Stem Cell Therapies for Myelin Disorders May Undergo Clinical Trials Soon

The highly-regarded journal Science has published a review article by University of Rochester Medical Center scientists Steve Goldman, M.D., Ph.D., Maiken Nedergaard, Ph.D., and Martha Windrem, Ph.D. that argues that stem cell researchers are very close to human application of stem cell therapies for a class of neurological diseases known as myelin disorders. Myelin disorders consist of a lengthy list of rather nasty diseases:

1) multiple sclerosis, which is a disease that affects the brain and spinal cord and damages the myelin sheath that surrounds and protects nerve cells. This damage slows down or blocks messages between the brain and body, which leads to the symptoms of MS, which include visual disturbances, muscle weakness, trouble with coordination and balance, sensations such as numbness, prickling, or “pins and needles,” and thinking and memory problems.

2) white matter stroke, a lack of blood flow to white matter, which is quite severe, since blood flow to the white matter far less than that of gray matter.

3) cerebral palsy, a group of disorders that involve the brain and nervous system functions, that include movement, learning, hearing, seeing, and thinking. There are several different types of cerebral palsy, including spastic, dyskinetic, ataxic, hypotonic, and mixed. Cerebral palsy is caused by injuries or abnormalities of the brain. Most of these problems occur as the baby grows in the womb, but they can happen at any time during the first 2 years of life, while the baby’s brain is still developing. In some people with cerebral palsy, parts of the brain are injured due to low levels of oxygen (hypoxia) in the area. It is not known why this occurs. Premature infants have a slightly higher risk of developing cerebral palsy. Cerebral palsy may also occur during early infancy as a result of several conditions, including: bleeding in the brain, brain infections, head injuries, infections in the mother during pregnancy, severe jaundice.

4) certain dementias, and

5) rare but fatal childhood disorders called pediatric leukodystrophies. Leukodystrophies are a varied group of diseases that primarily affect the white matter of the central nervous system (CNS). These diseases include both primary myelin disorders, axonal/neuronal degeneration and inflammatory disorders. There are two types of leukodystrophies: dysmyelinating diseases, which usually results from inherited defects in an enzyme pathway or organelle function that causes abnormal formation, destruction, or turnover of myelin, and demyelinating disorders that result in abnormal destruction of normal myelin and/or axons.

According to Goldman, “Stem cell biology has progressed in many ways over the last decade, and many potential opportunities for clinical translation have arisen. In particular, for diseases of the central nervous system, which have proven difficult to treat because of the brain’s great cellular complexity, we postulated that the simplest cell types might provide us the best opportunities for cell therapy.”

Myelin disorders share a common pathological factor and that is a cell that makes myelin, called an “oligodendrocyte.”  Oligodendrocytes arise from a cell found in the central nervous system called “glial progenitor cells (GPCs).  GPCs give rise to oligodendrocytes and “sister cells” called “astrocytes.”  Both cells serve rather critical functions in the central nervous system.

Astrocytes

Oligodendrocytes produce myelin, a fatty substance that insulates the fibrous connections between nerve cells that are responsible for transmitting signals throughout the body. When myelin-producing cells are lost or damaged in conditions such as multiple sclerosis and spinal cord injury, signals traveling between nerves are weakened or even lost.

Oligodendrocyte

Astrocytes are the unsung heroes of the central nervous system.  They were largely neglected for some time, but are now coming into their own as one of the main glial (support cells) in the brain.  Astrocytes secrete a cocktail of growth factors that keep neurons and oligodendrocytes healthy and help them properly signal to other cells.

Because they give rise to cells that are so central to the function of so many other brain cells, GPCs and their offspring represent a promising target for stem cell therapies.  An added bonus of using GPCs is that (unlike other cells in the central nervous system) they are rather homogeneous and are also don;t mind being manipulated and cultured.  Consequently, they are easy to transplant.  In fact, several animal studies have established that transplanted oligodendrocytes will disperse and repair or “remyelinate” damaged nerves.
“Glial cell dysfunction accounts for a broad spectrum of diseases, some of which – like the white matter degeneration of aging – are far more prevalent than we previously realized,” said Goldman. “Yet glial progenitor cells are relatively easy to work with, especially since we don’t have to worry about re-establishing precise point-to-point connections as we must with neurons. This gives us hope that we may begin to treat diseases of glia by direct transplantation of competent progenitor cells.”

Several key technological advances have made these recent advances in neural stem cell protocols possible.  First of all, superior imaging technologies with more advanced MRI scanners that provide sharper and more magnified images can provide more precise information about the specific damage in the central nervous system that result from myelin disorders.  Additionally, once cells are transplanted, these new scanning techniques allow scientists to more precisely trace their implanted cells and determine what those cells are doing.

Another important advance are the major obstacles that have recently been overcome in recent work.  First, there have been significant advances in the manipulation and handling of GPCs and their progeny.  Goldman’s lab  has pioneered the techniques for GPC manipulation.  He and his colleagues have determined the precise steps used in the induction of GPC differentiation into either oligodendrocytes or astrocytes.  Goldman’s lab has produced GPCs by reprogramming skin cells and he has also identified specific cell surface molecules that act as markers for GPCs.  The identification of markers is a huge advance because it allows him and his co-workers to isolate the differentiated cells away from those that might potentially cause tumors.

The Nedergaard lab has done a tremendous amount of work to understand the structure of the neural networks of the brain and their functional contributions to the brain as a whole.  Together, these two labs have developed models of normal human neural activity and brain disease that are based on laboratory animals that have been transplanted with human GPCs.  This enables the human neural cells to operated within a living brain instead of a culture dish.  Such experimental has already opened several new strategies for modeling and potentially treating human glial diseases.

The authors contend that these advances have accelerated research to the point where human clinical trials for myelin disorders will probably occur soon.  As an example, patients with multiple sclerosis would benefit from the invention of a new generation of stabilizing anti-inflammatory drugs, since multiple sclerosis results from thsensitizatione  of the immune system to the myelin sheath.  However, such drugs always have nasty side-effects.  If you do not believe me, just examine this short list of side effects for one of these drugs.  Instead MS patients would definitely benefit from a progenitor-based cell therapy that could repair the now permanent and untreatable damage to the central nervous system that results from this disease.  Also several childhood diseases of white matter are also excellent candidates for cell-based treatments.
“We have developed a tremendous amount of information about these cells and how to produce them,” said Goldman. “We understand the different cell populations, their genetic profiles, and how they behave in culture and in a variety of animal models. We also have better understanding of the disease target environments than ever before, and have the radiographic technologies to follow how patients do after transplantation. Moving into clinical trials for myelin disorders is really just a question of resources at this point.”

Induced Pluripotent Stem Cells Lead Neuroscientists to the Cause of Neuron Loss in Parkinson’s Disease

Salk Institute scientists have made induced pluripotent stem cells (iPSCs) from patients with early-onset Parkinson’s disease (PD) in order to study precisely what goes wrong in the brains of PD patients. Their findings may lead to new ways to diagnose and even treat PD.

At the Salk Institute for Biological Studies in La Jolla, CA, Juan Carlos Izpisua Belmonte and his colleagues have examined the effects of mutations in a gene that encodes the leucine-rich repeat kinase 2 (LRRK2) protein on cultured neurons. LRRK2 mutations are responsible for approximately 2% of all inherited and sporadic cases of PD in North American Caucasian populations and up to 20% of all PD cases in Ashkenazi Jewish patients and approximately 40% of all PD cases in patients of North African Berber Arab ancestry. Therefore, the LRRK2 gene product plays a central role in PD pathology.

When iPSCs derived from PD patients who carry LRRK2 mutations, they were differentiated into neurons that were cultured in the laboratory. Cultured neurons from PD patients show profound disruption of the nuclear membrane and this undoes all nuclear architecture, which leads to cell death.

According to Dr. Izpisua Belmonte, “This discovery helps explain how PD, which had traditionally been associated with loss of neurons that produce dopamine and subsequent motor impairment, could lead to locomotor dysfunction and other common non-motor manifestations, such as depression and anxiety. Similarly, current clinical trials explore the possibility of neural stem cell transplantations to compensate for dopamine deficits. Our work provides the platform for similar trials by using patient-specific corrected cells. It identifies degeneration of the nucleus as a previously unknown player in PD.”

Izpisua Belmonte and his colleagues were also able to confirm that these disruptions of the nuclear membrane also occur in brain tissue from deceased PD patients. While it is still unclear if these disruptions to the nuclear membrane are the result of PD or are a cause of PD, Izpisua Belmonte’s lab used gene replacement techniques that were initially developed and perfected in work with mouse ESCs to fix the mutation in the PD patient-derived iPSCs. When they fixed the mutation, the disruptions to the nuclear membrane failed to form. Belmonte thinks that this could open the door for drug treatments of PD patients, although he did speculate as to how a pharmacological agent might mitigate abnormal nuclear architecture.

These results underscore the power of using iPSCs to model genetic diseases. As Belmonte noted, “We can model disease using these cells in ways that are not possible using traditional research methods, such as established cell lines, primary cultures and animal models.”

Another finding that nicely comports with data from clinical observations of PD patients is the tendency for patients to become progressively worse as they age. Likewise, in their cultured neurons differentiated from that were iPSCs derived from PD patients, Belmonte and his group observed progressively greater deformities in the nuclear membranes of the cells as they aged.

“This means that, over time, the LRRK2 mutation affects the nucleus of neural stem cells, hampering [sic] both their survival and their ability to produce neurons. It is the first time to our knowledge that human neural stem cells have been shown to be affected during Parkinson’s pathology due to aberrant LRRK2. Before development of these reprogramming technologies, studies on human neural stem cells were elusive because they needed to be isolated directly from the brain,” said Belmonte.

Belmonte further opined that dysfunctional neural stem cell populations that are afflicted with LRRK2 mutations might also contribute to other health issues associated with this particular form of PD, which includes depression, anxiety, and the inability to smell.

Modeling diseases with iPSCs also has an added bonus, since this model system can effectively recapitulate the effects of aging. Since unique dysfunctions result from aging, there are very few ways to model such events. However, using cultured cells made from iPSCs can bypass this problem, since the age-related pathologies will typically show up in culture.

