- Welcome everyone to Wednesday Nite at The Lab. I'm Tom Zinnen. I work here at the UW-Madison Biotechnology Center. I also work for UW-Extension and Cooperative Extension. On behalf of those folks and our other co-organizers, Wisconsin Public Television, the Wisconsin Alumni Association, and the UW-Madison Science Alliance. Thanks again for coming to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight, it's my pleasure to welcome back to Wednesday Nite at the Lab David Gamm. He's a professor of ophthalmology and visual sciences. He's also with the McPherson Eye Institute and with the Waisman Institute, and with the Stem Cell and Regenerative Medicine Institute. He was born in Colorado Springs, Colorado, went to high school in Grand Rapids, Michigan, and got his undergraduate degree at the University of Michigan in molecular biology and cell biology. Got a PhD at the University of Michigan, and then got a MD at the University of Michigan, and then he came over to the light side. (audience laughs) And came to UW-Madison to do his ophthalmological residency, and there are two Ls on ophthalmology, is that correct?  -   (David)   Ophthalmology, correct?  - That's I always went down on in the spelling bee. And he also did a fellowship with pediatric eye surgery.  That's why I became a plant pathologist. And now he's going to talk to us tonight about making sense of stem cells, progress toward the treating of blinding disorders. Please welcome me in joining David Gamm back to Wednesday Nite at The Lab. (audience applauds)  - Thanks Tom.  Well, thank you very much. I really enjoy the opportunity to come and talk to you. I talk to lay audiences quite a bit, and this is a very sophisticated lay audience, so I'm trying to bridge my talks between the different levels that I try to hit. And I have a great deal of titles there. I debated whether or not I wanted to put all of them up there, but I think it's important that I do, because they all play a big role in what I can do here at the university, and why I am at this university. I came here for the a residency, as Tom mentioned, and stayed here because there were just way too many opportunities in Madison for me to leave. Then I've stayed here ever since, for reasons that I think would become obvious to you as my talk goes along. So, I'm director of the McPherson Eye Research Institute, so we're at one of the premier vision research institutes in the world. I'm also a part of the Department of Ophthalmology and Visual Sciences, which is one of the top clinical and research entities in vision science in the nation. And the Waisman Center is a wonderful place that looks at developmental disabilities. And so, my interest in blinding disorders fits in well with them. And then finally, the Stem Cell and Regenerative Medicine Center has been mentioned by many as being the mecca of stem cell research in the world. I'm privileged to be in a unique place on the planet to study what I'm going to talk to you about today. So, what does my lab do? So, my lab has a very clear mission, and I try to bridge what I do and see in the clinic with what I want to ultimately make changes in in terms of therapies for the future. Ultimately, what I'm interested in is using stem cell technology to try to build new treatment for blinding disorders that otherwise fall between the cracks, diseases that for which we don't have any traditional therapies that can help patients. And so, stem cells in general evoke a lot of questions in people's minds, and they're all very valid questions that deserve a lot of attention. And those include fields of ethics, politics, as well as medicine and science. In the interest of time, I'm going to be concentrating on the medical and scientific aspects of stem cell research, but that's not at all to belittle the other ones. I think they are all very important sources of discussion that need to be addressed in public forums, as well as in scientific conferences. And so, I'm happy to discuss those later on if there are specific questions. Speaking of ethics, I need to disclose a couple of things right off the bat, so that you can decide whether or not what I'm telling you is the up and up, or whether I have some bias. I do have a patent. We patented the ability to create three dimensional human retinal structures in a dish. And that has led to the establishment of a company, Opsis Therapeutics here in Madison that collaborates with another company, established by Jamie Thomson, and that's Cellular Dynamics International. I do want you to be aware of that upfront. So, what I'd like to go through today is basically two sections. The first is a lot of introduction. So I'd like to give you some background on cell biology in general, just enough so that I can then talk to you about the eye and the retina, what are its parts, what does it do, and then move into degenerative diseases of the retina, the specific types of diseases that I'm interested in, and then lead into a discussion of what is stem cell biology. And then that'll form the basis for which we can go into a longer discussion about how we're trying to use stem cell technology to combat some of these diseases. And then lastly, I debated whether I wanted to put this upfront, because I get the privilege of talking to you about a lot of his work, but there are tremendous number of people in my laboratory, collaborators here on campus and in other institutions, that play a tremendous role in advancing this technology. And so, while I appear to get the credit by talking about it, I want to make sure I have plenty of time to acknowledge the folks that a  So, as an ophthalmologist, we like to dilate pupils, we like to look inside of eyes, and when we do that, this is what we see. So, this is a normal retinal. The whitish, yellowish circle towards the left is the optic nerve. this is where all of the nerve