BROADCASTS.com

Published: April 14, 2015, 12:22 a.m.

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Host \u2013 Dan Keller

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Hello, and welcome to Episode Thirty-Seven of Multiple Sclerosis Discovery, the podcast of the MS Discovery Forum. I\u2019m your host, Dan Keller.

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This week\u2019s podcast features an interview with Jeanne Loring, who works with human induced pluripotent stem cells in a mouse model of MS. But to begin, we\u2019d like to tell you about one of the most useful features of the MS Discovery Forum.

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Each week somewhere between 30 and 110 papers related to multiple sclerosis are published in the scientific literature. At MSDF, we endeavor to list them all, publishing links to a curated set of each week\u2019s new papers every Friday at msdiscovery.org/papers.

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The first step in curating this list is an automated PubMed query that pulls all papers containing the terms multiple sclerosis, myelin, optic neuritis, acute disseminated encephalomyelitis, neuromyelitis optica, transverse myelitis, experimental autoimmune encephalomyelitis, cuprizone, neurodegeneration, microglia, and several related terms. This query returns many false positives. MSDF editors read all the titles and most of the abstracts and make judgments about which papers are directly relevant to MS or related disorders. Last week, for example, the query returned 139 papers and, in our judgment, only 58 of them \u2013 42% \u2013 were truly MS-related. Some weeks the proportion is even lower than that.

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The query terms neurodegeneration, myelin, and microglia are responsible for most of the false positives. Neurodegeneration, in particular, returns many references related to other neurodegenerative disorders, such as Alzheimer\u2019s disease, Parkinson\u2019s disease, amyotrophic lateral sclerosis, stroke, and hypoxia, to name a few. Editorial judgments on which articles are relevant are often subjective, and we frequently struggle with those decisions. It\u2019s easy to decide relevance when an article actually mentions multiple sclerosis. It\u2019s harder when it mentions only myelin or only Th17 cells. If you think we\u2019ve missed an important MS-related article\u2014or if you think we\u2019ve included an irrelevant article\u2014I hope you\u2019ll let us know by emailing us at editor@msdiscovery.org. And we\u2019re open to suggestions on how to adjust our PubMed query to decrease false positives and false negatives.

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Once we\u2019ve chosen which of the articles to include in the week\u2019s list, we select between two and four of them as Editors\u2019 Picks. Those are the week\u2019s articles that seem to us to be the most important or interesting or intriguing. Once again, we invite readers to take issue with our choices. We\u2019d love to hear about important articles that we have not designated Editors\u2019 Picks or, on the contrary, Editors\u2019 Picks that don\u2019t deserve the honor.

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Now to the interview. Dr. Jeanne Loring is Professor of Developmental Neurobiology and Director of the Center for Regenerative Medicine at the Scripps Research Institute in San Diego. She and her collaborators have been testing human neural precursor cells derived from embryonic stem cells in a mouse model of MS. The cells are injected into the spinal cords of immunocompetent mice with a model of MS induced by a neurotropic hepatitis virus. The cells are rejected within a week, but in that time they suppress the immune system and induce remyelination.

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Interviewer \u2013 Dan Keller

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In terms of how you came upon your most recent finding about human pluripotent stem cells in the mouse model of MS, could you give me a little bit of the back story?

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Interviewee \u2013 Jeanne Loring

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Oh yeah, sure. It was really interesting. So Tom Lane and I set out to try to develop a stem cell therapy for MS using human cells. So as a control experiment, we took human pluripotent stem cells, in this case embryonic stem cells, and turned them into neural precursors; differentiated them just a little bit. And then we transplanted them into Tom\u2019s mouse model of MS. These mice were not immunosuppressed, and so we expected the cells to be rejected. And this was just our first experiment. But the results were not what we expected. After the cells were rejected, the mice started getting better, and their clinical scores improved. And then after several months, these mice were almost indistinguishable from normal mice.

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The first thing we thought was that we\u2019d gotten the cages mixed up, and we were looking at something different. But we\u2019ve repeated the experiment now more than a hundred times, and about 75% of the time we get the same result. So what this tells us is that these cells that we put into the animals are having some effect during the seven days that they exist in the animals that leads to both immunosuppression and remyelination and a clinical improvement which is quite remarkable.

