Breakthroughs on the Brink: Turning the Tide on MS

By Patrick Perry

Richard Burt, M.D., chief of immunotherapy for autoimmune diseases at Northwestern University’s Feinberg School of Medicine, and his research team appear to have reversed the neurological dysfunction of early-stage multiple sclerosis patients by using the patients’ own adult stem cells, thereby “resetting” their immune systems.

In May one of the study participants, Edwin McClure, walked across the stage to receive his degree after completing a rigorous graduate program at Virginia Commonwealth University. The young man appeared strong, healthy, and confident.

The scene was in stark contrast to four years earlier when the high school star football player was battling a severe cold, fatigue, and inexplicable visual changes.

“It was like someone turned down a dimmer switch,” he recalls. “My mom thought the problems were due to sinus pressure and would eventually go away, but when I got over the cold and still had difficulty seeing, she took me to an optometrist.”

When nothing surfaced during visits to an optometrist and an ophthalmologist, McClure was referred to a neurologist for follow-up.

After a series of tests and an MRI scan, the doctor delivered the diagnosis - multiple sclerosis (MS). The visual changes the young man was experiencing were due to optic neuritis, an inflammation of the optic nerve that occurs in approximately 50 percent of patients with the disease.

McClure was placed on steroids and interferon injections?-?a regimen that successfully controlled symptoms for two years. But when the MS started to break through, his physician switched to another medication.

“Over the course of four months, I started to develop an allergic reaction to the drug,” McClure says. “Meanwhile, my disease was still progressing.”

McClure was at a crossroads: begin medications with significantly greater risk of side effects or, as his neurologist suggested, investigate a promising clinical trial underway at Northwestern University in Chicago.

He chose the latter, qualified, and enrolled in Dr. Burt’s study. McClure was one of the 21 patients in the trial, ages 20 to 53, who had relapsing-remitting MS for an average of five years and had not responded to at least six months of treatment with interferon beta. After an average follow-up of three years posttreatment, 17 patients (81 percent) improved and none got worse, according to Dr. Burt, whose findings were published in the March issue of The Lancet Neurology.

Resetting the Immune System

“The concept is that your immune stem cells - your blood stem cells - could be used to regenerate a new immune system in virtually any autoimmune disease,” Dr. Burt tells the Post. “If we treated patients in the early relapsing-remitting phase of MS who were experiencing frequent acute attacks despite the use of interferon, patients got better. Six months after the procedure, they were even better. By two years, they seemed to have reached their peak improvement in neurological function. Most people tend to be early- to mid-range in their disability, and that’s when this therapy is really effective. But if you treat MS in a later stage, called secondary progressive MS, it doesn’t really help. In this stage, patients experience a steady worsening of irreversible neurological damage.”

In the procedure, Dr. Burt and colleagues first push immune stem cells from the bone marrow into the blood by using a growth factor and a drug called Cytoxan (cyclophosphamide). Ten days later, they harvest cells from the blood via catheter. The cells are then separated, frozen, and cultured to ensure that none are contaminated with bacteria during the process. Next, the patients are treated with drugs to inhibit the old immune system, and then the frozen stem cells are thawed and infused back into the patients to make a new immune system.

Reversing the Tide

“I started to feel improvement while I was in the hospital,” McClure says. “I realized that I didn’t need my glasses to see. At home my parents noticed that my balance was improving and that I didn’t seem as fatigued as before. Honestly, these changes started within the first month after coming home. My life continued to improve. By the third month, I was actually going to the YMCA to exercise.”

Three years after treatment, McClure remains off medication and now experiences no symptoms of MS.

Like McClure, the majority of trial participants experienced benefits.

“We’ve seen patients who have had marked improvement in symptoms,” notes Dr. Burt, principal investigator of the clinical trial. “Your nervous system controls everything, so the part of the brain attacked by MS determines where you have a problem. Some patients had trouble walking - falling down and having to hold on to things - but after the procedure, they had marked improvement. Others had issues with incontinence, and that’s gone away. If you’re worried about incontinence, that’s quite remarkable. Numbness, tingling, inability to feel things, visual problems - blurred and double vision - can all reverse. Basically, any type of deficit can reverse.

In some patients, we actually had complete reversal - everything went away, and they were completely normal in all functional exams. In others, symptoms never completely reversed, but improved dramatically.”

The study participants are also off all conventional disease-modifying medications now used to slow the rate of disease progression.

While the small trial is only a first step, the results offer a completely new way to treat MS. “This is the first time in the history of any therapy used to treat MS where it actually reverses neurological deficit,” stresses Dr. Burt.

“All other therapies were studied or approved for their ability to slow the rate of progression - in terms of clinical deficits or MRI load of lesion burden - but nothing has, up to this time, reversed deficit. That’s what’s exciting. However, I want to stress that we cannot say it is a cure and current results with three years of follow-up are encouraging.”

Dr. Burt and colleagues are enrolling patients in a larger trial to test the procedure in a randomized setting. “If the results of the trial hold up, I believe it will help open the door for it to be accepted as standard therapy,” adds Dr. Burt.

At present, clinical trials are underway at the University of Calgary in Canada, the University of Sao Paulo in Brazil, and at Northwestern University. If interested in learning more about the trial, e-mail d-spahovic@northwester.edu.

A Different Approach

After undergoing conventional therapy for MS for several years, Fort Worth police sergeant Preston Walker learned about a new treatment for autoimmune disorders. Researchers were utilizing adult stem cells derived from cord blood at The Institute of Cellular Medicine in Costa Rica. Walker inquired about the potential of the treatment for multiple sclerosis.

“We knew that if the treatment worked, the potential benefits for multiple sclerosis patients could be limitless,” says Walker.

Dr. Neil Riordan, CEO of the Institute, suggested a therapy under consideration - using stem cells derived from a patient’s fat tissue. In May 2008, Walker flew to the clinic where doctors removed samples of his abdominal fat through a mini-liposuction, drawing out stem cells, which were later re-injected. According to Dr. Riordan, Walker and a colleague were the first to undergo this treatment protocol. “My quality of life has improved significantly,” Walker told the Post. “The problems with depression, fatigue, and balance have been corrected. I feel really good.”

