 Good morning. Good morning, everyone. Thank you for coming. I think we can get started now. It's a great pleasure and honor for me to be able to introduce Maria Grant, who's really a consummate clinician scientist and whose research has taught us more about diabetic retinopathy, angiogenesis, stem cells, circadian-clock gene regulation, and vascular repair. And you'll hear more about her work at the 11 o'clock lecture this morning if you're able to attend as well. But right now, she's going to present us with stem and progenitor cells. Do they have a future in retinal vascular repair? So please join me in welcoming Maria Grant. Good morning, everyone. And thank you, Emmy, for the invitation. It's a beautiful, beautiful state. It's my first time in Salt Lake City. And so it was just gorgeous. Emmy and her husband were very kind to show me around this yesterday a little bit, so. Well, I'm very happy to be here and talk to you a little bit. Can you hear me all right? Can you hear me? OK, good. I'm a diabetologist by training, even though I have spent most of my career working on the retina. And from a diabetic perspective, we have wonderful tools to correct macrovascular disease. We have stenting. We have revascularization procedures. But the major problem in diabetes really isn't macrovascular, isn't large vessel disease, even though large vessel disease, as you can see here in this patient that has a peripheral artery occlusion, leading to one of the most devastating complications, which is chronic wounds in the diabetic patient. But in the retina, you can see here there's an area of macular ischemia. But all of this really leads to the idea that we can fix a bit big vessels, but we don't really have the tools to go in and treat the microvascular disease. And that's really what diabetes is about. Diabetic microvascular complications are really the most disabling of all the complications of diabetes. And as you know, in the retina, after prolonged ischemia, you get compensatory angiogenesis. But what you may not know is there's equally, and perhaps harder to find, compensatory angiogenesis in the kidney. So there is a systemic ischemia that leads to pathology. So in much of what I'm going to talk to you about today, it'll be about how we can potentially correct early end disease rather than wait until we have the very complicated, very end stage aspects of microvascular disease. And as you know, I primarily work in the retina, ocular models to test our hypotheses. So this is work from a collaborator, Patricia Parsons-Wittaker, and she's a NASA bioengineer. And what Patricia did several years ago was to take the typical fluorescein angiogram and look at it in much more detail. And if you can, sorry, I have the wrong glasses for everything, sorry. You know, my pointer here, is it worth, okay, there we go, there we go, that's better. Patricia did was take patients in different categories, mild non-proliferative, moderate, severe non-proliferative and PDR patients. And she went and looked at the arteries, she looked at the veins, and she looked all the way down to the very small level, such as the sixth to ninth branch points. So these are the tiny, tiny little vessels. And what she found, which is really interesting, is that unlike the way I, well, perhaps you thought about the disease, is that in diabetic retinopathy you have progressive vasodegeneration, eventually get acetylcapillaries, you lose parasites, you lose endothelium, and then ultimately you get so much ischemia that you get angiogenesis. But what she saw was that early on, when the patient, this is normal, when the patients had moderate non-PDR, there actually was a burst of small vessels. And most of you, I'm sure the residents of fellows, I think of the burst of vessels as in the later stages when you have ocular neovascularization. But she found it early on. And Patricia presented this in an NIH diabetic retinopathy workshop. And the only thing I could think of was, wow, this is the retinous attempt at regeneration early on, and we missed it. So this was really inspiring to me and to hopefully other people. And now we're doing a collaborative study with her to take the patient over time in these various groups and trying to assess some of their circulating progenitors. And so it's trying to determine whether your vascular repairative cells can actually be a biomarker of what is happening in the retina and throughout the body. So this is a stem cell talk. And I know that it's early in the morning and I know there's a lot of enthusiasm, I hope, for stem cell research. But the stem cell story, as you probably know, has been kind of one of a lot of ups and downs, a lot of excitement, a lot of disappointment. And not very long ago, American Advanced Cell Technologies had a exciting, well, exciting results on two patients where there was evidence, at least the paper, suggested that there was some improvement in injecting embryonic stem cells to repair the RP layer in these patients. And that they did have some, potentially, the patients, at least they suggested that they had some improvement. One patient said that she could see color is better, the other patient said that she could actually thread a needle. And so these are all interesting. However, this, in Lancet, it was not a placebo-controlled study. So, and it was very, very controversial. It raised a lot of ups and downs. And at the same time, probably in the same week, Geron stopped their neural stem cell trial, basically embryonic stem cells for spinal cord injury. So, because of side effects. So this was the, some recent studies, there's also, as you probably know, there's much work now in IPS cells. And IPS cells are very, very exciting. And Yamanaka received the Nobel Prize because of this finding where he basically was able to identify four transcriptional factors that could reprogram differentiated cell back to a de-differentiated state. And the enthusiasm for his work led to a clinical trial being initiated for AMD using IPS cells in Japan. And in our own country, Susie Anna Parks, who's a clinician scientist at UC Davis, has a study where she's taking bone marrow-derived CD34 cells, a progenitor population. We're gonna talk about more in a few minutes, and using a patient's own cells. But she has it for pretty much every ocular condition, AMD, branch band occlusion, branch artery occlusion, diabetic retinopathy. So it's a very open inclusion criteria. However, she also has some pretty rigorous exclusion criteria. It's a phase one feasibility safety study. She's not really looking to see efficacy. However, she did indicate, she presented, she came