 Hello, everyone. We're going to get started with Grand Rounds. Welcome to Grand Rounds. Today, we're having two presentations. First, we're going to hear from Renee Choi. He's one of our new first year residents. I think this is actually his intern presentation, though, because he's scheduling it up doing it now. So he gets two Grand Rounds this year. But he's talking about seeing his believing in retinal regeneration. OK. Thank you, Brian, for that very kind introduction. So, well, today, I'm going to be talking to all of you about a topic that I'm particularly interested in. And I came across this the past couple of years that I've been to ARVO. And that's on the topic of retinal regeneration. And what I'm going to provide for you today is an overview of what's known in the literature regarding this field of retinal regeneration. So to begin this story, we have to first define what exactly are stem cells? They are a specialized class of cells that are not committed toward any particular cell fate. They have this limitless potential to proliferate as well as self-renew. And they also have the ability to develop or differentiate into many different cell types. Now, aside from obtaining stem cells from an embryo, there are many areas in the body, the human body, that we naturally have endogenous stem cells. Some of them include the bone marrow stem cells, corneal stem cells in the limbus to replace the epithelium. And there are also neural stem cells in the subventricular and subgranular zone. And what these serve as, these endogenous stem cells serve as is they essentially replenish all lost cells in a specific organ from normal wearing care. But to date, there are no identified endogenous stem cells in the human retina. And this is quite unfortunate because there are a number of blinding diseases affecting specifically the retina. And they impair vision by reducing the elements that allow us to process a visual scene. So some of them include retinitis pigmentosa and cone rod dystrophies, which primarily affect the photoreceptor layer. Also you have age-related macular degeneration, which affects the RP as well as the photoreceptor layer. Diabetic retinopathy, which is known to affect, affect all cell types in the retina. And glaucoma, you have the ganglion cells. Now to emphasize the gravity of this disease or these diseases, I'd like to demonstrate to all of you how retinitis pigmentosa manifests in a patient who's afflicted with it. So here is a beautiful picture of the Tetons from Teton National Park. And all of us who are not afflicted with retinitis pigmentosa, we can see this image in its entirety. However, somebody who has retinitis pigmentosa, they eventually lose their peripheral vision until they reach irreversible blindness. And to date, there's no effective treatment for retinitis pigmentosa. That's myself. There are all these diseases that specifically kill off specific retinal cells in the retina, right? So what if there were an endogenous stem cell source that can replenish these lost cells? As occurs, for instance, with our corneal epithelium. So to begin this journey, we've got to start with the history. History of the field of regeneration and how it all started. Starting in 1744, when Abraham Tremblay, he was a Swiss naturalist, and he was the first to discover that the Hydra was capable of regenerating different parts of its body when it was surgically resected. Now it wasn't until 1781, Charles Benet, many of you may be familiar with him because he discovered also Charles Benet syndrome. He discovered that Newt's had this remarkable capability of regenerating their eyes if a small portion of it was removed. This discovery essentially led to further investigations in lens and retinal regeneration as key experimental systems for the next two centuries. Now to the main part of the talk, is there evidence essentially of retinal regeneration in the literature? And the answer is yes. And it depends on where damage occurs. Also it depends on the species. And if damage occurs in the central parts of the retina, the amphibians have this remarkable capability of regenerating the retina and the putative stem cells are known to be the retinal pigmented epithelial cells. Now in the fish, it's been identified as the mulliglia. Now before we talk about this, I want to cover this one area known as the ciliary marginal zone, the CMZ. So what exactly is the CMZ? It's an area that lies between the ciliary epithelium and the endogenous retina. And it consists of retinal progenitor cells that are constantly cycling and they generate new neurons throughout the life of an animal. Now this occurs in amphibians as well as fish. Now what's remarkable about the CMZ is that in amphibian models such as Xenopus lavis, the South African clawed frog, as well as newts, multiple groups have found that once you damage the retina and it could be many different damage paradigms. It can either be from mechanical damage from a retinectomy or even neurotoxic damage, right? The CMZ can respond by increasing its rate of proliferation of cells. What's even more remarkable about the CMZ is that if you ablate a specific type of cell in the retina, for instance if you ablate an amocrine cell, the CMZ has a feedback mechanism where it increases the selective production of that cell type. It's actually quite remarkable. Now what about CMZ in mammals or more importantly humans? Unfortunately this does not exist. Now the limitation of the CMZ is that it proliferates and generates new retinal progenitor cells for regeneration only in the peripheral areas in the retina. They don't have the ability to migrate into the central areas of the retina if they're damaged. So now I'm gonna cover about the central