In June 2009, Walker, who continues to take Avonex as a maintenance drug, plans a return trip to Costa Rica for a “tune-up,” as he puts it. “I’m curious to see if they can further improve my cognitive abilities.”

Control of pathological immunity in multiple sclerosis may be accomplished (at least in part) by antigen-specific vaccination, by administration of immune modulators such as Interferon Beta, or by depletion of activated effector T cells using antibodies. 

Immune modulation by hormones offers a new method of addressing multiple sclerosis.  For example, it is known that mesenchymal stem cells have therapeutic effects in animal models of multiple sclerosis, and that these effects seem to be mediated both by immune modulaton but also by stimulation of regeneration.  Interestingly, hormones such as progesterone have been demonstrated to stimulate immune modulatory activities of mesenchymal stem cells.

A recent paper (Theil et al. Suppression of Experimental Autoimmune Encephalomyelitis by Ghrelin. J Immunol 2009 Jul 20) described the ability of the “hunger hormone” ghrelin to inhibit the mouse model of multiple sclerosis, experimental allergic encephalomyelitis (EAE).

Ghrelin is a hormone made by the pancreas and stomach cells that stimulates the feeling of hunger.  It is also known to stimulate growth hormone release.  Some have compared ghrelin as the opposite of leptin, a hormone known to inhibit hunger.  Interestingly leptin has been associated with induction of inflammation of autoimmunity.  For example, administration of leptin has been demonstrated to augment mouse multiple sclerosis (Matarese et al. Leptin potentiates experimental autoimmune encephalomyelitis in SJL female mice and confers susceptibility to males. Eur J Immunol. 2001 May;31(5):1324-32).

In the paper we are discussing, EAE was induced in B6 mice by administration of MOG peptide (myelin oligodendrocyte glycoprotein 35-55) and treated groups were administered ghrelin after immunization with the autoantigen.  As compared to vehicle-controls, the treated groups had a profound inhibition of EAE induction as assessed by the disease severity index.  Additionally, suppression of the inflammatory triad of TNF, IL-1, and IL-6 was observed at the mRNA level in cells that have infiltrated the spinal cord, as well as resident microglial cells.  In vitro treatment of microglial cells by ghrelin resulted in suppressed ability to produce inflammatory trial cytokines after stimulation with lps.

These data suggest that ghrelin itself may be useful for the treatment of multiple sclerosis, as well as the possibility of using it in combination with other agents that block microglial activation.  For example, the endocannabinoid anandamide has previously been demonstrated to inhibit microglial inflammatory activity.

Suppression of microglial-based inflammation is important because the microglia are activated by cytokine producing T cells and are critical components of multiple sclerosis neurodegeneration, not only by inflammatory mediators, but also by glutamate excitotoxicity.

Treatment of multiple sclerosis patients with their own fat derived stem cells has generated promising results which we recently published.  There are several mechanisms by which this treatment may mediate therapeutic effects.  One particular mechanism that we will discuss here is production of soluble factors by fat derived mesenchymal stem cells that may be protective to neurons.

In the paper (Wei et al. Adipose stromal cells-secreted neuroprotective media against neuronal apoptosis. Neurosciences Letters. 2009 Jun 21) conditioned media from fat derived mesenchymal stem cells was assessed for therapeutic activity in terms of ability to protect neurons from cell death.  The idea being that the fat stem cells produce factors that can inhibit apoptosis of brain cells.

The investigators used a model of neuronal cell death by culturing cerebellar granule neurons in absence of serum and potassium in order to induce apoptosis.  A dose-dependent inhibition of neuronal apoptosis was observed when adipose stem cell conditioned media was added to the cultures.  Inhibition of caspase-3 activity was observed in the protected neurons (which would make sense since caspase-3 is involved in the vast majority of apoptotic signalling).  Additionally, the adipose stem cell conditioned media was capable of stimulating the anti-apoptotic akt signalling pathway. 

In order to investigate what specific molecules were in the adipose stem cell conditioned media that induced protective effects on the neurons, it was found that neutralization of insulin growth factor (IGF)-1 resulted in loss of anti-apoptotic activity.

This study brings up several interesting questions. 

Firstly, would the supernatant from the adipose derived stem cells also have effects on oligodendrocytes?  In multiple sclerosis there is eventual neuronal death, however, activation of oligodendrocytes is very important in terms of re-myelinating the injured tissue.  We do know that bone marrow cells are capable of inducing remyelination under specific conditions, so the question would be if adipose stem cell supernatant may also mediate such an effect.  This would be relatively easy to test in an in vitro system, or using the experimental allergic encephalomyelitis model.  However the issue would be whether the supernatant would also mediate immune modulatory properties that may mask the effects of potential remyelinating activity.

The second question is whether the adipose stem cell supernatant would have effects on other models of neuronal apoptosis.  In the context of multiple sclerosis, neuronal apoptosis mediated by excess glutamate seems to be an important factor.  Alternatively, the adipose stem cell supernatant may increase astrocyte uptake of glutamate.

The third question would be whether various conditions can be applied to the adipose stem cells so as to enhance secretion of protective factors in the conditioned media.  For example, there are publications demonstrating that mesenchymal stem cells cultured in the presence of low oxygen actually increase secretion of various therapeutic factors, including IGF-1.  This may be because the mesenchymal stem cells “feel” the lack of oxygen and as a natural reparative mechanism start secreting factors that protect other cells.  This may be possible given that mesenchymal stem cells have been demonstrated to inhibit neuronal cell death in vivo in models of stroke.  Alternatively, another way to “stress” mesenchymal stem cells in vitro may be treatment with inflammatory cytokines such as TNF-alpha.  At the recent FOCIS meeting in San Francisco, data was presented demonstrating that mesenchymal stem cells secrete higher levels of antiinflammatory factors after exposure to immunological stress, as in the form of a mixed lymphocyte reaction.  Collection of supernatant of stem cells under various conditions of stress may be one interesting way to increase efficacy of the approach described in the current paper.

