 So our next presenter is Dr. Michael Burrow. He's going to be talking about stem cell therapy for corneal scarring. All right. Good morning everybody. I'm really excited to be here. Like Lisa, my name is Mike Burrow. I'm one of the new interns this year and I'm excited to talk to you a little bit about the research that I did at the University of Pittsburgh where I did medical school. Another direction of Dr. James Funderberg, so. So I'm going to skip past a few of these things just taking into account the audience here. But I want to talk about the cornea. We all know that the cornea is first tissue through which light enters. It's about two-thirds of the isofaracic power and there's five distinct layers, including three cellular layers. I'm going to focus a little bit on the cornea stromal today, which is the bulk of the cornea and made up of these tightly packed, orthogonal, collagen fibers and also a quiescent parasite population. Importantly, there are these hydrophilic proteoglycans, which help to maintain the spacing between these collagen fibers, which helps to maintain the transparency of the cornea, which is, I mean, this is critical to that transparency. As you can see in these pictures, when there's a disorganized and irregular spacing of those fibers, we go from a very transparent transparent tissue to a more hazy and opaque tissue. One of the things that can cause that disorganization or disruption is corneal scarring due to things like corneal trauma, foreign bodies of abrasion and infection. And treatment really varies, as I know all of you are well aware. But basically, if it gets bad enough, the options are limited to some type of transplant, whether that's total penetrating carotoplasty or some type of lamellar carotoplasty. With PKP, it's a common procedure and it's often very successful, specifically here in the United States. But there are some drawbacks to PKP and one of them is just the availability of tissue, which might not be such an issue here in the United States. But I'm going to focus a little bit more worldwide and specifically in India for the reason that during my research time there, we actually were lucky enough to have a visiting surgeon and researcher, Dr. Siam Basu, from the LB Prasad Eye Institute in Hyderabad come and visit with us. And so it kind of hit home for him and we talked a lot about this and this is where he eventually has now taken this research and tried to apply it clinically. But there are contraindications for the use of donor tissue and one of the major problems around the globe, not only in India, is just the availability of that tissue. Here are just some statistics that, I guess, highlight that point a little bit more. And I think we're maybe all aware of that on the surface that, you know, the donor tissue isn't available. But some of these statistics I thought were pretty striking. For example, compared to the U.S., you know, corneal blindness is about 14% of all blindness in India. They have trouble with a budding network of eye banks, not enough tissue availability, and an inadequate corneal specialist. But even more than that, the etiology of this corneal scarring, about 5% compared to about 5% here in the U.S., it can be due to infection and scarring. And then just to give, again, a little highlight to the total number needed, which is pretty striking, there's about 1.8 million people with corneal blindness. And about 30% of those are children and there's 30,000 out of this annually in India. Some more of the drawbacks that maybe apply a little bit more, I mean, certainly worldwide, but a little bit more here in the U.S. is where everyone, no matter where they get their transplants done, has to deal with the facet of rejection. And here's just a capital-mire survival curve showing, you know, if it's the first graph, second graph, what kind of the median survival is, or the overall survival is. And then, of course, other complications, surgical complications, post-op astigmatism, demand supply. So that brings me to the point here, you know, why, that's hopefully a good background on to why we thought this is such an important area of research, is can we use stem cells for the treatment of corneal fibrosis? Stem cells, whether they're all autologous or allogenic, I guess, have different pros and cons. Autologous, obviously, would be the ideal situation since they have a decreased risk of rejection and there's less manipulation required in vivo, especially if we were to harvest these cells from somewhere like the corneal stroma of the contralateral eye. However, there are, there is the possibility of doing allogenic stem cell transplants, and there's various areas where those cells can be harvested as well. And if the patient isn't a good candidate for the contralateral eye, we can still do a metologous stem cell harvest from, or in the way of mesenchymal stem cells, and then with some in vitro manipulation, which many, many, many groups have been studying for many years or decades at this point. Just a few that I highlighted that have been studied by Dr. Funderberg and his group are collection from bone marrow, from adipose tissue, or even dental pulp stem cells. So, back in 2005, Dr. Dew, with Dr. Funderberg, had identified through facts a population, a side population of cells, which expressed stem cell markers in this area here, similar to where we find stem cells in epithelium in the Palisades of Vogue region, right out at the Limbus. And deep to that, in the stroma, they were able to identify the side population of cells, which also expressed these stem cell markers. They took those cells and over the subsequent years, they retrieved a mouse model, a murine model of corneal hay. So this was a lumigan knockout model, which a lumigan being one of the proteoglycans necessary to maintain that regular space in the cornea. So in the lumigan knockout mouse, you can see that the cornea is much thinner and over here on the right, there's that haze or that opacity to the cornea. They took the side population of cells, which they had harvested from human donor tissue, either after they'd already been used for the purposes of donation with PKP, or if they were unsuitable for human use. They were able to harvest the stem cells, the population which we talked about in the previous slide, and inject those cells into this murine lumigan knockout model, which then restored the transparency to its kind of wild-type baseline. So they then wondered, can we use these human corneal stromal stem cells to apply to a scar model? So the idea is that if we take an eye that has a scar, really anywhere, but just for I guess to give it more importance, we