to Indiana University last spring. And she did indicate that there was some evidence of improvement on visual acuity with two of the patients in the study. I think she's done five so far. And then, our colleagues in Ireland, in Belfast, Alan Stitt, he has initiated some interesting work, once again, using progenitor populations. And these are all autologous, so the patient's own cells. And the intention here is to use it for the vasodegenerative phase of diabetic retinopathy. So treating these patients early when there's evidence of macular schemia, when there's evidence of retinoschemia. And then, there's, in Mexico, I think it's probably one of the more exciting studies because the Mexican FDA is unique in that you can ask for a specific IND per patient. So if you can make a good case for this patient potentially benefiting from a therapy, then there's a chance to move forward. And so they actually, we're with a company that I'm working with, BetaSTEM, right now. We're trying to use, we're trying to move forward in Mexico rather than in the U.S. Okay, so why STEM cells? Well, and why cell therapy? For one thing, you have to, I'd like you to think about the possibilities of early intervention in trying to stabilize the vasculature, trying to intervene before there's a lot of, excuse me, pathology. And the idea is that their vasculoprogenitors can regenerate blood vessels. These cells are able to do this. And then, not only can they repair the retinal vasculature, but they can then provide nutrition and support to the neural retina. So the pioneers, and actually the individuals and researchers that have done more to move the field forward are actually the cardiologists, the interventional cardiologists, because they've been using bone marrow drive cells for nearly a decade. And the results is overall very encouraging. Even though, as you can probably imagine, when you can't always clearly define a population, which is a problem with these vasculoprogenitors, there's a lot of mixed results. And there's a, and you think about it, you're taking some of the very sickest patients that have failed conventional medical regimens, such as revascularization procedures, they're maxed out on their medication. And these are the patients you're studying. So you're already at a disadvantage. So even despite all that, there is evidence that these cells improve left ventricular remodeling. And there was a large meta-analysis about almost greater than 2,600 patients suggesting that it reduces the incidence of death as well as major adverse events. Finally, because of the fact that we've been doing bone marrow transplantation studies for greater than 35 years now, there's a lot of knowledge about how to use these cells, these bone marrow drive cells. And right now, there's 1,800 plus clinical trials using bone marrow drive cells. So it is part of our armamentarium of care for our patients. And hence, exciting. So why in the retina? Well, for one thing, cells can adapt, they can respond to a changing environment. And you can all appreciate how much that occurs on our patients, they do change. And not only like we try to target with one agent like an anti-veg or anti-growth factor of some sort, these cells produce numerous factors. And the other thing that is interesting is that most of the anti-angiogenics and lasering, et cetera, really inhibit late and it's a different type of issue. They're blocking hypoxia-driven neovascularization rather than facilitating or improving the retina. And it's destructive when you put 3,500 burns to a retina. So what this slide talks about is a little bit of the different populations. I'm gonna tell you a little bit about, some of it is our work and some is from other labs just because I feel that I'd like to give you kind of a fair representation of what's out there. IU Indiana University is one of the leaders in adipocyte-derived stem cells. And these cells can make parasites. Marty Freelander almost a decade ago showed some studies that these CD-45 high cells can make microglia and support the vasculature. My lab spent most of our time trying to understand the mechanisms by which CD-34 cells can repair. These cells produce primarily paracrine factors to support the resident vasculature. Indiana University is the home where the first discovery of the endothelial colony forming cell. These are interesting cells and we'll talk a lot about them in the next few minutes. So all those different cells have different functions. And as you know, most of you know that the model of retinopathy prematurity is a model developed by Lois Smith and others in many, many years ago. It's kind of a gold standard model. And I'm gonna show you some data where we use this model. And actually inject the cells at different time points to see if we can prevent vaso obliteration, to see if we can prevent the compensatory neovascularization because when these mouse pups get out of the high oxygen, the retina senses it is relative hypoxia. This is not a model of human disease, but it's a convenient model that we can use to test our hypotheses. And in this model, Mendel et al, did a very interesting study. They, thinking clinical trials here, they used human adipocyte drive stem cells. And these are basically mesenchymal stem cells that are obtained from fat. And most patients have some fat that you can, and you don't need a lot. So you probably need about six to seven MLs of a fat slurry. So most patients can provide that easily. And they did do this with human cells. And what they showed here is that if you, this is the saline injected. So these are the controls. And they injected at P2. So really when the pups are just born in P12, two different time points to see whether these cells could prevent vaso obliteration. And what they do is they actually did some, there's some improvement, there's less vaso obliteration in the pups that receive the human cells. And there's less vaso obliteration when you injected, this is at P12. And so the idea here is that, yes, it appears that these cells may participate in vascular repair, and vascular stabilization. These cells don't form endothelial cells, but typically just form parasites. So stabilizing the vasoculture by promoting parasite growth. Now, this is what these cells look like. They really do take an abluminal position. And this is distinct from some of the other work I'm gonna show you in a minute. So these are clearly parasite locations. And this is Marty Friedlander's work. I bring it up because just so you realize there's a potential for many, many different types of cells to have utility. And same idea here, these are cells, bone