Sepsis is a condition where the inflammatory response occurs at such a high level that grave damage is caused to the body, causing millions of deaths each year.  Developing therapeutics for sepsis is also considered a graveyard for biotech companies due to the high rate of failures.  Although recombinant activated protein C (Xygris) has had some benefit, overall little therapies are available for this condition.

When we discuss stem cell therapy, we normally think about the stem cells regenerating the body, that is, a more chronic process.  When we think of sepsis we think of an acute medical event, that must be treated with rapid-acting procedures. 

This is why we were so shocked when we read a paper (Nemeth et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production.  Nat Med 2009 Jan;15(1):42-9) in the high profile medical journal Nature Medicine, describing the successful use of bone marrow mesenchymal stem cells in the treatment of this condition.

We know that mesenchymal stem cells possess antiinflammatory properties.  This, of course, is one of the reasons why we offer stem cell therapy for multiple sclerosis using mesenchymal stem cells.  These properties are mediated by the ability of mesenchymal stem cells to secrete factors such as LIF, HLA-G, and IL-10, all of which inhibit inflammation directly or indirectly.  However, it was always believed that mesenchymal stem cells mediate their effects in more chronic situations, not in situations where if the problem is not solved within hours the host perishes.

The investigators of the study we will discuss, used a mouse model of sepsis called the “cecal ligation and puncture model” in which the cecum is made to leak and the mouse dies within 24-48 hours if left untreated.

Treatment of mice with mesenchymal stem cells of the same genetic background as the mouse, or of a different genetic background inhibited mortality by about 50% ! 

More specific examination revealed that administration of mesenchymal stem cells was associated with preservation of liver and kidney function, two organs that are targets of the septic process.

The injection of mesenchymal stem cells was associated with rapid (3 hours !) induction of interleukin 10 production and suppression of the elevated TNF-alpha and interleukin 6 that are characteristic of the septic process.

The next question is whether the injected mesenchymal stem cells actually needed other cells in the body to mediate their effects, or whether they were inducing protection directly on their own.  To address this, T cells, B cells, and NK cells were depleted by antibody or genetic means before induction of sepsis.  Neither of these depletions affected ability of the mesenchymal stem cells to protect from sepsis.  So the next question was whether macrophages were involved. 

Depleting macrophages by administration of the chemical clodronate via liposomes resulted abrogation of the beneficial effects of the mesenchymal stem cells.  It was found that macrophages produce IL-10 after administration of the mesenchymal stem cells, and it is this IL-10 that protects against sepsis.  This was proven since inactivation of circulating IL-10 or blocking of its receptor, took away the protective effects of the administered mesenchymal stem cells.

So how would the mesenchymal stem cells induce production of IL-10 by macrophages?  It was found that the mesenchymal stem cells secrete PGE-2, which induces a biological cross-talk with the macrophages resulting in selective IL-10 release.

These data support the overall notion that mesenchymal stem cells are antiinflammatory in general, and specifically can act at the level of the macrophage.  Since macrophages are critical for multiple sclerosis progression in the CNS, it will be interesting to evaluate the mechanisms by which protective effects of mesenchymal stem cells are mediated in animal models of multiple sclerosis.  Additionally, these data provide yet another interesting method by which mesenchymal stem cells modulate inflammation and immunity.

We previously discussed a paper demonstrating augmentation of T regulatory cell (Treg) activity in patients with relapse-remitting multiple sclerosis after initiation of interferon beta therapy.  The possibility that one can increase activity of these cells whose physiological function is to prevent autoimmunity is very intriguing.  Therefore, we thought it may be worthwhile to see what else has been published on Treg cells in the area of multiple sclerosis patients, so we will talk about a publication (Viglietta et al. Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. J Exp Med 2004 Apr 5;199(7):971-9) from Dr. David Hafler’s group at Harvard investigating differences between healthy volunteers and patients with multiple sclerosis in terms of Treg activity. 

Before we begin discussing the paper, let us ask ourselves, how would one measure Treg activity?  If you think about it, it is actually very difficult if done in the most pure fashion.  What we mean is that theoretically, the cells that are protecting the body from immunological attack would have a specific receptor, a specific T cell receptor (TCR) that recognizes the myelin basic protein and that stops the “conventional” T cells, or “T effector cells” which also are recognizing the myelin basic protein from attacking the myelin sheath.  In other words, quantification of the suppressive activity of all the Tregs in the body may or may not be the best way to assess whether the Tregs are working or not.  The most important Tregs are the ones that inhibit the attack on the myelin, the other Tregs, that prevent attack against, say, collagen II (antigen in rheumatoid arthritis), or GAD65 (antigen in type I diabetes) are not important for the situation of multiple sclerosis.

Unfortunately, we dont know all of the antigens that the T cells are attacking in multiple sclerosis, and it is difficult to measure only the Treg cells that are specific for antigens that we do know.  When one is trying to quantify effector cells, there is something called “tetramer technology” in which peptides are bound to labelled proteins that resemble the MHC complex, and flowcytometry can be used for assessment.  I wonder why we dont really see this being done with quantification of Treg cells.  My guess is that they are found in much smaller numbers than the effector cells and thats why its difficult.  Just to give you an idea, Treg cells comprise approximately 5% of the CD4 population, with the other 95% being conventional T cells, or T effector cells.

So in the publication, assessment of Treg function was performed by adding increasing numbers of Treg cells (defined as CD4+ CD25+) to a fixed number of T effector cells (CD4+ CD25+) and providing an activation signal (anti-CD3 monoclonal antibody) that nonspecifically activates the T cell receptor of both the Treg and the T effector cells.  Activation of the cells can easily be measured by the rate at which the cells divide, as well as cytokines that they make.

So one would expect that if only T effector cells were mixed with the anti-CD3 antibody, there would be proliferation, and with the increasing number of Treg cells added to the mix, there would be a suppression of the proliferation.  As seen in the figure below, with increasing number of Treg cells there is an increase in suppression.  Most interestingly the addition of Tregs from MS patients did not seem to suppress the anti-CD3 stimulated proliferation as well as the Tregs derived from healthy volunteers.  The data is representative of a total of 21 healthy controls and 15 patients with multiple sclerosis.

