneration


Researchers at Wake Forest Baptist Medical Center’s Institute for Regenerative Medicine have hit upon a new strategy for tissue healing: mobilizing the body’s stem cells to the site of injury. Thus harnessing the body’s natural healing powers might make “in body” regeneration of muscle tissue is a possibility.

Sang Jin Lee, assistant professor of Medicine at Wake Forest, and his colleagues implanted small bits of biomaterial scaffolds into the legs of rats and mice. When they embedded these scaffolds with proteins that mobilize muscle stem cells (like insulin-like growth factor-1 or IGF-1), the stem cells migrated from the muscles to the bioscaffolds and formed muscle tissue.

“Working to leverage the body’s own regenerative properties, we designed a muscle-specific scaffolding system that can actively participate in functional tissue regeneration,” said Lee. “This is a proof-of-concept study that we hope can one day be applied to human patients.”

If patients have large sections of muscle removed because of infections, tumors or accidents, muscle grafts from other parts of the body are typically used to restore at least some of the missing muscle. Several laboratories are trying the grow muscle in the laboratory from muscle biopsies that can be then transplanted back into the patient. Growing muscle on scaffolds fashioned from biomaterials have also proven successful.

Lee’s technique overcomes some of the short-comings of these aforementioned procedures. As Lee put it, “Our aim was to bypass the challenges of both of these techniques and to demonstrate the mobilization of muscle cells to a target-specific site for muscle regeneration.”

Most tissues in our bodies contain a resident stem cell population that serves to regenerate the tissue as needed. Lee and his colleagues wanted to determine if these resident stem cells could be coaxed to move from the tissue or origin, muscle in this case, and embeds themselves in an implanted scaffold.

In their first experiments, Lee and his team implanted scaffolds into the leg muscles of rats. After retrieving them several weeks later, it was clear that the muscle stem cell population (muscle satellite cells) not only migrated into the scaffold, but other stem cell populations had also taken up residence in the scaffolds. These scaffolds were also contained an interspersed network of blood vessels only 4 weeks aster transplantation.

In their next experiments, Lee and others laced the scaffolds with different cocktails of proteins to boost the stem cell recruitment properties of the implanted scaffolds. The protein that showed the most robust stem cell recruitment ability was IGF-1. In fact, IGF-1-laced scaffolds had four times the number of cells as plain scaffolds and increased formation of muscle fibers.

“The protein [IGF-1] effectively promoted cell recruitment and accelerated muscle regeneration,” said Lee.

For their next project, Lee would like to test the ability of his scaffolds to promote muscle regeneration in larger laboratory animals.

Patient’s Own Stem Cells Treat Rare Neurological Disorder


Stiff-Person syndrome is a rare neurological disease that, for all intents and purposes, looks like an autoimmune disease. It is characterized by muscular rigidity that tends to come and go. This rigidity occurs in the muscles of the trunks and limbs. Patients with Stiff-Person syndrome also have an enhanced sensitivity to stimuli such as noise, touch, and emotional distress, and various stimuli may cause the patient to experience painful muscle spasms that cause abnormal postures and stiffening. Stiff-Person syndrome or SPS is more common in women than in men and SPS patients often suffer from other autoimmune conditions in addition to SPS (for example, pernicious anemia, diabetes, vitiligo, and thyroiditis). Unfortunately, the precise cause of SPS is not known, but again, it looks like an autoimmune condition.

A research team at Ottawa Hospital Research Institute has made a breakthrough in the successful treatment of SPS using bone marrow stem cell transplants. The medical director at the Ottawa Hospital Research Institute, Dr. Harold L. Atkins, who is also a physician in the Blood and Bone Marrow Transplant Program at The Ottawa Hospital and an associate professor at the University of Ottawa has used bone marrow transplants to two female SPS patients into remission.

SPS can leave patients bedridden and in severe pain, but thanks to Atkins and his team, the progression of the disease in these women has ceased, allowing both women to regain their previous function and leaving them well enough to return to work and normal everyday activities.

Adkins and his group published this case study in JAMA Neurology, which is produced by the Journal of the American Medical Association. This is the first documented report that taking stem cells from a person’s own body can produce long-lasting remission of stiff person syndrome.