Protein Induction of Pluripotent Stem Cells Made More Efficient

Clinicians and stem cells scientists have been hopeful but also quite cautious about the use of induced pluripotent stem cells iPSCs in human treatments. One of the primary concerns in the use of viral vectors that insert themselves into the genome of the cells they infect. Such insertions can create activating mutations or insertional inactivation mutations that can transform cells into tumors.

However, scientists at Stanford University School of Medicine have designed a safer way to make iPSCs that is also very efficient. This method is an extension of a protocol that has already been tried; treating the cells with recombinant proteins that can pass through the cell membrane and transform the cells into iPSCs without the use of viruses. Unfortunately, this protocol has proven to be rather inefficient relative to methods that use genetically engineered viruses.

The Stanford researchers discovered that viruses were not simply burrowing into cells to deposit genes. According to John Cooke, MD, PhD, professor of medicine and associate director of the Stanford Cardiovascular Institute and senior author of this work: “It had been thought that the virus served simply as a Trojan horse to deliver the genes into the cell. Now we know that the virus causes the cell to loosen its chromatin and make the DNA available for the changes necessary for it to revert to the pluripotent state.”

The derivation of iPSCs does not require the destruction of embryos. and therefore, offer an ethical alternative to embryonic stem cells (ESCs). Instead of using embryos, iPSCs are made from adult cells that have been genetically engineered to overexpress four different genes (Oct4, Sox2, Klf4 and c-Myc). These four genes are heavily expressed in ESCs and by transiently overexpressing them in adult cells, the adult cells revert to an ESC-like state.

The derivation of iPSCs from adult cells was discovered by Shinya Yamanaka and his colleagues, and Yamanaka won the Nobel Prize for this achievement.

The research of Cooke and his colleagues, however, provides an important clue as to how this reversion to the embryonic state occurs. Cooke noted, “We found that when a cell is exposed to a pathogen, it changes to adapt or defend itself against a challenge. Part of this innate immunity includes increasing access to its DNA, which is normally tightly packaged. This allows the cell to reach into its genetic toolbox and take out what it needs to survive.”

It is this loosening of the structure of DNA in adult cells that allows the pluripotency-inducing proteins to modify the expression pattern of the cell and transform it into an ESC-like cell.

This type of response to viral infections that causes the DNA of cells to loosen up has been termed “transflammation” by Cooke and his team. They think that this finding could easily simplify and increase the efficiency of iPSC derivation.

Cooke’s laboratory initially tried to increase the efficiency of cell-permeable proteins that can reprogram adult cells into iPSCs. These proteins can bind to their target sequences on DNA and can also enter the nucleus when they pass into the cell. Why were these proteins so inefficient when compared to viral-based techniques?

To answer this question, Cooke’s lab examined the gene expression patterns of cells treated with iPSC-inducing viruses or iPSC-transforming proteins. They discovered that the gene expression patterns differed extensively. This led Cooke to hypothesize the virus itself was causing some sort of change in the adult cells that was necessary for iPSC derivation.

To test this hypothesis, they repeated the experiment with recombinant proteins but also concomitantly treated the cells with an unrelated virus. This dramatically increased the rates of pluripotency transformation. The increased rate of transformation was also linked to a signaling pathway called the toll-like receptor-3 (TLR-3) pathway.

Toll-like receptors (TLRs) have been established to play an essential role in the activation of innate immunity by recognizing specific molecular patterns normally found on microbial components. Each TLR recognizes a different set of microbial-specific molecules, and TLR-3 binds to double-stranded RNA molecules. Therefore, these cells activate those pathways that are normally turned when they are infected by viruses.

According to Cooke, “These proteins are non-integrating, and so we don’t have to worry about any viral-induced damage to the host genome.” Cooke also pointed out that cell-permeable proteins can allow the researchers to exert greater amounts of control over the reprogramming process. This, essentially could speed the use of iPSCs in human therapies. Cooke continued: “Now that we understand that the cell assumes greater plasticity when challenged by a pathogen, we can theoretically use this information to further manipulate the cells to induce direct reprogramming.”

Therefore, to sum up, the elimination of TLR3 reduces the efficiency and yield of human iPSC generation, but if TLR3 is activated, it enhances human iPSC generation by cell permeant peptides. Also, TLR3 activation enables changes to the structure of DNA (epigenetic changes), and these changes promote an open chromatin state that makes iPSC generation much more efficient.

Human Neural Stem Cell Line From StemCells Inc. Makes Myelin in Shiverer Mice

Physicians and research scientists at the Oregon Health Science University in Portland, Oregon have used banked neural stem cells to make myelin in mice that have a severe disease that prevents the synthesis and deposition of myelin around nerves. This proof-of-concept experiment shows that such a treatment strategy is feasible for human patients.

In previous posts on this blog site, I have discussed the importance of the myelin sheath that surrounds and insulates certain nerves. I will not reiterate those points here, but simply refer you to those older posts.

In humans, myelin loss is not noticed until the patient begins to show symptoms.  Myelin disorders are quite disabling and even fatal in some cases.  Such disorders include cerebral palsy in children born prematurely, multiple sclerosis, and others.

Myelin loss has also been found to play an important role in age-related senility.  Researchers at the Oregon Health and Science University Doernbecher Children’s Hospital used very advance Magnetic Resonance Imaging to study myelin (white matter) in the brains of adults of all stages.  They discovered that widespread changes in the white matter, and damage to the myelin of the brain were highly correlated with progressive senility (See Black SA, et al., “White matter lesions defined by diffusion tensor imaging in older adults.” Annals of Neurology, 2011 Volume 70, Issue 3, pages 465–476).

Stephen Back and his colleagues at OHSU examined the ability of human stem cells to make myelin and heal the sick animals.  To test this possibility, Back used a mouse called the “Shiverer immunodeficient” mouse, that develops progressive neurological damage because of its inability to make myelin.  Remember that small regions of demyelination that cover particular segments or even patches of nerves are followed by repair, regeneration, and complete recovery of neural function.  However, extensive demyelination or myelin loss is typically followed by degeneration of the axon (the extension of the neuron that conducts nerve impulses to other neurons) and also the neuron cell body.  Neuron death and axonal death are often irreversible.

The use of the Shiverer mouse presented some unique challenges.  Most neural stem cell experiments utilize newborn rather than adult mice.  Back explained:  “Typically, new-born mice have been studied by other investigators because stem cells survive very well in the newborn brain.  We, in fact, found that the stem cells preferentially matured into myelin-forming cells as opposed to other types of brain cells in both newborn mice and older mice.  The brain-derived stem cells appeared to be picking up on cues in the white matter that instructed the cells to become myelin-forming cells.”

Back collaborated with StemCells Inc., to make use of their proprietary neural stem cell line.  His initial experiments showed that implanting these neural stem cells made myelin sheaths in presymptomatic newborn animals.  However, these experiments did not indicate whether or not these stem cells would survive after transplantation into older animals that were already showing symptoms and declining in health.  Black, therefore, wanted to perform a much more difficult experiment by transplanting the neural stem cells into very sick adult animals that showed the horrific symptoms of demyelination.

MRI studies confirmed that implanted neural stem cells did in fact make new myelin within weeks after transplantation.  However, the detection of something such as myelin in mice usually requires the use of dyes of some other agent that fills the thing you want to detect in order to see it.  Many of these Shiverer animals are so sick that they cannot survive MRI experiments.  Therefore OHSU used a very sophisticated piece of equipment to solve this problem:  ultra-high field MRI scanners that could detect myelin without the use of dyes.

Back further explained:  “This is an important advance because it provides proof of principle that MRI can be used to track the transplants as myelin is being made.  We actually confirmed that the MRI signal in the white matter was coming from human myelin made by the stem cells.”

This study is in combination with a clinical study at the University of California, San Francisco that examines the use of this same neural stem cell to myelinate the nerves of children with severe demyelination diseases.  Back’s group provides the crucial pre-clinical work that serves as the foundation for this clinical study.

Back noted:  “These findings provide us with much greater confidence that going forward, a wide variety of myelin disorder might be candidate for therapy.  Of course, each condition varies in terms of severity, how fast it progresses and the degree of brain injury it causes.  This must all be taken into consideration as neurologists and stem cell biologist [sic] work to make further advances for these challenging brain disorders.”

Neural Stem Cell Found in Skeletal Muscle

Scientists at the Wake Forest School of Medicine have more fully characterized a stem cell that was isolated from muscle, but does not differentiate into muscle. Instead, this stem cells expresses several genes normally found in cells that inhabit the nervous system. These cells might serve as a source of material for the treatment of neurodegenerative diseases.

Osvaldo Delbono, professor of internal medicine at Wake Forest University and the senior author of this study said this: “Reversing brain degeneration and trauma lesions will depend on cell therapy, but we can’t harvest neural stem cells from the brain or spinal cord without harming the donor.”

Delbono continued, “Skeletal muscle tissue, which makes up 50% of the body, is easily accessible and biopsies of muscle are relatively harmless to the donor, so we think it may be an alternative source of neural-like cells that potentially could be used to treat brain or spinal cord injury, neurodegenerative disorders, brain tumors and other diseases, although more studies are needed.”

In 2011, Delbono and his colleagues isolated a stem cell from skeletal muscle that expressed several genes that you usually find in very young nervous tissue (the early neural marker Tuj1, light and heavy neurofilament for those who are interested). These cells did not express genes normally expressed in other tissues, such as smooth muscle or blood vessels.

Upon further characterization, the muscle-derived stem cells were able to respond to the neurotransmitter glutamate. This strongly intimates that these stem cells express the types of ion channels normally found in neurons. Also, these neural-like stem cells from muscle were clearly not derived from muscle satellite cells (another muscle stem cell population that produces skeletal muscle in response to muscle injury). Instead this stem cell is  interspersed in between muscle fibers. These cells were also able to proliferate and survive in culture (see Birbrair, et al., PLoS ONE 6(2): e16816. doi:10.1371/journal.pone.0016816).

In this new publication, Delbono’s group isolated muscle-specific neural stem cells from non-human primates and aging mice and injected them into the brain. The injected cells not only survived in the brain, but also migrated the those areas of the brain where neural stem cells are located.

The next issue they addressed was whether or not these stem cells will induce tumors upon injection. Neither stem cells from non-human primates nor those from aged mice produced tumors upon injection into the brain or when injected under the skin.