These data seem to suggest that Treg cell function is compromised in patients with multiple sclerosis.  The question is, what could be compromising it?  There are many things that inhibit function of Treg, or example, ligation of the protein GITR-ligand has been demonstrated to abolish Treg activity.  Interleukin-6 in some situations has also been demonstrated to inhibit Treg activity.  Additionally, inflammatory stimuli such as activation of various toll like receptors has also been associated with suppression of activity.  However, none of these factors really come to mind when one thinks of multiple sclerosis patients. 

One other question that is posed by these data is whether multiple sclerosis patients would be predisposed to other autoimmune diseases?  Clinically multiple sclerosis seems to present as a distinct entity.  So if the immune attack is only against nervous system tissue, specifically the myelin sheath, why would ALL the Tregs seem to have deficient function? 

Another question is whether the lack of Treg activity is a cause of disease or whether it is a symptom.  For example, it may be that the intial immune reaction against the myelin sheath may stimulate systemic changes and inflammation that could in turn somehow modulate Treg activity.  In fact, systemic inflammatory mediators such as serum amyloid A protein is actually increased in patients with multiple sclerosis (Boylan et al. Interferon-beta1a administration results in a transient increase of serum amyloid A protein and C-reactive protein: comparison with other markers of inflammation. Immunol Lett 2001 Jan 15;75(3):191-7). 

The ability of mesenchymal stem cells to reduce systemic inflammation is best demonstrated in clinical studies of patients with steroid refractory GVHD which seem to respond after administration of non-matched bone marrow derived MSC (www.osiris.com).  It therefore makes sense to see some of the animal and early human data demonstrating activity of mesenchymal stem cells in multiple sclerosis.

The question of adult stem cells differentiating into needed tissue is not based on opinion but on over 2 decades of solid peer reviewed research at international academic institutions.  To state there is controversy regarding the ability of adult stem cells to therapeutically augment natural healing processes, is like saying there is controversy whether red blood cells transport oxygen. 

 

There is no question as to the scientific rationale of therapeutic benefits.  The only question is the extent of the benefits and the selection of patients for undergoing stem cell therapy. 

 

In order to understand these issues, we must first begin by understand a little bit about stem cells.  We will begin with the bone marrow stem cell.

 

1.  What is the bone marrow stem cell?

 

Traditionally, the bone marrow was viewed as the source of all blood cells, being responsible for production of trillions of cells per hour.  Essentially one type of cell in the bone marrow, called the hematopoietic (“hematopoietic” means “blood making”) stem cells, possesses the unique ability to make copies of itself, but also, depending on the needs of the body, to make other blood cell types as well.  At a molecular level we know that the human bone marrow hematopoietic stem cells expresses the markers CD34 and does not express markers of lineage commitment such as HLA-DR, CD38, CD11, CD31, etc [1].  This essentially means that bone marrow hematopoietic stem cells can be isolated and studied as a discrete entity. 

 

The bone marrow hematopoietic stem cell adapts to the needs of the body and accordingly produces specific cell types when the body needs them.  For example, when a person goes up to live on the mountains, the person’s body needs more red blood cells than usual since there is less oxygen at high altitudes.  In order to compensate for this, the kidney starts to produce more erythropoietin (a hormone that travels throughout the body), which instructs the hematopoietic stem cell to produce more blood cells [2].  The same occurs in situations of infections in which the body needs more white blood cells (such as neutrophils) to protect itself against the external pathogens.  In response to various molecular signals (G-CSF, GM-CSF) generated by the immune system of the person, the bone marrow hematopoietic stem cell starts to generate more white blood cells [3].

 

In addition to containing the cells that make blood, the bone marrow contains cells called “stromal cells” that support the hematopoietic stem cells.  Essentially, stromal cells are comprised of various actual cell populations The stroma includes adipocytes, osteoblasts, and mesenchymal stem cells [4].  The primary function of mesenchymal stem cells in the bone marrow is to control proliferation of the hematopoietic stem cell, through provided growth factor support.  This is why certain scientists are using mesenchymal stem cells to accelerate blood formation after administration of hematopoietic stem cells in patients [5].

 

Bottom line:  There are two main types of stem cells found in the bone marrow: 1) hematopoietic stem cells that make blood and 2) mesenchymal stem cells that provide support for the hematopoietic stem cell.

 

2.  Bone Marrow Transplantation: The first application of stem cell therapy.

 

Stem cell therapy is not new.  Ever since the discovery of the hematopoietic stem cell by Drs Till and McCulloch in the 1960s, the use of these cells for transplantation into patients with defective bone marrow was envisioned.  The first hematopoietic stem cell transplant, or bone marrow transplant, was performed in 1956 by Dr. E. Donnall Thomas using bone marrow cells isolated from an identical twin donor for a recipient who had leukemia.  The idea was that if the patient was irradiated with high doses, then the radiation would kill all of the leukemia cells.  Unfortunately, the radiation would also destroy the healthy bone marrow stem cells.  So the idea was to utilize donor bone marrow to replenish the recipient with healthy hematopoietic stem cells.   Dr. Thomas, along with Joseph E. Murray, won the Nobel Prize in 1990 for this discovery.  The usefulness of transplanting the hematopoietic system was not limited to leukemias, in 1968, bone marrow transplantation was performed successfully on a patient with a genetic mutation called severe combined immunodeficiency.  In this disease the bone marrow stem cells are deficient in ability to generate T and B cells, as a result the patient is immune compromised and is forced to live in a sterile environment.  The administration of healthy bone marrow cells resulted in the child being able to function normally as a result of a non-mutated hematopoietic stem cell that is capable of making T and B cells.

