We previously discussed the concept of DNA vaccination for antigen-specific modulation of the immune system in conditions of autoimmunity such as multiple sclerosis.  In essence, it appears that DNA-based administration of the same proteins that are targets of the immune system during autoimmunity seem to suppress the immune response in a specific manner.  This is different than other immune modulators such as anti-CD25 antibody or Tysabri which suppresses in a non-specific manner.

A published Phase I/II trial of BHT-3009, Bayhill Therapeutics DNA vaccine for myelin basic protein in 30 patients with either secondary progressive or relapse-remitting multiple sclerosis was performed in which antigen-specific immune modulation was seen, and safety of the vaccine was demonstrated.  Here we will discuss a larger trial by the same group that was recently published (Garren et al. Phase 2 trial of a DNA vaccine encoding myelin basic protein for multiple sclerosis. Ann Neurol 2008 May;63(5):611-20).

This trial was much larger than the previous trial (267 patients), and was limited to patients with relapse-remitting multiple sclerosis.  The patients were randomized to receive either placebo, or two doses (0.5 mg and 1.5 mg) of the DNA vaccine (BHT-3009).  DNA vaccination was performed intramuscularly at the timepoints of initiation, after 2 weeks, after 4 weeks and subsequently given for every month until the 44th week. 

The data demonstrated that in comparison to patients receiving placebo there were no positive effects of the 1.5 mg dose.  In contrast, the 0.5 mg dose caused a 50-61% reduction in new lesion formation as detected by MRI and profound reduction of anti-myelin antibodies.

These data support further expansion of the DNA vaccine approach into phase III clinical trials.  These data, as well as other antigen-specific tolerogenic vaccines may be one of the reasons why entered a $350 million deal with Genetech.

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.

The immune system self-regulates itself through many mechanisms, for example, thymic deletion of autoreactive cells, the need for co-stimulation for activation of T cells, and also through T regulatory (Treg) cells that recognize proteins that belong to the body and stop other T cells from attacking these proteins.  Another way in which activated T cells control themselves is by increasing expression of a molecule called CTLA-4.

This molecule works through several means.  One is outcompeting CD28 for access to CD80 and CD86 on the antigen presenting cell, the other is by sending a suppressive signal to the antigen presenting cell.

To activate a T cell, generally speaking, one requires 2 main signals, the T cell receptor (TCR) has to recognize a major histocompatibility complex (MHC) molecule that has a peptide inside of it.  If the TCR appropriately “fits” into the MHC-peptide complex, then the TCR sends a signal into the T cell.  However this first signal is not enough to activate the T cell.  The T cell requires another signal from the antigen presenting cell.  This second signal, also called costimulatory signal, is usually in the form of CD80 and/or CD86 from the antigen presenting cell, which both bind to CD28 on the T cell.  The activation of CD28 and the TCR on the T cell results in T cell activation.  When only the TCR is activated without CD28, then the T cell either goes into a state of prolonged unresponsiveness (called anergy), or in some situations actually can start expressing immune suppressive properties.  CTLA4 is turned on by the T cell after the T cell is activated. CTLA4 binds with much higher affinity to CD80 and CD86 than does CD28.  Therefore, after T cell activation, the T cell can “turn itself off” by expressing CTLA4.

Studies have demonstrated that activated T cells expressing CTLA4 can bind to dendritic cells (most potent antigen presenting cells), and that the CTLA4 on the activated T cell sents a negative signal to the dendritic cell, inducing the dendritic cell to secrete immune suppressive proteins and upregulate expression of the enzyme indolamine 2,3 deoxygenase (IDO) which in turn suppresses other T cells.

Interestingly CTLA4 is expressed not only by activated T cells but also by Treg cells.

Given the ability of CTLA4 to suppress immune responses via at least 2 mechanisms (more are known), scientists have tried to use CTLA4 therapeutically.  To make a soluble form of CTLA4 that can be administered, a pharmaceutical form has been developed by fusing the protein CTLA4 with the immunoglobulin domain of IgG, to make a chimeric molecule called CTLA4-Ig.  Currently CTLA4-Ig is sold by Bristol Myers Squibb under the name Abatacept (Orencia) for the treatment of rheumatoid arthritis that is nonresponsive to TNF-alpha inhibitors such as Remicade.

Given the immune modulatory properties of CTLA4-Ig, one question is whether it may inhibit suppress autoimmune responses in patients with multiple sclerosis.  A recent study (Viglietta et al. CTLA4Ig treatment in patients with multiple sclerosis: an open-label, phase 1 clinical trial. Neurology 2008 Sep 16;71(12):917-24) assessed this in a small clinical trial.