set it right in the middle of the cornea there. We take a biopsy from the contralateral eye. We can harvest the stromal stem cells in a xenofree autologous culture. We can then reintroduce those cells to the eye and hopefully restore the transparency which exists in the first place. The benefits this we alluded to before, no risk of immune rejection if they're autologous. There's no need for donor corneal tissue, and then some of the post-stop complications, no astigmatism, no suture-related infections, and it can be a fairly quick procedure done under topical format. So these are the steps we had to assess. We had to figure out the way to do the corneal tissue biopsy, then expand the cells in autologous or xenofree human sera, which wasn't really done. You know, most studies in vitro are done using some sort of We use fetal bovine serum, for example. But we need to make sure that culturing these cells in a human serum would not alter the stem, the stemness, if you will, or the differentiating ability of those cells. We do good quality control assays and then also come up with a delivery method for these stem cells. So this is just a series of pictures showing kind of the way that we harvested the limbo area and then digested the tissue. So we'd first strip off the conditiva. As you can see, this was, for example, a donor eye, which had been used for PKP, but it was still left with the limbus intact. So we're able to harvest the limbus, cut that out, and then cut it into sections, which we would then put in a collagenase or a disc paste and then be able to harvest those stem cells in further culture in a human serum versus a fetal bovine serum. And I didn't go into the details here. We're going to gloss over this a little bit, but we were able to find that there was no difference between culturing these cells with human serum versus fetal bovine serum. Again, this is a lot of graphs showing that the cells, whether they were culturing fetal bovine serum or human serum, maintained their stem cell markers as well as after they differentiated in this orange grafts. They also maintained their ability to produce in a regular keratocan and other corneal keratocyte markers. Finally, in in vitro, when these cells were cultured on a particular environment, so we used an aligned nanifier substrate, they produced an aligned and 3D stromal-like tissue and also secreted keratin-sulfate protein-aclycans. So that brings us to the SCAR model. Luckily, in a prior study that we had done, we had identified a way to mouse SCAR models. So we were working with a group who was trying to use OCT to be able to quantify SCARs and amurine cornea. So we worked with them doing that, but also developing this model of a mouse SCAR. So we used an algebra brush, which is basically a mechanical rotating burr. We'd do one pass over the mouse corneal epithelium to a debris of the epithelium, then go deeper into the stroma. And that in and of itself was we were able to use as a very nice SCAR model in this cornea. So then after that with the delivery model, we could just right after we've done that second pass to create that further debris, and we could add a suspension of those human corneal stromal stem cells in fibrinogen, and then we could solidify that or create a gel that would stay there a little bit easier by adding some thrombin. And then we could follow those over time, whether that fibrinogen contained the stem cells or as a control did not contain the stem cells. It was just a gel control. Oops. And to analyze this, there were kind of four main facets we want to look at is markers of fibrosis and healing, which we did using QPCR, immunohistochemistry, stem cell differentiation, collagen organization, and then light scattering corneal opacity. So this was the opening picture, just showing that we were able to identify the human corneal stem cells that were labeled with DIO, and they existed in the murine corneal up to this. This is four weeks, but we followed them up to six or even nine months afterwards, and they still were viable on the murine corneal. This is showing that after we wounded the mice, we added either the gel only or the gel plus the human corneal stem cells, and then we had the unwounded control mice. So this is showing spark and collagen three. These are just markers of fibrosis that in the gel only group, those markers were much higher, whereas in the unwounded and the gel plus stem cells, there was no significant difference found between the two. Similarly, just in vitro using immunohistochemistry, we were able to identify that these stem cells did in fact produce collagen one and keratocan, other markers of differentiation in the mouse cornea. So interestingly, this is again human corneal stem cells, which are producing human collagen one and human keratocan, but they are quite well tolerated in the mouse cornea. We then did electron microscopy of the cornea to show that this is a normal sort of normal corneal stroma. This is the cornea after fibrin gel only, and then the cornea after fibrin gel with the stem cells. And you can see that while it's not a perfect match, the diameter and organization of the gel plus the stem cells was largely maintained and was much closer to the average normal fibral diameter than in the fibrin gel only group. And finally, perhaps most striking is the light scatter, which we were able to assess using this model with OCT, showing the gel only group. You can see the light scatter lights up a lot better here than it does on their cell treated counterparts. Have a few different ways to show this. We also have the graph showing actual scar volume, which we were able to identify. And this just shows, kind of rotated out of view there, but it shows that the cornea here was much more transparent than the one up above. Have some other ways to view that here. We're also able to quantify by setting a threshold of the light scatter and then measuring the voxels involved after that threshold to show that in the gel only group, there was much more light scatter than in the gel plus the stem cells. So this was really able to tell us that the stem cells were able to prevent scarring in a mass model of corneal fibrosis, which is important because obviously we're not all carrying around a little suspension of our own corneal stem cells that at any point we can add to our eyes if we have an injury or anything like that. So can this really be applied in a patient population where we're coming in after the fact they already have established scars? What can we do about this? Is it clinically relevant? Basically it's the question and we think it is and it is. Dr, like I said, Sayama