“We approach these cases very carefully and are always aware that there have just been a few patients treated and followed for a short time,” says Dr. Atkins. Atkins and his extracted bone marrow stem cells from each woman, and then used chemotherapy to eliminate their immune systems. Once their immune system were reliably eliminated, both women had their own stem cells returned to their bodies in order to reconstitute their immune systems. This procedure essentially gives the immune system a “do-over.”.

“By changing the immune system, one hopes to put the stiff person syndrome into remission,” adds Dr. Atkins. “Seeing these two patients return to their normal lives is really every physicians dream.”

This very procedure, which is known as an “autologous stem cell transfer” or ASCT has been used to successfully treat people who suffer from autoimmune diseases such as multiple sclerosis, scleroderma, and systemic lupus erythematosis. Atkins and his team used high-doses of chemotherapy and antibodies that specifically bind lymphocytes to rid the women’s bodies of their rogue immune cells before their immune systems were regenerated using their own stem cells. Adkins an his colleagues viewed this as a viable treatment option based strategies that had been used to treat other autoimmune diseases.

Patient 1 was diagnosed with stiff person syndrome in 2005 at age 48 after experiencing leg stiffness and several falls. After her treatment, her symptoms disappeared and she was fully mobile again six months after receiving the stem cell transplant procedure in 2009.

Patient 2 was diagnosed with stiff person syndrome in 2008 at age 30. She had stopped working and driving, and had moved back in with her parents before her stem cell transplant in 2011. Also, she has been able to return to her work and previous activities, and has not had any stiff person syndrome symptoms in more than a year.

“The results achieved by Dr. Atkins and his team through this innovative treatment show how research at The Ottawa Hospital can lead to life-changing and, even life-saving care,” says Dr. Duncan Stewart, Chief Executive Officer and Scientific Director of the Ottawa Hospital Research Institute. “Translating research into better care for patients is what we’re all about at the research institute.”

Inhibition of signaling pathway stimulates adult muscle satellite cell function


Stem cell researcher Michael Rudnicki and his team from the University of Ottawa in Ontario, Canada has done it again. Rudnicki works on muscle stem cells and his work has greatly expanded our understanding of muscle satellite cells.

Muscle satellite cells are found in skeletal muscle, and they are a prime example of a “unipotent” stem cell, or a stem cell that can differentiate into only one cell type. Muscle satellite cells can only form skeletal muscle, but they can be isolated from skeletal muscle and grown in culture. When muscle is injured by exercise or shear forces, satellite cells move into action and divide to form muscle cells that fuse with existing muscle cells and firm them up. Lifting weights will also increase the activity of satellite cells and they will divide and contribute to the formation of new muscle fibers.

As we age, our capacity to regenerate damaged muscle slows way down. As someone who lifted weights in high school and then on and of after high school, I can attest to this as I have entered my later years. My joints get sore faster and I cannot handle heavier weights any more. Also, I do not get big from lifting anymore. This is due to the reduction in muscle repair and I have become older.

Rudnicki and others have identified a reduced capacity in adult mice to repair their muscles, and this reduction in muscle regenerative ability has been directly linked to reduced muscle satellite cell activity. Aged mice have muscle satellite cells that show a diminished ability to contribute to muscle regeneration and repopulate themselves.

In a recent paper published in the journal Nature Medicine, Rudnicki and his colleagues compared used gene expression profiles in the satellite cells of older and younger mice. Curiously, they identified the genes that encode the components of a cell signaling pathway called the “JAK-STAT” pathway that are more highly expressed in the satellite cells of older mice than in those of younger mice.

These data suggested that inhibition of the JAK-STAT pathway in the satellite cells of older mice might lead to higher satellite cell activity in older mice. Fortunately, there are drugs that will inhibit the JAK-STAT signaling pathway.

Knockdown of the activity of the Jak2 or Stat3 proteins significantly stimulated satellite stem cell divisions in culture (the satellite cells were grown in cultured muscles). When Jak2 of Stat3 were inhibited genetically (by introducing loss-of-function mutations in these genes), the isolated satellite cells showed a markedly ability to repopulate local satellite cell populations after they were transplanted into a wounded muscle.