The investigators performed a Phase I trial, meaning that the primary endpoint was toxicity and ability to identify therapeutic dose for subsequent Phase II investigations.  A total of 20 patients with relapse remitting multiple sclerosis were treated with intravenous infusions of CTLA4-Ig and monitored for 3 months. 

CTLA4-Ig administration correlated with suppression of interferon gamma production and proliferation in response to myelin basic protein ex vivo.  The treatment elicited no serious treatment associated adverse events, although some mild adverse events were reported.

This study demonstrated feasibility of CTLA4-Ig administration in relapse remitting multiple sclerosis, and provides support for further Phase II trials.

Unfortunately CTLA4-Ig is still a non-specific immune suppressant in that it globally inhibits activation of T cells and theoretically could predispose to infections.  One important question is whether the CTLA4-Ig administration may help to “reprogram” the immune system so as to promote eventual tolerance to the myelin antigens while leaving immunity to extracorporal antigens intact. 

A possible approach would be to use CTLA4-Ig in combination with antigen-specific approaches such as vaccination with autoreactive T cells.  Additionally, it will be important to assess the effects of CTLA4-Ig on Treg cells.  For example, we know that interferon beta, which induces therapeutic effects in multiple sclerosis stimulates Treg activity.  We also know from animal studies (Salomon et al.  B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. 2000 Apr;12(4):431-40) that administration of CTLA4-Ig can actually block Treg generation, so if interferon beta works through Treg generation, the combination may not work.

We previously discussed that interferon beta therapy is associated with upregulation of T regulatory cell (Treg) activity in patients with multiple sclerosis, and that this may be one of the mechanisms by which it mediates its therapeutic effect.  We also discussed the point that current methods of measuring Treg activity are somewhat limited in that they do not measure the specific Treg that protect the body against the specific autoreactive T cells that are attacking the myelin, but measure activity of all the Tregs as a whole.

Here we are going to discuss a publication (Andersson et al. Impaired autoimmune T helper 17 cell responses following DNA vaccination against rat experimental autoimmune encephalomyelitis. PLoS One 2008;3(11):e3682) that seems to demonstrate, at least in the rat model, that protection from multiple sclerosis (well actually EAE) by DNA immunization with autoantigen is dependent on production of interferon beta by the immunized animal.

To induce EAE (experimental allergic encephalomyelitis) the investigators vaccinated the rats with peptide 91-108 of the myelin oligodendrocyte glycoprotein in Complete Freund’s Adjuvant.  This stimulates an autoimmune reaction against the rat myelin, and rats develop a progressive disease that resembles multiple sclerosis. 

When rats were administered a DNA vaccine encoding the same peptide that the animals were immunized with to induce EAE, the progress of the disease was potently inhibited.  This is similar to what we discussed about in multiple sclerosis patients who were administered the DNA vaccine made by Bayhill Therapeutics against myelin basis protein.  In that situation, specific inhibition of the autoimmune responses was also observed.

What is interesting about the current study is that experiments were performed to determine through what molecular mechanism the DNA vaccine was mediating protection against disease.  It was found that T cells isolated from animals at the peak of disease had a much higher expression of the cytokine IL-17 when restimulated in vitro with the antigen myelin oligodendrocyte glycoprotein 91-108.  Animals that were treated with the DNA vaccine had a lower amount of IL-17 production.  Since IL-17 is associated with multiple sclerosis in both mice and human, it would make sense that a protective vaccine would reduce IL-17 production.  As a quick aside, the anti-diabetes drug metformin, which inhibits EAE, also suppresses production of IL-17 by autoreactive T cells.  Additionally, vitamin D has been demonstrated to reduce IL-17.

With the knowledge that the DNA vaccine seems to reduce ability of autoreactive T cells to produce the “bad” cytokine IL-17, the investigators next wanted to see what exactly is it that is responsible for this reduction in IL-17 producing cells.  So the DNA microarray technique was used to analyze 6240 genes at once to randomly see expression of which genes goes and which goes down after DNA vaccination. 

What was found was that the gene for interferon beta, as well as other genes that are associated with interferon beta were upregulated after immunization.  We know that interferon beta is part of the current-day treatment of multiple sclerosis, but also that it has several systemic side effects.  Therefore this study may be suggesting that DNA vaccination seems to be stimulating the body to make its own interferon beta.  This would be a much more attractive approach as compared to what is currently being performed for two reasons.  Firstly, the DNA vaccination encodes a specific antigen, therefore in addition to receiving interferon beta, the vaccination may offer the possibility of inducing tolerance to the autoantigen selectively.  Secondly, the production of interferon beta stimulating by the DNA vaccine would hypothetically be released at smaller and more localized concentrations, thus not having to “flood” the body with interferon beta from the outside and hopefully reducing the side effects.

