DR. KLASSEN’S PRESENTATION ON RETINAL STEM CELLS – MARCH 9, 2010
My current interest is in stem cells–and I’ve gotten kind of fond of those cells– but my main goal is repairing the retina. The stem cell just turns out to be a really great tool for approaching something that’s a very difficult, daunting task. This is because the retina develops as part of the brain and, although it’s in the eye, it really is connected to the brain and it develops as part of the brain, and if you look at it in scientific detail it is literally part of the brain, even though it is in the eye. Like the brain, it’s very hard to repair the retina if it’s injured. The retina doesn’t have the ability to heal the way your skin does, but it’s very crucial to daily activities because, as everybody knows, if your retina’s impaired, you have visual problems that can’t be corrected with the therapeutic options we have available now.
To repair the diseased retina, we actually have to replace neurons, which are the nerve cells of the central nervous system. It’s like putting cells back into the brain. In fact, that’s how I got my start. My research was originally directly along the lines of “Let’s build a new brain.” I guess I am one of those overly gung-ho types and I started working on building a new brain, specifically in the visual system doing retinal transplants. From this early work we found out that if you time everything just right, you could actually get a retinal transplant that could grow and integrate into the rat visual system to the point where–if you shine a light on that transplanted retina in the brain–the host eye would actually constrict its pupil, indicating that the transplant “saw” the light, conveyed this information to the host brain, and the signal said, “Hey, there’s light on somewhere out there”, whereupon the host system for pupillary constriction took over and said, “Oh yeah, there’s bright light. We’d better constrict the pupil to shade the retina.” The funny thing is that the light wasn’t coming into the host eye at all. It was aimed at the transplant, which was in the brain.
So that result was very inspiring, and I wanted to keep going with it. When I thought about it a little more realistically, I said wait a minute. I don’t think we’re ready to repair the brain quite yet. Maybe if we concentrate on this retina, then we’ll be able to develop this into a clinical modality. A significant advantage for testing new therapies in the eye is that everybody has two of them. Maybe we can work on one, and replace just a few cells — like the photo receptors that are lost in retinitis pigmentosa or macular degeneration.
Maybe this is an approachable set of diseases to work on. They’re very disabling, but we could potentially replace just one or two cell types and make a huge difference to these patients, whereas now there’s little in the way of effective treatment really available. So that’s what got me headed down the path of working on retinal diseases.
So when we look at retinal repair, we’re dealing with this complicated tissue, we’re trying to figure out how to replace cells or do something to make the situation better at the level of nerve cells, and there are two real strategies that we can bring to bear on this going forward. One of them is advanced drug delivery where we’re trying to preserve the cells and keep them from dying and give them a little more function back. The other is to actually go in there and replace the cells that are already gone. And that’s where the stem cells really come in, because if you can imagine a situation where the cells are so detailed in their structure and their connections relative to each other, you think about it this way: A muscle basically attaches to the bone and it has to attach in the right general place to move the arm. If it’s a little bit off, your arm moves a little funny, but it still moves.
In contrast, your retina has to have a pixel-perfect view of the world. Every little photo receptor has to be in its proper location right next to the other one, and the information coded this way has to stay very organized all the way back to the brain. If anything gets scrambled, then the image is all warped and disorganized and just doesn’t mean anything. The amount of precision you need in the organization of the retina is incredible. That’s the hard part. These cells don’t grow back, and they have to be very precise. That’s one of the amazing things about stem cells, is they get in there and actually replace cells without the experimenter or a surgeon having to tell them what to do to or put them in exactly the right places. We just let them “do their thing”, and they do it.
Which is why I jumped on stem cells; why I dropped everything and said this is an amazing kind of breakthrough, because nothing I had seen up until that point, even putting retinas in the rat brain, nothing looked like it could actually replace cells one by one with the cells doing the hard work, doing the heavy lifting, and figuring out what to do by themselves.
To reiterate, we’ve got drug delivery where hopefully we can perk up the cells in the retina, maybe keep them from degenerating, maybe attempt to get a little function rolling again. And then there’s the situation where you have stem cells that can hopefully replace cells of the retina that have died.