impulses that are collected in the retina from looking at objects gets collected and sent back to the brain. The red lines that you're seeing are the retinal blood vessels that feed the inner portions of the retina. And then the rest of it that looks kind of orange, the retina itself is really transparent like cellophane. The reason why it looks orange is that there's a very vascular layer right behind it. And so, that red blood shining through the rest of the tissues makes it look orangish. So that's a healthy retina. And just like everyone tells you, you only use 10% of your brain. For the most part, when us as humans, we use the central portion of our retina called the macula. And that's just to the right of optic nerve in that kind of area where it looks like there are no blood vessels. So that's the very important part of our retina that we use to recognize faces, to read. It gives us our highest acuity vision and our color vision. The rest, everything else around that is for peripheral vision. And there are some cones and some color sensation too, but that's more for night vision and peripheral vision, but it affords us very poor acuity. So, the high acuity vision that we rely on so much is right there in the center. And so, this would be then a normal visual field that someone might have with a healthy retina. So, now let's delve into this a little bit more. If we take that same eye and we cut it in half, and we look at it from the side, we get this sort of schematic here. So, on the left side, that's a cross section of your cornea. It's clear. It's part of the refractive portion of the eye that bends light. The oval to the right of that is the lens, light passes through that, and then that gets refracted even more, and that's actually a dynamic structure so it can refract light. Well, I'm starting to lose that ability, but when you're young, you can refract light, and then adjust the focus of objects onto your retina. And then on the very back, everything that looks orange is a chamber filled with what's called vitreous fluid, so that's kind of a jelly that's in there. And then along the back of the eye, like a wallpaper, is where the retina lies. It's like film in the camera. And so, if you take a little piece of that retina and blow it up, and look at it on the side, so now we're looking at that piece of cellophane on its edge, and we've blown it up under a microscope, and we see that it's really not just a boring piece of cellophane, but it's actually a very elegant layer cake with multiple different cell layers, each of which does a very particular job. And it's easiest to think about the retina as a simple brain. That has essentially three neurons or three cells arranged in a circuit. Deepest in the retina, and this is why it's transparent, but deepest in the retina, closest to the blood supply that makes the retina look orange, because it's such a highly metabolic cell type, is our photoreceptor cells. These are the rods, which detect gray vision, white and black, and then cones, which are your blue, your red, and your green cones. They're deepest within the retina in a layer called the outer nuclear layer. And they exist right up next to but another layer of cells called the retinal pigment epithelium. And these are kind of like the Batman and Robin of vision. So, photo receptors get all the credit because they're the rods and the cones that detect light. But the retinal pigment epithelium are their handmaidens. They do a lot of the work. They're there to provide all the nutrients so the photoreceptors can stay healthy and active. And they also take away all the waste products that photoreceptors produce in large abundance, and pull them away and deposit them into that blood supply behind it just so they can get carried away. So then you have photorece  On the right side, this is a patient that has something similar to the left, but also super imposed upon that has experienced a bleed, And this is what patient with advanced AMD would see. They wouldn't go blind entirely, because their peripheral vision is unaffected, but they lose that central portion, and so you're left with essentially very little ability to, essentially, to drive, to recognize faces, or to read, all these things that you really look forward to doing once you retire. So, that brings us to some-- There we are. If we have loss of cells, what are our choices? Just theoretically thinking about this. If you've lost cells in the retina that are critical for detecting light, you essentially have two options at that point. You can either bypass the whole system. So you say, "Okay, well, the photoreceptors are gone, so we have no natural ability to detect light, which means we're going to not have vision in the area where the photoreceptors are lost. So can we take some of the downstream cells, like the bipolar cells and ganglion cells, and get them to detect light? That's something called optogenetics. Or can we directly stimulate the ganglion cells with a chip? And that's also available these days. Second Sight has a chip that will directly stimulate. And that has helped patients who are totally blind get flashes of light, detect movement, provide them some sort of vision. And that's, while crude, something that has not been available decade or more ago. And I bring this up to mention that there are other places you can turn, besides stem cells. What I think stem cells bring to the table is because it's a natural replacement, the ceiling is a lot higher. And if we can get it right, we're more likely to restore a more natural type of vision, as opposed to an artificial light on, light off. Okay? So, my laboratory is interested in the idea of replacement. And so, that brings up the next question, which is, okay, where are the spare parts going to come from? So, if you're going to replace photoreceptors or RPE cells that are lost