But here is the catch…how do we know that the interferon beta being produced as a result of the DNA vaccination is actually responsible for suppression of disease progression?  In order to address this, the investigators created a DNA plasmid for vaccination that not only induced expression of the myelin oligodendrocyte glycoprotein peptide, but also short interferon RNA to target gene expression of interferon beta. 

In other words, the investigators repeated their experiments using two sets of DNA plasmids, one that has previously been shown to protect from disease by inducing expression of the myelin oligodendrocyte glycoprotein peptide, and another one in which the peptide is expressed but interferon beta gene is silenced (actually they used more controls, but these are the main ones to make the point).  It was found that suppression of the interferon beta gene expression resulted in loss of the therapeutic effect !  Also, recall response to peptide in terms of IL-17 secretion was augmented 20-fold if interferon beta was silenced.

What does all this mean?

Firstly, why would interferon beta be made by the simple procedure of DNA vaccination?  After all, DNA vaccination is performed as an alternative delivery of antigen.  Instead of injecting protein or peptides of the protein with adjuvant (which in the case of myelin oligodendrocyte glycoprotein results in disease) one injects DNA encoding the protein or peptide so that the cell generates the protein or peptide internally.  So if its the same protein or peptide being made, then why would one cause disease (direct injection of protein or peptide) but the other (DNA vaccination of the same thing) cause protection from disease and interferon beta production?

The authors state that one likely explanation is that the DNA vaccine backbone (the other parts of the DNA plasmid that do not encode the protein or peptide) has CpG motifs.  CpG motifs are parts of DNA that are recognized by human cells as being foreign and induce production of interferons, including interferon beta.  So it may be the interact effect of the CpG motifs that is responsible for protection.  In fact, the authors cite several papers, such as (Lobell et al. J Immunol 2003 Feb 15;170(4):1806-13) that demonstrate removal of CpG motifs from plasmid backbone used to “immunize” animals from autoimmunity, results in loss of effect.

In conclusion, the study discussed seems to provide a very interesting concept…that DNA vaccination can concurrently induce a cytokine that is therapeutic for multiple sclerosis, while at the same time antigen-specifically modulating an immune response.  It will be very interesting to see how the trials of Bayhill Therapeutics progress using a similar antigen-specific DNA-”vaccine” based approach.

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.

Autoimmunity, such as multiple sclerosis, is characterized by the immune system attacking components of the body.  Normally the body has numerous mechanisms of protecting itself from this, which we have previously discussed.  One way that the immune system “self regulates” itself is by a special type of T cell, called that T regulatory (Treg) cell.  The T cell receptor (TCR) of Treg cells is usually activated by recognition of proteins of the body.  This is an interesting point.  “Normal” T cells are usually turned on when their T cell receptor recognizes parts of proteins (called peptides) that are found on components that do not belong in the body.  Unfortunately in situations such as multiple sclerosis, rheumatoid arthritis, or Type 1 Diabetes, the T cells that are suppose to be activated by foreign peptides are activated by peptides that belong to the body (in multiple sclerosis the T cells are attacking components of the myelin basic protein which acts as an insulator around axons of the nerves).  The Treg cells, which recognize myelin basic protein are activated by the myelin basic protein components as well as by the presence of the conventional T cells being activitated.  What occurs is that the Treg cells attempt to suppress the destruction of the myelin by the normal T cells, through producing various chemical mediators, called cytokines, that suppress the effects of the conventional T cells.

So to try to state it in another way:  Conventional T cells activate the immune response and cause damage.  In the healthy situation, the conventional T cells cause damage to bacteria, virus infected cells, and cancer cells.  In the healthy situation Treg cells act as a protective mechanism so that in the cases that the conventional T cells start attacking components of the body, the Treg cells then inhibit the conventional T cells from doing this.  So another way of thinking about it is that the Treg cells are a “safety backup” so that the body does not attack itself.

So the question then becomes, what is going on in autoimmunity in general and specifically in conditions such as multiple sclerosis?  Are these Treg cells not doing their job properly?

There is a recent paper that was published (Korporal et al. Interferon beta-induced restoration of regulatory T-cell function in multiple sclerosis is prompted by an increase in newly generated naive regulatory T cells. Arch Neurol. 2008 Nov;65(11):1434-9) assessing Treg activity in 18 healthy volunteers and 20 patients with relapse-remitting multiple sclerosis. 

When comparing Treg activity between the healthy volunteers and the relapse remitting multiple sclerosis patients they found that ability of Treg to suppress immune response was deficient in the multiple sclerosis patients as compared to controls.