marrow derived, you inject some in the eye. Marty is very, very high level, it's like a million cells. I'm gonna show you data where we're using 10,000 cells. So I'm not sure how well these cells would work if you use less. These are the vehicle treated. And this is just in the interest of time. I'm gonna be very brief here. You can see in the same model that these cells reduce central vaso obliteration, which typically if you prevent this phenomena, you correct it and you prevent the neovascularization. And once again, these are abluminal. These cells, the microglia are abluminal. And he was able to show, he did some studies which were very important. He did studies showing that there was no toxicity of these cells after six months. That's the kind of data the FDA wants. They wanna know that there's no deleterious effect. They don't care about benefit. They really care about their issues or safety. So this is a study that we did in collaboration with advanced cell technologies back almost a decade ago where we took embryonic stem cells, human embryonic stem cells, and we injected them into a diabetic model. And these cells are pretty fascinating in that we attempted to generate a hemangioblast, a cell that could make all the blood elements as well as endothelial cells. So going back to a true embryological cell type. And these cells, as you can see here, they are GFE positive. They were genetically engineered to be GFE positive. And they also expressed the endothelial marker CD31 in the human. These are human cells in a rodent model. And you can see how these cells are so smart. They home to areas of injury and they try to fix the vessels. We did some long-term studies up to three months, not six months. And we did not have any issues with teratoma formation or any side effects of those. And this is in another model because I think it's very important to validate everything we're doing in multiple models. And the cells, the blood cells, basically if you look here, this is the control, this is Hanlon's chemia model, and this is the injured limb. You can see that if you inject locally, these cells can improve perfusion. So we could not test that in the retina. We couldn't look at blood flow in the retina. We didn't have the expertise at the time. That's something we're actually working on now to show that the vessels we generate are actually good vessels. They're perfusing. They're actually bringing blood to the retina. This is what we did here because we have the tools to do it. And this is a study by Jerry Luddy. This just came in, Jerry Luddy's group and others at Wilmer, they generated IPS cells from cord blood. And the interesting thing they found is that they tried to make IPS cells, used IPS cells from fibroblasts and other cell types, but the cord blood, which is not surprising because these are basically robust young cells, could, were better. And he was able to show that you can generate two types of cells, an endothelial cell that has a certain very characteristic markers, CD105 high, 144, and CD140 negative, and basically the opposite cell is a parasite precursor. And what he's able to show is that if you can see this, I don't know, it's a little hard to see, but in the ischemic reperfusion model, there's areas of vasodegeneration. And this model is when you put pressure, I don't know how familiar you are. It's a glaucoma model that Tim Kern's lab characterized that, wow, it causes ganglion cell death, but also causes endothelial. And parasite loss. And this model, Jerry's group injected the cells and they act normal. They home to these areas that you can see that there's no, there's a capillary tube there because he stands with collagen four and you don't see any collagen four standing here. So that would suggest that it's like a tube being lost. Or I'm sorry, this is CD31 he used here. So the idea here is these cells home to these areas where there's no capillaries endothelial cells and degenerate capillaries. And then if you inject them typically, intravitrally they form parasites, but if you inject them systemically, you get a cell that is intraluminal. So he was able to show that the route at which you administer the stem cell helps dictate the phenotype. But these cells are very plastic cells. And the cells I'm gonna talk to you about next are really less plastic. They are really more what we call a multipotent, not pluripotent, these cells can pretty much make anything. The multipotent tend to make fewer different types of cells. And they tend to be the case with adult cells. But these are cord blood derived IPS cells. Very interesting work. So what about the adult cells? The adult cells I think it's really important to appreciate with a stem cell therapy that the FDA wants you to use the patient's own cells. That's the safest. And where did all this start with? It started from work that was done out of Jeff Isner's lab back in the late 90s in a very talented postdoctoral fellow by the name of Nakayama Asahara. And Asahara identified in a patient population that had very bad peripheral vascular disease, these patients were part of Jeff Isner's clinical trial where he was giving virus into the, basically a virus that overexpresses VGF into the legs of these patients. And as part of monitoring what's going on in the periphery, they were looking for growth factor expression, they were looking for soluble VGF, they were looking for all sorts of things, mostly VGF expression. So if you inject into these patients that basically are gonna have an amputated limb if nothing is done. He found that there were a lot of these cells. And it was very odd, he didn't know exactly what they were because they didn't meet the usual criteria of white cells. And so that embarked on this great adventure where he was able to show that some of these cells that are mobilized by this VGF therapy actually can form blood vessels in if you inject them into a mouse. And here you can see these are fluorescently green labeled cells, this is staining for the vasculature CD31. And you can see that they associate. And this is really the beginning of the field that of using autologous cells for vascular repair. Originally there were six papers in 1999, nearly 15,000. And now, so these cells are very, very important part of our cure and interest for our patients. So let me tell you about where they come from and what's known. These cells are bone marrow derived, the hematopoietic stem cell can generate a cell, a hematopoietic progenitor that then conform what's called now a circulating angiogenic cell. And this is a big controversy. We don't call them endothelial progenitor cells