 

Since the initial bone marrow transplant procedure was developed, several hundred thousands procedures have been performed, literally giving a new lease on life to many patients whose diseases were previously considered lethal.  Variations on the theme of bone marrow transplantation have also been performed in order to increase efficacy.  For example, patients with some leukemias are known to have a higher probability of relapse (leukemia coming back) after the transplant.  In order to fight off the relapse, clinicians have started infusing lymphocytes from the donor as a type of cellular therapy.  These Donor Lymphocyte Infusions are currently part of the accepted medical practice for treatment of post-transplant relapsed CML[6].  Additional modifications to the transplant procedure have included the use of G-CSF mobilized donor stem cells.  Instead of performing puncture of the iliac crest for bone marrow aspiration, a gentler procedure that was developed involved “telling the bone marrow stem cells” to leave the bone marrow and enter systemic circulation, by the administration of the drug G-CSF.  The stem cells are collected from the blood using a procedure called leukopheresis [7].  Another advancement in the area of hematopoietic stem cell transplantation has been the use of cord blood as a source of blood-forming stem cells.  Cord blood stem cells express higher regenerative potential on a per-cell basis compared to bone marrow stem cells.  Although cord blood stem cells are generally safer in the sense that they do not evoke graft versus host (a side effect of transplantation) with the same severity as bone marrow stem cells, cord blood stem cells are available in small numbers and therefore their use in adults is still limited to certain patient subgroups [8].

 

Conclusion:  Stem cell therapy in the sense of hematopoietic stem cell transplantation, has been occurring since 1956 for treatment of disturbances of the blood making components of patients.  These disturbances can be caused by genetic abnormalities (ie severe combined immunodeficiency, sickle cell anemia, etc) or induced as a side effect of treatment (ie dose radiation for clearing leukemia).  The hematopoietic-reconstituting stem cell therapy should not be confused with stem cell therapy for regenerative medicine.  In regenerative medicine the stem cells are administered in absence of the destruction of the recipient’s bone marrow hematopoietic compartment. 

 

3.  Bone Marrow and Regeneration: The Bone Marrow Cells Can Become Different Cells besides Just Blood Cells

 

As we described above, transfer of bone marrow stem cells has been performed for decades.  Scientists have wondered, if the bone marrow stem cell possesses the potential to differentiate into all the different types of blood cells, maybe it can also differentiate into other cells as well.  This process was originally termed “transdifferentiation”.  The first report of transdifferentiation to appear in the major medical literature was a paper by Orlic et al. [9], in which mouse bone marrow derived stem cells were injected into mice that were given an experimental heart attack.  The interesting thing about this experiment was that the bone marrow stem cells used were labeled to glow green.  The donor animals were genetically engineered to express the green fluorescent protein (GFP) gene throughout their bodies.  This essentially means that all cells derived from the GFP donor mice were green.  Additionally, the experimenters purified the mouse equivalent of the human CD34 bone marrow hematopoietic stem cell.  The molecular markers used where positivity for stem cell antigen (SCA-1) and negativity for the lineage markers (lin negative).  Following induction of a heart attack by ligation of one of the coronary arteries, the researchers implanted the cells in the area of infarct.  The mice which received implanted hematopoietic stem cells, but not control cells, had increased pumping ability of the heart and decreased levels of heart damage.  Most interestingly, when mice where sacrificed, green cells were observed throughout the area of damage.

 

This paper served as a strong indication in animals that bone marrow derived cells are capable of differentiating into heart tissue and helping to repair injury.  Eventually scientists started finding that bone marrow stem cells can differentiate into other tissues.  For example, human bone marrow derived CD34 cells have been demonstrated to differentiate into cells expressing liver markers, and can actually generate human liver proteins when injected into animals [10].

 

The concept of bone marrow cells differentiating into cells other than hematopoietic cells has subsequently been demonstrated in numerous laboratories for many types of tissues.  The table below summarizes some of the tissues.  We will include both hematopoietic and mesenchymal stem cells in the analysis since both are found in the bone marrow.  It is important to make a note that some researchers believe embryonic stem cells are the only cell types capable of differentiation into different tissues.  While it is true that embryonic stem cells indeed are “totipotent”, these cells are very far from clinical use.  Injection of embryonic stem cells into animals causes a type of aggressive tumor called “teratomas” [11], and furthermore, the differentiation of these cells is difficult to control.  In other words if you want to generate livers cells from embryonic stem cells, and you add liver-induction factors, such as hepatocyte growth factor, some cells become liver, but other cells still become neurons, kidney cells, and skin cells, thus until science advances, adult stem cells still seem to be superior for clinical applications. 

 

4.  Importance of Bone Marrow Resident Stem Cells for Natural Repair of Tissue Injury

 

The examples above, as well as the published literature, demonstrate in an unequivocal manner that bone marrow derived cells have a certain amount of plasticity, or ability or ability to differentiate into non-hematopoietic tissues. 

 

The main idea being that bone marrow can differentiate into a variety of tissues.  Now why would the bone marrow have this ability? One theory is that the bone marrow acts as a reserve of “regenerative cells” that are important in healing the body as the body ages.  The ability to regenerate is most potently seen in the salamander, which can regenerate whole limbs, based on potent stem cell activity.  Although humans do not have regenerative activity that potent, there are still evidences that bone marrow stem cells do in fact contribute to regeneration.  First we will provide an in vivo example of bone marrow “transdifferentiating” into other tissues in the human, and then we will provide examples of bone marrow helping in healing processes.

 

As we discussed above, bone marrow transplantation is performed for hematological disorders, one of which is leukemia.  Now if a female receives a male bone marrow transplant, one would imagine that the circulating blood cells of the female have the Y chromosome, which is correct.  What is more interesting is whether tissues in the female’s body actually start to express the Y chromosome.  In a very interesting study [12], researchers performed autopsy on female patients who had received male bone marrow transplants.  When they examined the heart tissue, the female heart tissue contained cardiac muscles that had the Y-chromosome !! This conclusively demonstrates that in normal situations bone marrow derived cells differentiate to become different parts of the body.  Similar findings were also reported in liver, pancreas, and kidney.

 

Now what about in situations of heart attacks?  One would imagine that during a heart attack, bone marrow derived stem cells would migrate into the heart.  If this is indeed the case, then taking blood samples of patients after heart attacks should demonstrate increases in stem cells.  Indeed numerous studies have shown this to be the case.  Below is an example from one study [13].