Alexander Birbrair, a postdoctoral student in Debono’s laboratory and the first author in this paper said, “Right now, patients with glioblastomas or other brain tumors have a very poor outcomes and relatively few treatment options.” Birbrair continued: “Because our cells survived and migrated in the brain, we may be able to use them as drug-delivery vehicles in the future, not only for brain tumors but also for other central nervous system diseases.”

Delbono’s team is also investigating whether these neural-like cells also have the capability to differentiate into functional neurons in the central nervous system.

See Alexander Birbrair, et al., Skeletal muscle neural progenitor cells exhibit properties of NG2-glia. http://dx.doi.org/10.1016/j.yexcr.2012.09.008,

One Embryo – Three Parents?

The web is alive with reports that scientists at the Oregon Health & Science University have managed to make embryos that contained genetic material from two mothers and one father. There has been a certain amount of “creepiness” applied to this experiment, but there are various reasons why this experiment was done. I will fully admit that there is a degree of creepiness to this experiment and the destruction of these embryos is also deplorable. However, this is a strategy to cure some genuinely nasty genetic diseases. Therefore, the research is not for nothing.

Deoxyribonucleic acid or DNA is the molecule all living organisms use to store genetic information, with the exception of some RNA viruses, but there is a debate as to whether or not viruses are actually alive. DNA is housed within the nucleus and is organized into linear molecules of DNA known as chromosomes.

However, there is another compartment in human cells that also houses DNA. The power-generation structure of the cell is called the mitochondrion. Mitochondria are enclosed by two membranes; and inner and outer mitochondrial membrane. There is also an internal network of membranes called cristae. Embedded in the membranes of the cristae are the components of the electron transport chain that are used for energy production.

Directly inside the mitochondrion is a soluble region known as the mitochondrial matrix. Soluble enzymes are found in the matrix as are metabolites and other small molecules. Another large molecule found in the mitochondrial matrix is the mitochondrial genome, which consists of multiple copies of small, circular molecule of DNA.

The mitochondrial genome encodes several genes necessary for the energy production machinery of the mitochondrion. The vast majority of the energy production machinery components are encoded by the nuclear genome, but the small number of mitochondrial components encoded by the mitochondrial genome are crucial for energy production.

Replication of the mitochondrial DNA is accomplished by a DNA replication system that is specific to the mitochondrion.  Unfortunately, this DNA replication system is less accurate than that used in the nucleus.  Therefore, mutations in mitochondrial DNA are relatively common.  Loss of function mutations in mitochondrial genes can compromise the ability of the mitochondrion to make chemical energy, and such mutations have dire consequences for several different organ systems.

The list of genetic diseases causes by mutations in mitochondrial DNA is long.  Here is a short list:

1.  Kearns-Sayre Syndrome – weakness or paralysis of the eye muscles, impaired eye movement and  drooping eyelids, loss of vision, abnormalities of the electrical signals that control the heartbeat, coordination and balance problems, abnormally high levels of protein in the fluid that surrounds and protects the brain and spinal cord, muscle weakness in their limbs, deafness, kidney problems, or a deterioration of cognitive functions (dementia). Affected individuals often have short stature and suffer from diabetes mellitus.

2.  Leber hereditary optic neuropathy – first sign is blurring and clouding of vision, and over time, vision worsens with a severe loss of sharpness and color vision.

3.  Leigh Syndrome – first signs are seen in infancy and are usually vomiting, diarrhea, and difficulty swallowing, eating problem, an inability to grow and gain weight at the expected rate, severe muscle and movement problems, weak muscle tone, involuntary muscle contractions, and problems with movement and balance, loss of sensation and weakness in the limbs.

4. MELAS – mitochondrial encephalomyopathy lactic acidosis, stroke-like episodes – signs and symptoms appear in childhood and may include muscle weakness and pain, recurrent headaches, loss of appetite, vomiting, and seizures. Stroke-like episodes beginning before age 40, and often involve temporary muscle weakness on one side of the body, altered consciousness, vision abnormalities, seizures, and severe headaches resembling migraines.  Strokes can progressively damage the brain, leading to vision loss, problems with movement, and a loss of intellectual function.

5.  MERRF – myoclonus epilepsy and ragged-red fibers – characterized by muscle twitches (myoclonus), weakness (myopathy), and progressive stiffness (spasticity).

6.  MILS – maternally inherited Leigh syndrome – a progressive brain disorder that usually appears in infancy or early childhood.  Affected children may experience vomiting, seizures, delayed development, muscle weakness, and problems with movement. Heart disease, kidney problems, and difficulty breathing can also occur in people with this disorder.

7.  Pearson Syndrome – a fatal disorder of infants with anemia and exocrine pancreatic insufficiency.  It is now known to be a rare, multisystemic, mitochondrial genetic disease, with anemia (low red blood cell count), neutropenia (low white blood cell count), and thrombocytopenia (low platelet count), as well as variable liver, kidney, and endocrine failure. Death usually occurs early in life.

8.  Progressive external ophthalmoplegia – Weakness of the eye muscles, drooping eyelids (ptosis), weakness or paralysis of the muscles that move the eye.  Affected individuals may also have general weakness of the skeletal muscles particularly in the neck, arms, or legs that may be especially noticeable during exercise.

9.  NARP – neuropathy, ataxia, retinitis pigmentosa – Beginning in childhood or early adulthood, numbness, tingling, or pain in the arms and legs; muscle weakness; and problems with balance and coordination; also vision loss learning disabilities, developmental delay, seizures, dementia, hearing loss, and cardiac conduction defects.

None of these diseases sounds terribly pleasant, and there are no known cures or effective treatments for them.

The severity of these diseases depends upon the proportion of the mitochondria that possess the mutated version of the mitochondrial genes.  Typically, mitochondria contain multiple copies of their genomes, and mutant versions of these genomes are mixed with normal copies.  When mitochondria divide, the copies of the genomes are randomly distributed between the two daughter mitochondria.  Therefore, some mitochondria will have mainly copies of the mutant version of the genome while others will have mainly copies of the normal version of the genome.  This condition is called heteroplasmy, and how widely these mutant versions are distributed throughout the body determines the severity of the mitochondrial genetic disease.

Mitochondria are inherited from the mother.  This is due to the fact that the egg, which is supplied by the mother, contains a large quantity of mitochondria, whereas the sperm that fertilizes the egg, only has relatively a few mitochondria.  Therefore, mitochondrial genetic diseases will only be transmitted through the mother, and if a mother is known to have a mitochondrial genetic disease, she will pass that disease onto her children, regardless of the health of the father.

This is the main reason for the technology tested in this paper: Masahito Tachibana, et al., Towards germline gene therapy of inherited mitochondrial diseases, Nature (2012) doi:10.1038/nature11647.  In this paper, scientists from the Division of Reproductive & Developmental Sciences at the Oregon National Primate Research Center in Oregon Health & Science University, used a technique that extracts the nuclear genome from the egg and transplants it into the egg of a donor, after which the egg is fertilized with normal sperm.  This technique would bypass the mitochondrial mutations in the mother’s eggs and replace that genome with a new genome that does not carry such a mutation.

The technique used in this paper is called “spindle transfer.”  This technique takes an oocyte donated by a woman who carries and suffers from a mitochondrial genetic disease and isolates and transplants the chromosomes (nuclear genetic material) from the patient’s unfertilized oocyte into the cytoplasm of another donated, enucleated egg, that contains healthy mtDNA as well as other organelles, RNA and proteins.  Such a child born a result of this spindle transfer procedure will be the genetic child of the patient but will carry healthy mitochondrial genes from the egg of the donor. Prior studies in a monkey model showed not only the feasibility of the spindle transfer (ST) procedure but also that ST is highly effective and completely compatible with normal fertilization and birth of healthy offspring (see Tachibana, M. et al. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461, 367–372 (2009)).  This strategy might have an important future as a therapy to avoid transmission of serious mitochondrial diseases.

In this paper, seven volunteers (aged 21–32 years) donated a total of 106 mature eggs, and 65 eggs were used for the ST procedure and 33 served as non-manipulated controls.  Of the 64 ST eggs, 60 of them survived intracytoplasmic sperm injection (ICSI; 94%) and 44 formed showed the early signs of successful fertilization (73%).  These results were comparable to those found in the non-manipulated eggs; 32 oocytes survived ICSI (97%) and 24 (75%) formed pronuclei .  However, when these embryos were further observed, 48% (21/44) of the ST eggs were normal, but 87% of the non-manipulated embryos were normal.  Therefore, these manipulations can decrease the efficiency of fertilization.

If fertilization occurs normally, the ST embryos seem to be able to form blastocysts as well as the normal controls.  Blastocyst formation rate in the normally fertilized ST group (13/21, 62%) was statistically similar to controls (16/21, 76%).  Embryonic stem cell derivation rates were higher in the normal embryos (56%)  than in the ST group embryos (32%).

This paper uses an ingenious technique to potentially help women with a genetic disease.  That should give us some hope.  However, what I find reprehensible in this paper is the destruction of all these embryos.  These were young human lives that were snuffed out for the sake of convenience.

Wesley Smith at the Human Exceptionalism Blog has a different take on this technique.  Here are his words:  “Also note, that preventing illness is just the key that opens the door to many of these Brave New World technologies. Eventually–given the way things go these days–if the procedure ever becomes doable, it will go quickly from the “medical” to the “consumerist,” e.g., facilitating lifestyle choices and personal preferences.  That’s what happened with IVF, after all, which is no longer restricted to treating the infertile. Indeed, if we ever normalize polyamory, one could see the technique as a way for three partners to have biologially related children.”

Smith has a good point.  However, given the devastating nature of these mitochondrial genetic diseases, it seems to me that using this technique to prevent such horrific diseases from being passed on is a good thing.  However, we should certainly not let this technique be a license into another foray into experimental lifestyles.  Could we use this technique for medical purposes only?  Smith seems to think that the answer to this question is “No.”  I am certainly sympathetic to his caution, but I am also unwilling, at this point, to prevent mothers with these diseases from using this technology to have healthy babies that do not die at a young age.  If there is another way to purge such diseases from the mother’s eggs, then I am all ears, but for now this seems to be the best and only way.

