Stem cells may be attracted to the injured central nervous system of patients with multiple sclerosis by virtue of the molecule known as SDF-1, which is expressed at the onset of disease.  Indeed, we do know that stem cells selectively home to the central nervous system, at least from animal studies, in which adult mesenchymal stem cells are selectively found associated with areas of injury.  But could there be other injury signals that attract stem cells? 

We discussed previously that receptors associated with pain-related peptides, such as the kinin receptor B1, have the ability to make the disease worse or better depending on inhibition or activation, respectively.  An interesting molecule called Substance P, is a peptide neurotransmitter that is released in various situations of tissue injury.  We will discuss a recent paper (Hong et al. A new role of substance P as an injury-inducible messenger for mobilization of CD29(+) stromal-like cells. Nat Med. 2009 Apr;15(4):425-35)  demonstrating that Substance P is associated with homing of stem cells.

The investigators describe a model system in which injury induces mobilization of a mesenchymal stem cell-like population that expresses CD29 and is involved in acceleration of wound healing.

They  demonstrate that administration of Substance P in absence of injury in either mice, rabbits, or rats, induces mobilization of the CD29 cells from out of the bone marrow and into systemic circulation.  

To demonstrate that the mobilized CD29 cells actually had regenerative activity, they harvested CD29 cells that were mobilized, and injected mobilized cells, together with substance P in a rabbit wound model, in which the wound is induced by alkaline injury. Engraftment of the transplanted cells, as well as acceleration of healing, was observed. 

In order to make the case for clinical relevance of these observations, the investigators performed a series of experiments using human bone marrow mesenchymal stem cells as a model system for in vitro study.  It was observed that Substance P augmented the rate of transmigration, induced nuclear translocation of beta-catenin, triggered cell proliferation, and stimulated the activation of ERK1 and ERK2 pathways.

The authors conclude with the statement that: “This finding highlights a previously undescribed function of substance P as a systemically acting messenger of injury and a mobilizer of CD29(+) stromal-like cells to participate in wound healing”

If indeed new stem cell mobilizers can be identified in addition to G-CSF and the Anormed compound, perhaps one day it may be possible to simply redistribute your stem cells between body compartments so as to not need to take stem cells from outside of the body.

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.

 

 

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.

Multiple sclerosis is an autoimmune disease in which the patient’s immune system begins attacking the lining of the nerves called the myelin sheath. 

 

There are two main problems that need to be addressed in MS: a) how to stop the misdirected immune attack and b) how to repair the damage that has already been caused. 

 

Currently available therapies are directed towards non-specifically suppressing the immune system so that ongoing nervous system destruction is prevented. The drawback being that immune suppression exposes the patient to various other pathogens. 

 

Mesenchymal stem cells (MSC) have been demonstrated in numerous animal models and clinical trials to inhibit pathological immune responses while stimulating regeneration of damaged nerve tissue.  In clinical trials, bone marrow derived MSC have passed Phase I safety studies and are currently in a number of Phase II and III efficacy studies. 

 

Stemnow.com offers the therapy using the patient’s own fat derived cells which contain not only MSC but also several therapeutic cell populations.

 

Background:  In healthy people, the immune response is fine-tuned so as to specifically be able to destroy foreign pathogens, while at the same time have the ability to know that it should not turn against the body.  For reasons that are not completely understood, some patients developed “autoimmune” diseases in which the immune system starts attacking tissues of the body.  In multiple sclerosis this is manifested through destruction of the myelin sheath that insulates nerves in the white matter of the brain.  This destruction is observed by MRI in the form of plaques, and functionally is manifested by the patient experiencing the various characteristics of MS ranging from visual (e.g. optic neuritis, nystagmus, etc), motor (e.g. paresis, spasticity, etc), sensory (e.g. paraesthesia), balance (e.g. ataxia, vertigo) and cognitive (depression, cognitive dysfunction) alterations. 

 

There are 4 main types of MS: 1) Relapse-remitting MS is the condition which the majority (about 85-90%) of MS patients are initially diagnosed with.  As the name indicates, this type is characterized by relapses followed by periods of remission in which disease activity subsides.  It is believed that during remission the oligodendrocytes “fix” the neurons by producing new myelin;  2) Secondary progressive MS usually occurs as a progression of the relapse remitting type at the point where remissions decrease in frequency and eventually the debilitating characteristics continually progress.  On average it takes about 19 years for MS to convert from relapse-remitting to secondary progressive; 3) Primary progressive is characterized by patients presenting with MS in which no remissions are seen; and 4) In the progressive relapsing form, a continuous increase in symptoms is seen, however spikes of accelerated disease activity are interspersed in the progression of the condition.