anymore because they don't make endothelium well. They really serve to support vascular repair by producing paracrine factors. This same cell can make all the blood elements, all the T cells, the lymphocytes, the B cells, and all the granulopoietic components. And in many years ago, we did a study where we took healthy cells from a non-diabetic patient, put them in the ischemia reperfusion model. We fluorescently labeled these cells, injected them into the eye. And once again, you can see that these cells home. So these are human cells at home to areas of vascular injury. And then the field is really fraught with some controversy. And this is the beginning of it. If you take peripheral blood that CD34 labeled, which is how we characterize our cells, you then can further characterize them. Trust me, we have for many of these markers. And these circulating angiogenic cells are called early endothelial progenitor cells. There's a lot of nomenclature issues. But they have their hematopoietic in origin. And this is the essence of this big controversy. The cells that we study primarily are bone marrow derived. And these are how they're characterized. Merv Yoder, about nearly 10 years ago, identified a non-hematopoietic cell. It's CD45 negative, meaning it's non-hematopoietic. It's actually vascular wall-derived. And you can see there's a lot of the same markers here. They're actually the same. You can see there's some differences. This is not nothing. You have to think about a cell as being very dynamic. And again, it can express one marker, one men, and another marker the next. So it's not that this is a set in stone that this is what these cells are expressing, all of these markers. This is markers that are representative of what these cells express. But Merv found by basically trypsinizing the vasculature that there's some very robust progenitor cells within the vasculature itself. They're not bone marrow. Their source is the actual endothelium. And everybody in this room has them. And as you get older, you deplete them. And if you're sick like you're diabetic, you also deplete them because you're constantly using them in an attempt to repair. And we studied over 100 diabetic patients. And the only patients that we could isolate, diabetic patients we could isolate these cells from were patients that had no complications. So if you're gonna use this cell therapy, this for autologous cell therapy, you have to obtain it when the person's newly diagnosed and bank the cells and then use them later because you're not gonna be able to get them when that patient 20, 30 years down the road has peripheral vascular disease and may need them. So anyhow, let me tell you a little bit about how they're isolated. The colony-forming cells, which is a type of early endothelial progenitor, is basically you just take mononuclear cells from the blood and you plate them. And then the cells that don't adhere, so the non-adherent cells, they're then replated on fibronectin. And then after about five days, you get these pretty little colonies. And you can actually enumerate these. Most, 99.9% of the clinical trials done in Europe and in the United States in the 90, or actually probably the early 2000s and up to today are with these cells because you can isolate them. They typically isolate them from the bone marrow because you can get more of them and you can grow up enough of them to be able to re-inject them into a patient five days after their heart attack. These are the cells that myrviota at Indiana University identified. These are endothelial cells that are sloughed off into the blood. You need about 200 ml of blood from a patient. You cannot get these from the bone marrow. You have to get them for a full blood or fat. I'm gonna tell you about that in a minute. And these cells grow out after about 21 days. Like I said, if you're healthy, you can grow these out. If you have a disease that really causes damage to your vasculature, you're not gonna be able to grow them because your body's already attempted to replace the damage of vasculature by using up these cells. So these are just some characteristics. The major characteristic and where myrviota and his colleagues have really been very critical of the early progenitor work is that the progenitor cells, the CD34 cells that are 45 positive, they do not form in vivo vessels and they aren't as robust in forming vessels and they aren't as good for expanding in vitro. And these are all true. There's nothing arguable about it. It's true. The cellular origin is typically the marrow or the blood for these cells, whereas the origin for the ECFCs is the vessel wall. And the gold standard that is used to show that they do indeed make blood vessels is when you take the human cells and plant them into a skid mouse and you take the human cells, kind of mix them up in a collagen, fibronectin gel, and you actually implant them and you can see that after a period of time, the human cells form blood vessels with the mouse cells. And this is the gold standard. And this is important work. It has its limitations and while we were publishing work saying you can't grow ECFCs from diabetic patients, Merviota found very similarly that patients that need these cells the most, you can't obtain them. So what he's done is now used cord blood and generated IPS cells that instead of making endothelial cells, make these progenitor cells. So, and that paper is just coming out in nature of biotechnology. So what about these cells? I've talked a lot about their good and their bad points, but let's see them do something. And so here I'm gonna show you some of our data where we've combined used exogenous and CD34 cells and these ECFCs and to see how they work in this ROP model. And the whole idea was that if you think about it from a sort of an ideal perspective, the CD34 cells are little powerhouses of growth factors whereas the ECFCs are of cells that can actually make endothelial cells. So if you combine them, you might make better, stronger blood vessels. So that was our hypothesis. And so we injected in the ROP model and unlike the levels that Marty Freelander, we use really low levels. Why did we use these? Because we showed that individually these, this number of 10,000s of the CD34 and Alan Stitt and others have shown that 100,000 of the ECFCs can actually make an impact on this model. So we combine them and here is the data. So if you look at the areas of vasoblideration are shown up in yellow just to make it easier. What's interesting is all these cells, compared to the saline group, all of them do a pretty good job at preventing vasoblideration when you inject them