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MSDF

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When you say 75% of the time, does that mean you get almost no effect 25 % of the time? Or does it mean that 75% or the mice? Because that would say whether you\u2019re making your stem cells right or not.

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Dr. Loring

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It\u2019s 75% of the mice.

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MSDF

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How do you explain it at this point, or where do you go from here to find a way to explain it?

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Dr. Loring

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So once we\u2019d realized that we had a phenomenon that was repeatable, we realized that there was something special about these cells. And we tested other cell types, like the pluripotent stem cells that they were derived from, and human fibroblasts, and discovered that neither one of those was effective. And since then we\u2019ve also tried other ways of making neural precursor cells, and those cells aren\u2019t effective either. So it\u2019s something extremely special about the cells that we used in these experiments, which is very lucky when you think about it.

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So we\u2019ve now, both Tom and I \u2013 even though we\u2019re not in the same place \u2013 we\u2019ve set out to try to find out what it is about these cells that gives them these properties. Our first sort of cut on this \u2013 our hypothesis \u2013 is that the cells are secreting something that has a lasting effect. Our sort of big picture idea is that there are probably more than one protein or glycoprotein being secreted. And together they suppress the immune system so they act on the inflammatory response so that they increase the number of regulatory T cells that are produced and decrease the other T cell types. And they induce remyelination.

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So Tom is now working on trying to identify what factors these cells make that are inducing the T regulatory cells. And on my side, we\u2019re trying to identify what it is that makes them remyelinate.

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MSDF

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When you make these cells, how do you know you got a good batch? Can you characterize them? Are there biomarkers, and you can say, \u201cWe did it right this time?\u201d

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Dr. Loring

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Yes. In fact, that turns out to be really important because we did it wrong a few times. And we have a gene expression signature. It\u2019s essentially diagnostic assay for this particular cell type. We\u2019ve boiled it down to a set of qRT-PCR markers. And, because we have collaborators in Australia, we had to be able to transfer this quality control assay to them. So far it seems like those markers, I think it\u2019s a group of 10 or 12 markers, seem to be predictive of the cells\u2019 working in the animals.

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MSDF

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And just to clarify, that\u2019s real time quantitative polymerase chain reaction? How are you going about characterizing what they\u2019re doing? I mean, are you doing cytokine measurements or you\u2019re looking at cells that get produced in the mice?

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Dr. Loring

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Tom is really handling the cells that get produced in the mouse. He\u2019s doing the T cell analysis. What we\u2019re doing, we developed an in vitro cell culture method to look for the effect of these cells on maturing oligodendroglia in culture. And we found that something secreted by these cells which shows up in their culture medium actually induces maturation of oligos \u2013 of OPCs \u2013 in vitro. I guess that\u2019s another result that we didn\u2019t expect to be quite so clear. So that shows that there\u2019s something that is secreted by the cells. I mean, that\u2019s the most likely idea. And on Tom\u2019s side, he\u2019s shown that the conditioned medium from the cells induces T-reg generation. And on our side, we\u2019ve shown that conditioned medium from the cells induces oligo maturation.

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So now we\u2019re trying to figure out what it is in that conditioned medium because now we think we can do a cell-free therapy for MS if we can identify what the factors are. It would be much simpler for us to do even a protein therapy for MS than it is to do a cell therapy. So both sides are taking sort of a candidate gene approach in which we\u2019re identifying the proteins that are most highly specifically expressed in the cells that work in the mice. We have a list of those proteins, and we\u2019ve sort of snatched a few candidates out of that group, and we\u2019re testing to see whether each one of those proteins in purified form has the same effect as the conditioned medium.

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The other approach, which is more tedious but more likely to actually tell us what\u2019s going on is for us to fractionate the medium into different sized proteins and then test each one of those fractions. We\u2019re in the process of doing that right now.

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MSDF

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But it sounds like these cells are pluripotent. Not pluripotent in the normal sense of a stem cell leading to different lineages, but they have a couple of effects. One is the immunomodulatory, the other is regenerating oligodendrocytes. Do you think it really requires the gamish of proteins? If you fractionate them, will you possibly lose the signal? And that\u2019s a big matrix to put back together again.