Preliminary Results of Stem Cell Treatment for Stroke Show No Major Side Effects

A collaborative effort between physicians and scientists at the University of Texas Health Science Center in Houston and other centers, has spawned a clinical trial to test a stem cell treatment for stroke patients.

The lead researcher, Sean Savitz, professor of neurology and the director of the stroke program at UT, presented the first results from 10 stroke patients who were treated with stem cells at the World Stroke Congress in Brasilia, Brazil. This clinical trial is the only randomized, double-blind, placebo-controlled intra-arterial clinical trial in the world for ischemic stroke. The goal of this trial is to test the safety and efficacy of a therapy developed by a company called Aldagen Inc. (a wholly-owned subsidiary of Cytomedix Inc.) that uses a patient’s own bone marrow stem cells to treat stroke patients.

In this clinical trial, after a patient has suffered a stroke, the bone marrow stem cells are administered between 13-19 days after the stroke. This therapy, which is known as ALD-401, uses a technology developed and owned by Aldagen to isolate cells from bone marrow that express very high levels of a particular enzyme. This enzyme marks the cells that express it as stem cells. Pre-clinical studies with these isolated cells in mice showed that mice that had suffered from a stroke showed enhanced recovery when given intra-arterial infusions of these stem cells.

All patients infused with these stem cells will be monitored for 12 months after the infusion. The patient’s mental and physical functions will be closely watched, and any side effects from the infusions will be noted and treated.

According the Dr. Savitz, “We have been approved by the Data Safety Monitoring Board (DSMB) to move the study into the next phase, which will allow us to expand the number of sites in order to complete enrollment.”

Since the 10 people treated in this study have not shown any adverse side effects, Savitz wants to eventually enroll 100 patients. According to the submitted protocol for this study, the initial study only placed 10 patients at risk for this untested treatment. Therefore, before more patients could be enrolled in the clinical trial, the Food and Drug Administration had to review the safety data on the first ten patients before more could be enrolled. The FDA has approved the move to the next phase of this clinical trial.

In pre-clinical trials, some of which were conducted at the UTHealth Medical School, bone marrow stem cells promoted the repair of the brain after an ischemic stroke. Savitz and his colleagues induced stroked in rats and measured the amount of oxygen that flowed into the brain by means of Magnetic Resonance Imaging or continuous laser Doppler flowmetry. In rats that made been given injections of bone marrow-derived stem cells, the oxygen flow to the brain was significantly better than in rats that had suffered a stroke but had not been given the stem cell treatments. Savitz’s group also showed that a molecule that dilates blood vessels called nitric oxide was necessary to keep the vessels open and to allow entry of the stem cells into the brain so that they could repair the damage. When Savitz and his group prevented nitric oxide synthesis with an inhibitor called L-NAME, the infused stem cells were unable to enter the brain and fix it, and oxygen flow to the brain tanked. It was the strength of these pre-clinical studies that convinced the FDA to approve this present human clinical trial that tests this same procedure in human patients.

Ischemic strokes result from blood clots in the tiny vessels in the brain, which starves portions of the brain for oxygen, thus killing off brain cells. Stroke is the leading cause of disability in the United States and the fourth most common cause of death, according the statistics provided by the Centers for Disease Control and Prevention in Atlanta, Georgia.

A New Way To Screen Safe Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are derived from adult cells. Genetic engineering techniques that place specific transcription factors into adult cells turn on genes necessary for early development and turn off genes required for terminal differentiation. This reverts the adult cells to a more incipient state, and the become similar to embryonic stem cells.

Because iPSCs can be made from a patient’s own adult cells, they do not have as great a chance of being rejected by the patient’s immune system. Unfortunately, iPSCs show a robust ability to form tumors when transplanted into mice. The ability of iPSCs to cause tumors varies greatly from cell line to cell line and even individual cells within a particular iPSCs cell line vary in their ability to cause tumors. Some iPSCs show a tumorigenicity that exceeds that of embryonic stem cells, but others do not. Is there a way to screen for those cells that are prone to cause tumors and weed them out?

The laboratory of Timothy Nelson at Mayo Clinic in Rochester, MN has devised an ingenious technique that does exactly that. Alyson Smith and colleagues in Nelson’s laboratory used a cancer drug that destroys cancer cells that have acquired so many mutations that they can no longer effectively repair their DNA. Such drugs destroy cells that lack robust DNA repair, and tumor-causing iPSCs have exactly this problem. Thus treatment of the iPSC, with the drug etoposide purges the culture all of the rogue cells and generated and iPSC culture that no longer effectively forms tumors when transplanted into laboratory animals.

This technique does not affect the ability of the iPSC culture to differentiate into various cell types, but it does eliminate the cancer-causing cells and, therefore, the risk of using such a cell line for therapeutic purposes. This is a remarkable find that promises to revolutionized regenerative medicine.

See Alyson J. Smith, et al., Apoptotic Susceptibility to DNA Damage of Pluripotent Stem Cells Facilitates Pharmacologic Purging of Teratoma Risk. Stem Cells Trans Med 2012 vol. 1 no. 10 709-718.

Nanotubes Mediate Functional Switch in Mesenchymal Stem Cells to Cardiac Muscle-like Cells

Stem cell scientists from Dublin, Ireland have exploited the electrical properties of a material that is widely used in nanotechnology to grow cells that can more efficiently regenerate the heart.

In Ireland, heart disease is the leading cause of death. Heart attacks damage the heart muscle, and the adult heart has very little ability to heal itself. Presently, there are no approved methods for repairing damaged heart muscle.

New work from a research team at the Regenerative Medicine Institute (REMEDI) at the National University of Ireland, in collaboration with Trinity College Dublin brought together the skill of materials scientists, biologists and physicians.

Cell-based therapies for heart disease have been the subject of intense research over the last ten years, and there have certainly been some very hopeful clinical trials in the last few years. This new approach, led by Drs. Valerie Barron and Mary Murphy at the REMEDI, capitalized on an observation of carbon nanotubes. Carbon nanotubes are reactive to electrical stimulation. These nanotubes were then used to modify the activity of mesenchymal stem cells from bone marrow.
According to Dr. Barrow, “The electrical properties of the nanomaterial triggered a response in the mesenchymal (adult) stem cells, which we sourced from human bone marrow. In effect, they became electrified, which made them morph into more cardiac-like cells.” She continued: “This is a totally new approach and provides a ready-source of tailored cells, which have the potential to be used as a new therapy. Excitingly, this symbiotic strategy lays the foundation for other clinically challenging areas such as in the brain and the spinal cord.”

Mesenchymal stem cells have a deep history as a source of cells for treating heart attack patients. Mesenchymal stem cells (MSCs) have the capacity to improve the heart if implanted after a heart attack, but the mechanism by which they do this is multifaceted and somewhat mysterious. The therapeutic capacity of MSCs is improved if they are pre-conditioned or genetically modified to survive better in the hostile environment of the heart after a heart attack. However, MSCs have only a very limited ability to differentiate into heart muscle cells, and this is one of the largest limitations MSCs as therapeutic agents for heart attacks.

This new work suggests that MSCs can be shifted into a more heart muscle-like state by means of electrical stimulation. Nanotube-mediated stimulation seems to be even more effective for such a shift, and this work might be the beginning of a new strategy to augment the therapeutic capacities of MSCs for treating heart attacks.

Stem Cell Therapy for Inflammatory Bowel Disease in the Works

Stem cells from umbilical cord blood have the ability to migrate to the intestine and integrate into the tissues. This integration allows umbilical stem cells to contribute to the cell population of the gastrointestinal tract. This biological property of umbilical cord stem cells might make them ideal treatments for inflammatory bowel disease (IBD).

One million Americans have IBDs such as Crohn’s disease or ulcerative colitis. Crohn’s disease can affect the small and large intestine, whereas the ulcerative colitis is usually restricted to the colon (large intestine). Also Crohn’s disease displays patchy lesions whereas ulcerative colitis consists of continuous stretches of inflammation. These disease are characterized by frequent diarrhea and abdominal pain. Patients who suffer from ulcerative colitis also tend to have bloody stools, and if left untreated, the blood loss can be extensive. Ulcerative colitis only affects the upper layer of the large intestine, whereas Crohn’s disease can affect multiple layers of the intestine.

There are no cures for IBDs, but there are drug treatments. In the case of ulcerative colitis, the drug prednisone is used to calm down fulminant outbreaks and then mesalamine (5-aminosalicylic acid) or sulfasalazine are used to maintain the disease in a calm or quiescent state. Mesalamine is present in an oral form marketed as Asacol or Pentasa. The difference between Asacol and Pentasa is in the outer chemical coating, since Pentasa packages its drug in coated microgranules, which enables a prolonged release of the active substance throughout the intestinal tract, from duodenum to the rectum. Therefore Pentasa is more useful for Crohn’s patients. Asacol is a delayed release enteric-coated tablets that releases the active ingredient only in the colon. Mesalamine is also available in an enema form (Rowasa)

If these drugs do not work, biologic treatments such as Infliximab (Remicade), adalimumab (Humira) and Golimumab (Simponi) are commonly used to treat patients with Ulcerative Colitis, but these drugs suppress the immune system and can raise the risk of severe illness. Corticosteroids are also used, but long-term use of these drugs also causes severe side effects.

Thus, if the drugs do not work, the treatment can be as bad as the disease itself. Certainly a treatment that regenerates the bowel is preferable, and a stem cell treatment seems to fit the bill.

In an article in the journal Hepatology, the senior author, Graca Almeida-Porada, a professor at Wake Forest Baptist Medical Center’s Institute for Regenerative Medicine, and her colleagues argue that a special stem cell population known as endothelial colony-forming cells, found in umbilical cord blood and bone marrow and circulating blood, can play a definite role in the treatment of IBDs.

Almeida-Porada said, “These cells are involved in the formation of blood vessels and may prove to be a tool for improving the vessel abnormalities found in IBD.”

In 1997, scientists discovered that these endothelial colony-forming stem cells contribute to the formation of blood vessels in embryos, and adults. This study initiated investigations of the capacity of endothelial colony-forming cells as potential therapeutic agents. Clinical studies have shown that endothelial colony-forming cells can improve reduced blood flow to limbs and can also treat heart disease.