at P12 and sacrifice the antler euthanize them at P17. As you'd expect, if you prevent the vasoblideration you're gonna also prevent neovascularization which is exactly what we saw. And we didn't see that anyone that the combination was better. And this is a different experiment. We injected at P6. So this is before the puffs go into high oxygen and then we looked at them right shortly after we took them out of oxygen long enough to sacrifice them or euthanize them. And you could see that yeah, there's a little bit of significant but it's not real dramatic. So these cells didn't prevent part of the pathology. They helped prevent it but it's not dramatic. Still significant but not dramatic. And then this is sort of the combination of the data of showing where it's really better if you try to translate this into like a patient or baby if you took their cord blood cells and you could isolate the CD34s or the ECFCs from an infant you could wait till there was actual retinal pathology to inject the cells and see some improvement. You don't have to kind of pretreat anticipating which babies and it's sometimes it's very difficult to identify which babies are gonna go on and have ROP and others that will not. And here's just a confocal image of we engineer the GFP positive ECFCs just to track them better. And truly they do go to the endothelium. They do form the interesting part of within making endothelial cells. They're not parasites. They have a very intravascular location even if we inject them intravitrally. So they home and then they actually become endothelial cells within the vasculature of this model. And then if this is just some more of the same you can see looking at the different vascular beds it tends that what happens is at this time point in the pup they're really forming the deep and intermediate layer that's where those cells are going. They're not in the superficial layer they're more in the deep layer where the actual more remodeling is occurring. So that's all I'm gonna talk about as far as ROP and I'm going back to diabetes because first of all I think before we go to diabetes just the idea that a baby, a little infant could actually a premature infant you can obtain the cord especially it's very easy to do. You can isolate the cells very easily. Most medical centers have GMP facilities not all for cell preparation but they can be sent to a facility that does. You can take and literally have the cells ready and available the baby's own cells and they're really you can make like a stock of them so that baby has cells the rest of their life and you can actually think about yes you can start with the eye but the baby has damage in the lungs the baby has damage in the brains and these are a source of cells that can be used throughout to repair all the vasculature. So going back to the diabetes in the CD34 cells there's a lot of problems with these cells. The problem is that they don't work well and diabetic cells if you're gonna use the patient's own cells to repair they are non-functional and the data here is old data but I think it shows it better than anything. All the healthy individuals in this audience should have a typical biphasic response to stromal drive factor one if you look at migration. So your CD34 cells should have this kind of pattern. A diabetic patient pretty much has a flat response. They do not sense this factor. They have their receptor but there's no downstream signaling for this receptor to promote migration and it's not just SDF1 but VGF so these are profound migratory defects and what as you can appreciate wherever you inject these cells or wherever they come from they need to go to an area to repair so they come from the bone marrow go into the circulation then they go to the tissue whether it's the ratten or the heart, the ischemic limb and they home there and migrate to that region to repair so if the cells are not capable of migrating then they can't fulfill their function so we spent a lot of time trying to understand what makes these diabetic cells so dysfunctional and one thing that we identified is their levels of bioavailable nitric oxide is very low and if you consider the fact that here is the typical flat response this is a diabetic patient if you give them an NO nitric oxide donor even for 10 seconds it makes these cells migrate better and then if you take and put an NO scavenger to take away the NO donor that you use it brings them back to normal so the levels of bioavailable nitric oxide are critical for their migration and we've done a lot of self signaling and it's not easy because you have to work with very small numbers of cells but basically if you restore the levels of bioavailable nitric oxide that improves it you can do that by reducing oxidative stress by inhibiting NADH oxidase and I'm gonna show you a little bit it's the TGF beta in the last few minutes work that we did in collaboration with Julia Busek's lab is that in diabetes you have profound abnormalities in lipid metabolism and acid sphing myelinase is critical to eliminating ceramide which is very detrimental and if you inhibit this you inhibit ceramide formation and that helps the cells also the nuclear receptor LXR work that we published in diabetes last year if you activate this receptor this is a receptor that takes cholesterol out of a cell and then moves it back into the circulation and so you're improving systemic cholesterol metabolism that can also improve the function of these cells interestingly the cells themselves have this nuclear receptor and they have the transporters to get the cholesterol out of the cell so you're actually the CD34 cells when you activate it with a pharmacological agonist you can move the cholesterol out of the cell and that tends to make the membrane of the cell more fluid it moves better and then it can migrate better because diabetic cells are like little stiff tennis balls and you really want to be kind of like a deflated tennis ball because a deflated tennis ball can move in and out of spaces better and interesting LXR activation or inhibition of acid semi-malonase does that and then the study that we're doing now with Patricia Parsons-Wittiger we're looking at the renin angiotensin access within the CD34 cells themselves to see if you can predict which patients are going to progress and which ones aren't and so far we have really exciting data that if a patient has more of the protective arm this is angiotensin N1-7 and the mass receptor the receptor for angiotensin N1-7 that they migrate better and that these patients don't appear to develop retinopathy that the other side of the coin is the everybody has