 

A similar argument can be made in stroke.  Interestingly, in stroke, there has been reported an association between higher degree of mobilization and improved recovery as assessed by the NIHSS score [14].

 

From the examples presented above, as well as numerous other publications, endogenous bone marrow stem cells have been demonstrated to play a regenerative role in humans.  The next question is whether the administration of bone marrow stem cell therapy can actually induce a clinical effect?

 

5.  Clinical Examples of Bone Marrow Stem Cell Therapy for Regeneration

 

Bone marrow stem cell therapy for regenerative (non-hematopoietic) purposes originally started with Japanese research when bone marrow cells were injected into the heart muscle of patients undergoing bypass surgery.  The idea was that the injected bone marrow cells will stimulate production of new blood vessels and thereby increase oxygenation to the heart [15].  The procedure, although highly invasive, was associated with no treatment related adverse effects and 3 out of the 5 patients had increased blood vessel production as assessed radiologically, as well as improved cardiac function.  This first demonstration in 2001, was repeated by numerous investigators.  In 2003, the study was repeated using CD133 purified bone marrow stem cells and published in the prestigious journal Lancet [16], reporting positive results.  Subsequently numerous studies have been conducted in the area of cardiology demonstrating that administration of a patient’s own bone marrow is associated with positive outcome.  For example, there are published pictures of myocardial activity before and after stem cell therapy from a clinical study [17].

 

Convincingly, statistically significant improvements in left ventricular ejection fraction have been observed in double blind trials.  For example there is published from a 200 patient trial [18].

 

In addition to the heart, bone marrow stem cells have been used for numerous other indications clinically.  One interesting indication is critical limb ischemia, which involves occlusion of blood flow to the lower limbs and is associated with need for amputation.  Below is a representative angiogram from a patient before and after bone marrow stem cell therapy as part of a double blind trial [19].  Additionally, in the same trial, clinical endpoints such as ankle brachial index (how much blood flows to the leg), pain-free walking, and transcutanous oxygen where all increased in a statistically significant manner.

 

Bone marrow stem cells have also been demonstrated to be effective in regeneration of other damaged/degenerated organs.  In a clinical trial with 9 patients suffering from liver failure [20], the administration of their own bone marrow cells intravenously elicited restoration in albumin production as seen in the figure below.  Each line in the figure represents one patient.  As well as overall decrease in clinical severity of disease (Child Pugh Score).

 

6.  Bone Marrow Derived Mesenchymal Stem Cells

 

The clinical examples above were aimed at fresh, non-cultured bone marrow derived stem cells.  Since it is difficult to enforce patents on medical procedures, numerous companies have developed “universal donor” off the shelf, stem cells that have been cultured in vitro and can be sold as “medicines”.  Of the companies activity in this area (Neuronyx, www.neuronyx.com in Phase I clinical trials, Pluristem, www.pluristem.com in late preclinical, and Osiris Therapeutics, www.osiris.com, 2 phase IIIs, several phase II trials), Osiris therapeutics has been the clear-cut leader, anticipated to have a product on the US market in the near future.  Below is a summary data of Osiris’s bone marrow mesenchymal stem cells used to treat Crohn’s Disease. Survival figures in patients treated with mesenchymal stem cells for a lethal inflammatory disease called are well published in the case of GVHD [21].

 

7.  Conclusion: Bone Marrow Stem Cell Therapy…Enhancing the Body’s Regenerative Potential

 

Dr. Thomas would be proud. From the initial bone marrow “cell therapy” transplant in 1956, the use of bone marrow stem cells has expanded to cover almost any indication one could imagine.  One only has to look at the www.clinicaltrials.gov database to see that clinical applications of bone marrow cells are being tested in conditions ranging from heart disease, to autoimmunity, to neurological conditions.

 

The wide applicability of bone marrow stem cells to so many diseases comes from the fact that stem cell therapy is only an augmentation of the natural regenerative processes.  It is established that injured or damaged tissue releases distinct factors that “call in” bone marrow stem cells.  By administering the cells in high concentrations intravenously, or locally, the bone marrow stem cell therapist is only helping the body to do what it is trying to do…to heal itself.

 

References

 

1.         Srour, E.F., et al., Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis. Blood Cells, 1991. 17(2): p. 287-95.

2.         Berglund, B., High-altitude training. Aspects of haematological adaptation. Sports Med, 1992. 14(5): p. 289-303.

3.         Lee, K.Y., et al., Varying expression levels of colony stimulating factor receptors in disease states and different leukocytes. Exp Mol Med, 2000. 32(4): p. 210-5.

4.         Krebsbach, P.H., et al., Bone marrow stromal cells: characterization and clinical application. Crit Rev Oral Biol Med, 1999. 10(2): p. 165-81.

5.         Koc, O.N., et al., Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol, 2000. 18(2): p. 307-16.

6.         Dazzi, F. and C. Fozza, Disease relapse after haematopoietic stem cell transplantation: risk factors and treatment. Best Pract Res Clin Haematol, 2007. 20(2): p. 311-27.

7.         Levesque, J.P., et al., Mobilization of bone marrow-derived progenitors. Handb Exp Pharmacol, 2007(180): p. 3-36.

8.         Riordan, N.H., et al., Cord blood in regenerative medicine: do we need immune suppression? J Transl Med, 2007. 5: p. 8.

9.         Orlic, D., et al., Bone marrow cells regenerate infarcted myocardium. Nature, 2001. 410(6829): p. 701-5.

10.       Wang, X., et al., Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells. Blood, 2003. 101(10): p. 4201-8.

11.       Nussbaum, J., et al., Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response. FASEB J, 2007. 21(7): p. 1345-57.

12.       Deb, A., et al., Bone marrow-derived cardiomyocytes are present in adult human heart: A study of gender-mismatched bone marrow transplantation patients. Circulation, 2003. 107(9): p. 1247-9.

13.       Muller-Ehmsen, J., et al., The mobilization of CD34 positive mononuclear cells after myocardial infarction is abolished by revascularization of the culprit vessel. Int J Cardiol, 2005. 103(1): p. 7-11.