Inhibition of Jak2 and Stat3 activity with drugs also stimulated the engraftment of satellite cells in a living animal. If these same rugs were injected into the muscle of older laboratory mice, these mice showed marked enhancement of muscle repair and force generation after injury.

Thus, these results from the Rudnicki lab show that they is an intrinsic property of satellite cells that separate the satellite cells of younger animals with those of older animals. These results also suggest a promising therapeutic avenue for the treatment of muscle-wasting diseases.

Stem Cells Aid Muscle Strengthing and Repair After Resistance Exercise


University of Illinois professor of Kinesiology and Community Health, Marni Boppart and her colleagues have published experiments that demonstrate that mesenchymal stem cells (MSCs) rejuvenate skeletal muscle after resistance exercise. These new findings, which were published in the journal Medicine and Science in Sports and Exercise, might be the impetus for new medical interventions to combat age-related declines in muscle structure and function.

Marni Boppart
Marni Boppart

Injecting MSCs into mouse leg muscles before several bouts of exercise that mimic resistance training in humans and result in mild muscle damage caused increases in the rate of muscle repair and enhanced the growth and strength of those muscles in exercising mice.

“We have an interest in understanding how muscle responds to exercise, and which cellular components contribute to the increase in repair and growth with exercise,” Boppart said. “But the primary goal of our lab really is to have some understanding of how we can rejuvenate the aged muscle to prevent the physical disability that occurs with age, and to increase quality of life in general as well.”

MSCs are found throughout the body, but several studies have established that MSCs from different tissue sources have distinct biological properties. Typically, MSCs can readily differentiate into bone, fat, and cartilage cells, but coaxing MSCs to form skeletal muscle has proven to be very difficult. MSCs usually form part of the stroma, which is the connective tissue that supports organs and other tissues.

Because of their inability to readily differentiate into skeletal muscle, MSCs probably potentiate muscle repair by “paracrine” mechanisms. Paracrine mechanisms refer to molecules secreted by cells that induce responses in nearby cells. Not surprisingly, MSCs excrete a wide variety of growth factors, cytokines, and other molecules that, according to this new study, stimulate the growth of muscle precursor cells, otherwise known as “satellite cells.” The growth of satellite cells expands muscle tissue and contributes to repair following muscle injury. Once activated, satellite cells fuse with damaged muscle fibers and form new fibers to reconstruct the muscle and enhance strength and restore muscle function.

“Satellite cells are a primary target for the rejuvenation of aged muscle, since activation becomes increasingly impaired and recovery from injury is delayed over the lifespan,” Boppart said. “MSC transplantation may provide a viable solution to reawaken the aged satellite cell.”

Unfortunately, satellite cells, even though they can be isolated from muscle biopsies and grown in culture, will probably not be used therapeutically to enhance repair or strength in young or aged muscle “because they cause an immune response and rejection within the tissue,” Boppart said. But MSCs are “immunoprivileged,” which simply means that they can be transplanted from one individual to another without sparking an immune response.

“Skeletal muscle is a very complex organ that is highly innervated and vascularized, and unfortunately all of these different tissues become dysfunctional with age,” Boppart said. “Therefore, development of an intervention that can heal multiple tissues is ideally required to reverse age-related declines in muscle mass and function. MSCs, because of their ability to repair a variety of different tissue types, are perfectly suited for this task.”

Repairing Muscles in Muscular Dystrophy Depends on the Degree of Muscle Deterioration


Pier Lorenzo Puri, M.D., an associate professor at Sanford-Burnham Medical Research Institute (Sanford-Burnham), has led a research team that work in collaboration with Fondazione Santa Lucia in Rome, Italy, to characterize the mechanism by which a class of drugs called “HDACis” drive muscle-cell regeneration in the early stages of dystrophic muscles, but fail to work in late stages. These findings are integral for designing HDACis drugs for Duchenne muscular dystrophy (DMD), which presently, is an incurable muscle-wasting disease.