Basu, he is really a fantastic surgeon and oh, that's all I'm going to show. So this is a picture of him in a patient. He's started just very preliminary human studies in patients that have these established corneal scars where he's actually going in and just trying to start from baseline again. So using a tree find there to kind of measure out an area of scar that he's going to then debris mechanically and then here you can see him adding that suspension of the patient's human corneal stem cells, which I had another video showing that but just chose not to show it where he had done the biopsy. He adds that and then he'll place just a contact lens over it to aid in the healing and rehabilitation process. So I spoke with him just a few weeks ago. These are still obviously very early in the trials but I thought this was really exciting work that he's doing over there. There's just been, you know, a handful of patients that have kind of been enrolled in these preliminary trials and these are some of the pictures. Just kind of draw your attention to this bottom one, most importantly, because the other ones on the post-op pictures, you know, aren't staying like this. So, but this is one week post that stem cell therapy, which was kind of showing in the video. So you can still obviously note the haze and the scar in here, but it's certainly not nearly to the extent that it is in this pre-op picture. This is some nine month outcome. So, like I said, he's been doing this for a while but it's still just in the very preliminary stages. Pre-op versus post-op and then some associated imaging to just show the improvements in the light scatter and transparency. So that's basically it. I just want to just give thanks to the lab back in Pittsburgh, especially Dr. Funderberg, who was my primary investigator, and then also Andrew Hertzenberg. So he is the doctoral student that was working on this while I was there and just let me kind of jump right in and he's now graduated and working in Boston and then there's the rest of the lab and then here's my funding. So I thank everybody for your time and I'd be happy to take any questions or any comments, especially Dr. Olson. So the feeding is that even without removing the scar, that the stem cells will result in a free organization to enhance clarity. I mean that's generally we think of what you've got to the level of scar that you're so disorganized in regards to your core corneal physiology that the idea that you'd be able to regenerate the cornea without a transplant, you know, is something but this is this is quite, you know, interesting to see. It would be it's going to be interesting to see, you know, how much that happens and whether it's good or good enough. I think those are going to be some really important questions that we need to discover on that. The big areas stem cells obviously are ways or means that for those with epithelial stem cell failure, how we could use even small areas or you can take where you've got profound changes in both eyes where you can take some stem cells elsewhere, turn them in the corneal epithelial cells. I know there's a lot of work going on at UCSF and Procter and that kind of area. And then we can also figure out how to regenerate endothelial cells and avoid having to do a transplant. But this one, that's pretty interesting. How many clinical cases have they done, you know, where they've actually just gone down to the level of the scar, added their own stem cells and it clears up and we're talking dozens by now? No, no. Yeah, when I spoke with Dr. Rasoud just a few weeks ago, he said just a handful, so five to ten patients. And have they all responded or just some responded? They have. They've all responded. You bring up an excellent point, you know, that, you know, is this going to be good enough. Some of the problems that they're running into is despite this, you know, increase in clarity of the corneal transparency. Yeah, I bet you the acuity is probably not improving. Exactly. It's not improving as much as maybe they expected and they're attributing some of that to the irregularity of the corneal surface. Yes. We've got, remember, you've got transparency, but regularity is even more important. And if you don't believe that, I mean just realize how what looks like a pretty good corneal grossly with soot lamp that you realize that there's a lot of epithelopathy from whatever caused dry ivy. And your vision can be, you know, you can drop vision substantially just associated with regularity. So if it isn't producing a, you know, a regular corneal surface out of this, then it may look good, but, you know, not necessarily really help the patient that much. But fascinating work and obviously a whole new way of thinking about corneal scar. Thank you. And just to kind of add on to that, one of the thoughts that they had is, you know, this is kind of a, you know, not a very, I guess, delicate precise way to remove that scar. So one of the thoughts they've had is maybe introducing the use of, you know, laser to be able to hopefully create a scaffolding for those cells to work on, which might make it a little more regular. Obviously, as I've been tried, and there's a long way to go. And then contact lenses, you know, especially, you know, rigid permeable or gas permeable lenses have helped increase that visual acuity afterwards. But you're certainly right. It's not to the extent as kind of they had hoped in these preliminary phases. Well, the fact that they got that, the change you showed on the picture, I was, I would not have expected that from all the scarring, even that way. Well, what's interesting about the stem cells is they're inherently preprogrammed to go ahead and to lay down this stroma. And the nice thing if you're getting them from the, you know, the area of the limb is whoever you're getting from in the patient, you don't have to induce them with other, you know, materials like you have to do with stem cells from elsewhere to make them become stromal stem cells. And so they're just preprogrammed to do it. And it's very interesting in that you just get them, you grow them, you put them in the gel and put them on there. And they just automatically do what they're supposed to do. And so that is a very much advantage of using the stem cells from the patient themselves, or at least from another eye or something as opposed to getting them from elsewhere in the body and having to induce them. And so this is exciting, especially in a place like India where, you know, you said, what, 60% or due to, you know, infectious scarring. So this would be a huge advance. Yeah, I think so too. Thank you. Thank you. Okay, thank you.