diabetic patients on angiotensin ACE inhibitors ACE inhibitors tend to block the ACE1 receptor which is a deleterious arm that automatically if you block the bad arm of the renin angiotensin you kind of bolster the good arm it's just sort of the yin and yang of the system and I don't really have a lot of time if anybody's interested I'd be happy to send any articles on that but it's really, really fascinating that the cells are like a little biosensor of what's going on in the vasculature and it's really, really exciting to start using cells as biomarkers of disease progression. So in the last couple of minutes here I'm gonna tell you about a study this is a study that we're trying to move forward in Mexico diabetic patients and their cells have a lot of molecular defects and one, in addition to the lack of nitric oxide they tend to have very little ability to proliferate and basically they're very quiescent and what happens is part of a compensation of all the systemic inflammation TGF beta levels are increased within these cells and that is like the break that TGF beta is like the break for any progenitor cell to proliferate because in the bone marrow the levels of TGF beta are what high levels are what keep the hematopoietic cell quiescent. So if you have excess of this as part of a disease process such as diabetes these cells don't proliferate and you can do whatever you want but the only thing that fixes them is taking those high levels and normalizing them back to a lower level or more physiologic level. So what we found was the typical we did hundreds of patients and the TGF beta axis is associated with a low level of this CXCF4 which is the receptor for SDF1, low nitric oxide and inability of proliferate and if you inject diabetic cells into the ischemia reperfusion model the cells just stay on the surface of the vitreous they don't go anywhere because they have this migratory defect. So what we did is we used morpholinoantersense to these are the control morpholinos and this is the diabetic here, the TGF beta. What we did is we were able to we just exposed them overnight, 16 hours to the morpholinos and this is co-localization with the vasculature. So we took diabetic and healthy, healthy as yellow diabetic as purple. We treated them either with the morpholinos for the control ones or the TGF beta and then we injected them into the ischemia reperfusion model. So healthy, like I mentioned, healthy cells with a control PMO they can migrate from the vitreous cavity to the areas of injury in the ischemia reperfusion model. And as one would expect treating them with the TGF beta PMO they can also work but we're reducing levels that are already low so it doesn't really have much of an effect. It's, they're still good cells but the diabetic cells have high levels of TGF beta. They don't migrate. They just, this is what they look like on the surface of the retina. They're fluorescently labeled so you can see them but they just stay there on the surface. If you treat them with the PMO to TGF beta they actually can home and repair and act like normal cells. And we've done this in hundreds of animals with hundreds of patients. It's very consistent. Both type one and type two patients have higher levels of TGF beta and this is just a compensation. You can imagine, I know that we didn't talk about that the disease pathogenesis of diabetes but it's a very pro-inflammatory state. So the body tends, a lot of the diabetics have higher levels of stomach TGF beta and a lot of the cells are expressing higher levels of TGF beta. And it doesn't always have a deleterious impact. It has a deleterious impact in cells that have to migrate. So we looked at these, this is a different assay where we're looking at nitric oxide generation because this is critical to their migratory function and DAPH FM is a fluorescent marker. And so all the healthy cells, they release a good robust amount of nitric oxide that's shown in yellow. If you treat them with the TGF beta PMO as you get a good level. And here, if you block, this is AMD 3100 which blocks the CXCR4 receptor or the receptor for SDF1. You block nitric oxide generation. So that proves that, or at least suggests that part of how TGF beta works is through the CXCR4. And that CXCR4 is responsible in part for the generation of nitric oxide which is needed for these cells to migrate. So how do we translate this? This is the fun part as clinicians, I think you really wanna see how could we make this happen? Well, this is what we're trying to do in Mexico right now. If a patient comes in, they get leukophoresis meaning they get a fair amount of their peripheral blood cells isolated. There's a clinically validated model that's used to isolate CD34 cells. It's a clinic max. And you do this in a GMP facility because that's required by the FDA to for any sort of cell therapy. So then just look, if you look at this part, this is kind of what happens. You isolate the cells, you incubate them overnight with some sort of pharmacological agent here. TGF beta PMO is the one that we're interested in. And then you can then inject. And these cells should go back and facilitate revascularization of an ischemic eye. What conditions are we focusing on? Macular ischemia, the patients that have very severe macular ischemia is one of the conditions that we're trying to treat. As you know, there's no current treatment for that. And so going back to the babies, this is kind of the paradigm for them. The baby's cord blood is available. You immediately isolate the CD34 cells. You can freeze these cells down. That's not a problem. You can purify the 34s and then freeze them down. You can freeze down the whole cord and then you can isolate them. They're fairly robust cells. You can take within the cord. There's also the vascular progenitors from the umbilical cord. Those vascular progenitors then can be expanded and the baby can have these cells within a month. And then the idea is the purification expansion can be used to treat the conditions you combine them and this combination therapy may show more utility. Now it's interesting, our data that I showed you today doesn't really support the generic CD34 and the generic ECFC. They both work really well in the ROP model. So combining them didn't show a difference but work we're doing now on Neuropellan. Neuropellan is one of the surface receptors that is critical for the activation of VGF. And it appears that within all these populations, especially the ECFCs, there's a Neuropellan positive population that is really the cell we're interested in. It's the the most robust cell. So we're now isolating those cells and testing