 So, good evening everybody. My name is Himanshu Wadhwa and I'm a medical student at the Department of Ophthalmology doing some research there and grafting campus. And it's my pleasure to be able to share my research review today titled, Investigating the Use of Corneal Stem Cell Enriched Spheres and Karateconic Tissue Repair. So before I start, I'd just like to thank the Exposure Committee and judges and everybody for coming and for giving me this chance to present my research. So first of all, I would like to start with some background. So what is Karateconis? So Karateconis is a disease that affects the cornea, which is the front part of the eye. Indicated in white is the cornea and as we can see in the image on the left, the Karateconic cornea is almost forming a cone shape compared with the more spherical shape of the normal cornea in the image on the right. The word Karateconis, Karate comes from cornea and conus of course cone. So how does it affect people? So people who are diagnosed with Karateconis are often young adults aged in their 20s and 30s and this can cause vision loss in severe cases. And in those cases what we do is we treat them with corneal transplants. So we cut out the weak diseased cornea and replace it with a healthy cornea from a human donor. And this comes with its own risks and resource constraints. In New Zealand, Karateconis is the number one indication of corneal transplants. And New Zealand has one of the highest prevalence rates of Karateconis in the world. It predominantly affects Māori and Pacific Island people. So why does it happen? So normally the corneal cells, the creatocytes, produce matrix which is the scaffold that holds these cells together. And the matrix influences the corneal cells. But for complex reasons, not completely understood in Karateconis, the corneal cells die and there's also a loss of matrix. So what we thought is what if we could replace the lost cells? And so this is what my research is about. So first of all we took stem cells which live in the limbo area, which is the area between the clear cornea and the white sclera indicated by the red line. And we made spheres. So stem cells are a very special type of cells. These limbo stem cells are adult stem cells which normally become the other eye cells. And what's special about them is that they normally repair anything that damages the cornea. So let's say a child scratched the front of the cornea, these cells would go and repair the cornea. So we generated spheres. And as we can see, this is a well of spheres. And the little white speckles here are the spheres. Now if you're wondering why you can't see them, don't worry. It's because they're really small and it's really hard to see with the naked eye. So what I've done is I've magnified them for you. And these brown circles are the stem cell and rich spheres. So what we did is we took keratoconic corneas from donors and we implanted them with our spheres. And these are the things we assessed. So do the cells survive? So there's no point in planting spheres if they don't survive. Do they migrate? We'd like to see the cells spread out. Do the cells divide? So this is quite important in the sustainability of using spheres as potential treatments for keratoconus. And finally, do the cells differentiate? So do they become more specialized? Do they become wound healing cells? Do they become cells that produce matrix? What type of cells do they become? And this is what we saw. So this is a keratoconic cornea from a human donor. And it's about eight millimeters in diameter. And what we can see is that there's three spheres implanted, one, two, three. And this image, the green indicates the living cells. And what this image tells us is three things. A, that this spheres can survive in keratoconic tissue. They can adhere in keratoconic tissue. And they begin migrating. And what I'm about to show you illustrates the impressive migratory potential of spheres. So just three spheres, and at 10 days we can see that the cells have continued to migrate even further. So they're beginning to repopulate the disease keratoconic cornea. And what's interesting is that we also noticed that the spheres can communicate with each other when they're placed near to each other. So see here how the cells are forming a cellular bridge. So that's indicated between the white arrows. But more interesting than this is whether the increased number of cells that we see is only due to cell migration or are cells dividing as well. So remember that is important for the sustainability of using stem cell spheres as potential treatments. And so to check this, what we did is we stained our cells with a special dye, which causes cells to light up red when they're dividing. And so as we can see, these are cells within the sphere. And because of the lighting, it's not as clear, but here's a few cells that are lighting up red. So as we can see, some cells are able to divide when they're in the sphere. And interestingly, these cells are not within the sphere. And what we can see is that cells don't need to be within the sphere to be able to divide. So some cells are still lighting up red, even though they're outside the sphere. So cells are able to migrate, cells are able to divide, our cells able to differentiate too. So let's go back to the image at day 10 and take a closer look at this cell here. And what we can see is that this cell doesn't normally appear like a limbo stem cell because it's got these long kratocytic projections. And remember kratocyte is a specialized cell that's seen in the middle layer of the cornea, the front part of the eye. And so what this tells us is that cells are beginning to become specialized. They're beginning to differentiate. But we had to be sure, so we had to check. And