14.       Dunac, A., et al., Neurological and functional recovery in human stroke are associated with peripheral blood CD34+ cell mobilization. J Neurol, 2007. 254(3): p. 327-32.

15.       Hamano, K., et al., Local implantation of autologous bone marrow cells for therapeutic angiogenesis in patients with ischemic heart disease: clinical trial and preliminary results. Jpn Circ J, 2001. 65(9): p. 845-7.

16.       Stamm, C., et al., Autologous bone-marrow stem-cell transplantation for myocardial regeneration. Lancet, 2003. 361(9351): p. 45-6.

17.       Stamm, C., et al., Intramyocardial delivery of CD133+ bone marrow cells and coronary artery bypass grafting for chronic ischemic heart disease: safety and efficacy studies. J Thorac Cardiovasc Surg, 2007. 133(3): p. 717-25.

18.       Schachinger, V., et al., Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med, 2006. 355(12): p. 1210-21.

19.       Tateishi-Yuyama, E., et al., Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet, 2002. 360(9331): p. 427-35.

20.       Terai, S., et al., Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Cells, 2006. 24(10): p. 2292-8.

21.       Ringden, O., et al., Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease. Transplantation, 2006. 81(10): p. 1390-7.

 

 

There is some evidence to suggest that pregnant women with multiple sclerosis experience a diminished frequency and severity of relapse in the last trimester of pregnancy.  This has prompted investigators to assess whether hormones such as progesterone are capable of inhibiting multiple sclerosis in animal models.  Indeed this seems to be the case. 

For example, Garay et al (Steroid protection in the experimental autoimmune encephalomyelitis model of multiple sclerosis. Neuroimmunomodulation 2008;15(1):76-83) used the B6 mouse model of multiple sclerosis (immunized with peptide from myelin oligodendrocyte protein 40-54) to demonstrate that administration of progesterone before induction of pathology led to suppressed disease severity index, inhibition of demyelination and increased expression of the sodium-potassium-ATPase gene in motor neurons.  Another study, (Correale et al. Steroid hormone regulation of cytokine secretion by proteolipid protein-specific CD4+ T cell clones isolated from multiple sclerosis patients and normal control subjects. 1998 Oct 1;161(7):3365-74) demonstrated that culture of T cells in progesterone upregulated ability to generate interleukin-4, a Th2 cytokine.  This is shown in the figure below.

Now we on the one hand we know that hormones affect immunological cells, but do hormones such as progesterone alter the ability of stem cells to modulate immune responses?  It appears that they do.  Ivanova-Todorova et al published (HLA-G expression is up-regulated by progesterone in mesenchymal stem cells. Am J Reprod Immunol. 2009 Jul;62(1):25-33) that treatment of mesenchymal stem cells with progesterone increased expression of the immune modulatory protein HLA-G.  This implies that ex vivo treatment of mesenchymal stem cells with progesterone may be useful in augmenting their ability to alter immune responses.  Additionally, it would be interesting to see if in vivo synergy may be obtained by treating patients with hormones and concurrently administering stem cells.

The ability to augment therapeutic activity of mesenchymal stem cells is very appealing since these cells are already in Phase III clinical trials by the company Osiris Therapeutics for treatment of Graft Versus Host Disease.  Once these cells are approved for marketing purposes (anticipated to be next year), then physicians will be able to use them on a more widespread basis and in many situations for off-label uses.  This will cause a great interest in methods of augmenting their efficacy, including methods as mentioned above.

A point of confusion regarding “stem cell therapy” for multiple sclerosis comes from two very different types of approaches that both use stem cells.  The first approach is the use of stem cells WITHOUT depleting the recipient immune system, the second approach involves administration of stem cells AFTER depleting of the immune system.

The first approach usually involves mesenchymal stem cells, such as found in the patient’s own fat, but has also been performed with cord blood.  The rationale for using stem cells in the absence of immune suppression is that the stem cells may help to regenerate the injured tissue, and also that they may suppress the autoreactive immune response.  Another interesting feature of mesenchymal stem cells is that they specifically “react” to their environment.  In other words, mesenchymal stem cells produce a certain level of antiinflammatory compounds when they are grown in tissue culture by themselves, however, when they are treated with compounds that cause inflammation, such as TNF-alpha, then they markedly upregulate their production of antiinflammatory agents such as IL-10, and also start producing more growth factors such as IGF-1.  This is believed to be because the mesenchymal stem cell normally acts as a “repair cell”.  That is, when there is tissue injury in the body, the mesenchymal stem cells naturally migrate to the injury (by virtue of proteins called chemokines) and then play a fundamental role in the healing process.

The second approach to treatment of multiple sclerosis by stem cells focuses on “reprogramming” the immune system.  Stem cells used for this are the stem cells that make blood, called “hematopoietic” stem cells.  In other words, we know that in multiple sclerosis there are numerous T cells that are attacking the body, and specifically the myelin sheath of the central nervous system.  These “bad” T cells have not only been identified but vaccines have been made with them.  Well instead of selectively killing some specific T cells, or only killing the activated T cells (like daclizumab does), the process of using stem cells with immune depletion involves first killing ALL immune cells, and secondly restoration of the immune cells by administering the patient’s own purified hematopoietic (blood making) stem cells.  By readministering the patient’s own stem cells in absence of T cells, the body is left to make its own T cells again from scratch.  Theoretically this is very appealing.  Practically there are a couple of problems.  First of all, the period of time from when the patient’s immune system is destroyed artificially (usually by chemotherapy and/or irradiation), to when the administered stem cells make new immune cells, leaves the patient exposed to many bacteria and viruses.  Secondly, there is a phenomena called “homeostatic expansion” in immunology.  This is explained in this video.  Essentially, when a small number of immune cells are placed in a host that lacks immune cells, the few immune cells start to multiple aggressively and lose ability to be regulated by normal mechanisms that stop the body from attacking itself.  In autologous transplantation with immune depletion, purified stem cells are reintroduced to the patient, so this should not be a problem, however, even a small amount of T cell contamination could potentially cause exacerbated disease.  The third danger with this approach is that when the stem cells are given to the patient that is lacking an immune system, the new T cells need to be made in the same way that the T cells were made from stem cells when the patient was young.  See, T cells only get made before you are born, primarily because of bone marrow hematopoietic stem cells migrating to the thymus and making new T cells in the thymus.  After puberty the thymus becomes much smaller and loses a lot of its functional ability.  This is because almost no new T cells are made after puberty.  So when you destroy the immune system and “ask” the hematopoietic stem cell to regenerate a brand new population of T cells, these T cells are made in a thymus that is severely atrophied.  Therefore the new T cells may have many potential abnormalities.