them in the same paradigm because we think we'll see then that the ECFCs are gonna be really really good cells for vascular repair. Once again, these are all available from the baby's own blood so it's not a problem from the cord. The other issue is that once again it's otologous. The FDA is probably gonna be more difficult about twice two different type of isolation protocols. Yet in many ways what was given to the patients were ex vivo expanded and it was a mixed population. So as long as once again there's no toxicity demonstrated and what we're doing now is doing, we're refining the populations we wanna use and doing long-term toxicity studies in the ischemia reperfusion model because we take human cells into a skid mouse that has this injury and then we have to show for the FDA six months that the cells haven't caused any damage. I'm really surprised if they would. I don't think they would. Susanna Parks, we helped her with that study. The CD34 cells will not, I'd be surprised that the ECFCs would cause any damage. So that's kind of ongoing right now in preparation to try to get an IND at some point for use of these cells in the babies. So the last slide is just to review and just to review the possibilities the data, I didn't talk a lot about these edipocyte derived stem cells but clearly, at least in my diabetic population, a source of fat is never gonna be a problem. So and we're actually trying to see if within that same mass of fat you get these mesenchymal stem cells that are perivascular cells but amazingly, thank God for them, there's also endothelial cells or there's these ECFCs. So from the same mass of fat, we call it the stromovascular faction, you can get the parasite type cells mesenchymal cells and you can also get ECFCs. I don't know, I think they're gonna be dysfunctional just like all the diabetic ones but we isolated them in the past from the blood. So now we're isolating them from the fat of the diabetic and I don't know if the fat would protect them but sometimes you just have to do the experiment rather than guess that oh, they're gonna be not good either. So we're doing that, we're in the process of doing that, trying to understand. This is the cells that Marty Friedlander studied in death, these cells make microglia, microglia are support to the vascular. Embryonic stem cells, I don't think there's a much future in these cells because IPS cells have really gained popularity because they're taking a differentiated cell and de-differentiated rather take an embryonic cell and differentiating it but embryonic stem cells really give you an unlimited supply but every cell line that's characterized has to be tested for teratoma formation and rigorously examined before they can be used in humans but there are like I mentioned there's the AMD trial using these cells, there's trials for ALS, there's trials for different type, interesting conditions, a lot of neural damage, different embryonic stem cell populations but each of them have to be rigorously tested. The IPS cells, you know, there's some future, they're in California, the SIRM is using IPS cells, if you take IPS cells and you don't do anything to them, they make RP cells, they just love to make RP cells, basically that's their default program and there's a clinical trial now at UCLA using IPS-derived cells for severe geographic atrophy. Then these are the cells, I've talked to you about great length as well as ECFC, I don't know about combination synergy, maybe you have to optimize these two populations to get the synergy because they're too good on their own. So at least the model is a limit, the model doesn't help refine the cells. So we're probably gonna have to go to another model of ocular disease rather than the ROP model which is a great model to just evaluate vaso obliteration and urvascularization and then diabetic cells, the future really is about protecting the patient's microvasculature. Sadly, the diabetic vasculature has exhausted its reparative potential and these cells, the bone marrow, you know, they're close to, it's an amazing, like 30 billion cells produced out of the bone marrow, even in an aged patient, that's your body generates so many cells. So the bone marrow is a lifetime source of cells and that's why I continue to work with these cells despite all the issues. The ECFCs are gonna be exhausted. They're not really a viable opportunity. I think that's why Merviota made such heroics to generate these cells from IPS cells. So take a patient's fibroblast, program them through IPS technology back to ECFCs to have good repair. I think this is a viable option, but it's gonna have to be done on a patient-by-patient basis. Maybe they can come up with a generic ECFC that you can use to treat patients. That's the goal. Everybody wants to just reach on a shelf and use that same cell population to treat everybody. The FDA still is not really excited about it. It's gonna be a while before this technology moves forward, I think, into big clinical trials because it takes a lot of time, effort, and resources to make IPS cells from one of your cells. Just one cell line would probably be about $5,000. Now that doesn't seem like a lot, but that's just to generate the cells. And then you have to test them. I'm sure the FDA is gonna require rigorous testing before that same cell drive from that patient can go back to that patient. So I think it's interesting. I'm not sure how California got away with it, that they're doing the IPS work, but that's for patients that are basically blind with AMD. And so I think it's a compassionate basis protocol. So I think the most important part, are all the great people that really did this work, the folks in my lab. I'm really blessed to have an outstanding group of people. Sergio Cavalero has been with me for 26 years and done a lot of the animal model work. Sergio Licazi did the work that you saw on the most recently, he's at IU. Sergio is still in Florida. They're still working together though. And a lot of my graduate students that have been outstanding previous members, Sugata Hazra, she's a postdoc here at the University of Utah. She was my graduate student, really great girl. She did a lot of the LXR activation work. And then my collaborators, Merv Yoder, who used to be my most rigorous competitor. I used to be feared that he would read my grants because he didn't believe in the cells that I worked on. It was like, now I'm working with him. So life is very funny and things just turn around. So, but I think you've become, having to confront your competitors daily and defending your research makes you a better scientist. And also it makes you