 

Depending on the type of MS, various treatments are routinely used.  These include steroids, immune suppressants (cyclosporine, azathioprine, methotrexate), immune modulators (interferons, glatiramer acetate), and immune modulating antibodies (natalizumab).  The general treatment concept is to rapidly treat relapses so as to minimize permanent damage, as well as to prevent onset of relapse or progression to more advanced forms of MS.  Unfortunately long term efficacy data is not available for many of the current approaches used clinically.  At present none of the MS treatment available on the market selectively inhibit the immune attack against the nervous system, nor do they stimulate regeneration of previously damaged tissue.  Experimental approaches in clinical trials are trying to use a peptide vaccine to specifically inhibit anti-myelin immune responses by the company BioMS, however even if successful this approach will not induce regeneration.  Other experimental approaches include the use of bone marrow stem cells in combination with chemotherapy to destroy the original immune system of the patient and subsequently attempt to restart it.  Needless to say this procedure has the possibility of adverse effects.  We use another type of stem cell therapy that does not involve chemotherapy or destruction of the immune system, instead it involves a “re-education” through administration of mesenchymal stem cells that concurrently inhibit pathological immunity while stimulating regeneration of damaged neural tissue.

 

Rationale: Mesenchymal stem cells are a type of cell that possesses potent immune regulatory activity.  It has been demonstrated in clinical trials that patients suffering from deregulated immune responses, such as in Crohn’s Disease or Graft Versus Host Disease go into remission after administration of mesenchymal stem cells (www.osiris.com). 

 

Unlike conventional immune suppressive drugs that act everywhere in the body, mesenchymal stem cells possess the ability to “home” to areas of inflammation and specifically shut off immune responses that are harming the body.  Various molecules secreted by injured tissues such as FGF-2 are known to chemoattract mesenchymal stem cells.  Additionally, during pathological immune responses, such as in multiple sclerosis, the molecule interferon gamma is produced.  Mesenchymal stem cells “sense” interferon gamma production and become more immune suppressive in response to it (Ryan et al. Interferon-gamma does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells. Clin Exp Immunol. 2007 May 22). 

 

One way that mesenchymal stem cells locally shut down pathological immune responses is through blocking T cell proliferation.  This occurs through a variety of mechanisms, one being activation of the immune suppressive enzyme indolamine 2,3 deoxygenase (Meisel et al. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2,3-dioxygenase-mediated tryptophan degradation. Blood. 2004 Jun 15;103(12):4619-21). 

 

Additionally, mesenchymal stem cells are known to induce generation of an immune suppressive population of T cells called T regulatory cells (Maccario et al. haematologica 2005; 90(4).  These cells are known to protect the body against autoimmunity and act in a specific manner to inhibit pathological but not healthy immune responses.  Specific inhibition of pathological immunity in mouse models of multiple sclerosis has also been reported by mesenchymal stem cells (Zappia et al. Blood, 1 September 2005, Vol. 106, No. 5, pp. 1755-1761). 

 

In addition to blocking such autoreactivity, mesenchymal stem cells have been demonstrated to:

 

a) Accelerate remyelination (Keilhoff et al. Transdifferentiated mesenchymal stem cells as alternative therapy in supporting nerve regeneration and myelination. Cell Mol Neurobiol. 2006 Oct-Nov;26(7-8):1235-52);

 

b) Prevent neuronal apoptosis (Caplan et al. Mesenchymal stem cells as trophic mediators. J Cell Biochem. 2006 Aug 1;98(5):1076-84);

 

c) Directly differentiate into neurons (Muñoz-Elías et al. Marrow stromal cells, mitosis, and neuronal differentiation: stem cell and precursor functions. Stem Cells. 2003;21(4):437-48)

 

d) Induce endogenous neural stem cells activation to regenerate new neurons (Caplan et al. Mesenchymal stem cells as trophic mediators. J Cell Biochem. 2006 Aug 1;98(5):1076-84). 