But the investigators then took the study a step further.  They assessed the Treg activity in patients before and after starting to take interferon beta therapy (Avonex is a type of interferon beta).  They found that both at 3 and 6 months after administration of interferon beta the suppressive activity of the Treg was restored to normal levels.  See figure below. 

Given that mesenchymal stem cells have been demonstrated to induce Treg activity, and that adipose stem cells actually have high concentrations of Treg, two interesting questions arise.  Firstly, can interferon beta therapy synergize with stem cells?  Secondly, can autologous adipose stem cell therapy serve as a substitute for interferon beta therapy? 

Other thoughts come to mind as well, such as, can if indeed Vitamin D is associated with Treg activity, would it synergize or antagonize the effects of interferon beta on the Treg and actually on the clinical situation?

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.

 

 

We have previously discussed that one of the mechanisms by which mesenchymal stem cells may have therapeutic activity on multiple sclerosis is through stimulation of T regulatory cells.  In our publication we previously demonstrated that adipose derived cells, which are known to contain mesenchymal stem cells also contain high concentrations of T regulatory cells.

A recent publication (Royal et al. Peripheral blood regulatory T cell measurements correlate with serum vitamin D levels in patients with multiple sclerosis. J Neuroimmunol 2009 Jun 16) assessed circulating levels of 1, 25-dihydroxyvitamin D (1, 25-(OH)2 vitD) and 25-hydroxyvitamin D (25-OH vitD), which are metabolites of vitamin D.  Specifically, 25-OH VitD  (also called calcidiol) is made by chemical modification (hydroxylation) of VitD3 by the liver. Calcidiol made into the active form of vitamin D (calcitriol) by the kidney in a process mediated by the enzyme 25(OH)D-1 alpha hydroxylase. 

So there are two forms of vitamin D: a) Calcidiol and b) Calcitriol. 

The paper demonstrated a positive relationship between high levels of calcitriol and number of T regulatory cells were seen.

The possibility that Vitamin D is related to inhibiting multiple sclerosis comes from some other sources as well.  For example the paper (Correale et al. Immunomodulatory effects of Vitamin D in multiple sclerosis. Brain 2009 May;132(Pt 5):1146-60) makes the following interesting points:

1.  There are reports of diminished multiple sclerosis risk associated with sun exposure and use of Vitamin D supplements.

2.  Circulating levels of vitamin D have been associated with reduced risk.

3.  Out of 60 controls and 132 patients with multiple sclerosis the levels of Vitamin D, both calcitriol and calcidiol were lower as opposed to control.

4.  Patients during relapse had lower vitamin D.

5.  Calcitriol inhibited proliferation of T cells in vitro, stimulated IL-10, and reduced number of cells making IL-17.

6.  T cells can make calcidiol into calcitriol.

7.  Calcitriol increases indoleamine 2,3-dioxygenase activity.

8.  Calcitriol increases Treg cells in vitro.

For stem cells to mediate their effects they must either be placed locally at the point of damage, or they must find their way there.  One of the questions that people ask is “how can intravenously administered stem cells home to where they need to go?”  To answer this, lets first think about the stem cell therapy that has been used for more than 4 decades: bone marrow transplantation.

In bone marrow transplantation the stem cells are injected intravenously into the recipient.  So how do they find their way to the bone marrow?  One of the may ways that this occurs is because the bone marrow expresses a protein called stromal derived factor (SDF)-1, which is also known as CXCL-12.  Specifically, bone marrow stromal cells are known to constitutively make this protein, which is what keeps the hematopoietic stem cells in the bone marrow.  So when donor hematopoietic stem cells are injected into a recipient, they selectively home to the bone marrow because of expression of SDF-1.  We know that SDF-1 is important for this process because if you block the interaction of SDF-1 with its receptor on the stem cell, called CXCR4, in a healthy person, then the healthy person’s bone marrow stem cells enter the blood.  The clinically used stem cell mobilizer mozobil works by interrupting this pathway.

We also know that SDF-1 is important for attracting stem cells because after heart attacks, this protein is produced by the injured heart muscle in large quantities, which attracts the patient’s own bone marrow cells to the area of injury.

A recently published study (McCandless et al. IL-1R Signaling within the Central Nervous System Regulates CXCL12 Expression at the Blood-Brain Barrier and Disease Severity during Experimental Autoimmune Encephalomyelitis. J Immunol. 2009 Jun 17) demonstrates that SDF-1 is expressed during the initial phases of disease progression in the mouse model of multiple sclerosis.

This study may provide one important clue as to how stem cells home into the central nervous system of patients with multiple sclerosis.  However this is a controversial area since, although mesenchymal stem cells prevent disease in animal models, some studies suggest that the stem cells do not need to actually home to the area of injury to inhibit multiple sclerosis, but instead may do this through immune modulation.

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.