open-minded because you realize there's, for the stem cell work, there's many, many opportunities for great stem cell options. And then my other collaborators at IU, Julia Busek has been a longtime collaborator. She's really helped with all the work with our lipid metabolism. And that's an area of diabetes research that's really underappreciated. And I'm really grateful for our collaboration and our funding sources. And thank you for your time and attention. I tried to end her a little early, sir. So, that was fascinating and obviously a subject that's gendered a lot of excitement. First an observation and then a question. It's my feeling that one of the biggest things has hurt this field and sadly a couple. One issue that I see over and over. Yeah, I think it's a great question. You know, I go back to, I always defend that, you know, the diabetic CD34 cell is so quiescent. It never proliferates. They should proliferate. So you're really taking, you know, the TGF beta levels are really high. You're not making the cell a malignant cell. You're just taking it and bringing the levels down to a normal range. So I think if you identify a key factor like TGF beta and you can normalize it, I think that's very safe. You know, we did a lot of work with the embryonic stem cells like 2005, 2007. And they are very, very potent cells. And they, my fear is with those cells. And I, at this point, you know, I don't want to be a heretic because I spend a lot of time working on them. And I don't feel that they have much of a future because the IPS cells, you can make IPS cells from without a virus. So you can do it chemically and that's safer. And I think there's a lot of hype about IPS cells. And so far, you know, so far there hasn't been any, you know, to my knowledge, at least the literature that I've read, there hasn't been any really deleterious outcomes of the IPS cells. I still think, and maybe it's just because I take care of patients and I realize, you know, I want something now. I don't want to wait 10 years. You know, and I would like any patient to have that opportunity. There is so much work in developing an IPS cell line that it's, you know, there's gonna be a lot, there's gonna be limited the number of patients that can have that. There's gonna be a lot of money. It's gonna be a very expensive way to deal with things. So, you know, I still think autologous, minimal manipulation of a patient's own cells to kind of normalize them. I still think that's a really good strategy. You know, like I said, I'm in Indiana now. They're really very pro adipocyte-derived stem cells, which are very mesenchymal. Mesenchymal cells tend to make smooth and luscious cells in parasites. So, I think taking the fat out and isolating, you know, the cells from that, both two populations I think is gonna be great because nothing works in isolation. So, you're kind of synergized. I really like the idea of synergy of using combination therapy. So, I think the concerns are very, very legitimate. I think the days of embryonic stem cell research are, I mean, that's, and not because of the legal issues or the moral issues. We just have safer options, you know? So, yes. Thanks for the great talk. Thank you. I was wondering if you take these cells and make sure of whatever very important cells you have, would that same hyperglycemic milieu still eventually make a better generation than that square one? It's a very good question. And, you know, there's, will you take care of patients? You know how hard it is to get them to be compliant. But I think if a patient gets a stem cell therapy, I think they're gonna be more compliant to systemic management. I just think so. I mean, maybe I'm naive about it, but I would, you know, I would exactly, we would target the idea of lowering systemic TGF beta levels where the drug like Lusatin actually works to do that. So, I, you know, you're absolutely right. You can take a good cell, put it in a bad environment. How long is it gonna stay healthy? We're doing some hypoxia preconditioning experiments because that's kind of like, think about it. You're putting a cell, the cell that you're preparing under normaloxic conditions and then you put it in a horribly toxic environment. You should think about preparing it for that. It's like you prepare yourself for an exam, you study or, you know, you prepare yourself for, you know, working all day by getting a good night's sleep. You gotta optimize the situation and we don't do that typically. So I think there's gonna be a lot of, a lot of that in the future that's gonna be needed is to prepare the cell before, for exactly that environment. I would hope, you know, I think diabetic patients are amazing when there's some of them or so will do anything to help because they'll, you know, participate in research studies, they'll give their time, their effort, their, you know, everything. And, you know, those same patients, I think they wanna see something done. I'm hoping that they will be, those typically will become compliant. When they start losing their vision or have had a heart attack or something bad, it usually frightens them into compliance, sadly. You know, so, anyhow. The same concern, actually, is macrosgeneration. Yeah. And the reason why there's advanced geographic factors. About putting the cells in. Putting stem cells in that area. Yeah. That's likely to make them. Yeah. And I know a lot of our research has never. Right. I mean, we're doing some work with AMD and the hematopoietic stem cells, you can actually program them to become retinal pigment epithelial cells and they home, giving them systemically back into the eye. And I think, once again, I think you can't wait till the patient has such bad geographic atrophy that that microenvironment has lost all the signals. You have to treat it early. But it was nothing. Yeah, exactly. Yeah. And that's gonna be too late because they're very context, you know, sensitive. And I think, I think, I mean, as physician scientists, we need to kind of think ahead. We can't be doing the same thing over and over again. And that's what we're doing. You know, we're not really changing the way we treat AMD patients. You know, so the whole that we're trying to do now is try to identify whether we can do the same things we did with the mice with human cells. So once again, bone marrow drive cells, program them to RP cells. They actually differentiate once they get to the eye. But we've used human cells in eye models, you know, in rodent models. So whether it'll work in the patient or not. And why nature doesn't do it on its own, you know? So, oh, you're very welcome. Thank you. Thank you.