Another thing I’ll just point out is that stem cells can have another effect, namely, the stem cells can themselves make the same kinds of chemicals that rescue the retina, which are called growth factors. So stem cells may also be a way to perk up the retina, and that’s something that might be beneficial without even having to rely on them for replacing photoreceptors. Stem cells may function at multiple levels in terms of treatment. They might deliver?growth factors early on, and then move into the retina and replace cells.
So what are stem cells? Let’s just back up a little bit and explain, because everybody has heard of stem cells. You get used to the idea, “Yeah, stem cells. I think I know what those are.” But what are they, you know, if somebody came and asked you the question. In other words, take a bone cell, take a fat cell, take any cell, and ask the question: “Is it a stem cell?”
To be a stem cell, it really has to have two fundamental properties. One is self-renewal. That means the cell has to be able to divide, and when it divides, it has to make a clone of itself, a twin. It has to be able to make identical twins of itself, and that’s how it’s going to make more of itself so it can continue the process of regeneration into the future. And the other thing it has to do is to differentiate into the target cell type, the mature cell that you’re trying to make. Remember that a stem cell is immature. It has all this flexibility, but at the end of the day, you’re made out of mature cells that have a specific job to do, part of an intestine, a liver, etc. Stem cells are actually a very small minority population within the body, and the body is made out of almost exclusively mature cells that do the job they’re supposed to do. Most of those cells don’t divide; so the stem cells are a small little nest of cells that still divide and repopulate and regenerate.
If your skin is cut, the skin has to grow back. There must be a stem cell in the skin that allows skin to grow back. If the brain gets injured, it doesn’t repair very well. That suggests that the number of stem cells in the brain has gone down as opposed to, say, early in fetal development when–if a brain is damaged–it can repair itself to an amazing degree. As a person gets older, that stem-cell quality tends to diminish. Scientists have looked in human brains and found that the brain stem cells go down rapidly in number as we age. So that kind of explains that. Just remember, a stem cell renews itself, and having done that, it can make those cells that go on and make mature cells of all the different types of tissue in the body. Looked at that way, there are many different types of stem cells.
So you say, “Okay. That’s the definition of a stem cell. What are all these things I’ve heard about? They say there are fat stem cells and umbilical stem cells and embryonic stem cells. What’s going on here?” It turns out the embryonic stem cells, the ones that all the commotion was about — those are the true stem cells. They can replicate themselves indefinitely, and they can make all the different tissue types in the body. So they’re a true stem cell.
Then there are “adult” stem cells. We can regenerate our skin. We can make new intestinal lining after we’ve had that hot Thai food. You know, different parts of the body can regenerate. Your blood cells have to regenerate all the time. Red blood cells only last about 4 months; so you have to keep making those. Your immune system keeps regenerating itself. There are stem cells in the bone marrow that we all have, and we need them, and if we don’t have them, we die pretty quickly.
So there are stem cells in the body, and the tissues that repair themselves have more stem cells. But these stem cells aren’t true stem cells. They can’t repair everything, at least not normally, and they can’t self-renew forever. They self-renew for a while and–like in the brain–gradually “time out” and diminish in number. Very often they’re focused on regenerating the tissue that they’re in. So bone marrow stem cells make blood. Brain stem cells make brain. You don’t really cross over much. You don’t have brain stem cells starting to chuck out blood cells. You don’t start making brain in your bone marrow. Which is good, you know, that it doesn’t work like that.
