In this video we discuss the paper Bai et al. in which the immunological and regenerative activities of human bone marrow derived mesenchymal stem cells were assessed in two different animal models of multiple sclerosis.
Treatment of multiple sclerosis patients with their own fat derived stem cells has generated promising results which we recently published. There are several mechanisms by which this treatment may mediate therapeutic effects. One particular mechanism that we will discuss here is production of soluble factors by fat derived mesenchymal stem cells that may be protective to neurons.
In the paper (Wei et al. Adipose stromal cells-secreted neuroprotective media against neuronal apoptosis. Neurosciences Letters. 2009 Jun 21) conditioned media from fat derived mesenchymal stem cells was assessed for therapeutic activity in terms of ability to protect neurons from cell death. The idea being that the fat stem cells produce factors that can inhibit apoptosis of brain cells.
The investigators used a model of neuronal cell death by culturing cerebellar granule neurons in absence of serum and potassium in order to induce apoptosis. A dose-dependent inhibition of neuronal apoptosis was observed when adipose stem cell conditioned media was added to the cultures. Inhibition of caspase-3 activity was observed in the protected neurons (which would make sense since caspase-3 is involved in the vast majority of apoptotic signalling). Additionally, the adipose stem cell conditioned media was capable of stimulating the anti-apoptotic akt signalling pathway.
In order to investigate what specific molecules were in the adipose stem cell conditioned media that induced protective effects on the neurons, it was found that neutralization of insulin growth factor (IGF)-1 resulted in loss of anti-apoptotic activity.
This study brings up several interesting questions.
Firstly, would the supernatant from the adipose derived stem cells also have effects on oligodendrocytes? In multiple sclerosis there is eventual neuronal death, however, activation of oligodendrocytes is very important in terms of re-myelinating the injured tissue. We do know that bone marrow cells are capable of inducing remyelination under specific conditions, so the question would be if adipose stem cell supernatant may also mediate such an effect. This would be relatively easy to test in an in vitro system, or using the experimental allergic encephalomyelitis model. However the issue would be whether the supernatant would also mediate immune modulatory properties that may mask the effects of potential remyelinating activity.
The second question is whether the adipose stem cell supernatant would have effects on other models of neuronal apoptosis. In the context of multiple sclerosis, neuronal apoptosis mediated by excess glutamate seems to be an important factor. Alternatively, the adipose stem cell supernatant may increase astrocyte uptake of glutamate.
The third question would be whether various conditions can be applied to the adipose stem cells so as to enhance secretion of protective factors in the conditioned media. For example, there are publications demonstrating that mesenchymal stem cells cultured in the presence of low oxygen actually increase secretion of various therapeutic factors, including IGF-1. This may be because the mesenchymal stem cells “feel” the lack of oxygen and as a natural reparative mechanism start secreting factors that protect other cells. This may be possible given that mesenchymal stem cells have been demonstrated to inhibit neuronal cell death in vivo in models of stroke. Alternatively, another way to “stress” mesenchymal stem cells in vitro may be treatment with inflammatory cytokines such as TNF-alpha. At the recent FOCIS meeting in San Francisco, data was presented demonstrating that mesenchymal stem cells secrete higher levels of antiinflammatory factors after exposure to immunological stress, as in the form of a mixed lymphocyte reaction. Collection of supernatant of stem cells under various conditions of stress may be one interesting way to increase efficacy of the approach described in the current paper.
Sepsis is a condition where the inflammatory response occurs at such a high level that grave damage is caused to the body, causing millions of deaths each year. Developing therapeutics for sepsis is also considered a graveyard for biotech companies due to the high rate of failures. Although recombinant activated protein C (Xygris) has had some benefit, overall little therapies are available for this condition.
When we discuss stem cell therapy, we normally think about the stem cells regenerating the body, that is, a more chronic process. When we think of sepsis we think of an acute medical event, that must be treated with rapid-acting procedures.
This is why we were so shocked when we read a paper (Nemeth et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med 2009 Jan;15(1):42-9) in the high profile medical journal Nature Medicine, describing the successful use of bone marrow mesenchymal stem cells in the treatment of this condition.