Unfortunately, few studies have examined the ability of endothelial colony-forming cells to home to different organs and integrate into their circulatory systems. Thus, Almeida-Porada wanted to examine the ability of endothelial colony-forming cells to integrate into the intestine. Also, since abnormal blood vessels are a hallmark of IBDs, they might be a potential treatment for IBDs.

In this experiment, fetal sheep at 59-65 days gestation were injected with human endothelial colony-forming cells (EPCs). At 11 weeks gestation, the fetal sheep were examined to determine if the human cells had integrated into the fetal sheep tissue. Researchers found that the infused cells had migrated into the intestine and had made significant contributions to the cell population of the bowel.

According to Almeida-Porada: “The study shows that the cells can migrate to and survive in a healthy intestine and have the potential to support vascular health. Our next step will be to determine whether cells can survive in the ‘war’ environment of an inflamed intestine.”

Interestingly, Almeida-Porada’s team found that endothelial colony-forming cells also colonized the liver of the fetal sheep. Although smaller numbers of cells reached the liver as opposed to the intestine, new strategies might enhance the therapeutic potential for these cells with respect to the liver.

Neural Stem Cells Produce Myelin in Human Clinical Trial

Neurons are the cells in the brain that conduct nerve impulses. Nerve impulse conduction is the result of ion movements across the membrane of neurons, and these ion movements are mediated by ion channels embedded in the cell membranes of the neurons. Neurons consist of a main cell body that houses the nucleus, and two sets of extensions: axons that conduct nerve impulses away from the cell body and dendrites that conduct the nerve impulse toward the cell body. The axons of some neurons are coated with a layer of insulation that increases the speed at which neural impulses are conducted. This insulating layer is called the “myelin sheath,” and damage to the myelin sheath can decrease conductivity through these neurons and decrease nervous system function.

Spinal cord injury damages the myelin sheath that insulated spinal nerves. Also diseases such as multiple sclerosis can damage the myelin sheath of nerves and cause neural degeneration. Drug treatments can only delay the inevitable, but replacing the lost myelin sheaths is one of the holy grail goals of regenerative medicine.

We might be closer to such a goal than previously thought. A Phase 1 clinical trial at the University of California, San Francisco that was sponsored by Stem Cells Inc. has shown that a neural stem cell line can be safely transplanted into the brain of patients who suffer from demyelination diseases. Furthermore, these patients were devoid of side effects from the transplant one year after the procedure. However even more exciting is that these transplanted cells seem to have successfully engrafted into the brains of these patients and have produced new myelin sheaths.

This investigation was designed to determine the safety and preliminary efficacy of implanted neural stem cells and the results are extremely encouraging, according to the principal investigator for this trial, David H. Rowitch, MD, PhD, who is also professor of pediatrics and neurological surgery at UCSF, and chief of neonatology at UCSF Benioff Children’s Hospital and a Howard Hughes Medical Institute Investigator.

The co-principal investigator for this trial, Nalin Gupta, MD, PhD, associate professor of neurological surgery and pediatrics and chief of pediatric neurological surgery at UCSF Benioff Children’s Hospital, said: “For the first time, we have evidence that transplanted neural stem cells are able to produce new myelin in patients with a severe myelination disease.” Gupta continued: “We also saw modest gains in neurological function, and while these can’t necessarily be attributed to the intervention because this was an uncontrolled trial with a small number of patients, the findings represent an important first step that strongly supports further testing of this approach as a means to treat the fundamental pathology in the brain of these patients.”

In this trial, human neural stem cells that had been developed by Stem Cells, Inc., a Newark, California biotechnology company, were directly injected into the brains of four children who had been diagnosed with an early-onset, fatal form of a condition known as Pelizaeus-Merzbacher disease (PMD). PMD is a genetic disease that typically occurs in males and affects brain-specific stem cells known as oligodendrocytes that construct the myelin sheath that insulate the neurons of the central nervous system. Defective oligodendrocytes prevent deposition of a functional myelin sheath and without a myelin sheath the white matter neural tracts are unable to correctly propagate nerve signals. This results in neurological dysfunction and neurodegeneration. Patients with early-onset PMD can neither walk nor talk and also have trouble breathing and undergo progressive neurological deterioration leading to death between ages 10 and 15.

All the PMD children who participated in this clinical trial were given standard neurological examinations and developmental assessments before and after the transplant procedures, which were conducted from 2010-2011. All patients also underwent magnetic resonance imaging (MRI) in order to assess myelin formation.

After the neural stem cells had been transplanted, Rowitch and his collaborators found evidence that the stem cells had successfully engrafted into the brains of the children. There was also indication that they were receiving blood and nutrients from the surrounding tissue and integrating into the brain. Rowitch likens stem cells engraftment to a “plant taking root.” This is a very significant finding because the engrafted cells were not the patients’ own stem cells. The implanted cells were not rejected by the patients.

The MRIs provided another very exciting piece of evidence, albeit and indirect piece of evidence, that the transplanted stem cells had become oligodendrocytes and were producing myelin. According to Rowitch: “There is no non-invasive way to test this definitively, but our MRI findings suggest myelination in the regions that have been transplanted.”

These neural stem cells have the capacity to differentiate into a wide variety of neural cell types. The differentiation of the neural stem cells appears to be greatly influenced by the environment into which the cells find themselves. The sites chosen for the Phase I study were determined by pre-clinical experiments done with animals. Investigators, at Oregon Health & Science University’s Papé Family Pediatric Research Institute published their animal work in the same issue of Science Translational Medicine. Stem Cells Inc’s neural stem cells were injected into mice and differentiated into oligodendrocytes and formed myelin. “The animal study is consistent with the MRI findings from the clinical trial and further supports the possibility of donor-derived myelination in human patients,” said Rowitch.

Dr. Arnold Kriegstein, who is the director of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research at UCSF said: “This is a landmark study for the field. Without such studies in human patients, we won’t really know how transplanted cells behave –whether they disperse or migrate, whether they engraft or degenerate and die, whether immune-suppressing regimens really work or not. It’s only through these investigations that we will be able to refine the necessary procedures and technologies and make progress toward cell-based therapies for this disease and related disorders.”

Another Update on Lance Armstrong

Sports Illustrated and the Wall Street Journal as well as a whole host of newspapers have been running multiple articles about Lance Armstrong’s fall from grace.

One week after the United States Anti-Doping Agency (ASADA) released their bombshell report containing detailed evidence that Armstrong had not only taken performance enhancing drugs (PEDs), but had instigated their use and intimidated his teammates to do so as well, Armstrong was dumped by several sponsors. Nike, Anheuser-Busch, RadioShack, and other sponsors severed their relationship with Armstrong, and Armstrong also stepped down as chairman of his beloved cancer-fighting charity, Livestrong.

The clothing and footwear company, Nike issued a rather harsh statement, citing what they company described as insurmountable evidence that Armstrong had participated in doping and had misled Nike for over a decade. Nike did say that they would continue to support Livestrong and carry Livestrong-branded products.

Armstrong began his relationship with Nike in 1996, and Nike stood by Armstrong even after allegations emerged that he had doped. Since 2000, Nike pushed Armstrong to the forefront as a clean, ethical athlete who trained and raced hard without the benefits of doping. In 2000, Nike aired commercials showing Armstrong taking a blood test in front of reporters. The commercial then included this refutation of doping allegations: “What am I on? I’m on my bike, six hours a day, busting my ass.” It was as public an asseveration as Armstrong could make that he was racing clean and not doping.

Nike has a reputation for standing by its endorsements no matter what. For example, even after the issuance of the USADA report, Nike released a statement that it was standing by Armstrong. Slate Olsen, the general manager of cycling club Rapha North America and a former Nike employee who worked on the Livestrong brand in the 2000s said this about Nike: “We used to joke that they were the untouchables—[Michael] Jordan, Tiger [Woods] and Lance—the upper echelon of Nike. After the bracelets launched around 2004 there was even talk about trying to move the corporate color from Nike orange to Livestrong Yellow.”

Examples of Nike’s corporate fidelity are seen in the case of golfer Tiger Woods and basketball superstar Kobe Bryant. A torrid sex scandal led other companies to dump Tiger Woods as an endorser in 2009, and Kobe Bryant was accused of sexual assault in 2003; even though the charges were eventually dropped. Nike stood by both athletes despite their moral shortcomings.  Nike, however, did dump football star Michael Vick, who pleaded guilty in 2007 to dog-fighting charges and served almost two years in prison. After his sentence, however, Nike re-signed him as an endorser last year, saying, “Michael acknowledges his past mistakes. We do not condone those actions, but we support the positive changes he has made to better himself off the field.”  Nike also did not renew its deal with baseball player Jason Giambi after he admitted to steroid use.

Davie-Brown Entertainment is a member of the Omnicom Group Inc that tracks the status of celebrities as marketing symbols.  Davie-Brown Entertainment uses on-line consumer polls to determine the appeal of celebrities.  Apparently, Armstrong was as sure an investment bet as one could make.  In June 2008, Mr. Armstrong was ranked as the 60th most effective product spokesperson.  He was on par with such advertising luminaries as swimmer Michael Phelps and actor Brad Pitt.  However, as of September 2012, Armstrong ranked 1,410th, which puts him alongside rapper Nicki Minaj and actor Jeff Goldblum.

How did Armstrong beat the drugs tests?  According to former Armstrong teammate Tyler Hamilton in his book “The Secret Race,” It took drug-testing authorities several years and millions of dollars to develop a test to detect EPO (erythropoietin, a hormone that increases the quantity of red blood cells in circulation and therefore, increases aerobic capacity) . . . It took Ferrari about five minutes to figure out how to evade it.”

Dr. Michel Ferrari, an Italian physician, was found guilty of “sporting fraud” and “illegally acting as a pharmacy,” but his convictions were overturned on a technicality.  He remains banned from working with cyclists.  In 2005, Armstrong testified in a US court case that there had been no professional contact between himself and Ferrari since his public break with Ferrari was announced on October 1, 2004.  However, USADA reported that Armstrong paid Ferrari some $210,000 after he had publicly claimed to have severed all ties with Ferrari.  In fact, USADA spoke with 15 professional cyclists, six of whom were former Armstrong teammates, who individually confirmed that Ferrari supervised Armstrong’s doping program.  Financial records obtained by USADA show that Armstrong paid Ferrari over $1 million between 1999 and 2006, which is the time during which Armstrong won his Tour de France titles.  In his 2003 memoir, “Every Second Counts,” Armstrong also wrote, “Michele Ferrari…was a friend and I went to him for occasional advice on training…He wasn’t one of my major advisers.”  The evidence amassed by the USADA investigation shows that this is not true.