And that brings us to retina. So during the development of the retina, which happens when you’re a little fetus, you start with this tiny little eye cup, and then it slowly becomes an eye, or actually rather quickly if you think about the job being done. These cells are dividing, and they’re dividing very quickly to make more and more so that your eye can get big enough so that you can see something. Because you start with a tiny little eye, microscopic, and then it gets bigger and bigger until it gets to the normal size. As the retina is getting bigger, it’s adding more cells. Those cells have to be dividing. So there must be a retinal stem cell in there that’s making more of itself and then, as it matures, those cells start to turn into the mature retinal cells like rods, cones, ganglion cells. But it starts as a stem cell, and then it matures. By the time you’re born, there’s very little in the way of stem cells lodged in your retina. And as you get older and older, there’s probably few if any retinal stem cells left. We don’t know for certain, but that’s probably why the retina doesn’t repair itself, because the stem cells already did their job, and now they have turned off and differentiated. So we work with retinal stem cells, or more accurately: retinal progenitor cells since they’re limited compared to embryonic stem cells. Limited in that they only make retinas, and you can’t get them from every tissue. You have to go to the retina early in development. But the good news for these cells is they’re focused on making retinal cells. They’re very good at it, and no one’s better. That is why it can be good to use adult stem cells. Because they’re more mature and they’re more directed, they’re a little bit behaved better than, say, the earlier stem cells like embryonic stem cells that have much more potential, like children, but they more easily run into trouble.
In terms of stem cells from the eye, scientists have tried different types of stem cells for treating the eye, either in animals or sometimes in patients. The form of stem cells that usually comes up in discussion with the eye are called corneal or limbal stem cells. You may have heard of them. Now, these kinds of cells are present in all of us. So when I said there weren’t cells in the retina, that’s true, but we do have stem cells in the front of the eye, for the cornea. So if you ever scratched the front of your eye, it’s a painful experience, but you know that the surface that was injured grows back in a few days, very quickly. There must be a stem cell there somewhere. There is. It’s the limbal stem cell. It’s around the white part of the eye, and these grow out and can repopulate the surface of the cornea in a few days. So that’s great. And now people can transplant those between patients or between eyes in the same patient and help regenerate the cornea, whereas in the old days, maybe that eye would just be blind forever if there was a failure of the stem cells.
For instance, if you got an acid or alkali burn in that one eye, you would be blind unless you could take some stem cells from this eye and move them over there, and they can clear up the cornea and repopulate the stem cells. So that’s a wonderful technology, but it only applies to the front of the eye, you need to realize that. To get the retinal stem cells, we’ve got to go early in development and get those cells and then grow them in culture and expand them and use them. Here I am referring to the retinal progenitor cells. They don’t repopulate forever. They pretty much divide the way they would during development, and then time-out. That is a limitation in terms of supplying the cells, but it’s a safety feature in terms of risks like tumor formation, which can be an issue with the embryonic type of stem cell. Those just divide forever. Well, that could be a problem. You want them to divide up to a point, and then you want them to stop. The progenitors do that naturally. It’s all part of their natural behavior. We like that! The big striking result was that–after these cells are transplanted to the eye–they migrate in a directed manner to wherever the injury is. They can migrate into the retina. They can migrate right through the jelly of the eye and move through the retinal tissues. When they get to the spot, they just stop and start to mature and send out processes that look very much like the normal architecture of the retina.
In contrast, you can put other cells in the retina that will also migrate into the retina, but they’ll just tear it up. You can put a fibroblast skin cell into the retina, but it’s not a good thing. Just because a cell can migrate through a tissue isn’t necessarily good. But a stem cell does it in a way that doesn’t disrupt the tissue and then gets to a place where it just stops and fits in so well that if it wasn’t fluorescently labeled, you wouldn’t even realize that it wasn’t part of the normal tissue. That’s the kind of finding that got me all excited about stem cells. And the way we spotted these cells was that we tagged them with fluorescent proteins. If you’ve been to the Long Beach Aquarium, and seen the display of jellyfish, and how they shine ultraviolet light on these jellyfish that are floating around, then you know that jellyfish put out these magnificent fluorescent colors, and you might think, “Wow, that’s some weird lighting effect”, but actually the jellyfish really do this. They have fluorescent proteins in them. So those crafty scientists took the genes for these fluorescent proteins, they turned them into different colors of the rainbow, and now can insert the genes into animal cells. The transgenic animal cells will also make the fluorescent protein. If you put them in stem cells, that means you have fluorescent stem cells that you can transplant. When you want to find them, you shine the right light and the fluorescent tag stands out like a beacon on a dark night.