Puri’s research was published April 15th, 2014 edition of the journal Genes and Development. In their paper, Puri and his colleagues used mouse models of DMD to show how special cells known as “fibro-adipogenic progenitor cells” or FAPs, direct muscle regeneration. FAPs reside in the spaces between muscle fibers and detect those cues that indicate that muscles have been damaged. In response to muscle damage, FAPs direct muscle stem cells, known as satellite cells, to rebuild muscle.

 HDAC inhibitors (HDACi) promote muscle regeneration in a mouse model of Duchenne Muscular Dystrophy at early stages of disease by targeting fibro-adipogenic progenitors (FAPs). Staining of FAPs from muscles of HDACi-treated young mdx mice reveals presence of differentiated muscle cells (green) at the expense of fat cells (red). Nuclei are stained in blue. Image: Lorenzo Puri, M.D.

HDAC inhibitors (HDACi) promote muscle regeneration in a mouse model of Duchenne Muscular Dystrophy at early stages of disease by targeting fibro-adipogenic progenitors (FAPs). Staining of FAPs from muscles of HDACi-treated young mdx mice reveals presence of differentiated muscle cells (green) at the expense of fat cells (red). Nuclei are stained in blue. Image: Lorenzo Puri, M.D.

“HDACis create an environment conducive for FAPs to direct muscle regeneration—but only during the early stages of DMD progression in mice,” said Puri. “At some point, DMD progresses to a pathological point of no return and become permanently resistant to muscle-regeneration cures and to HDACis.”

Indeed, Puri’s research showed exactly that; namely that FAPs embedded in muscle that was in the earlier stages of muscular dystrophy responded robustly to HDACis and upregulated a wide range of muscle-specific genes. In contrast, FAPs from late-stage dystrophic muscles were resistant to HDACi-induced muscle-specific gene expression and failed to activate satellite cells.

HDACis stands for histone deacetylase inhibitors. These are epigenetic drugs that regulate the accessibility of those genes that code for muscle proteins. HDACis ensure that the DNA within cells is open and easily accessible to the gene expression machinery. In the presence of FAPs, in particular, rev up their support for muscle regeneration. Under conditions of normal wear and tear, FAPs direct stem cells within the muscle to regenerate and repair damaged muscle. However in patients with DMD, the persistent breakdown of muscle cells creates a chaotic environment that overwhelms the ability of the FAP’s to direct muscle regeneration.

Puri collaborated with Italian colleagues at Fondazione Santa Lucia, Italfarmaco, and Parent Project Muscular Dystrophy, an advocacy association. The goal of this research is to develop HDACis for the treatment of DMD. To that end, Puri and others have launched a clinical trial with DMD boys.

“Our study is important because it provides the rationale for the clinical development of HDACis to treat DMD,” said Puri. “And, now that we understand the mechanics and sensitivities of the muscle-regeneration system, we have the rationale and can use new tools to select patients most likely to benefit from HDACIs based on their FAP profile, predict outcomes, and see how long patients should remain on the therapy.”

“Duchenne muscular dystrophy patients and their families rely on important research such as that performed by Dr. Puri,” said Debra Miller, Founder of Cure Duchenne, a patient advocacy group. “Our efforts at Cure Duchenne are to support leading scientists in the world to bring life-saving drugs to help this generation of Duchenne boys, and our vision is to cure Duchenne muscular dystrophy. Every added piece of knowledge about the disease brings us closer to realizing our goals.”

The Puri paper also shows why trying to regenerate muscle cells in severely affected individuals is not feasible, since the dystrophic muscles have deteriorated to the point of no return. This will definitely influence the construction of treatment strategies for patients with muscular dystrophy.

New Method Derived Skeletal Muscle Cells from Pluripotent Stem Cells


A University of Wisconsin research team led by Masatoshi Suzuki has devised a new protocol for the production of large quantities of skeletal muscle cells from pluripotent stem cells.

Suzuki and his team used embryonic stem cells lines and induced pluripotent stem cells to generate large quantities of muscles and muscle progenitor.