in disease, you have to find some place that you can get them. Living donors, so you can say, "What about a kidney? "Can't you take something from my other eye "and put it into this eye?" Just like in the brain, when you mess with the eye, you're going to blind somebody. So, you don't have the ability to reach in and take your twin's retina and try to replace something that's been lost. What about a deceased donor or a cadaver. Again, that's been tried, and it just doesn't hook up very well. If you take a mature retina that's already been fully formed and try to pull it a part, they're so fragile at that point that they tend to die, and they don't hook back up again. What about non-human? We used pig valves. And saw other animal parts sometimes we use for other tissues. That has not been directly tested in the eye, but the same thought would generally apply. But if you take something that's fully formed, a fully formed part of the retina, just like a part of the brain, that those brain transplants, these transplants are fully formed neurons just don't do a good job in these patients. So, what about then lab manufactured? So, can we actually-- Can we make these cells in a dish, usher them to an appropriate stage where we think they might conducive to installing them and getting them to hook back up into a deceased retina, so that we can restore vision. And that was a great thought, and many people worked towards that, but there really was no source material for that. Until in the late '90s, this person Jamie Thomson, collaborator of mine, and somebody that helped get me into the field, discovered a way to take a part of a five-day-old blastocyst, called the inner cell mass, and grow that in a dish. These were embryonic stem cells. Everyone knew that they existed, because ultimately all of the tissues in our body emanate from this mass of cells. But the ability to grow it in a dish in a laboratory had never been shown prior to Dr. Thomson's work. And embryonic stem cells are just one  and eggs that are going to be discarded, and they're shown here. So each one of those circles there in the center in the square are thousands of individual embryonic stem cells. The other cells that are rounded are feeder cells. We generally don't have to use those very much anymore, but those very bland-looking colonies of cells are what all the fuss is about. And they do indeed have the, at least, theoretical capacity to produce all the different cell types in a human, but it can't make a human, but it can make all the different cell types. And then along around 2006, 2007, there came about another type of pluripotent stem cell called an induced pluripotent stem cell. And Dr. Thomson here at UW-Madison as well as Shinya Yamanaka in Japan, both almost concurrently figured out a way to take somatic cells, so cells from an adult human, and to genetically reprogram them back to a state where they're very similar to, if not identical to, embryonic stem cells. So, because these are cells that were induced to become pluripotent, instead of being that way to begin with, they are called induced pluripotent stem cells. And this was a wonderful advancement, because it allowed us to make, essentially, stem cells, pluripotent stem cells from any human being on the face of the planet. And here's what they look like. So they're grown in the exact same way. They grow in these colonies that are relatively bland looking. And ultimately, they can, at least theoretically, produce all the cell types of a human body. But because they're produced from adults, we can take, for instance, a patient that has a particular disease and through a skin or blood sample, reprogram those cells. So, these days, you can take a blood sample. Note, not much dissimilar from a sample you might send for a cholesterol check, and isolate certain cell types, genetically reprogram them back to this IPS cell or stem cell state. And at that point, you have that particular patient's genetic material encapsulated in their own stem cells. And if you have the ability then to push those, forward engineer those to, in our case, replacement retinal cells, then perhaps you could put that back into the patient. But along the way, you're also going to have to probably correct if there's a genetic defect. You want to correct that, otherwise you're just going to have the same disease occurring again. The other option would be to take anther source of IPS cells from a different individual who doesn't have that particular disease, who perhaps is closely immune matched, and we'll talk about that at the very end. Now, something else that you could do with these IPS cells that's important to point out, but I won't talk about, is something called disease modeling. In this case, you have these IPS cells from the patient. And if you can create the cell types that are affected by that disease, you automatically now have an unlimited supply of that patient's affected tissue in a dish. And it has the same genetic disease. It has all the other genetic makeup that that patient has, And so, you can study that disease, that patient's disease in a dish, and then perhaps test drugs, test gene therapies, test more traditional approaches to therapy to see whether or not you can affect the course of that disease. And that way, you can use IPS cell technology to help the patient without having to put that cell necessarily back into the patient. So, I'm not going to go into that, but it's a very powerful application of the technology that does not require transplantation, so keep that in mind. About half of my lab works on this type of disease modeling project. But in both cases, like I said before, the trick is you have to make the cells. You haave to ultimately get these stem cells to create the cells that you need to study or to place back into the patient. So, how do we do that? In almost every case, we