When we first saw these fluorescent stem cells migrating into the degenerating retina and taking up position just like they were normal cells, it was so amazing that if they hadn’t been specifically tagged with the green fluorescence, we wouldn’t have believed what we were seeing. We would have thought “Those are just normal retinal cells we are looking at. Where did our transplant disappear to?” But in fact, it disappeared by fitting in. So that’s really why, from my perspective, stem cells are a really important tool and crucial to developing reconstructive therapies for the retina.
People have put these fluorescent proteins into mice, and they have a range of differently colored mice. You can get them from laboratory supply companies. You can order up green mice, red mice, blue mice, yellow mice, and as they take it further, you should be able to get them such that just the eyes are red, and things like this. It’s all about the genetics and there are scientific reasons why you might want these sorts of mice, but for us, it’s mainly about the colored stem cells that we can transplant.
They’ve also made a green pig. We took advantage of that to take this project beyond the rodent model. We isolated green fluorescent pig stem cells and put those into a regular pink pig to see if we could replicate the kinds of findings that we saw in the rodents in the larger animal. You’ve all heard how many times they’ve cured cancer in a mouse, and yet there’s still cancer. And the problem is that a lot of times the mouse, although it’s a great vehicle for testing many things, doesn’t always carry over to humans reliably. A lot of times there are a few hiccups along the way, whereas something that works in a pig, that’s putting it on a different level. Of course, it’s more expensive and all that, but we wanted to do it because we wanted to confirm that our work was of potential utility in humans and not just limited to curing rodents.
There is also a red cat. The cat is a slightly different story because the cat was domesticated probably in Africa or the Middle East thousands of years ago, and you all know that the Egyptians had cats, and their cats look a lot like the modern Abyssinian breed. It turns out that the Abyssinian breed has genes that give it RP-like conditions. A subset of Abyssinian cats go blind, and we can try to treat those animals with stem cells. So there may be veterinary applications here. Interestingly, it was recently discovered that some Siamese cats have the same genetic defect as the Abyssinian, probably indicating that those Abyssinians were the root stock of domestic cats that actually spread in the direction of the Far East a very long time ago, bringing this blindness gene along with it. Perhaps we finally can treat some of these animals, and we are working on that.
In the lab we grow the stem cells, we do all kinds of arcane molecular analysis, we’re really into which genes are actually making these cells function as stem cells. Then we can look at some of the usual suspects like the genes that control cell division. Obviously, being a stem cell requires the ability to divide, and we can look at all the things that control that. We want to also look at the mechanisms by which the stem cells will become an adult cell. We want to see if we can direct it or keep it from going astray and how efficiently we can get the photoreceptors, rods and cones, to do their work. We also want to look at what growth factors they’re making. Remember I said these retinal stem cells probably have a protective effect when you transplant them. Even before they integrate, they secrete these growth factors. We want to categorize what the specific growth factors are to get a better handle on this.
We’ve also looked at functional testing. This was most developed in the mouse where we did running wheel tests. Anyone who’s had a hamster remembers how they make noise all night on the running wheel, running and running and running. And that’s all night, and people get tired of it, like my dad did, and go out there and flip the light on, and the hamster would stop their running immediately. But when he’d turn off the light and go back to bed, it started up again. So the point here is that this is a predictable behavioral phenomenon. The onset of light suppresses the running behaviors. The running behavior is active in the dark and suppressed by light. Well, you can hook the wheel and the light up to the computer and test if the animal sees light. And that’s what we did. We transplanted the mice with the stem cells, had them running in the dark, and then we interrogate their visual system with light of different intensities to see how bright it had to be before they stopped running. And that gives you a measure of their sensitivity to the light. We compared the blind mice and the transplanted mice to normal-seeing mice. We found that the transplant was conferring an advantage in terms of light sensitivity.