Suzuki adapted a technique used to make brain cells to derive his muscle cells in culture. He grew the stem cells as floating spheres in high concentrations of two growth factors: fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF). This combination of growth factors directed the stem cells to differentiate into skeletal muscle cells and muscle progenitors.

To replace damaged or diseased muscles in the clinic, physicians will require large quantities of muscle cells. Therefore, there was an ardent search to design a technique that was efficient, but also fast and relatively simple. Even though several protocols have been devised to differentiate pluripotent stem cells into muscle cells, not all of these protocols are practical for clinical use. For example, some protocols are simply too cumbersome for clinical use. Still others make use of genetically engineered cells that have not been approved for clinical use.

Earlier, Suzuki transplanted lab-engineered skeletal muscle into mice that had a form of amyotrophic lateral sclerosis. These animals had better muscle function and survived better than the control animals.

The muscle progenitors generated in Suzuki’s laboratory could potentially play a similar role in human patients with Lou Gehring’s disease. Suzuki’s method can grow muscle progenitor cells, which can grow in culture, from induced pluripotent stem cells, which are derived from the patient’s own cells. Such cells could be used as a model system to study the efficacy of particular treatments on the patient’s muscles, or they could be used to treat patients who have muscle defects.

“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,” said Suzuki.

The advantages of Suzuki’s protocol are manifold. First, the cells are grown in a defined medium devoid of animal products. Secondly, the stem cells are grown as spheres, and these grow faster when grown as spheres than they do with other techniques. Third, 40-60 percent of the cells grown in this culture system differentiate into skeletal muscle cells or muscle progenitor cells. This is a very high proportion of muscle cells when compared to other protocols.

Suzuki hopes that by toying with the culture system, he and his colleagues can increase this proportion of muscle cells that form from the initial stem cell culture. This would enhance the potential of using these cells for clinical purposes.

Duke University Tissue Engineering Team Grows Self-Healing Muscle in Laboratory


Scientists have grown living muscle in the lab. While this is nothing new, this new advance has succeeded in making muscle that not only looks and works like genuine skeletal muscle, but also heals by itself, which is a significant advance in the field of tissue engineering.

This ultimate goal of this research is to use lab-grown muscle repair muscle damage in human patients. To date, preclinical trials have shown that lab-grown muscle properly regenerated damaged muscle in laboratory mice.

This research comes from Duke University, and the research team responsible for this work thinks that their success was due to the culture environment that they have created to grow muscle in the laboratory. Their well-developed contractile muscle fibers also contained a pool of satellite cells, which are an immature stem cell population in skeletal muscle that are activated when the muscle is damaged. Satellite cells can divide and differentiate into normal muscle tissue in order to heal muscle damage.

Cultured Muscle

Laboratory tests showed that the lab-grown muscle was as strong and good at contracting as muscle isolated from living organism. Also, the laboratory-grown muscle was able to use its satellite cell population to repair itself when the muscle was damaged with toxic chemicals.

Muscle satellite cells

When it was grafted into laboratory mice, the muscle properly integrate into the rest of the surrounding tissue and functioned beautifully when called upon to do so.

The Duke team, however, stresses that more tests must be conducted before this work can be translated into human patients.

The lead researcher for this work, Nenad Bursac, Associate Professor of Biomedical Engineering at Duke University, said: “The muscle we have made represents an important advance for the field. It’s the first time engineered muscle has been created that contracts as strongly as native neonatal [newborn] skeletal muscle.”

UK expert in skeletal muscle tissue engineering Prof Mark Lewis, from Loughborough University, said: “A number of researchers have ‘grown’ muscles in the laboratory and shown that they can behave in similar ways to that seen in the human body. However, transplantation of these grown muscles into a living creature, which continue to function as if they were native muscle has been taken to the next level by the current work.”

Tissue engineering seeks to use stem cells to fashion new organs and tissues from cultured stem cells. Tissue engineering and stem cell biology will certainly transform regenerative medicine, and in many ways it is already doing so. Scientists have already made mini-livers and kidneys in the lab using stem cells, and others are using stem cells to heal damaged heart muscles. Even though some cures and treatments are still some years away, advances continue to pile up. The future of medicine is upon us.