Ferrari taught US Postal Service riders how to administer EPO intravenously rather than subcutaneously.  The intravenous form would be less easily detected, since it is degraded as soon as it enters the bloodstream, whereas, subcutaneous EPO is absorbed slowly and broken down slowly.  EPO becomes undetectable within 19 hours after intravenous administration as opposed to 43 hours after a subcutaneous injection.  Another trick to prevent detection of EPO use was to infuse saline into the bloodstream to bring down the hematocrit (i.e., how much space in the blood is occupied by red blood cells).  Since EPO boosts the hematocrit, abnormally high hematocrits are viewed as indications of EPO doping.  Saline infusions artificially lowered the hematocrits of the riders and prevented them from getting pinched for EPO use.  Team physician Pedro Celaya used this technique on Armstrong at the 1998 world championships, according to an affidavit by rider Jonathan Vaughters.

Ferrari also showed Armstrong and his riders how to microdose to ensure that any trace of the drug would disappear quickly.  Testosterone, for example, could be microdosed with a patch or by taking it under the tongue.  Former Armstrong teammate Floyd Landis recalls Armstrong giving him testosterone patches.  Another trick was to take the dose of testosterone at night so that the drug would perform its magic but would be metabolically degraded by the body by the next morning.

In a comment to the Associated Press, Armstrong said, “People are smart.  They will say: ‘Has Lance Armstrong ever tested positive? No.'”  This one assertion in Armstrong’s defense, is not strictly true.  A June 4 1999 letter from UCLA’s Olympic Analytical Laboratory to USA Cycling documents eight of Armstrong’s testosterone tests from 1991 to 1998 (there was a gap in 1997 when he being treated for cancer).  These tests examine the ratio of testosterone to epitestosterone.  Epitestosterone is an inactive epimer of testosterone that results from the biosynthesis of testosterone.  In the vast majority of people, the testosterone to epitestosterone ratio is ~1:1.  A ratio that is greater than one can occur naturally, but a T/E ratio greater far greater than 1 is evidence of testosterone doping.  According to USADA, a ratio of over 4 is evidence of doping, and a sample taken from Armstrong on June 23, 1993 had a T/E ratio of 9.  Three of these samples are at 6, and six of the eight samples are over 4.  The lab director Don Catlin said that two of the tests could not be confirmed, there is no evidence that the other high samples were ever retested.

Armstrong also tested positive for corticosteroids in 1999, when he won the Tour for the first time.  According to Emma O’Reilly, a soigneuse (masseuse) for the US Postal Team and who is not an athlete and has no reason to lie or be jealous of Armstrong’s success, Armstrong secured a backdated prescription for therapeutic steroid cream for saddle sores from the team physician  Dr. Luis Garciá de Moral.  O’Reilly also testified that she had disposed of Armstrong’s used needles, covered his needle marks with makeup, and retrieved pills for his in Spain when Armstrong was in France.  Armstrong sued O’Reilly for libel and a settlement was reached, but O’Reilly paid no money.  Here is a witness with a lot to lose and nothing to gain but grief for telling the truth.

There was another time, when Armstrong tested positive for EPO in 2004, but he told teammate Tyler Hamilton “No worries dude.  It’s all taken care of.”  According to Floyd Landis, Armstrong had told him that he and his team manager Johan Bruyneel made a deal with the International Cycling Union (UCI) to conceal the positive test for a $125,000 donation from Armstrong.  While UCI denies this, testimonies from Armstrong confidants show that this is exactly what happened.

There is nothing about this story that sits well.  The sport of professional cycling has been besmirched, the careers for several professional athletes have been adversely affected, and Armstrong’s life has certainly been ruined.  Armstrong raced at a time when doping was rampant in cycling, and one could make the argument, that he was simply one of many who dopes, but still managed to win.  There is some merit to this argument, but we must remember that not everyone responds to PEDs the same.  Stephen Swart, Armstrong’s Motorola teammate during the 1995 Tour de France told Sports Illustrated that he took EPO at Armstrong’s urging but that “straightaway, it actually made me perform worse.”  Biological differences between people mean that some will respond much better to PEDs than others.  Therefore, it will give some athletes a huge advantage, and others none at all and PEDs will even make some perform worse.  The fact remains that the best way to do sports is or all athletes to race or play cleanly, but making sure that this ideal is achieved is not easy and will continue to be a challenge for years to come.

Platelet-Rich Plasma for Torn or Degenerated Rotator Cuffs?

The Regenexx Stem Cell Blog reports on a study that shows that platelet rich plasma (PRP) injections into torn rotator cuffs is better than placebo.

PRP has been used by the Centeno clinic and several others to treat knee and foot  injuries, but there is less known about the efficacy of PRP on rotator cuffs. In this study patients with rotator cuff problems in both shoulders were given dry needle injections on one side and PRP on the other. The PRP shoulders showed noticeable improvement whereas the dry injected shoulders did not.

Centeno has been using PRP for knee treatments for several years and with some success. If this treatment can also work for shoulders, then this will certainly augment the possible treatment options for patients with rotator cuff injuries.

SCIPIO Clinical Trial Shows Improved Heart Function

In an earlier post, I discussed the initial results of the SCIPIO clinical trial. This clinical trial extracts cardiac stem cells from the upper chambers of a heart attack patient’s heart. The cardiac stem cells are then cultured and reinjected back into the heart. The initial report was published in an internationally acclaimed medical journal called the Lancet.

The results were remarkable. After a heart attack, the heart usually deteriorates and undergoes remodeling. Remodeling consists of an enlargement of the heart, followed by congestive heart failure. However, those patients in the SCIPIO clinical trial who received transplants of their own cardiac stem cells (CSCs) showed significant improvements in heart function over those who received the placebo. Heart attack patients who had received CSC transplants showed shrinkage of their cardiac scars and greater ejection fractions.

Now a follow-up paper by the same research group has extended and confirmed these results.

In this paper, 33 patients were enrolled and of these patients, 20 were treated with their own CSCs and 13 served as controls. CSCs were isolated from each patient by means of a heart biopsy from the right atrial appendage during cardiac arterial bypass graft (CABG) surgery. These cells were harvested and processed during CABG surgery, but the harvesting of the CSCs did not increase the time required for CABG surgery.

CSC-treated patients showed a marked increase in the ejection fraction of the left ventricle. The ejection fraction is the percentage of blood pumped out of a filled ventricle by a heartbeat. After 4 months, the ejection fraction increased from 27.5±1.6% to 35.1±2.4% [P=0.004, n=8]. By 12 months after the procedure, the ejection fraction increased further to 41.2±4.5% [P=0.013, n=5]. This means that the efficiency with which blood is pumped by the heart increased after CSC infusions.

Secondly, the size of the infarct size was measured by a technique called “late gadolinium enhancement.” Gadolinium is a somewhat rare metallic element that us very useful because it is sensitive to electromagnetic resonance. Traces of it can be injected into the body to enhance the MRI pictures. Gadolinium also nicely outlines the noncontracting areas of the wall of the heart. MRIs after Gadolinium infusions revealed that the heart scars in those patients that had received CSC infusions had shrunk by -6.9±1.5 g [-22.7%] at 4 months [P=0.002, n=9] and -9.8±3.5 g [-30.2%] at 12 months [P=0.039, n=6]. The regions of the left ventricle (the main pumping chamber of the heart) that were dead also shrunk in CSC-treated patients. The dead regions shrunk -11.9±2.5 g [-49.7%] at 4 months [P=0.001] and -14.7±3.9 g [-58.6%] at 12 months [P=0.013]. Likewise, the total living mass of the left ventricle increased in CSC-treated patients: +11.6±5.1 g at 4 months after CSC infusion [P=0.055] and +31.5±11.0 g at 12 months [P=0.035].

This study confirms and extends the results of the initial report of SCIPIO. The isolation of CSCs during heart surgery is feasible and does not adversely affect CABG surgery. Also cardiac MRIs also reveal that CSC infusion produces a striking improvement in both global and regional function of the left ventricle. The CSCs also cause a reduction in infarct size, and an increase in viable tissue. These benefits persist for at least 1 year and are completely consistent with cardiac regeneration as a result of CSC infusion.

This is great news for heart attack patients.

See: Chugh AR, Beache GM, Loughran JH, Mewton N, Elmore JB, Kajstura J, Pappas P, Tatooles A, Stoddard MF, Lima JA, Slaughter MS, Anversa P, Bolli R. Administration of Cardiac Stem Cells in Patients With Ischemic Cardiomyopathy: The SCIPIO Trial: Surgical Aspects and Interim Analysis of Myocardial Function and Viability by Magnetic Resonance. Circulation. 2012 Sep 11;126(11 Suppl 1):S54-64.

Stem Cells Made from the Brains of Cadavers

Shinya Yamanaka won the Nobel Prize this year for his discovery that the application of genetic engineering to adult cells can revert them into embryonic-like stem cells. Such cells, known as induced pluripotent stem cells (iPSCs) are made from adult cells when four different genes are transiently expressed in an adult cell. These four particular genes are all transcription factors that bind to DNA and activate the transcription of particular genes that are necessary for the acquisition of the embryonic state. Once they are derived, iPSCs can grow in culture and differentiate into any adult cell type, although the efficiency with which they do this is very cell line-dependent.

The discovery of iPSCs gave new hope to the notion that patient-specific stem cells could be used to treat genetic diseases. However, a research group has actually managed to extract live cells from dead bodies and use those cells to derive iPSC lines.

Fibroblasts are cells found in connective tissue, and they are very common in the skin and brain. Fibroblasts can be collected from cadavers and subjected to a protocol that will convert them into iPSCs. These reprogrammed stem cells can differentiate into a multitude of cell types, including the neurons found in the brain and spinal cord. Because microorganisms can colonize the body and degrade it after death, such a culturing process is tricky to carry out successfully.

Scientists from the laboratory of Thomas Hyde at the Lieber Institute for Brain Development, Johns Hopkins Medical Campus in Baltimore, Maryland have used fibroblasts from scalps and the linings that surround the brain (dura later) from 146 human brain donors and used them lake iPSCs.