 

Since mesenchymal stem cells promote nervous system repair through several synergistic mechanisms, these cells possess a much higher probability of success as opposed to other gene or cytokine therapies in which only one reparative mechanism is being activated.  In fact, these cells are known to produce a wide number of trophic growth factors themselves which assist in neural regeneration.

 

Clinical Implementation: The dual role of mesenchymal stem cells for blocking pathological immunity while inducing regeneration of neural tissue that has already been damaged has already been described (Uccelli et al. Stem cells in inflammatory demyelinating disorders: a dual role for immunosuppression and neuroprotection. Expert Opin Biol Ther. 2006 Jan;6(1):17-22).  Animal studies and case reports are supportive of this approach.  In fact, a clinical trial is currently ongoing at the University of Cambridge utilizing mesenchymal stem cells for multiple sclerosis.  We have treated numerous patients with mesenchymal stem cells with no evidence of adverse events or immune reactions.  Some of the patient experiences in our hands can be seen in our recent publication.

Why would the immune system “turn” on patients?  I figured I would write a couple of thoughts regarding the cells and mechanisms involved in how the immune system functions.  For many of you this may be obvious, but I thought I would review some of the concepts here at a very high level, with the hope of stimulating ideas/discussions.

The immune system shares with the nervous system the features of: memory, self-nonself recognition, specificity, and “knowing” when to respond or not respond based on quantity of stimulus.

For example, the immune system is does not “blindly” attack everything that is foreign.  As you know, sometimes the immune system does not even attack just what is foreign but alson turns against the body, such as in autoimmune conditions like multiple sclerosis, or type 1 diabetes, or rheumatoid arthritis, where the immune system attacks myelin basic protein, GAD, and collagen I, respectively.

Usually the immune system selects what it should attack or not attack based on recognition of what is “self” and what is “non-self”.  Classically, this process is thought to occur before birth.  The main cells of the “adaptive immune system”, the part of the immune system that learns based on exposure, are the T cells and the B cells.  The T cells can either be killer T cells (CD8), which recognize other cells that are virally infected or cancerous, and CD4 T cells, which provide chemical signals (cytokines) for recruitment of other immune cells (T helper cells).  Of CD4 cells broadly speaking, there are also T helper 1 cells, that contribute to immunity to cancer and viruses, Th2 cells that fight parasitic infections, and Th17 cells that cause inflammation.  There are also T suppressor/T regulatory cells, which inhibit ongoing T cell responses.  The B cells, on the other hand, are responsible for making antibodies, proteins that bind to bacteria or other cells and inhibit their activity or kill them. 

Lets first discuss how the T cells tell “self” from “non-self”.  The process is called thymic selection.  Each T cell has the capability of making approximately 10(10)-10(15) different types of receptors, which can each recognize different “antigens” or in other words, molecules found on other things.  This means that in the thymus T cells are made that can recognize pretty much anything in the universe !  So how is it then then the T cells that are made in the thymus do not attack the body? 

What happens is that in the thymus, many of the proteins or chemical compositions that are present in the body, or will be present in the body in the future as the person ages, are actually expressed in the thymus before birth.  So T cells that are generated before birth, when they encounter proteins in the thymus, they are instructed to die.  This causes the T cells that escape the thymus (because they didn’t recognize “self”) to be able to recognize everything else that is not the body.

So if T cells are deleted that recognize the body, then why do some people get autoimmunity?

There are a couple reasons.  Firstly the protein in the thymus that causes expression of every single body protein, called AIRE (stands for “autoimmune regulator”), is sometimes mutated.  Patients with a mutation in AIRE do not express all of the body proteins in the thymus and as a result autoreactive T cells enter the body and eventually cause autoimmunity.  Mind you, this is a pretty rare cause of autoimmunity.  AIRE mutations are found primarily in patients with the disease Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) which has autoimmunity against tissues that make hormones.

Another cause of autoimmunity is that the T cells that escape from the thymus, although at time of escape do not recognize “self”, upon encounter with a protein that is “non-self” they start to mutate so as to recognize “self”.  The ability of the T cell to mutate is critical since many pathogens mutate…so if the T cells could not recognize the mutated pathogens, then they would eventually kill the host.