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Dr. Loring

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Yes, it is. And obviously, if we get no signal from our fractions, we\u2019ll put our fractions back together again and try to find out whether \u2013 there are only three fractions, really, right now. So we\u2019ll try different combinations of these fractions to try to find out if we can reproduce the effect. The effect is quite robust. We essentially get no maturation in medium conditioned by other cell types, but we get very strong maturation when we use conditioned medium from this particular neural precursor cell.

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MSDF

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If you only have three fractions now, is it because you just have chosen not to fractionate it even more until you know what\u2019s going on?

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Dr. Loring

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Yes, we\u2019re trying to hone in on it. So we don\u2019t want too many different things to look at right away. I\u2019m hoping that we find that only one of those fractions works, and that we can discover everything is within that fraction, but I really can\u2019t predict what\u2019s going to happen.

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MSDF

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It sounds like the approach would be to put everything in except one each time as opposed to keep adding back. You\u2019ve got to find the one critical one missing that makes the thing not work.

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Dr. Loring

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Yes, and eventually we will do that with specific antibodies, but right now that is, since we don\u2019t really have our candidates narrowed down enough, that isn\u2019t a viable approach. But you\u2019re absolutely right. We want to find out if that\u2019s missing, whatever the things that are that are missing. And I\u2019m hoping it\u2019s not so complex that it\u2019s five or six or seven proteins, because that\u2019ll make it much harder for us.

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MSDF

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How do characterize the condition of the mice?

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Dr. Loring

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So that\u2019s Tom\u2019s area of expertise. It\u2019s essentially an observation of the mice over time. We have a movie which I can show you, but I can\u2019t actually do it in a recording. It\u2019s quite obvious when the mice \u2013 they\u2019re blind scored so the person who looks at the mice and sees how well they\u2019re walking around doesn\u2019t know whether they\u2019re controls or experimentals. If you just see the movies that are selected at particular times after the cells have been transplanted, it\u2019s quite dramatic. They have a much better clinical score. Essentially, they\u2019re almost normal after six months.

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MSDF

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And how are you sure that the cells you injected into the spinal cord are gone, that they\u2019ve been rejected completely?

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Dr. Loring

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That\u2019s a good question. We used a method for live imaging of cells in which we use luciferase to label the cells. And then we used an instrument which allows us to image the cells in mice \u2013 in living mice \u2013 over time. So we did this in individual mice and saw that they disappeared over time. And after eight days we couldn\u2019t detect them anymore. That doesn\u2019t mean there isn\u2019t one or two left because the resolution isn\u2019t that high. We will go back eventually and look through sections of the spinal cords and see whether we can detect any. The other thing we can do is (skip 13:37) a human-specific markers. So we can just take a section of the spinal cord and find out if there\u2019s any human cells in it at all, or any human genes in it at all. But we haven\u2019t done that yet.

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MSDF

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Do the cells have to be gone? Have you tried injecting a second time?

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Dr. Loring

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No, we haven\u2019t. We don\u2019t know. We really don\u2019t know. It would be very interesting if it reversed the effects. Then we\u2019d really have a problem to solve.

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MSDF

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What else is there important to add or that we\u2019ve missed that\u2019s important to this kind of research?

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Dr. Loring

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So our dream is that we will identify a group of proteins and the concentration of those proteins necessary to have these two effects in this mouse model. And then we will do some biological engineering. We\u2019ll be putting the cells into these little spheres and matrix that allows slow release of these proteins or controlled release of these proteins. And then, instead of putting cells in, we\u2019ll put these beads in. And I don\u2019t know whether that would end up being the final product or not, but there are lots of ways to deliver proteins, and this one I find rather attractive because it doesn\u2019t require pumps or syringes. And I think that\u2019s certainly the direction we\u2019re going to try to go in. And so Tom Lane and I have just gotten an NIH grant for five years of funding, which seems like a very long time to me. So in five years we will have discovered the best way to deliver these things. We\u2019ll discover what they are and the best way to deliver them. Tom has put conditioned medium into the mice, and it also works.

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MSDF

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Because I was going to ask, had you encapsulated the cells just to see that the supernatant does it without cell contact?