use development. So, developmental biology is our guide. So, stem cells are, during the course of their lifetime, normally, have to go down different pathways, are affected by different cues   In some cases, there's like natural clock inside the cells that tells them when it's time to make a decision, but there are also other cues in the environment, and they could be diffusible factors or chemicals that are made by the embryo. They can be physical interactions where something pushing on another thing or touches another thing that it influences what it's going to become ultimately. And so, it's a tall order, right? We're going to try and somehow recapitulate this in a dish. It turns out we're pretty lucky, because a lot of it kind of does it on its own. And so, we do have to nudge it in certain critical times, but what we found is with just perhaps a few key cues or manipulations, then we could really get these cells to respond in a way that we want to, whether that be Tim Kamp here trying to make cardiac myocytes for heart disease or Shusen Zhang trying to make dopaminergic neurons for Parkinson's, or us trying to make photoreceptors for retinal diseases. So, we're going to go through just kind of on a high level what we do to get them to towards retina. Here's, again, a colony. We have a couple of colonies of IPS cells. We pick those up off the plate, and let them grow in a three-dimensional structure. As it turns out, anything that wants to be brain likes to grow on these free-floating structures. So that's by itself a step that helps us to get in the right direction, just a simple picking it up off of a plate so that it can grow by itself in a suspended aggregation. We also add certain cues to push towards an early brain. And then over a period of time, we then played them back down, so that they go from being a suspended aggregate to being stuck down onto a plate. We switch the cues that we provide them, again, trying to mimic development. And over time, we see them form these kind of very interesting radially oriented cellular colonies with almost like a little hole in the center. And we could take those off. We can actually pick those off, Like this. And so, when we pop them off, we leave behind all the cells that stay stuck, and we pick off these little circular structures which we call golden cheerios, and they're actually vesicular little aggregates of cells, and they're about perhaps the size of a coarse grain of salt, if you look at them in your hand. So, these are small. And so, we can pick them up, grow them on mass and flasks. And if you look first at what's left, so instead of looking at those golden cheerios, which is what we'll spend time with for the rest of my talk, if you look at what's left behind, so that's the hole from where we picked off one of these vesicular type structures. And if we let it go over time and we treat it just right, we'll find that, that skirt around where those golden cheerios used to be will become retinal pigmental epithelium. And they will form the retinal pigment epithelial cells, not only in terms of what they look like, but they also can do all the same functions that we see in a normal eye. And we can make these, and this is now being done by many laboratories around the world. It's also in clinical trials now, and I'll talk about that at the end. But it's really remarkable, we can make it, essentially, an unlimited supply of retinal pigment epithelium. So, the spare part, at least for that support layer is not that hard to make, it turns out. and it kind of wants to do it. You have to almost make a mistake not to get this. So, that's nice. Not the same for photoreceptors as I'll show you. So let's go back then to the little golden cheerios, the little vesicular structures that we picked up. So these are hollow balls. These is under a microscope, so it looks kind a phase bright and golden on the outside, but envision that they're just hollow. They're hollow balls. But are these really optic vesicles? Are these really those early structures that I showed you earlier in my talk that are budding off of the developing brain? And to be able to show that, I've have to be able to follow these over time, and see what they make. And so, let's take a look at one of  Beth Capowski and Joe Philips deserve special recognition. Beth did a lot of the work on the three-dimensional structures that you saw, and Joe runs our transplant program. Without these two individuals, there's no way I would be anywhere close to where we are right now. And of course my laboratory is just one of hundred plus that are part of the McPherson Eye Research Institute. I have them all interconnected here, because we talk with one another, we exchange ideas, we exchange resources and expertise. And that allows us all to move ahead faster and safer, and that's the important thing. And so, our motto is to pioneer science, medicine, and art of saving sight. We're very serious about our support of art. Because ultimately, what are we trying to save? We're not trying to save the ability to see a white dot on a black background. We want to save or restore the ability of people to appreciate the visual world around them. And I have collaborations across campus. These are just some of them. Jamie Thomson is there, Bill Murphy, Sarah Gong. I can't name them all, but just tremendously talented people that are willing and enthusiastic to work with us. And of course we have a lot of sources of support that I'm very grateful for, particularly, I mentioned Dr. Monroe and Sandra Trout, and David and Nancy Walsh who have been very instrumental in getting a lot of this work going, as well as our other source of funding. And with that, I want to thank you for listening to that. And I'll take any comments and questions you folks have. (audience applauds)    - Welcome everyone to Wednesday Nite at The Lab.