Those cells extracted from the dura mater were 16 times more likely to grow successfully than those from the scalp. Hyde explained that he expected this disparity in growth potential since the scalp is prone contamination with bacterial and fungi after death. Such contaminants can inhibit the growth of fibroblasts in the laboratory.

However, scalp cells grew more rapidly than dura mater cells. “Since the skin is constantly renewing, while the turnover in dura mater is much slower,” such a result makes sense, explained Hyde.

The derivation of iPSCs from cadavers might play an important role in developing future stem cell therapies. Cadavers can provide brain, heart and other tissues for study that researchers cannot safely obtain from living people. The derivation of iPSCs from cadavers provides scientists with an excellent source of material for research that cannot be obtained elsewhere. As noted in the paper, “These tissues may be accessible through existing brain tissue collections, which is critical for studying disorders such as neuropsychiatric diseases.”

“For instance, we can compare neurons derived from fibroblasts with actual neurons from the same individual,” Hyde said. “It tells us about how reliable a given method for deriving neurons from fibroblasts is. That can be crucial if, for example, you want to create dopamine-making neurons to treat someone with Parkinson’s disease.”

Also using iPSCs made from patients who died from developmental abnormalities can greatly enlighten scientists on those maladies that are due to malfunctions in development.

“We’re very interested in major neuropsychiatric disorders such as schizophrenia, bipolar disease, autism and mental retardation,” Hyde said. “By understanding what goes wrong with the brain cells in these individuals, we could perhaps help fix that.”

Citation: Bliss LA, Sams MR, Deep-Soboslay A, Ren-Patterson R, Jaffe AE, et al. (2012) Use of Postmortem Human Dura Mater and Scalp for Deriving Human Fibroblast Cultures. PLoS ONE 7(9): e45282. doi:10.1371/journal.pone.0045282

Induced Pluripotent Stem Cell Transplant Claims Debunked

When this report first appeared, it seemed too good to be true and it turns out it probably was. Nobel Laureate Shinya Yamanaka at Kyoto University announced his remarkable discovery of induced pluripotent stem (iPS) cells in 2006. However, another Japanese researcher, Hisashi Moriguchi, made an even more earth-shaking claim earlier this year. Moriguchi, who was a visiting researcher at the University of Tokyo, claimed to have modified iPS technology to treat a person with terminal heart failure. The patient was allegedly surgically treated in February, 2012, according to a front-page article in the Japanese newspaper Yomiuri Shimbun. The article also said that the patient was healthy. If this was true, this would certainly be an earth-shaking result. An unidentified head of a Tokyo-based organization devoted to helping children with heart problems, told Yomiuri Shimbun, “I hope this therapy is realized in Japan as soon as possible.”

The Nippon News Network had posted a video of Moriguchi presenting his research at the New York Stem Cell Foundation, but they have since removed this video.

Unfortunately, once the journal Nature was altered to this report, they contacted Harvard Medical School and Massachusetts General Hospital (MGH), where Moriguchi claimed to have performed this work. Both institutions denied that Moriguchi had even done such a procedure. According David Cameron, a spokesperson for Harvard Medical School, “No clinical trials related to Dr Moriguchi’s work have been approved by institutional review boards at either Harvard University or MGH.” Likewise, the public affairs officer for MGH, Ryan Donovan, said “The work he is reporting was not done at MGH.”

There are other problems with Moriguchi’s work. Moriguchi reported that he had invented a method to reprogram cells using just two chemicals: a small molecule that inhibits a small RNA called “microRNA-145” and another molecule that binds the TGF-β receptor. However, a University of Tokyo stem-cell researcher, Hiromitsu Nakauchi, said that he has never “heard of success with that method.” Nakauchi even said that before this week he had never heard of Moriguchi.

Another bizarre claim made by Moriguchi was that he could differentiate iPS cells into heart muscle cells by utilizing a ‘supercooling’ method that he had invented. Nakauchi said that this was “another weird thing.”

Moriguchi never published his technique in a peer-reviewed journal, but in a book about advances in stem-cell research (see Moriguchi, H., Mihara, M., Sato, C. & Chung, R. T. in Embryonic Stem Cells — Recent Advances in Pluripotent Stem Cell-Based Regenerative Medicine (ed. Atwood, C.) 359–370 (InTech, 2011)). In this book, there are paragraphs copied almost verbatim from other papers. For example, a section under the heading “2.3 Western blotting” is identical to a passage from a 2007 paper by Yamanaka (see Takahashi, K. et al. Cell 131, 861–872 (2007)). Furthermore, section 2.1.1 describes human liver biopsies but the information in this section matches the number of patients and timing of specimen extractions described in an earlier article, but the name of the institution has been changed (see Thenappan, A. et al. Hepatology 51, 1373–1382 (2010)).

Nature contacted Moriguchi and he stood by his publication. He told Nature, “We are all doing similar things so it makes sense that we’d use similar words.” However, he did admit to using other papers “as reference.”

With respect to his reported supercooling technique, Moriguchi cited a paper of his own in Scientific Reports, which is published by the Nature Publishing Group. Nature, however, noted that this paper describes supercooling of human ovaries for preservation (Moriguchi, H., Zhang, Y., Mihara, M. & Sato, C. Sci. Rep. 2, 537 (2012)). The paper has nothing to do with the differentiation of iPS cells into cardiac cells. Moriguchi said that a journal referee had recommended that he leave the latter experiment out of the paper “because it’s basically the same technology”.

Moriguchi said that he did most of the contentious work himself, including safety research in pigs. However, the initial surgery and some of a further five similar procedures in other patients that took place from August onwards, and while, according to Moriguchi, other researchers were supposedly involved in some of these procedures, he would not provide any names.

Where did Moriguchi acquire the surgical expertise to perform these procedures? Moriguchi initially told Nature that earned a medical degree at the Tokyo Medical and Dental University, and that he learned surgery there. However, in his later conversation with Nature, Moriguchi said that he has a nursing degree from the institution and not a medical degree.

The University of Tokyo confirmed that Moriguchi held a position there from 2006 to 2009, during which he studied “medical economics” and “evaluation of clinical technologies.” Currently, he is a visiting researcher at the university, working in the laboratory of Makoto Mihara in the university hospital’s cosmetic-surgery section, where, according to a secretary, he “comes in once or twice a week.”

Moriguchi also claimed to have a laboratory at MGH and Harvard Medical School, but these institutions only confirmed that Moriguchi was a visiting fellow at MGH in 1999–2000, but he has not been associated with the hospital or the medical school since then.

Nature asked Moriguchi who had funded his iPS cell procedures and where they had been carried out, where his ethical review had taken place and which good manufacturing practice (GMP) facility had produced the necessary clinical-grade iPS cells, Moriguchi referred again to MGH and Harvard Medical School, but he could not name the head of the ethical review board or any contacts at the GMP facility.

Jerome Ritz, co-director of the Connell O’Reilly Cell Manipulation Core Facility at Harvard Medical School, told Nature, “We have not produced any iPS cells for any patients in our facility. I can’t imagine what other facility might have produced these cells.”

What do we have? We have a Japanese researcher who is a liar and who has as much of a problem telling the truth as Barak Obama. This clinical trial clearly never happened and Moriguchi should be banned from further stem cell work.

Human Neurons Derived from Adult Brain Cells

A research group from Mainz, Germany have discovered a protocol that can reprogram a particular type of brain cell from human brains into new neurons.

Within the brain, neurons are the cells responsible for nerve impulses. Learning and memory, personality, volition and responses to stimuli are functions of neurons. When large numbers of neurons die, the patient suffers and their memory leaves them, their personality changes, or worse. Neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease cause the death of large numbers of neurons and it is the death of neurons that is responsible for the symptoms of disease like these.

Benedikt Berninger, a faculty member of the Institute of Physiological Chemistry, at the Johannes Gutenberg University Mainz, Germany, and the senior author of this research said, “This works aims at converting cells that are present throughout the brain but themselves are not nerve cells into neurons. The ultimate goal we have in mind is that this may one day enable us to induce such conversion within the brain itself and provide a novel strategy for repairing the injured or diseased brain.”

The cells used by Berninger’s laboratory are known as “pericytes.” Pericytes are found in close association with blood vessels and are important in maintaining the blood-brain-barrier. Pericytes have also been shown to play a role in wound healing in other parts of the body.

Berninger chose pericytes for his research because he wanted to “target these cells and entice them to make nerve cells,” so that he and his research team could “take advantage of this injury response.”

When the converted neurons were subjected to further tests, they produced the normal types of electrical-chemical signals usually found in neurons, and also extended their connections to other neurons. This provided evidence that the converted cells could integrate into neural networks.

In their paper (Karow, et al., Cell Stem Cell 2012 11(4): 471), Berninger’s team write, “While much needs to be learnt (sic) about adapting a direct neuronal reprogramming strategy to meaningful repair in vivo, our data provide strong evidence for the notion that neuronal reprogramming of cells of pericytic origin within the damaged brain may become a viable approach to replace degenerated neurons.”

Making Thyroid Tissue from Stem Cells

Belgium scientists from the Universite’ Libre de Bruxelles have developed a novel recipe that coaxes stem cells to efficiently form thyroid tissue. Such a finding could be used potentially to treat those patients who have defective thyroid function or suffer from abnormal thyroid gland development.

This research was led by Sabrina Costagliola, who used mouse stem cells for her model system.

The thyroid gland is located just over the voice box (larynx), and it synthesizes and releases thyroid hormone into the bloodstream. Thyroid hormone is the central regulator of basal metabolic levels, and patients who suffer from insufficient thyroid hormone levels tend to gain weight, are lethargic, and lack energy. Thyroid insufficiency tends to run in families and as people age, the effects of thyroid insufficiency tend to take their toll.

Thyroid hormone requires the incorporation of iodide, which is the main reason your mother always told you eat your fish. You need iodide to make thyroid hormone, but you can also get iodide from eating kelp or other types of foods.

1/3,000 babies is born with some form of thyroid insufficiency. Because thyroid hormone is essential for development of the brain, babies with an underactive thyroid are affected by irreversible mental retardation, and life-long hormonal treatment regime is necessary to maintain proper metabolic levels and normal growth patters.