so to do this, we looked at a few markers. And I'm going to show you one today, which is an important marker. And the name of the marker is Alpha Smooth Muscle Acton. Rather than the name, which is not important, is the fact that it indicates a myofibroblast. Again, the more important thing is that this cell is a wound healing cell. So it's commonly seen in wound healing. It's a repair cell. And so what we can see here is these cells have migrated away from the sphere. And these green lines here are the individual filaments inside the cell. In fact, these filaments are Alpha Smooth Muscle Acton. So the cells that have these green lines are positive for that marker. And that means that these cells are the repair cells. And so what this means is that some cells are able to differentiate as they move away from the sphere. And so in summary, what we've shown is that spheres can survive, they can adhere, and they can maintain their stimulus and cratoconic tissue. And cells can migrate from spheres and appear to become more differentiated as they do so. This is the first time that we've been able to culture stem cell enriched spheres and cratoconic tissue. And our research shows real potential for treating cratoconus in the future, potentially even being a cure. So currently, on the wait list, there's about six months booking in advance for corneal transplants in public and in private, even up to February. So there's quite a lot of people on the wait list. And if we can use spheres, we can cut down the number of transplants that are needed. So our future research is to get some quantitative data to look at spheres in real time, to study the spheres in live tissue implants and, importantly, to study the functional aspects of these spheres. So if we can replace the cells, do they produce the matrix as well? I'd like to thank my team and a heartfelt thank to, heartfelt thank you to the corneal donors and their families for donating the tissue without whom this would, of course, not be possible. And all the people who have helped me in this, who I haven't listed because the journey would, because the list would be so exhausted. And thank you all for being in a tentative audience. Thank you. Please correct me if I'm wrong. So the methodology you proposed is not addressing the underlying pathological process. Is that correct? Sorry, what was that? You're not addressing the underlying pathological process. It's not clear why they were doing that. So one of the chances that this is milder than the cells, we'll just respond to the second page. That's a good question. So that's true. We don't completely understand the underlying process that causes the disease, correct? But these spheres that we implant are from normal human donors. So one of the things that we want to see is that the cells can behave normally when they're put in this disease matrix. And that's why we've got, I haven't been able to show you, but we've got controls and that shows that they seem to be behaving the same in the craticonic matrix. Another thing is that craticonus, although we don't completely understand it, we do understand that there's genetic influences and there's also environmental influences that influence the disease and its progression. So if you're implanting normal cells into a disease process, what we're seeing is that they behave normally at the moment. Of course we need to do some further work. At the moment, we've followed it for 14 days. That's a really good question. My work's not to look at the epidemiological or the causes, as per se. I was more focusing on a potential treatment. But at the moment what we think is just possibly genetic predisposition. Yeah. That's a good question too. So one of the things, there's many steps, but initially what we can look at is just look at a staining for, take a control that hasn't been implanted and take controls that have been implanted earlier and compare them all and as time permits you can stain for the collagen. So collagen is the main thing that is made up on the matrix. And we can just stain for it using immunohistochemistry. So some of the images I showed you is just proteins that are present and we've tagged it and then it fluoresces. So we can do that. Look to see if the amount of collagen increases over time. So especially compared to non-implanted and implanted. We can also do mRNA analysis. So before proteins comes genetics and we can look at that. Other thing is down the track we can look at some clinical tests. So like corneal thickness, which we can do. And yeah, at the moment that's what comes to mind. So there's no animal models at the moment that we can model keratoconus. So that's why studying keratoconus itself is quite a hard disease as well. I have had the privilege and the fortune of getting enough tissue from human donors who have received transplants to be able to do all my work in human corneas. But for functional studies I think if we just continue using these ex vivo implants and then just do the functional studies on them first that can maybe guide future potential studies. Yeah, that would be my best answer for that at the moment. We do have other work like so not in keratoconus but in another disease which is called limbal stem cell deficiency. So basically from chemical injury or a burn or something the stem cells themselves die and the eye goes white. So the person can't see. We've seen that in some of those patients where corneal transplants by themselves are not working, you can put these fares, you can put stem cells and limbal explants onto them and it seems to restore vision. So maybe that might be another functional way. So you know in the extreme cases where other treatments are not working we might be able to give them another like here we can try this. Yeah.