We know that mesenchymal stem cells possess antiinflammatory properties. This, of course, is one of the reasons why we offer stem cell therapy for multiple sclerosis using mesenchymal stem cells. These properties are mediated by the ability of mesenchymal stem cells to secrete factors such as LIF, HLA-G, and IL-10, all of which inhibit inflammation directly or indirectly. However, it was always believed that mesenchymal stem cells mediate their effects in more chronic situations, not in situations where if the problem is not solved within hours the host perishes.
The investigators of the study we will discuss, used a mouse model of sepsis called the “cecal ligation and puncture model” in which the cecum is made to leak and the mouse dies within 24-48 hours if left untreated.
Treatment of mice with mesenchymal stem cells of the same genetic background as the mouse, or of a different genetic background inhibited mortality by about 50% !
More specific examination revealed that administration of mesenchymal stem cells was associated with preservation of liver and kidney function, two organs that are targets of the septic process.
The injection of mesenchymal stem cells was associated with rapid (3 hours !) induction of interleukin 10 production and suppression of the elevated TNF-alpha and interleukin 6 that are characteristic of the septic process.
The next question is whether the injected mesenchymal stem cells actually needed other cells in the body to mediate their effects, or whether they were inducing protection directly on their own. To address this, T cells, B cells, and NK cells were depleted by antibody or genetic means before induction of sepsis. Neither of these depletions affected ability of the mesenchymal stem cells to protect from sepsis. So the next question was whether macrophages were involved.
Depleting macrophages by administration of the chemical clodronate via liposomes resulted abrogation of the beneficial effects of the mesenchymal stem cells. It was found that macrophages produce IL-10 after administration of the mesenchymal stem cells, and it is this IL-10 that protects against sepsis. This was proven since inactivation of circulating IL-10 or blocking of its receptor, took away the protective effects of the administered mesenchymal stem cells.
So how would the mesenchymal stem cells induce production of IL-10 by macrophages? It was found that the mesenchymal stem cells secrete PGE-2, which induces a biological cross-talk with the macrophages resulting in selective IL-10 release.
These data support the overall notion that mesenchymal stem cells are antiinflammatory in general, and specifically can act at the level of the macrophage. Since macrophages are critical for multiple sclerosis progression in the CNS, it will be interesting to evaluate the mechanisms by which protective effects of mesenchymal stem cells are mediated in animal models of multiple sclerosis. Additionally, these data provide yet another interesting method by which mesenchymal stem cells modulate inflammation and immunity.
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.
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An interesting study was published by Gordon et al (Human mesenchymal stem cells abrogate experimental allergic encephalomyelitis after intraperitoneal injection, and with sparse CNS infiltration, Neurosci Lett 2008 Dec 19;448(1):71-3) describing the use of human bone marrow derived mesenchymal stem cells in the treatment of EAE, a mouse model of multiple sclerosis.
Previously people have demonstrated that administration of mouse mesenchymal stem cells into mice with EAE results in remission of disease. In this current paper an interesting, and very relevant variation of the previous study was made….human stem cells were used. This is important since human mesenchymal and mesenchymal-like stem cells are being developed by companies such as Osiris and Medistem as “universal donor” cells. This means that theoretically these cells are not rejected by the immune system. So the authors of this paper wondered whether the human cells would survive in the mouse, and whether they would actually mediate a therapeutic effect.
The investigators induced EAE through administration of the peptide MOG 35-55 together with an adjuvant in order to elicit immune responses against myelin. They injected 1 million mesenchymal stem cells intraperitoneally and found a statistically significant reduction in disease score. Disease score is measured on a scale of 0-5 (0 – Normal; 1 – Tail flaccidity or hind limb weakness; 2 – Partial hind limb paralysis; 3 – Complete hind limb paralysis, spastic paresis, impaired righting reflex; 4 – Complete hind and fore limb paralysis; 5 – Dead).
On day 50 the disease score seemed to have went in remission (about 0.5) in the mice receiving mesenchymal stem cells but was still active (2) in the control mice. Interestingly tracking of the cells showed that few mesenchymal stem cells were found in the brain on day 50.
This paper was really nicely written and provides some good background references for people interested in this area. It is available for free online at this link.