Another interesting aspect of this work is the immunology. When anybody gets a transplant of a kidney or some other organ, unless they happen to get it from an identical twin, they probably had to go on to some immunosuppressant drug regimen. These are drugs that knock your immune system down so you don’t reject the new organ. Remember the original heart transplants, by Christian Barnard, way back when in South Africa? He was doing this meticulous work transplanting hearts in people, and then it would work great for a while, and then the organ was rejected. And so that pointed out the need for immunosuppressant drugs in order to get transplant recipients to survive reasonable amounts of time, and now people can survive well on immunosuppressants. But do you really want your immune system knocked down? And the answer is: not unless you have to! Because that immune system is there for a reason; right? I don’t have to talk about AIDS to show you what can happen when your immune system is knocked down too much or for too long.
So what’s really great about the cells we’re using, the retinal progenitor cells, is that these cells are incredibly well tolerated between members of the same species. You take the cell from one mouse and you move it to another, it’s not rejected. One thing is that the eye doesn’t reject foreign cells as much as most other parts of the body. There’s something called immune privilege in the eye. When we took our retinal progenitor cells and moved them to the kidney, a non-privileged site, they still weren’t rejected — not in mice. That was really strong evidence that these cells are simply not rejected the way that a heart or a solid organ can be. One of the reasons is they’re a pure population of cells. This is a little bit arcane, but when you transplant a kidney or a heart, it’s not really the heart or kidney tissue that initiates the rejection. It’s the white blood cells and the blood vessels inside of these organs. Our stem cell transplants don’t have any blood vessels or white blood cells in them at all. So those triggers, those immune targets, just aren’t present. Plus we looked at the cells at the molecular level, at the particular molecules that are known to trigger immune rejection, and they’re just not expressed. But when we looked at the human progenitors, one particular type of trigger is expressed on the human cell. So, again we’re sitting there thinking, well, it worked great in rodents and it worked fine in pigs too. We don’t know for sure yet if the grafts are going to be tolerated in humans, and I guess we can’t really know until we try it. I’ll be getting to that shortly.
I can say that we’ve got a lot of evidence now that these cells can turn into photoreceptors after transplantation. So that’s not in question. And luckily they do it on the right side of the retina. In other words, there’s a photoreceptor side of the retina and there’s an output side of the retina. You want your photoreceptors on the correct side, and that’s where the progenitors form them–they even orient in the right direction. They have a polarity, and the polarity’s right, and they’re in line with the other photoreceptors, give or take. So things are looking pretty good. You know, they could have developed backwards or randomly oriented. That wouldn’t be all that helpful. It would help you see light, but it wouldn’t help you see shapes or objects.
We’ve been able to get these kinds of cells from humans, grow them in the dish, and conduct the same kinds of molecular analyses. Like I said, we were questioning what the immune situation’s going to be. One thing we could do before we got to people is transplants the human cells into mice that have a problem with their immune systems. There are mice like the bubble boy. Bubble boy was born with no immune system. That’s why he was in the bubble — to protect him from the rest of the world. There are mice that have the same condition as the bubble boy, and they’re very useful in the laboratory because we can put human cells into these bubble-boy mice, and they’ll survive because that mouse is incapable of rejecting the cell. There’s an interesting side effect of these bubble-boy mice, these immune-incompetent mice also had retinal degeneration, but most people weren’t paying attention to that. They just thought, well, how useful these are that they have no immune system. Turns out they have a rapid form of retinal degeneration that had spread through all this strain. So we were able to put the human cell into the degenerating retina of bubble-boy mice and they survived. And what was great was they integrate into this degenerating retina. The reason you want to transplant to the degenerating retina is that when the retina is degenerating, the stem cells have a job to do. There’s something going on that they respond to. You put these stem cells into a normal retina, there’s nothing there to do. They just don’t do much. They respond to the need for some fixing up. It was great to see that these transplanted human cells differentiated into photoreceptors in the living retina of a mouse and made the rhodopsin and the right kinds of molecules needed to detect light. We are in the process of checking out the visual capability of these mice, but it looks — in related experiments–it does look like there’s some ERG activity coming from these types of cells, an indication that they are helping the retinal function and detect light. They can pick up the light.