The two stem cell approaches (without destruction of the recipient immune response and with it) are discussed in a recent publication (Muraro et al. Immuno-Therapeutic Potential of Haematopoietic and Mesenchymal Stem Cell Transplantation in MS. Results Prob Cell Differ 2009 Jan 23).

The authors review how hematopoietic stem cell transplantation (involving immune destruction) has been used for more than 40 years for treatments of leukemias, and is now expanding into the area of autoimmune diseases, not only multiple sclerosis, but also rheumatoid arthritis and type 1 diabetes.  According to the paper, the highest number of hematopoietic stem cell transplants for autoimmunity has actually been performed in multiple sclerosis. 

In multiple sclerosis, hematopoietic stem cell transplants appear to stop acute inflammation in the central nervous system and prevent relapses.  Unfortunately, limited to no effect is seen in patients with secondary progressive multiple sclerosis. 

The article then goes on to talk about non-immune depleting transplants, specifically focusing on mesenchymal stem cells.  It states that the original idea with using mesenchymal stem cells was that they can differentiate into myelin producing oligodendrocytes and neurons, but now most people believe that the mechanism of action of these cells is primarily mediated by modulation of the immune system. 

Currently clinical trials are being conducted using mesenchymal stem cells that have been culture-expanded and hematopoietic stem cells with destruction of the immune system before placement.  It will be interesting to see which ones have better effects.  Of course the period of immune destruction before administration of the hematopoietic stem cells has the potential to cause numerous adverse effects.  Therefore, places like StemNow.com exclusively offer therapies that do not involve destruction of the immune system before stem cell administration.

Bristol, UK -

An interesting study was published by Gordon et al (Human mesenchymal stem cells abrogate experimental allergic encephalomyelitis after intraperitoneal injection, and with sparse CNS infiltration, Neurosci Lett 2008 Dec 19;448(1):71-3) describing the use of human bone marrow derived mesenchymal stem cells in the treatment of EAE, a mouse model of multiple sclerosis.

Previously people have demonstrated that administration of mouse mesenchymal stem cells into mice with EAE results in remission of disease.  In this current paper an interesting, and very relevant variation of the previous study was made….human stem cells were used.  This is important since human mesenchymal and mesenchymal-like stem cells are being developed by companies such as Osiris and Medistem as “universal donor” cells.  This means that theoretically these cells are not rejected by the immune system.  So the authors of this paper wondered whether the human cells would survive in the mouse, and whether they would actually mediate a therapeutic effect.

The investigators induced EAE through administration of the peptide MOG 35-55 together with an adjuvant in order to elicit immune responses against myelin.  They injected 1 million mesenchymal stem cells intraperitoneally and found a statistically significant reduction in disease score.  Disease score is measured on a scale of 0-5 (0 – Normal; 1 – Tail flaccidity or hind limb weakness; 2 – Partial hind limb paralysis; 3 – Complete hind limb paralysis, spastic paresis, impaired righting reflex; 4 – Complete hind and fore limb paralysis; 5 – Dead).

On day 50 the disease score seemed to have went in remission (about 0.5) in the mice receiving mesenchymal stem cells but was still active (2) in the control mice.  Interestingly tracking of the cells showed that few mesenchymal stem cells were found in the brain on day 50.

This paper was really nicely written and provides some good background references for people interested in this area.  It is available for free online at this link.

Cleveland, Ohio -

The possibility of stem cells treating multiple sclerosis is very enticing. 

This comes from two angles.  The first is that various type of stem cells either directly can heal injured nervous system tissue or produce various growth factors that allow the injured tissue to heal itself.  For example, it has been published that mesenchymal stem cells can differentiate into oligodendrocytes, which make myelin.  It has also been reported that stem cells produce growth factors such as IGF-1, which when administered into injured central nervous system tissue cause its repair.  The second reason why stem cell therapy for multiple sclerosis is appealing is that various types of stem cells, such as mesenchymal stem cells, are known to have immune modulating properties.  In other words, since multiple sclerosis is mediated by an abnormal T cell response, there is a possibility that therapy using cells such as mesenchymal stem cells may actually not only heal the damage that has occurred, but also address the root cause of the damage.

There was a recent paper (Bai et al. Human bone marrow-derived mesenchymal stem cells induce Th2-polarized immune response and promote endogenous repair in animal models of multiple sclerosis) which used human bone marrow mesenchymal stem cells to treat mice which were induced to have a disease that is similar to multiple sclerosis.

The scientists used two types of mouse multiple sclerosis.  The first is a progressive type, in which the MOG peptide was used to ”immunize” B6 mice, and the second is a relapse-remitting type in which another myelin component called PLP was used to “immunize” SJL mice.  What this means is that the mice develop an immune response against components of the myelin, and subsequently exhibit a disease that resembles clinical multiple sclerosis.

Administration of human bone marrow derived mesenchymal stem cells into these mice resulted in reduction in disease progression, as well as healing at the cellular level.  Increased numbers of oligodendrocytes (the cells that make myelin) were observed in the mice that recieved stem cell therapy. Interestingly, the autoimmune response seemed to be suppressed, well at least the inflammatory component of it, since reduction of interferon gamma and interleukin-17 was seen, which are both associated with poor patient prognosis, whereas elevated levels of interleukin-4, an antiinflammatory agent were seen in the treated mice.

This paper was particularly interesting since it demonstrated that human mesenchymal stem cells work in mice, not only for modulating the immune system but also for accelerating repair.  Although not assessed, it is possible that the mesenchymal stem cells were also increasing levels of T regulatory cells.  This is something that should be performed in future studies.