To convert stem cells into thyroid tissue, Costagliola and her research team targeted two important thyroid-specific genes. By transiently expressing these genes in undifferentiated stem cells, they were able to differentiate these cells into “thyrocytes,” which are thyroid gland precursors.

In the next step, Costagliola and her team transplanted these thyrocytes into mice that lack functional thyroid gland. Four weeks after transplantation, the researchers noticed that the recipient mice showed normal levels of thyroid hormone and no longer had any symptoms of thyroid insufficiency.

These results are remarkable, but they are show that Costagliola and her team have designed an excellent model system to study and characterize thyroid development. Secondly, Costagliola’s model system might provide a therapeutic strategy to replace the thyroid gland of patients who have had their thyroid glands extirpated as a treatment for thyroid cancer.

The next step for Costagliola and her team is to replicate this research with human embryonic stem cells, and then with human induced pluripotent stem cells to determine if this protocol is feasible as a treatment regime for human patients.

EPO Improves the Efficiency of EPC Treatment After a Heart Attack

I have just written about the abuse of EPO (erythropoietin) by the professional cyclist Lance Armstrong. However, commercial EPO has legitimate medicinal uses. It is given to cancer patients who have very low red blood cell counts after chemotherapy, and to those who suffer from chronic anemia. EPO might also increase healing after a heart attack, and several experiments seem to provide evidence for this conclusion,

EPCs are stem cells programmed to become blood vessels. They repair blood vessels and make new ones. When EPCs are transplanted into an animal or human heart after a recent heart attack, they make new blood vessels, which help feed and repair the damaged heart muscle, and improve circulation of the heart in general. The problem with transplanted EPCs is that the vast majority of them die soon after transplantation.
EPO, however, has the useful capability of prevent cell death. EPO activates several signaling pathways inside the cell that increases cell survival and viability. Therefore, a Chinese group has examined the ability of EPO to increase the survival and therefore the utility of EPC treatment after a heart attack in laboratory animals.

Yan Cheng and colleagues at Jinling Hospital at Nanjing University School of Medicine in Nanjing, China used mice labeled with a gene that makes their cells glow. They isolated bone marrow from these mice and teased from this bone marrow EPCs, which is not as easy as it sounds. Then they induced heart attacks in a different mouse strain whose cells do not glow. Into one group, they transplanted 50,000 glowing EPCs. Into another mouse group, they transplanted only buffer as a control. Into the third group, they transplanted 50,000 EPCs and gave 20 units of EPO. Into a fourth group, they gave only 20 units of EPO. All cells were transplanted with a small needle, directly into the heart muscle.

The mice were assessed in several ways. The cells were viewed in the hearts of the mice to determine if they had survived. Mice were also given an electrocardiogram to determine if the electrical activity of the heart was normal. Hearts were also assayed to determine is the number of blood vessels increased and if the heart scar changed during the course of treatment. Finally, mice were checked to see how many cells had died in the hearts of the mice.

28 days after transplantation, no detectable EPCs were seen in those mice that only received EPCs. Apparently, all the transplanted cells have died. Those mice that had received EPO and EPCs, had detectable EPCS in their hearts after 4 weeks. Also, the EPC + EPO-treated mice had far more EPCs in their hearts after 7 days and the death of those cells was much less precipitous than in those mice treated only with EPCs and no EPO.

Another group of chemicals measured in the hearts of these animals are those that summon stem cells to the heart. 7 days after the transplantations, the EPO + EPC-treated mice had more of those stem cell-summoning chemicals in their hearts, but by 28 days after transplantation, those differences had disappeared. Also,

ECGs of mouse hearts confirmed that those mice treated with stem cells had healthier hearts than those who did not, but the mice that received EPO + EPCs had even better ECGs than the rest. This shows that the functional recovery of those mice that had received EPCs and EPO was accelerated relative to the other mice.

Finally, the level of scarring was decreased in all categories of mice relative to the controls, but the EPO + EPC-treated mice had the lowest amount of heart scarring. This demonstrates the ability of EPO to augment EPC therapy after a heart attack in laboratory mice.

This study suggests that there is a synergy between EPO and EPCs in healing a heart after a heat attack. EPCs have been used experimentally in human heart attack patients, but little has been done with EPO in heart attack patients in humans. Various commercial types of EPO have been dropped from the market because of their tendency to induce heart attacks. Increasing the red blood count sometimes increased the blood volume and that can be too much for heart patients, and high blood pressure and congestive heart failure are two possible side effects of erythropoietin. In kidney patients, high dose erythropoietin can prompt to onset of kidney failure. However, at the dosages of EPO used in these experiments, it is doubtful that this will be an issue. 20 units of EPO for a 22-28-gram mouse is less than the 300 units/kg body weight 3 times weekly dosage for clinical purposes. Therefore, it seems unlikely that this treatment would produce the side effects of continued high-dose EPO.

Therefore, we have a strategy for heart attack patients that seems to work well in mice. Further pre-clinical work is required, but should this pan out, human trials will hopefully evaluate this strategy in human patients dome day soon.

Update on Lance Armstrong Doping Case

The United States Antidoping Authority (USADA) released a 202-page report this Wednesday that includes testimonies from 11 former teammates of Lance Armstrong. The report is a blistering indictment of Armstrong and states that he was at the center of “a massive team doping scheme, more extensive than any previously revealed in professional sports history.” This report is the result of an extensive investigation that took years of gum-shoe-type work and examined the methods behind the success of one professional cycling’s greatest teams.

USADA issued this report in response to requests from the Swiss-based Union Cycliste Internationale (UCI) that they explain their decision to ban Mr. Armstrong from professional cycling in August. UCI is professional cycling’s international governing body, and they simply wanted an explanation of the charges USADA brought against Armstrong.

The USADA report cited testimony from former teammates of Mr. Armstrong on the U.S. Postal Service cycling team, including George Hincapie, Floyd Landis, Levi Leipheimer, Christian Vande Velde, Jonathan Vaughters and David Zabriskie. Each one of these riders have admitted taking banned substances.

The report includes very detailed events recounted by Armstrong’s former teammates. For example, Jonathan Vaughters, who is a very respected individual in American cycling and was a teammate of Mr. Armstrong, recounted an event that occurred one evening in Mr. Armstrong’s hotel room in Spain during the 1998 season. According to Vaughters, he watched while Lance Armstrong injected himself with a syringe used for EPO (erythropoietin) injections. After injecting himself, Armstrong said to Vaughters, “Now that you are doing EPO too, you can’t go write a book about it.” After this, Armstrong was open with Vaughters about his EPO use.

EPO is a hormone that is made by the kidney in response to low oxygen content in the blood. EPO boosts the production of red blood cells. Athletes use EPO to augment the number of red-blood cells in circulation, which gives them a competitive advantage in aerobic sports. EPO is banned in cycling and most other sports.

Vaughters was not the only Armstrong teammate to detail doping by Armstrong, George Hincapie, Armstrong’s close friend and teammate during all of his Tour de France wins, issued a statement on Wednesday in which he broke his silence and confessed to doping while competing professionally. He also acknowledged that he had provided testimony to investigators.

Another former Armstrong teammate, Levi Leipheimer, admitted to doping in a letter to The Wall Street Journal. Leipheimer also said that a doping culture was so ingrained in cycling during his time as a professional athlete that he believed cycling to be “a sport where doping was so accepted that riders from different teams—who were competitors on the road—coordinated their doping to keep up with other riders doing the same thing.”

The USADA report presented evidence that Armstrong and his associates had organized a large, organized network for doping, Armstrong “acted with the help of a small army of enablers, including doping doctors, drug smugglers, and others within and outside the sport and on his team. However, the evidence is also clear that Mr. Armstrong had ultimate control over not only his own personal drug use, which was extensive, but also over the doping culture of his team.”

Even though he could not be reached for comment, Armstrong continues to steadfastly deny that he ever doped during his career. Armstrong’s lawyer, Tim Herman leveled some fairly heavy criticism at the USADA report: “USADA has continued its government-funded witch hunt of only Mr. Armstrong, a retired cyclist, in violation of its own rules and due process.”

Herman also pointed our that Armstrong has passed 500 to 600 drug tests over the course of his career. However, the USADA report, however, also detailed the different tactics used by the riders to beat the French police and drug testers. Riders would bury drugs in the woods to hide them from police. They would also dump them off a ferry, and even text one another to warn of surprise visits from drug testers. Other strategies included injecting EPO into their veins instead of under their skin. This way the drug would leave their bodies faster, thus decreasing the chance of a positive test for EPO. Other EPO users diluted their blood with saline injections to mask the effects of EPO when drug testers drew blood to test their hematocrits (red blood cell count).

The team director, Johan BruyneeI told riders that if a drug tester arrived at the hotel room after they had just taken some EPO, that they should not open the door. USADA has accused Bruyneel of trafficking and administering prohibited substances to cyclists, and working actively to conceal rule violations.

Of particular interest in this USADA report is evidence that the relationship between Mr. Armstrong and Michele Ferrari did not end when Armstrong said it did. Dr. Ferrari is an Italian doctor who has been associated with some of cycling’s most notorious doping cases. Ferrari was formally accused by USADA in June of engaging in an a doping conspiracy in which he doped the US Postal team so that they would win the Tour de France.

Armstrong had made public statements in October 2004 that he had formally severed his relationship with Ferrari. He had acknowledged that Ferrari had been his trainer up until that time. However, the USADA report shows that bank statements demonstrate that Armstrong was still making payments to Ferrari after he had announced that he was no longer dealing with Ferrari. Bank records document payments from Armstrong to Dr. Ferrari’s Swiss company, starting in 1996 and most recently in 2006 that exceed $1 million.

In an affidavit from another former Armstrong teammate, Christian Vande Velde, Armstrong criticized him because he did not follow Dr. Ferrari’s program. Mr. Vande Velde said the conversation ensured him that the only way to escape Armstrong’s doghouse was to get fully on board the doping program.

Much of this report either corroborates earlier suspicions about Armstrong or introduces new information that suggests that the doping situation on Armstrong’s teams were far worse than thought before, Armstrong is painted as a bit of a bully who would intimidate cyclists into doping, otherwise they would be cut from the team. The picture is not a pretty one, and it seems more than likely that Armstrong doped and that professional cycling is fraught with a doping culture that resists being cleaned up.