areas of the retina. What are the primary cell type? What's the primary cell type that gives rise to these new neurons? Well, let's first cover amphibians. In amphibians it's been identified as the retinal pigmented epithelial cell. So let's talk a little bit about the retinal pigmented epithelium. So this is a specialized group of epithelial cells that sits above the photoreceptor layer. And they're involved in many different maintenance tasks for the retina. Some of them include absorbing light to protect the photoreceptors, recycling photopigment that's part of the phototransduction cascade, as well as phagocytosing the outer segments of the photoreceptors. Now various groups across the country as well as internationally have identified amphibians and two different species or subsects of amphibians, the aneuron amphibians, as well as the urodyl amphibians. They have this ability where you perform a retinectomy. So that's mechanical damage. When you physically scoop out the retina, you give these animals ample time to recover. They're able to re-establish the retina almost as if nothing ever took place. It's quite fascinating. So what does this process entail? It's known as a process called retinal pigment epithelial trans-differentiation. And it's broken up into two different steps. The first step is that the cells need to de-differentiate into an earlier precursor cell. So what they do is they re-enter the cell cycle. They begin to lose their pigment and then they start to express retinal progenitor specific genes. After that, the second step, they begin to re-differentiate into all the different cell types within the retina. Now up until now, we've been talking about mechanical forms of injury, at least in the amphibian. So if you look in the literature, there's actually evidence of disease models showing that they can actually they can regenerate lost retinal cell type. So here's one particular paper where they develop a transgenic line of retinitis pigmentosa and frog. They were able to specifically ablate all the rod photoreceptors. And then after doing so, so this picture right here, they got rid of all the rod photoreceptors. They gave the frog 30 days to recover and what they were able to determine was that there are these trans-ducin labeled cells which transducin is a specific marker to rod photoreceptors and they're also labeled with EDU. And EDU is a marker for cells that are newly generated. Basically, if they re-enter the cell cycle. Thus suggesting that these are brand new newly generated cells. And what about the molar glia? The molar glia have been identified as the stem cells in zebrafish as well as potentially mice. However, there's only circumstantial evidence showing that it occurs in mice and we'll cover that in a second. So I want to know in the field how exactly did they determine that the molar glial cells were the stem cells giving rise to new neurons after damage. It was, they figured out, it was a brilliant experiment done by Pamela Raymond's group from the University of Michigan. What she did was that she developed a transgenic fish line where she had the GFAP promoter which is a molar glial specific promoter that's driving green fluorescent protein. So essentially what that means is that every molar glial cell is labeled with green fluorescent protein. She essentially did a lineage tracing study here. Then she photo ablated all the cone and rod photoreceptors and found that over time, right, there are new rod and cone photoreceptors that were labeled with GFP. If you see here, here's GFP. Here's row 42 which is a marker specific for rod photoreceptors. And here's BRDU which is a, it's basically a marker for new cells that are generated and they're all cologalized. And you also have here cone photoreceptors. This is a ZPR-1. It's a marker specific for cones, co-labeled with GFP. Now how about mammals? Literature is very, it's very limited. So, but there's this one paper that was published by Tom Ray's group from the University of Washington. And what they determined was after neurotoxic damage, they specifically used neurotoxin NMDA which kills amicron cells as well as retinal ganglion cells. They found out was that there's a very limited proliferous response of the molar glial cells after injury. However, this response can be increased with exogenous factors administered such as FGF, fibroblast growth factor and epidermal growth factor. D-differentiation also took place when they administered these exogenous growth factors. However, these newly quote unquote stem cells had a limited differentiation profile meaning that they're only able to differentiate into amicron cells. They didn't develop into any other cell type. But the real question is whether or not the molar glial cells really are the stem cells for the mouse retina. And that question hasn't been answered yet because they didn't do a lineage tracing study like Pamela Raymond's group did in the zebrafish. Before I get to the last section of my talk, I just wanna cover briefly stem cell transplantation. So there are a number of groups trying to develop stem cell transplantation techniques for to heal or essentially as a form of treatment for blinding diseases affecting the retina. And obviously they do this because we lack this regenerative response. There's a seminal paper published by Flaren and colleagues out at the University of College in London where they were able to take rod precursor cells, transplant them into the sub-retinal space of various retinitis pigmentosa models in mouse. And these precursor cells were able to fully differentiate into rod for receptors as well as functionally integrate into the rest of the retina. And they were able to determine that they functionally