So why is it then that not everyone gets autoimmunity? One reason is that recognition of proteins by T cells is not enough to stimulate the T cell.  The T cell also requires what are called “second signals”.  These are signals associated with “Danger”.  For example, the immune system intrinsically “knows” that certain things are potentially harmful for the host.  Bacteria have unique components that are not found in mammalian cells, such as lipopolysaccharide, which stimulate cells of the body such as dendritic cells, to provide a “second signal” to the T cell.  So if a bacteria has a “non-self” protein on it, but is not stimulating the “danger signal”, then the immune response will not be activated.  Only if there is: a) a unique nonself protein; and b) a “danger signal”, will be immune system be activated.

One way in which autoimmunity occurs is when there is inflammation at a site in the body that expresses proteins that may be recognized by the “self-reactive” T cells.

Let me provide an animal model example that proves this point.  Pamela Ohashi, now at the Ontario Cancer Institute, was the first author of a paper (Ohashi et al. Ablation of “tolerance” and induction of diabetes by virus infection in viral antigen transgenic mice. 1991 Apr 19;65(2):305-17) in which the cellular basis for self-nonself recognition was elucidated.

Ohashi developed two types of mice.  The first type expressed a foreign protein called LCMV glycoprotein (GP) selectively in the pancreas.  She did this by genetic engineering of the mouse so that the “foreign” protein is present in the mouse from fetal stages all the way to adulthood.  How as we discussed before, T cells capable of reacting against GP may theoretically be deleted by AIRE in the thymus.  Even if these cells are not deleted, they theoretically would not be able to cause autoimmunity (in this case very easily measurable by insulin levels because the autoantigen is in the pancreas) because there is no “danger signal”.   When she examined these mice, they grew old and as expected did not develop autoimmunity.

She then developed a second strain of mice that have T cells that recognize the GP protein.  These mice, called T cell receptor (TCR) transgenic mice have a large quantity of their T cells capable of recognizing the GP protein.  When T cells from the TCR-trangenic mice are added to pancreatic cells that express GP, the pancreatic cells are destroyed in vitro.

So when she bred the mice, so that the offspring had: a) GP expressed in the pancreas; and b) Large numbers of T cell recognizing GP, the animals did not get diabetes !!

When she examined these mice, she found that because there was no “danger signal”, despite the large number of circulating autoreactive T cells, the mice did not get diabetes. 

When she infected the mice with the LCMV virus, which causes a potent “danger signal” then self-tolerance was lost and the animals rapidly developed diabetes.

Perhaps more representative of human autoimmunity are model systems in which administration of an autoantigen is given in the presence of a “danger signal”.  For example, if one wants to induce a disease that mimics multiple sclerosis, one takes myelin basic protein, or peptides of it, mixes it with adjuvant (serves as the “danger signal”) and administers it into mice or rats of certain genetic backgrounds.  The disease that follows resembles multiple sclerosis in terms of cellular pathology (infiltration of T cells and macrophages in the central nervous system), as well as physically in that it culminates in paralysis.  Some animal models of multiple sclerosis actually have a relapse-remitting component, thus further resembling human disease.

The interesting thing is that this sort of “induced” autoimmunity is not specific to multiple sclerosis but can be seen in other conditions.  For example, administration of type I collagen to mice on the SJL background together with adjuvant leads to induction of a disease similar to human rheumatoid arthritis, called “collagen induced arthritis”.  Even autoimmune cardiomyopathy can be induced (autoimmunity against heart muscle) by injection of myosin peptides together with adjuvant.

So the question is, how could multiple sclerosis actually start in people? and how could we even have a chance of curing it?

Of course the answer is not known.  There are some indications that various pathogens may be associated with initiation of disease.  Unfortunately, conclusive studies do not seem to support this.  One of the problems may be that a lot of pathogens are still not known because they can not be cultured.  Various cell wall deficient bacteria may be involved which are difficult to detect, or which cause a “hit and run” in the sense that they induce the autoimmune disease but are subsequently cleared by the immune system.

Some interesting work in this area was conducted at Vanderbilt University in the early 1990s.  There was speculation that Chlamydia pneumoniae was involved in multiple sclerosis because it was found in the cerebral spinal fluid of some patients, and other patients had high levels of antibody to it (Sriram et al. Chlamydia pneumoniae infection of the central nervous system in multiple sclerosis. Ann Neurol 1999 Jul;46(1):6-14).  So the group of Dr. Sriram actually starting infecting mice with Chlamydia pneumoniae and inducing EAE by immunization with self-antigen.  As seen in the figure below, administration of the live bacteria result in acceleration of disease.