So now I get to the moment you’ve been waiting for, and that’s putting these kinds of cells into humans. People inspired by this kind of research, people in China, Beijing, started to do some transplants of retinal progenitor cells in patients with optic nerve problems — and this was some years ago — and they gave the patients about three days of immune suppression, but after that nothing. And although patients with this kind of chronic optic nerve problems didn’t have a recovery of function, what I found really interesting when I looked at their data, which they were nice to share with me, was that the cells were still there where they transplanted. And remember, there was just three days of immune suppression. So this was giving me a kind of reassurance that the human cells were going to be tolerated when you transplant them between individuals, which is really important. Like I said, we’d rather not immunosuppress anyone if we can avoid it.
More recently another group has started to look into the possibility of transplanting into people with retinitis pigmentosa, which is a much better target than a chronic optic nerve problem because here you have a discrete cell population that is dying off and where we know the retinal progenitor cells can replace them. The cells might also have a beneficial growth factor effect. Meanwhile, I’d like to know for sure if they’re going to be immune-tolerant. So they did some transplants and no immune suppression other than three days of cortisone drops in the eyes, topical drops, which is not a particularly profound form of immune suppression. But I just mentioned that to be complete. And there were three patients who got grafts. None of them were related to the donor cells, and in all cases, the cells survived. I think this is pretty strong evidence. So far the grafts have survived five months. If you were going to reject something, typically it happens within a month. Now, that’s not to say you can’t have slower forms of rejection. But what I am saying is that the kind of really powerful, strong forms that would make you want to be on immune suppression — it doesn’t look like that applies. If we go out some time, there’s no telling whether maybe in a year or two or three years, maybe some kind of immunological event does occur. At that point, you could theoretically give somebody some temporary suppression, just like you would if a corneal transplant starts to reject. But what’s reassuring to me is that we probably won’t have to start patients before the surgery on these powerful drugs that suppress their bone marrow.
There were no complications related to the surgery. The clinicians have been monitoring these cells, always concerned to see whether there is proliferation going on. Again, I said with a retinal progenitor, I don’t think they’re going to proliferate unless some kind of mutation occurs. And so far, there are no signs of these cells increasing in number or forming any kind of mass. Again, this is what we would have predicted based on our years of large-animal studies. So far the human is stacking up very much like the pig — at least from our perspective. And the most interesting thing is that each of these patients has at one point or another in their early course expressed some kind of improvement — in other words, they were happy with what was happening. I’m still trying to get a handle on what this means, but I’ll just let you know that there seemed to be some improvement there. We’re not sure about what the mechanism of this will turn out to be, but it seems to relate to the procedure that was done. Measurements of their visual acuity in some cases — in fact all cases — got better, at least somewhat better, and in one case rather dramatically better. I’m not saying that the whole retina came back and everything was just like it used to be, but it is unusual to see improvement in a system where degeneration is the only direction that’s been happening from a biological standpoint.
My guess — and this is just a guess — when you have three patients, that’s not enough to make a lot of conclusions — but it seems like the cells are doing something beneficial, and I’m leaning towards that growth-factor thing. I know they make growth factors, and they’re going to secrete, and it’s going into an eye that is super hungry for growth factors. That’s what’s missing. The cone cells of the eye are in a crisis because the growth factors from the rods are being lost with rod cells dying. The cones didn’t do anything wrong. They’re innocent bystanders, but they’re losing that growth factor that keeps them going. If we’re putting these cells in and supplying some of that, it could be that that’s why the cones would perk up, come out of the kind of hibernation mode, and start to be able to do a little functioning in terms of seeing.
There’s some animal data that might relate to what I’m saying, and that is some people have looked into mice that have photoreceptors that are degenerating and found that you put the stem cells in, that the rhodopsin, the molecules that actually detect the light, instead of being scattered all over the cone cells, start to get collected into the part of the cell that’s supposed to actually function as the detector. So that’s microscopic evidence that’s in line with the clinical picture. But I warn you that this is all hypothetical, connecting the animal data and person data, and I can’t say for sure that that’s what’s happening. It’s very encouraging and I think very interesting. So maybe we’ll leave it at that.
Posted March 2010