integrated by doing light-devoked multi-fueled potentials as well as with pupillary or gauging pupillary responses. Now this is all very exciting. At this point of the literature search, I thought it was very exciting. The fact that people are trying to uncover the mechanisms that govern regeneration but also different techniques for these blinding retinal diseases. However, I was thinking if we were to take this approach and translate it to the clinic, what are some potential limitations? Now one of the major limitations is this concept of retinal remodeling. And many of you may be familiar with this because this has all been characterized by Robert E. Mark from our very own Moran Eye Center. So what Dr. Mark and his group discovered was that in various animal models of retinal degeneration, after de-affirmentation of the rest of the retina from the photoreceptors, there's this massive reorganization of the retina that occurs, okay? And it's characterized, or it's broken up into sequential phases. The first is characterized by rod photoreceptor death, cone outer loss, the molar glial cells begin to hypertrophy, undergo the gliotic process, as well as bipolar cell dendrite retraction. The second phase is characterized by the death of cone photoreceptors and the molar glial seal that actually forms. And that's when the molar glial cells extend their processes out into the sub-retinal space and they entomb any cells that are in the outer nuclear layer. The third phase is broken up into two sub-phases. The first sub-phase characterized by new micro neuroma formation, that's when these neurofibrillary tangles form from various processes from various cells, such as bipolar cells, ganglion cells, as well as amicron cells. Lastly, the late stage three, there's death of all cell types within the retina as well as cell translocations. So you'll see an amicron cell translocated to the outer nuclear layer where they don't belong, or you'll have a surviving rod photoreceptor in the inner nuclear layer. So I ask myself if there's this massive rewiring of the retina that's going on, it may not be the best idea to just reactivate these mechanisms that govern regeneration or take a stem cell transplantation approach in order to replenish lost cells because how do we know the rest of the retina is still intact in order to process information the same way that you and I would be able to if we don't, because we don't have these diseases. So if I were to take a clinical approach using stem cell transplantation or activating these regenerative mechanisms, I would first take a two-step approach. This is actually the part that the stem cell field is starting to get somewhat ignoring, most likely because they're trying to get their grants funded, but really, what happens is that I think we first have to at least, you know, it doesn't matter what the disease is that's affecting the retina. We have to first focus on elucidating the mechanism that governs the disease because only then can we identify specific steps to intervene on to halt the progression of the disease, prevent modeling of the retina, and then hopefully then we could focus on activating these regenerative mechanisms as well as using stem cell transplantation to one day allow somebody with retinitis pigmentosa to be able to see this image in that entirety. On that note, I'd like to thank everybody for coming to my talk and it's an honor to be here at the Moran. Thank you. Yes, Dr. Olson. First, I would like to acknowledge that I agree with everything Dr. Bernstein said. I think that if one were to take the stem cell transplantation approach to heal retinal diseases, I think it's in its infancy. The field is in its infancy and the reason why is because if you look at the literature, everybody is just using as a, it's almost a crude form of engineering. They're just shoving cells into the sub-retinal space or the intravitural space and hoping to God that they differentiate, functionally integrate, structurally integrate. But really we have to focus first on understanding all the different developmental steps of stem cell development but also differentiation. And that hasn't been mapped out yet. I think we have to understand that before we start throwing cells into an in vivo, you know, animal model. And they're doing it because the presence act in very advanced disease because of the level of the study. Well, we know that there's substantial... Dr. Warner, you're absolutely right about that because there have been some great developments from stem cell transplantation research. For instance, after taking stem cells transplanted to the retinal, they found that it could somehow, for some reason, it actually prevents the progression of retinitis pigmentosa. And they've identified it was BDNF. I think this was a group out in Bascom Palmer. And now they're trying to use, find some way to administer BDNF into retinas with afflicted retinitis pigmentosa. Very good, yeah. That's a very good question. So in amphibians as well as in fish, they've determined in fish, I believe they perform ERGs after regeneration, after photoablation of the cone and rod for receptors and then after they regenerated and they found that they were able to fully function just based on electrophysiologic data. In amphibians, there's one group that use a behavioral, a visually mediated behavioral assay where half the tank is white, half the tank is black. Usually they like to stay on the white side of the tank but after ablation of all the rod for receptors, that's scotop vision, 50% of the time they're on the white, 50% of the time they're on the black. Give them ample time to regenerate, they all swim back to the white side. So that's all I know. All right, thank you very much.