 

So at present it is still not known what causes multiple sclerosis in patients.  We do know that there are inflammatory components that appear to be associated with its onset, and also that there are certain genetic factors that contribute.

The next question is, even if we do not know how it starts, what type of treatments could work against it?

The obvious concept is that since MS is an immunologically mediated disease, it would make sense that suppression of the immune system would be therapeutically useful.  Indeed, this is how steroids work.  Numerous other agents that nonspecifically suppress the immune system are used in the treatment of MS, for example, cyclosporine, azathioprine and methotrexate.  These agents have various side effects in part because they knock-down the majority of T cell functions of the immune system.

Another class of agents used in the treatment of MS are biological response modifiers, the first generation being inhibitors of the protein Tumor Necrosis Factor-alpha (TNF-a).  Originally TNF was identified independently by Beutler and Cerami.  Beutler found that mice injected with the immune stimulant BCG made a protein that could cause necrosis of tumors, whereas Cerami found that mice bearing tumors had a protein that was responsible for weight-loss (cachexia).  Eventually it was found that both of them were working with the same protein.  Interestingly, while the protein ended up not have profound anti-tumor activity in humans, it was discovered that the protein played a significant role in the various inflammatory conditions that affect humans such as rheumatoid arthritis.  Interestingly antibodies to TNF-alpha instead of inhibiting multiple sclerosis actually appeared to accelerate the disease (van Oosten et al. Increased MRI activity and immune activation in two multiple sclerosis patients treated with the monoclonal anti-tumor necrosis factor antibody cA2. Neurology 1996 Dec;47(6):1531-4).  This led to the search for other modulators of the immune system.

Currently used biologics or biological response modifiers are interferon beta and glatiramer acetate.  Although their mechanism of action is not entirely known even today, they appear to reduce severity of multiple sclerosis.  Other agents include antibodies such as natalizumab (Tysabri).  These agents do not suppress all aspects of the immune system, so have a better toxicity profile as compared to chemotherapeutics, however, are still relatively nonspecific and have adverse effects.  Newer treatments such as anti-CD25 antibodies are also in clinical trials, but still are not antigen-specific.

Antigen-specific approaches towards treatment of multiple sclerosis would specifically involve the neutralization of the T cells that are causing damage to myelin but not other T cells.  The level of immune specificity in multiple sclerosis is very high, as explained in this video, that demonstrates only certain T cell clones act as “driver clones” in that they maintain the autoreactive phenotype.  One way of killing these autoreactive cells is to use the immune system to kill them by vaccination with expanded autoreactive cells.

Another way is to “trick” the autoreactive cells into believing that they are no longer necessary.  For example, the immune system intrinsically knows that if there is too high concentrations of a protein that it is attacking, more often than not, it should not be attacking it.  So what happens when the body is overloaded with an autoreactive antigen, the T cells “decide” that they should stop their reactions against it.  This is the concept of “high dose” tolerance.  An interesting study from the NIH demonstrated that repeated administration of myelin basic protein into mice after induction of experimental allergic encephalomyelitis actually inhibited disease progression (McFarland et al. Amelioration of autoimmune reactions by antigen-induced apoptosis of T cells. Adv Exp Med Biol. 1995;383:157-66).

The concept of adding to the body something that could exacerbate an autoimmune response sounds risky, however, this was successfully used by the Canadian company BioMS in that they have performed clinical trials using peptides from myelin basic protein and administering them intravenously.  The company is currently performing pivotal Phase III trials in patients with secondary progressive multiple sclerosis.

Treatment of multiple sclerosis would ideally involve the combination of immune modulation and regeneration.  Both of these are important since current approaches do not address the fact that after damage to the central nervous system has occurred, the natural regenerative capacity, while present, is still relatively limited.

We have previously published results of 3 multiple sclerosis patients being administered their own fat derived stem cells, and subsequently having positive responses.  This procedure, which was described in a collaborative publication with the University of California San Diego, appears to be working via concurrent immune modulation and induction of regeneration.