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Dr. Loring

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It turns out that the conditioned medium itself, you inject that into spinal cord, it\u2019s not as dramatic an effect, but you have a clear clinical improvement.

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MSDF

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Have you tried injecting it either IV or intraperitoneally?

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Dr. Loring

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Yes. Well, we didn\u2019t inject the conditioned medium. We did try injecting the cells, and they pretty much stayed where we injected them. These cells, unlike mesenchymal stem cells, they aren\u2019t very migratory. So they don\u2019t really have the receptors that cause them to move to areas of inflammation like CXCR4, for example; they don\u2019t express that on their surfaces. So that does not seem to be a good delivery method for these cells. They don\u2019t go anywhere.

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MSDF

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I was also thinking that if something they secrete is important, whether it circulates. Maybe they\u2019re not making enough concentration if you inject them outside of the central nervous system, but it seems like you\u2019re going to be faced with a little cumbersome problem in a clinical situation years and years ahead from now if you have to keep injecting proteins into the spinal cord as opposed to more peripheral.

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Dr. Loring

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I agree. And the solution to that is generally to look for peripheral effects and then try to suppress those when you do a therapy like this. That\u2019s a long time, and we could certainly imagine how we would do it. But we need to know what those proteins are before we can decide on whether we expect them to have effects peripherally or not. But I agree with you; delivering them intravenously would be far easier.

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MSDF

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I know you have a lot of work ahead of you now with this, but is there another animal model of MS \u2013 or even another mouse model of MS \u2013 where you can see if it works even in a mouse model different from this one?

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Dr. Loring

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Yes, we\u2019re actively pursuing that with our collaborators in Australia. And it\u2019s interesting because they\u2019ve gotten some positive preliminary results using the EAE model, but the approach to the EAE model I\u2019ve realized is quite different. Generally, what people do is they provide the therapy at the time that the pathology is developing, and they try to prevent it, which is a really different idea than what we had using the mice that are already paralyzed. So they have found that if you can deliver the cells at least close to the spinal cord, then you can see some effects. The problem is that in Australia, and this is one of those technical things we had not anticipated, they don\u2019t have permission to inject cells into the spinal cord. So they have to go through their animal rights people or their animal protection groups and try to get permission to do so. So with Craig Walsh at UC Irvine we have started doing parallel experiments with the EAE model. I\u2019m not necessarily sure that it\u2019s going to have similar effects. I mean, I really don\u2019t know.

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MSDF

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Can you describe how these mice in your experiments were made to have MS?

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Dr. Loring

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Yes, they were given a virus, a neurotropic virus, which kills off the oligodendroglia. They become demyelinated, and there is a secondary inflammatory response. So the mice are actually paralyzed in their hind quarters at least by the time we put the cells in. They have to be fed by hand. So this is not a trivial thing to do. But we\u2019re trying to reproduce the effects during the progressive form of MS, for example, or during an attack of MS. So we\u2019re trying to repair the mice or cure the mice that are in a condition which would be similar to the worst case scenario for people with MS.

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MSDF

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Do you think this may also have effects not only on the myelin, but also on damaged neurons?

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Dr. Loring

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We don\u2019t know, because the mice haven\u2019t really had enough time to get a lot of neuronal damage, but that\u2019s a very good question. We don\u2019t know yet.

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MSDF

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I appreciate it. Thank you.

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Dr. Loring

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You\u2019re welcome. It was a pleasure.

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[transition music]

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Thank you for listening to Episode Thirty-Seven of Multiple Sclerosis Discovery. This podcast was produced by the MS Discovery Forum, MSDF, the premier source of independent news and information on MS research. MSDF\u2019s executive editor is Robert Finn. Msdiscovery.org is part of the non-profit Accelerated Cure Project for Multiple Sclerosis. Robert McBurney is our President and CEO, and Hollie Schmidt is vice president of scientific operations.

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Msdiscovery.org aims to focus attention on what is known and not yet known about the causes of MS and related conditions, their pathological mechanisms, and potential ways to intervene. By communicating this information in a way that builds bridges among different disciplines, we hope to open new routes toward significant clinical advances.

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We\u2019re interested in your opinions. Please join the discussion on one of our online forums or send comments, criticisms, and suggestions to editor@msdiscovery.org.

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